PEER REVIEW CBS-KNAW 2008–2013 To Collect, Study and Preserve
Self-evaluation report 2008–2013 of The CBS Fungal Biodiversity Centre (CBS-KNAW)
Utrecht 2014 CBS directors Prof. Dr Pedro W. Crous, Director (2002-present) Dr Mariëtte A. Oosterwegel, Managing Director (2012-present)
CBS Fungal Biodiversity Centre (CBS-KNAW) Uppsalalaan 8 3584 CT Utrecht The Netherlands T +31 (0)30 2122600 www.cbs.knaw.nl [email protected]
Postal address: P. O. Box 85167 3508 AD Utrecht The Netherlands Preface
It is with great pleasure that we herewith present the self-evaluation of the CBS Fungal Biodiversity Centre of the Royal Netherlands Academy of Arts and Sciences. The report evaluates our research accomplishments over the period of 2008–2013.
As stipulated by the Dutch self-evaluation protocol, the report contains general documentation pertaining to the institute as whole, while the research programmes and future perspectives are also presented.
The preparation of this report required input from numerous members of staff, and we would like to take this opportunity to thank all of them for their time and dedication. The present self-evaluation presents a good overview of the past performance of the CBS, and also clearly establishes our exciting future research goals in fungal biodiversity.
April 2014
Prof. dr P.W. Crous (Director) Dr M.A. Oosterwegel (Managing Director) Contents
1. CBS Fungal Biodiversity Centre (CBS-KNAW) 1 2. Evolutionary Phytopathology Programme – P.W. Crous 15 3. Origins of Pathogenicity in Clinical Fungi Programme – G.S. de Hoog 24 4. Yeast Research Programme – T. Boekhout 33 5. Applied and Industrial Mycology Programme – R.A. Samson 44 6. Fungal Physiology Programme – R.P. de Vries 54 7. Bioinformatics Programme – V. Robert 62 8. Collection – G. Verkley 79 9. Appendix - Bibliometric analyses 91
Additional information online: www.cbs.knaw.nl/index.php/research e.g. access to PDFs of key publications. 1. CBS Fungal Biodiversity Centre (CBS-KNAW)
1.1 Objectives and research area
Introduction and history
The AIM of the CBS Fungal Biodiversity Institute (CBS) is to study all aspects of fungal biodiversity in three focused themes, namely Agricultural, Industrial, and Medical mycology. CBS is one of four life science institutes in the Royal Netherlands Academy of Arts and Sciences (KNAW). It employs around 70 scientific staff, plus 20–30 support staff, which it shares with the Hubrecht Institute, its neighbour institute on De Uithof in Utrecht. CBS has an annual Academy budget of approximately 3 million euros, and generates an additional 3–4 million euros from industry and research grants. The CBS was established in Baarn in 1904 to house a collection of fungal cultures following a decision taken at the 11th International Botanical Congress at Vienna. For some time between the two World Wars CBS was financially supported by the Royal Netherlands Academy of Arts and Sciences, of which it became an official institute in 1968. Professor F.A.F.C. Went, who was responsible for founding the culture collection, appointed the first female professor in the Netherlands, Prof. J. Westerdijk, as first director of the institute in 1907. In December 2000 the CBS moved to Utrecht where it was accommodated in a completely renovated building, previously occupied by the Hubrecht Institute. The complete staff and collection of the yeast department from T.U. Delft also joined the staff, followed by the Dutch bacterial culture collection NCCB, containing the wild- type collections of the T.U. Delft and the Phabagen collection of Utrecht University.
The CBS culture collections of micro-organisms are embedded in a scientific environment, which guarantees state of the art quality checks, and simultaneously allows for the development of scientific programmes to improve the quality of the material entrusted to them. CBS was actively involved in setting the international standards for modern long-term preservation as well as for data storage, and recognising the necessity of data exchangeability. It was also one of the first public culture collections to make its complete open catalogue available on the internet. The CBS Collection has been ISO 9001:2008 certified since 2007. The Collection is partner of the Microbial Resource Research Infrastructure (MIRRI). MIRRI aims to build one pan-European infrastructure for microbial collections and to establish links across the distributed microbiological resource centre community, its users, policy makers and potential funders. As member of the Consortium for the Barcode of Life (CBOL), International Barcode of Life (IBOL), and coordinator of the European Consortium for the Barcode of Life (ECBOL), the CBS has undertaken an ambitious programme to generate DNA barcodes (ITS, LSU) of all its holdings [80 000 strains of filamentous fungi (open and non-public collection), and 9 500 yeast strains], transforming it into a Genetic Resource Centre that can actively serve the research community of the future. The present self-evaluation covers the period 2008–2013 of the research activities of the CBS.
Vision, mission and objectives of the institute
Vision: CBS is one of the major microbial Genetic Resource Centres in the world. As such, our unique fungal collection and databases, linked to a strong research staff component, enable us to participate in diverse EU, national and international research programmes, focusing on innovative research in industrial, medical and agricultural mycology. Our vision is to collect, study and preserve as much of the world’s fungal biodiversity as possible, and to unlock this resource for the international research community, using current state of the art techniques. Initiatives such as MycoBank, EU-BOLD, and Open Access Journals (Studies in Mycology, Persoonia, IMA Fungus) are true to the CBS mission and focus, and will further strengthen the institute, and promote mycology as discipline.
Part 1 - CBS Fungal Biodiversity Centre (CBS-KNAW) 1 Mission: The CBS Fungal Biodiversity Centre was established more than 110 years ago with the specific mission to preserve the world’s fungal biodiversity. Presently the CBS includes the largest known public service collection of living fungi. The CBS is the world leader for the elucidation of fungal biodiversity, of which presently is estimated that less than 10% is known. From its fast growing culture collection, the CBS aims to initiate fundamental and applied research with other (inter)national research groups on the exploration of the vast potential that fungi represent with respect to agricultural, industrial and medical themes. As such, the CBS as genetic resource centre is an (inter)national hub for mycological research.
Objectives: CBS has chosen to transform itself from simply being an international culture collection to being the trendsetter and gold standard of mycology. Our core business is the collection, and our research programmes add value to that collection. Each research programme consists of several projects. While some projects represent “discovery science”, focusing on discovering biodiversity, others are focused on “understanding processes”, and thus on striving to unravel metabolomic, proteomic or genomic complexities of specific fungal groups or species. A third approach is “recognition science”, developing diagnostic tools for practical use in industry, plant pathology, and in the hospital. Our main objective, however, is to collect, study, preserve and unlock the world’s fungal biodiversity, enabling us to do innovative research in agriculture, human health and industry.
Research area
The CBS Fungal Biodiversity Centre is an institute of biosystematics, with the culture collection at its core. The primary aim of its research programmes is to enhance its unique living collection of fungi by adding valuable new data and cultures. Research activities and programmes are coordinated in three focus areas (based on national and international relevance, impact, service to society, and available expertise).
Table 1.1. Research themes and programmes. Theme Programme Leader Period Agriculture Evolutionary Phytopathology Prof. dr P.W. Crous 2002– Medical Origins of Pathogenicity in Clinical Fungi Prof. dr G.S. de Hoog 2002– Medical Yeast Research Dr T. Boekhout 2002– Industry Applied and Industrial Mycology Prof. dr R.A. Samson 2002– Industry Fungal Physiology Programme Prof. dr R.P. de Vries 2009– All themes Bioinformatics Programme Dr V. Robert 2009–
1.2 Composition
Structure
The institute has three research themes containing six programmes (Table 1.1, Fig. 1.1). Each programme has at least a group leader, and senior technician. Each group leader is expected to build his/her own group through acquisition of external funds. Biosystematics requires combined expertise, and thus various members from different groups, including the collection and the bioinformatics programme, frequently work together on specific projects between the various groups. The present evaluation presents condensed information pertaining to the various research programmes. Additional information, such as publications, can be found online. Research is supported by technical staff with different expertise (e.g. molecular, culture collection, bioinformatics). Although technicians are assigned to specific programmes, most provide support CBS-wide, as well as to visiting scientists and students, pending on their fields of expertise. Non-scientific support is provided by the Collection, and
2 the Central Management & Services staff (Financial Administration, Personnel & Organisation, Technical Services and Infrastructure, ICT, and Science Information and Communication Services). Besides attending to their curatorial and service-related tasks, the scientists in the collection also collaborate in research with staff of different programmes.
DIRECTOR SCIENTIFIC ADVISORY BOARD Pedro Crous
MANAGING DIRECTOR SECRETARIAT Mariëtte Oosterwegel
CENTRAL MANAGEMENT & LIBRARY SERVICES
BIOINFORMATICS COLLECTION SERVICE UNIT Vincent Robert Gerard Verkley & Marizeth Groenewald Martin Meijer & Tineke van Doorn
AGRICULTURE HEALTH INDUSTRY
EVOLUTIONARY PHYTOPATHOLOGY ECOLOGY OF CLINICAL FUNGI APPLIED & INDUSTRIAL MYCOLOGY Pedro Crous Sybren de Hoog Rob Samson
YEAST BIODIVERSITY FUNGAL PHYSIOLOGY Teun Boekhout Ronald de Vries
Fig. 1.1. Organisation chart of CBS.
Management
Presently the CBS is managed by a directorate consisting of a director (P.W. Crous, since 2002), a management director (M. Oosterwegel; shared between Hubrecht Institute/CBS, since 2012), and a managerial assistant (M. Verweij). Since the new directorate was established in 2004, monthly meetings occur with the programme leaders of the respective research programmes (Management Team; 3 h/mo). Furthermore, staff meetings occur weekly during the Monday Morning Seminar Series, where general announcements are made, and new research results are presented and discussed by all members of staff. To ensure that the finances are well managed, the budget was broken down into different parts for research groups (running expenses and equipment), publications, services, etc. Financial and personnel aspects, as well as other support services are shared between CBS and its neighbour, the Hubrecht Institute, and are under the direct management of the managing director. The institute has an active works council (OC) who critically follow the management of the organisation on behalf of all personnel. The council has regular meetings with the directorate. Certain matters (e.g. reorganisations or expansions) require their approval. The institute is managed independent from the KNAW, albeit within broad guidelines provided by the Academy. It has full responsibility for its financial, personnel and information policy, and the implementation thereof. The financial policy of the institute gets checked annually by an accountant. Personnel policy conforms to the rules of the CAO (Collective Labour Agreement of Dutch Universities). The system management and computer infrastructure is the sole responsibility of the institute, as is science communication and public relations (via the KNAW press office). The overall scientific programme of the institute is the responsibility of the director, determined in good collaboration with the respective programme heads, and discussed annually by the WeCo (Scientific Committee), who consist of six prominent scientists (two international and four Dutch). Programme heads are responsible for the strategy, work planning and output of their respective departments.
Part 1 - CBS Fungal Biodiversity Centre (CBS-KNAW) 3 Research input
Table 1.2. Staff at institute level in fte. Input 2008 2009 2010 2011 2012 2013 Tenured staff 8.6 8.6 8.6 7.0 7.0 7.8 Non-tenured staff 3.8 7.4 8.4 11.4 13.2 10.6 PhD candidates 4.0 5.0 7.0 8.6 7.6 13.6 Total research staff 16.4 21.0 24.0 27.0 27.8 32.0 Expenditure Technicians 18.7 21.6 23.6 27.2 31.0 27.8 Support staff 10.6 10.6 10.2 10.8 12.1 12.1 Total staff 29.3 32.2 33.8 38.0 43.1 39.9
Funding
Table 1.3. Input and expenditure at institute level. Input 2008 2009 2010 2011 2012 2013 Direct 5.0 30.5% 7.0 33.3% 7.0 29.2% 7.0 25.9% 7.0 25.2% 6.5 20.3% Research grants 1.0 6.1% 1.7 8.1% 2.1 8.8% 4.2 15.6% 7.1 25.5% 14.6 45.6% Contract research 10.4 63.4% 12.3 58.6% 14.9 62.1% 15.8 58.5% 13.7 49.3% 10.9 34.1% Total funding 16.4 100% 21.0 100% 24.0 100% 27.0 100% 27.8 100% 32.0 100% Expenditure Personnel costs 3178 67.0% 3635 70.6% 3711 67.2% 3578 58.7% 4454 66.4% 4857 69.3% Other costs 1563 33.0% 1514 29.4% 1809 32.8% 2517 41.3% 2253 33.6% 2148 30.7% Total expenditures 4741 100% 5149 100% 5520 100% 6095 100% 6707 100% 7005 100%
Income
Table 1.4. Total income of CBS-KNAW and its origin. Income (k€) 2008 2009 2010 2011 2012 2013 Direct 4329 4082 4359 4081 3999 4409 Research grants NWO/STW/etc. 48 78 102 254 412 739 Contract research EU/Min/Other 677 893 1063 1481 2480 1409 Total income 5054 5053 5524 5816 6891 6557
1.3 Research environment and embedding
The CBS is one of four life science institutes in the Academy, and houses the largest population of mycologists in the Netherlands, and probably one of the largest in Europe. The CBS in-house expertise, its unique databases and collection, and its investment in these resources, are unique in the world, and add a strong strategic value to the field of mycology. Research in CBS is conducted within various research programmes, where scientists, PhD students and technicians collaborate on specific projects. Such successful collaborative projects lead to synergy that stimulates coherent research on complex questions that extend past the abilities of individual scientists. Research programmes develop plans that extend for periods of five years, with each being evaluated by means
4 of a mid-term review. Highly successful research programmes can influence the direction of the general research being conducted in CBS, within the confines of its mission and vision. Research programmes, and the groups can, however, change in composition and focus over time. Presently it is our goal to have each research programme consisting of a programme leader, senior scientist, and technician (Table 1.2). Programme leaders have a specific role within the group, as they are the group’s representative in the management team, and are thus up to date with the CBS research policy, and management issues. They are responsible for the management of the programme and its finances (Tables 1.3, 1.4). They are expected to have a broad vision about the future of their subject area, and exciting research possibilities and options. Groups formally meet one to four times per month, and formulate their own goals, maximising collaboration by means of team building and training courses in new techniques and methodologies, thereby ensuring their own expertise in their research niche. Programme leaders are also expected to play a significant role in the group’s research, and should have co-authored and first authored papers in support of this fact. Scientists are thus given the freedom to develop their own programme without any top-down approach. In several cases, based on the specific research project, collaboration will also result between two or more different research programmes. Based on the nature of our research and the institute, all programmes have close ties and collaboration with the culture collection and the Bioinformatics programme. Finally, programme leaders are also expected to respond to new national and international research developments, and to be able to tap into new funding opportunities. CBS members of staff are encouraged to interact socially, and collaborate scientifically. Several scientific meetings are held at CBS during the course of the year where all staff members are welcome to attend (mycological meetings of the Dutch medical, mycological and microbiological societies, as well as the CBS Spring Symposium in April each year). Communication is logistically supported by means of social functions (at least 4–6 per year), seminars, internet, intranet, central facilities, newsletter, etc.
1.4 Quality and scientific relevance
A scientific performance analysis of CBS output in a national and international perspective is provided by The Netherlands Observatory of Science and Technology (NOWT). NOWT’s Science and Technology Indicators report 2010 show that CBS’s Citation Impact Score (2006–2009), like the other KNAW life science institutes, is well above the world, Dutch and university average. This is again confirmed by the latest analysis presented below in the section “1.5 Output” (Table 1.6). The quality of our research output is the result of dedicated, passionate, and highly motivated staff. CBS policy is to recruit and train the most talented young mycological researchers from institutions in and outside the Netherlands. Within CBS exists a healthy ambition to publish in the highest quality journals and to collaborate with strong (inter)national partners. This ambition, however, does not distract from CBS staff collaborating with students and young scientists from third world countries to assist them in achieving similar goals. We also strongly support scientists to publish in open access journals. The scientific output of all scientists is monitored on an annual basis. For scientific highlights and key publications, please consult the various research programmes.
1.5 Output
Table 1.6. Number of scientific publications of the CBS. Scientific publications 2008 2009 2010 2011 2012 2013 Total Refereed articles 82 88 89 133 130 158 680 Book Chapters/Proceedings 13 2 15 63 3 3 99 Total 95 90 104 169 133 161 752 Books 4 5 8 3 0 0 20
Part 1 - CBS Fungal Biodiversity Centre (CBS-KNAW) 5 Table 1.6. (Continued). Scientific publications 2008 2009 2010 2011 2012 2013 Total Ph.D. theses - internal 3 2 1 4 3 1 14 Ph.D. theses - external 2 0 3 1 1 1 8 Non refereed articles 0 0 1 2 0 1 4 Software development 0 3 4 0 2 4 13 Audiovisual Material 0 1 0 0 0 0 1
An independent research performance analysis on our output was conducted by The Centre for Science and Technology Studies (CWTS) in Leiden (see additional information online). The CWTS concludes that CBS shows a profile well above world average, at institute level as well as for each department. The entire CBS volume of output is above 1,73 (WoS) papers in 9 yr (2006–2013) and has increased significantly during this period. During this period the Web of Science covered 78 % of our scientific output. The high impact is shown by the high average number of citations and by the 106 CBS publications that qualify as being among the top 10% most cited of their different fields. With 20 % of highly cited publications it can be said that overall the CBS has produced double the number of highly cited publications than expected (i.e. 10%). CBS’s publication strategy is two-fold: publish in journals with the highest impact factor possible, but be sure to also publish in the most relevant specialist journals. This strategy has proven to be beneficial: from the 130 plus papers published per year (2011–2013), with around half of the papers were published in high-impact journals (IF > 3). Over the years CBS has published a steady number of papers in the highest IF journals, with 10 papers in journals with an IF > 10 in 2012. Journals with high IF in which CBS scientists publish include Clinical Microbiology Reviews, Genome Biology, Lancet Infectious Diseases, Molecular Biology and Evolution, Nature, Nature Biotechnology, Nature Immunology, PLoS journals, Science, etc.
The high impact of CBS publications is confirmed by the Thomson Reuters database ‘Essential Science Indicators’ (ESI) that outlined 35 CBS papers in the last 10 years as the highest cited papers in all categories. January -‐ August period 2009 2010 2011 2012 2013 CBS is in favour of open access to scientific publications and supports the “gold road to open access” for Abstracts 8759 11567 19466 69454 99753 our own journals, StudiesFull Text in Mycology HTML (Table 1.7),11225 Persoonia 14302 and IMA33471 Fungus , and51310 the “green 44830 road to open access” for papersTotal published PDF elsewhere. Research11726 papers16032 can be freely19196 viewed in27255 our own repository55686 (http://www.cbs.knaw.nl/index.php/research)Total Full Text . Our22951 repository 30334 can be searched52667 and harvested78565 via100516 Google, and the Academy repository via NARCIS.
120000
100000
80000 Abstracts
60000 Full Text HTML Full Text PDF 40000 Total Full Text
20000
0 Fig. 1.2. Studies in Mycology usage1 report2 graph3 period4 20095 (1) – 2013 (5).
http://hwmaint.studiesinmycology.org/stats/usage/200908_ytd_summary.html 6 http://hwmaint.studiesinmycology.org/stats/usage/201008_ytd_summary.html http://hwmaint.studiesinmycology.org/stats/usage/201108_ytd_summary.html http://hwmaint.studiesinmycology.org/stats/usage/201208_ytd_summary.html http://hwmaint.studiesinmycology.org/stats/usage/201308_ytd_summary.html Table 1.7. Studies in Mycology usage (2009–2013). January-August Period 2009 2010 2011 2012 2013 Abstracts 8759 11567 19466 69454 99753 Full text HTML 11225 14302 33471 51310 44830 Total PDF 11726 16032 19196 27255 55686 Total full Text 22951 30334 52667 78565 100516
1.6 Earning capacity
The CBS obtains its lump sum funding from the KNAW (3 M €, 1st funding stream), representing 42–44% of its total budget (6.7–7 M €, 2010–2012). Although we have in former years generated most of our external income directly from industry (2.5 M €), we have in recent years also been successful in obtaining competitive grants from the Netherlands Organisation for Scientific Research (NWO), and the Technology Foundation (STW) (Table 1.3). Several projects get funded based on competitive grant applications to NWO or STW, which are highly dependant on the level of innovation, quality and competitive ability of the researchers. We have also received personal grants from the NWO Innovation Research Incentive Scheme (Veni-Vidi-Vici), or bilateral grants with China (JSTP) (see research programmes). Research staff at the CBS have also been highly successful in the competitive international and European research arena, and presently we are actively involved in several major EU, national and regional grants (FES: Making the Tree of Life Work; STW: How mushrooms feed on sugars; FunTech, NGI: Zenith project on Fungal Diversity with respect to carbon utilisation, NWO: Biofuel production, in collaboration with CAS; Eurotransbio: CandIDazol project; SLOAN: indoor fungi; NUFFIC, KNAW/Chinese Academy: Bye-TOL project funded by KNAW and NSFC; barcoding medical fungi, one VENI- and VICI-grant, respectively; two NWO-JSTP grants with China; STW: heat resistance of food spoilage fungi, and biofilms for wood protection; Zon/MW: Bioprospecting for novel antibiotics, etc.). We also participate in several major 7th Framework Programmes (e.g. Q-Collect, i4Life, NEMO, EMbaRC, Cornucopia, NH&MRC: DNA Barcoding, MIRRI). We have also been successful in obtaining research equipment in exchange for research collaboration and data, e.g. Life Technology, Thermo Fisher, Bruker and Biomerieux.
1.7 Academic reputation
The CBS Fungal Biodiversity Centre is a well-known international research leader for fungal biodiversity studies. Our mycology courses and books are well acknowledged in the field of mycology, and our journal, the Studies in Mycology, has for several years had the highest ISI impact factor in the field “mycology”, with Persoonia being third. We have had considerable success in obtaining external funding for our research (more than 50 %), especially considering our field of study. In the past six years CBS researchers occupied influential appointments on the council of the World Federation for Culture Collections, President of ISHAM, secretary general of IUMS, and President of the IMA. Scientific staff also act as editors of numerous international scientific journals. The CBS is an honorary member of the Mycological Society of America, and the British Mycological Society. Several researchers have also been acknowledged by special awards such as honorary and distinguished membership (or honorary degrees) from the Mycological Society of America, from the American Society for Microbiology, the Southern African Society for Plant Pathology, Hungarian Microbiology Society, European Confederation for Medical Mycology, American Society for Microbiology, Swedish University of Agricultural Sciences in Uppsala, etc. Group leaders are also affiliated as honorary or extraordinary professors to numerous universities internationally (see research programmes for details), while some group leaders are also appointed as Research Fellows to the Institute of Microbiology, Chinese Academy of Sciences (IMCAS). An additional sign of recognition is the success which CBS scientists have had in obtaining personal
Part 1 - CBS Fungal Biodiversity Centre (CBS-KNAW) 7 grants from NWO, namely one NWO-Vici grant (R.P. de Vries), and one NWO-Veni grant (J. van den Brink). The International Peer review Committee that visited CBS in 2008 rated the CBS as a whole as “excellent”, with its research programmes as “very good to excellent”. In the past five years CBS has faced the challenge to maintain this high level of performance and, where possible, improve its research programmes even further. We are therefore extremely proud that the output and quality of our research publications has increased significantly since 2008 (see 1.5 Output). The academic reputation of the CBS builds on the combined efforts of its staff. We thus refer to the additional research publications online, and the bibliometric analysis (Appendix).
1.8 Societal relevance: quality, impact and valorisation
CBS delivers valuable in-depth knowledge about fungal biodiversity via MycoBank (20 000+ downloads per day) or any of the close to 100 websites that it maintains, ultimately benefiting governmental and applied research institutes, private industry and other stakeholders and end users. Our “core business” is preserving, researching and unlocking the world’s fungal biodiversity to improve quality of life, and leading to applications wherever possible. Our knowledge is freely made available to both the academic world and society, e.g. to help protect biodiversity, to adapt to global change, to design more sustainable agro-ecosystems, to explore possibilities for a sustainable biobased economy, to develop new products or services, to make companies more efficient, and to improve human health via rapid identification of disease causing organisms, or improve quality of food and beverages, and improve the indoor living environment. Because of the specifically chosen research themes, all research in CBS is focused on relevance and valorization. CBS plays a central role in gaining and disseminating new fundamental knowledge that directly benefits society and industry. Researchers at CBS increasingly participate in research consortia and private initiatives that connect basic and strategic research to application by end users (consult individual programmes, also for concrete examples of relevance). One of CBS’s objectives is to disseminate fungal biodiversity knowledge to the general public. Therefore, CBS maintains three journals in the open access model, namely Studies in Mycology (financially supported by CBS), Persoonia (financially supported by Naturalis Biodiversity Centre), and IMA Fungus (financially supported by the IMA). Via the numerous websites that we maintain for societies (IMA, IUMS, ISHAM, etc), the CBS also supports that dissemination of knowledge, and the popularisation of science. The CBS is strongly interlinked with international microbial biodiversity issues, and has (or had in the past 5 yr) members present or on boards of the OECD, WFCC, CETAF, NL-TAF, NL-BIF, IUMS, IMA, ISHAM, as well as most of the local societies like the Dutch Mycological, Microbiological, and Phytopathological (KNPV) societies. The numbers of cultures supplied to external CBS customers and internal research groups have increased considerably in the period 2008–2013 (Table 1.8), demonstrating that CBS has strengthened its position as a resource of reference material for researchers all over the world. In 2013 strains were sent to 60 different countries.
Table 1.8. Numbers of new aquisitions, delivered cultures, patent deposits and safe deposits, and culture identifications (fungi and bacteria). 2008 2009 2010 2011 2012 2013 Total number of cultures 5723 5058 5709 6304 5258 5400 delivered to external customers Total number of cultures 10737 20771 9795 19453 23024 15522 ordered for internal use (incl. barcoding) $
8 Table 1.8. (Continued). 2008 2009 2010 2011 2012 2013 Total number of lyophilized 7879 10683 4940 9745 11208 9796 cultures ordered for internal use (incl. barcoding) Patent deposits (accum. total 1068 1095 1127 1140 1152 1179 number of deposits) Safe & Maintenance deposits 293 285 292 299 332 358 Identifications § 445 346 308 295 319 390
$ Also includes cultures taken from LN stock for establishing a backup collection in a different location. § Service unit was established in 2005.
1.9 Viability
Human resource management
Human resources are managed by the KNAW P&O department, in conjunction with the CBS-Hubrecht Institute human resources department (shared between the two institutes). Together, tasks relating to personnel policy, training, education, job mobility, legal guidance, and settlement of personal issues are provided. All personnel are encouraged to further their career, and a special part of the annual budget (0.08 %) is reserved to facilitate this purpose. All employees are expected to contribute to the development and expansion of the research and international profile of the institute. Various ways in which this can be achieved are discussed with the staff during their yearly personal progress appraisals with their supervisors. PhD candidates have, in addition to daily supervision and progress meetings, an additional yearly appraisal, in view of the PhD defence that is scheduled four years after the onset of the study. As part of their Training and Supervision Plan (TSP), graduates also follow extra courses, and participate in activities of the Graduate Schools. These personal appraisals enable the evaluation of research based on the institute’s high expectations. In a number of cases this has in the past led to substantial actions pertaining to changing research lines, or tasks assigned to staff. Staff that work in the Collection and Bioinformatics programme are service orientated, spending 90 % of their time adding meta-data to the strains in the collection, make these data accessible via various databases, and ensure the quality, accuracy and viability of the strains in the culture collection. During the evaluation period the curators have also spent a lot of time on validation of in-house produced barcode sequences. The remainder of their time (10 %) is dedicated to research. In contrast, the majority of the tenured CBS staff in the research programmes are primarily conducting research. Several programme leaders are involved with the CBS mycology courses, and thus have teaching obligations, or are linked to universities as professors, and participate in teaching at their respective universities. Teaching obligations vary from the CBS mycology courses, to university undergraduate courses, to guest lectures given randomly in various courses throughout the year. Although its difficult to express time allocation as a percentage, the director spends 60 % of his time on management and organisational issues, and 40 % on matters related to teaching and research; programme leaders spend 80 % of their time on research and teaching, and 20 % on service. Post-doctoral candidates spend 100 % of their time on research (60 % in the case of those that manage major grants or European projects), and PhD’s 85 % on research, and 15 % on training.
Part 1 - CBS Fungal Biodiversity Centre (CBS-KNAW) 9 Financial resources
To maintain its leadership role in fungal biodiversity research, CBS requires substantial investments in high- quality personnel and infrastructure. With regards to the reorganization and financial restructuring in 2005 (6.4 fte), CBS was able to be more flexible, and address changing research and societal demands. The ecology programme was closed (this line of microbial research being conducted by the NIOO, the largest institute in the Academy), and the research focus was strengthened and changed (e.g. towards DNA Barcoding, genomics and bioinformatics). The financial position of the institute remained healthy until the end of 2013, when financial reserves were threatened by several unexpected setbacks (moving to a new financial system, Oracle, that did not support client services and required hiring expensive consultants, rising costs of support services, and a decline in externally funded projects linked to the financial situation in Europe). In 2015 the CBS will again restructure its research programmes into one research programme for each of the three themes (Agriculture, Industry and Medical Mycology). The CBS will also strengthen Medical Mycology, Bioinformatics, and build a new line of research in industrial mycology, incorporating novel product discovery.
Available infrastructure
The CBS has access to an extensive range of facilities and equipment to serve its specific needs for collection management and research. We have excellent facilities for: cryopreservation or freeze-drying, DNA-Bank (since 2006), media preparation and destruction, databases with associated software support and bioinformatics, collection and identification services, molecular facilities (high throughput DNA barcoding pipeline, Sanger and next generation sequencing, Q-PCR), shakers, incubators, serial incubator and incubation rooms, biochemical facilities (spectrophotometer, plate-reader for absorption, fluorescence and luminescence, small scale shakers for microtiter plates and sample tubes), HPAEC analysis system (Dionex) for detection of sugars, polyols, organic acids, aromatic compounds and oliosaccharides, cryo- scanning electron microscope, and numerous state-or-the-art light microscopes, Mass Spectrometry systems for fungal identification, course room and teaching facilities. CBS has access to most of the older literature (books, journals, and reprint collections), and also houses the library of the Dutch Mycological Society. Since 2007, all groups in CBS also have online access to library facilities (e-journals, books, web of science, current contents, etc.), via the University of Wageningen and Utrecht (linked to the appointment of the director to WUR and UU). Although CBS subscribes to various journals that are not available electronically, some are also obtained by swapping them for Studies in Mycology. Some genomics activities are outsourced to laboratories in NL, France, USA or China (sequencing, transcriptomics, metabolomics).
1.10 Next generation
The CBS does not have any formal educational tasks like universities, but scientists are involved in teaching masters and PhD-courses at different universities (Table 1.9). Furthermore, at least two tailor-made courses are taught to undergraduates at Amsterdam university (Medical Mycology), and Wageningen university (Fungal Biodiversity). The CBS also teaches three international courses, namely the General Course on Fungal Biodiversity, Course on Indoor Air and Food Mycology, and the Course on Medical Mycology, which also have their own text books. The latter courses are also listed in various research schools, e.g. Experimental Plant Sciences, Biodiversity (now Production Ecology and Resource Conservation), and are available to Dutch students, technicians, and researchers. Researchers are frequently affiliated to more than one research school. To enable PhD students to obtain a certificate from the research school at the completion of their degree, they need to set up (in coordination with their committee) a Training and Supervision Plan (TSP), which means that they follow additional courses (national and international) depending on their research topic, and educational background. Under the
10 TSP, PhD students are also required to attend PhD days of their research school and to present their research at these days and at national or international symposia / conferences. CBS actively monitors the training and performance of their PhD students, a go-no go decision judgement within the first year (that is also reported to the KNAW) and yearly appraisal interviews. The CBS also finances PhD students to attend workshops and courses.
Table 1.9. PhD success rate. 2008 2009 2010 2011 2012 2013 Graduations elsewhere 2 0 3 1 1 1 Graduations in the Netherlands 3 2 1 4 3 1
1. 11 SWOT
Strengths
The biggest asset of CBS is its rich culture collection, which compared to other collections on a global scale is the largest and represents the widest fungal diversity. CBS contributes to the development of internationally recognised quality standards for fungal preservation and culture collection management. A further strength is that CBS manages data pertaining to fungal names (nomenclature) via MycoBank (more than 20.000 downloads per day). This approach has led other international collections to approach CBS to also host their data on our servers, which proves that our system is well suited for this purpose. CBS also developed its own software (BioloMICS), which enables polyphasic online identifications and represents the future of taxonomic identification. CBS has converted the Studies in Mycology, Persoonia and IMA Fungus into open access journals, which enhances the image of the institute, along with its books, the CBS Biodiversity Series and the CBS Laboratory Manual Series. CBS has co-ordinated its research to fall in focus areas (Agriculture, Industry, Medical), where our high quality programmes can have maximum effect, and also be easily justified. CBS has embraced molecular and genomic techniques in its various research programmes. CBS has well-trained scientific staff. CBS has a professional support staff who facilitate the functioning of the institute. The high level of science at CBS, and its unique collection, attracts many international visitors, which lead to partnership/grant opportunities. CBS has embraced DNA barcoding as tool to enhance the quality of its identifications, and strains in the collection, the genomic DNA of which are also placed in a DNA bank once barcoded. CBS charges competitive rates for its services and cultures, making it more accessible to research staff from third-world countries. Members of the CBS staff serve on international councils, advisory committees, scientific committees of various societies and as editors of peer-reviewed journals. CBS is successful in exploiting the money follows people Veni-Vidi-Vici programme of NWO.
Weaknesses
The lump sum from the KNAW has not increased for 15 years, and is insufficient to support the growing fungal collection. The biggest weakness is that there are insufficient researchers to study all fungal groups, as would be expected of a Fungal Biodiversity Centre (we simply lack the funds to appoint enough scientists to
Part 1 - CBS Fungal Biodiversity Centre (CBS-KNAW) 11 cover all fields). This will negatively influence the quality of the collection, and it is one more reason to generate barcodes for all existing species, in order to have quick reference points for those taxa known in culture. CBS has limited funds to attract world experts. Two programme leaders have retired within the past three years, and need to be replaced by world experts. CBS needs to create more career opportunities for its younger scientists. Although CBS has managed to address its gender balance in the institute (62% female), we still lack female group leaders. Some core activities such as biosafety officer and work safety officer are handled as secondary tasks by permanent staff members after the last restructuring.
Opportunities
Given the unique collection, there are numerous research opportunities for CBS within the EU and abroad. The near future will also see more collaboration with companies that generate data (metabolomic and other) pertaining to the strains in the collection. New initiatives concerning DNA barcoding also fit CBS perfectly, and make the institute indispensable for future national and international research initiatives. The establishment of MycoBank gives CBS a central place in the field of mycology, as this service will be indispensable for mycological research in future. The CBS journal, Studies in Mycology (SIM), is now freely available online, and has become part of our culture acquisition policy. To publish in SIM, cultures need to be deposited in CBS. To deposit data in MycoBank, a culture number would be preferred, with CBS being the obvious choice. Regarding rapid pathogen detection, CBS is also involved with numerous different research groups, and employs a range of state of the art molecular detection techniques, and has an open exchange and memorandum of understanding with major centres such as the American Centre for Disease Control (CDC), Dutch National Plant Protection Organisation (NVWA, PD), etc. The move of CBS to its new location on “De Uithof” in Utrecht has opened new possibilities for collaboration, but more advantage is possible from this association, which could lead to increased benefits in the future. New sequencing technologies, which enable whole genome sequences to be generated in a day, will make collections with diverse strains of the same organism more important to investigate species variation in future. These technologies, and the associated bioinformatics approaches, present new opportunities for CBS (e.g. F-1000 genomes project with JGI). The increasingly important field of medical mycology presents an opportunity for CBS to further extend its relevance, national and international profile in future years. Although a DNA bank is present at CBS, it is not actively being marketed. Given the increasing amount of bureaucracy involved in shipping live cultures to some destinations, good quality genomic DNA provides a simple and relatively fast alternative. Currently, the classification of fungi (and yeasts) in biosafety or biorisk levels is not well defined. Although there are some lists available, these are frequently regional (e.g. per country) and often incomplete or outdated or sometimes even contradicting in classification level. The CBS could position itself as a world leader of these classifications and as repository of accurate and up to date classification lists. Ideally, the equivalent of a MSDS (material safety data sheet) should be developed for each micro- organism potentially harmful to humans. CBS is well positioned to play a significant role in new initiatives screening cultures for novel product production.
12 Threats
The Nagoya Protocol on Access and Benefit Sharing (CBD) is expected to enter into force soon. As the legal instrument for the CBD, it will greatly impact on day-to-day service operations and research in collections, and CBS will have to prepare itself to meet the challenges and comply. A decrease in numbers of strains deposited and sold is expected. It furthermore seems inevitable that Collection staff and other researchers will have to spend more time on administration of genetic resources they receive, use and distribute, in order to comply to Access and Benefit Sharing Regulations. Most countries are developing their own Biological Resource Centres, and given the problematic situation surrounding the CBD, and fears related to bio-piracy, it could lead to a potential reduction in new accessions. Individuals are wary of freely sharing their strains with others due to possible commercial exploitation. Activities with micro-organisms potentially harmful to humans require special handling under the Dutch labour laws. Although the CBS has special laboratories and work guidelines for such cultures, it is first necessary to know whether a culture is potentially harmful and to what extent (also see Opportunities). A further threat is bioterrorism. Biosecurity regulations hamper shipping of fungal isolates on the so-called “dual use lists” to e.g. the USA. To circumvent this problem we will move to a system where we can in some cases ship DNA (often this is what is needed for molecular analysis) (Also see Opportunities). The European economic crisis has affected the availability of government funding for research projects in the Netherlands. This has had a negative impact on our Dutch funding possibilities for basic research. NWO still does not reimburse overhead costs, and thus CBS suffers financially from its own success in obtaining NWO grants.
1.12 Future strategy: CBS Roadmap 2020
The CBS was established more than 110 years ago with the specific mission to preserve the world’s fungal biodiversity. Presently the CBS includes the largest known service collection of living fungi, a collection actively growing with approximately 3000 strains per year, and dispatches up to 17 000 strains per year to CBS research groups or external clients for research purposes. With its respective research programmes, CBS aims to add DNA Barcodes, genomes, physiological data, descriptions and illustrations to species in the collection, and to make these data available for international scientific study. This aim is achieved by selling fungal cultures for research purposes, accepting patent strains, DNA, culture and specimen deposits, the provision of fungal training courses, the control and management of several of the world’s largest fungal databases, and the publication of novel data in journals and books which are also freely available on the CBS web page(s), linked to various state-of-the-art databases. A further aim is to advise various national and international bodies on aspects related to fungi and fungal biodiversity. A final aim of the CBS is to maintain and expand its position as international centre of excellence pertaining to fungal biodiversity, preservation and research. CBS has chosen to transform itself from simply being an international culture collection of choice to being the trendsetter and gold standard of mycology.
CBS will retain its focus on the three themes, Agriculture, Industry and Medical Mycology, but try to strengthen these research groups. New areas that will be addressed include the following: • Medical Mycology – the retirement of G.S. de Hoog (group leader), and the repositioning of the RIVM on campus, as well as the close proximity of the hospital, suggest new opportunities. • Bioinformatics – the huge success of projects like FES plant health, QBOL, Q-bank, and MycoBank, suggests that our BioloMICS software hold lots of potential for the future, which is bound to be much more data-rich than the past, given the huge number of genomes becoming available. • CBS will be the first biological resource centre that will be entirely known from DNA data, which also creates huge opportunities for the future.
Part 1 - CBS Fungal Biodiversity Centre (CBS-KNAW) 13 • The acquisition of new laboratory facilities, and the expansion of the existing CBS building to strengthen industrial mycology, pave the way to move into novel product discovery, meaning that CBS, with a strong European and international partnership, can build a significant resource in this area of research. • CBS has strong strategic collaborations with Dutch universities, e.g. Amsterdam, Utrecht and Wageningen, and will foster even closer ties to them in future. • As is true in other Academy institutes, CBS has few internal career opportunities for young scientists. The CBS will thus strive to train students to guide them to the best positions internationally (post- doc positions, lectureships, professorships), as we have done in the past. • In the past five years, CBS has positively influenced the digital presence of mycology online, and also see this as part of our essential strategy for data dissemination and education in the future. • In the previous peer review our goal was to increase external funding (2006: 32 %, 2008: 40 %), to 56–58 %, a goal we reached in 2011–2012, but need to maintain in coming years. External funding is obtained via competitive grants (e.g. NWO and EU), which allows CBS to stick to its own research agenda, strategic vision and mission. • To address the loss of a permanent biosafety officer, such an overlapping function(s) between both the CBS and the Hubrecht Institute could possibly be filled by a central professional(s) serving both institutes in a dedicated way. • Within CBS the various research programmes strongly benefit from each other’s approach and experiences by cross-fertilisation of ideas and communal projects. The axenic or pure culture method (developed from the Dutch yeast school, Westerdijk, von Arx, Gams, etc.), the polyphasic identification and characterisation (Stolk, Samson, Yarrow, Roberts, etc.), linked to online databases of associated metadata (Stalpers, Stegehuis, Robert, etc.), were developed over several decades and have become standard in CBS. These protocols are shared among groups, as are the newly developed techniques such as DNA barcoding approaches, the screening of novel loci for strain identification using phylogenomics, and more recently, other genomic and bioinformatics approaches. We refer to the polyphasic approach as the “CBS method and work ethic”, which leads to a common ground among the various researchers, technicians and PhD students.
Conclusions
CBS has ambitious staff that publish high impact papers, and these frequently appear in highly rated journals. We are always open for collaboration with both national and international partners, but will retain our focus on relevance, products, and free access to published data. To ensure further growth in spite of retirements and a declining financial situation, CBS will again have to undergo some restructuring in medical and industrial mycology. We must, and will, put additional effort in securing external finances. Our goals remain dynamic and ambitious, and we believe that we are on the verge of launching the CBS and mycology as discipline into the future.
14 2. Evolutionary Phytopathology Programme
Programme leader Prof. dr P.W. Crous
2.1a Objectives(s) and research area
By 2050 the global population will rise to an estimated 9.7 billion from the current level of 7.1 billion. Global demand for food, feed and fibre will double, while global climate change may mean that certain crops will have to be replaced in specific climate zones. The importance of producing food sufficient in quality and quantity remains paramount for sustaining quality of life. Inadvertent introductions of phytopathogenic fungi have had dire consequences to nature and to cultivated crops on various continents in the past. The economic impact of such introductions can be seen in yield loss and in increased input costs for cultivation and disease control, as well as in social impact. To combat these diseases on an international scale, it is important to clarify whether the same species and genotypes occur in various countries, since each different species and genotype can be expected to have different patterns of attack, as well as different responses to fungicides and to climatological conditions. With such pathogens, it is also important to know what their host ranges and mating strategies are, and how this relates to different disease control mechanisms. Whether surrounding plants or crops can either act as reservoirs or sources of pathogen inoculum or assist in the survival of the pathogen during absence of primary host adds another complicating factor to the equation. The global movement of agricultural and forestry produce is inextricably cross-linked, and will continue to be so in future. Knowing which pathogens occur where and on which crops facilitates trade in agricultural produce. In this programme, we address these economically vital matters by investigating the speciation and host adaptation of various important phytopathogenic fungi, specifically focusing on Dothideomycetes, the largest class of Ascomycetes, which also contains the highest number of known phytopathogenic fungi.
2.1b Forward look
Since the onset of the programme, we focus on two areas where we expect to continue obtaining research grants and generating publications, namely species discovery (to uncover and understand novel fungal biodiversity, specifically plant pathogens and their substrates/hosts), and understanding processes (genomics of sex to unravel species boundaries and interactions). Since the last peer review we have focused our research efforts mainly on the Dothideomycetes. We are in the process of revising several major phytopathogenic genera and families of Dothideomycetes, resolving the status of 2000 plus genera of Dothideomycetes, and also generating genome sequences of two representatives of each family (F- 1000 project with JGI). We have also coordinated the fungal component in FES Plant Health, and the Quarantine Barcode of Life (“QBOL”, FP-7), and now co-curate the fungal database in Q-bank together with the Dutch National Plant Protection Organisation. Together with coworkers in China (Q&Q Fungi, JSTP-NWO), we continue to expand Q-bank as curated reference sequence database to facilitate global trade. European citrus markets have recently been closed to South African export due to the presence of Phyllostica citricarpa, which has led to the loss of 80 000 jobs in South Africa alone. We have since elucidated five Phyllosticta species that cause this disease, though some are restricted to Latin America or Asia. We have shown that several Colletotrichum species are associated with anthracnose disease of strawberries in Europe, and not C. acutatum as previously believed by the European Plant Protection Organisation, which has serious implications for trade. These, and numerous other examples are the results of our research, which are shared with the international community via Q-bank, facilitating trade based on molecular data linked to valorized names and specimens of plant pathogenic fungi.
Part 2 - Evolutionary Phytopathology Programme – P.W. Crous 15 Other than being successful in obtaining national and international funding for our research, we have also acquired NWO and other funding to further develop postdocs, steering them to various positions in industry.
2.2 Composition
Table 2.1. Research staff at programme level, 2008–2013. 2008 2009 2010 2011 2012 2013 Funding* Research staff Prof. dr P.W. Crous 1.0 1.0 1.0 1.0 1.0 1.0 1 Dr J.Z. Groenewald 1.0 1.0 1.0 1.0 1.0 1.0 1 Postdoc Dr L.-H. Zwiers 1.0 1.0 1.0 1.0 – – 1 Dr M. Binder – – – – 1.0 1.0 1 Dr G. Hunter 1.0 – – – – – 3 Dr H. Madrid – – – – 1.0 1.0 2 Dr L. Lombard 1.0 1.0 1.0 1.0 2 Dr U. Damm 1.0 1.0 1.0 1.0 1.0 1.0 2 PhD H. Brouwer 1.0 1.0 1.0 – – – 2 M. Aveskamp 1.0 1.0 – – – – 1 A. Cabral 1.0 1.0 1.0 1.0 – – 5 R. Cheewangkoon 1.0 1.0 – – – – 4 H. de Gruyter 0.2 0.2 0.2 0.2 0.2 – 2 J. Woudenberg – – – 1.0 1.0 1.0 2 W. Quaedvlieg – 0.5 1.0 1.0 1.0 0.5 6 S.I.R. Videira – – – 1.0 1.0 1.0 2 L. Lombard 1.0 1.0 – – – – 7 Chen Chen – – – – – 0.5 8 Carlos A.D. Bragança – – – – 1.0 – 9 F. Lui – – – – 1.0 1.0 10 J. Nguanhom – – – – – 1.0 4 T. Trakunyingcharoen – – – – 1.0 1.0 4 R.F. Alfenas – 1.0 1.0 1.0 1.0 – 9 Y.P. Tan – – – – 1.0 1.0 11 Technicians M. Starink 0.8 0.8 0.8 0.8 0.8 0.8 1 A. van Iperen 0.9 0.9 0.9 0.9 0.9 0.9 1 J. Bloem – – – 1.0 1.0 1.0 2 J. Woudenberg 1.0 1.0 1.0 – – – 2
*1 = Royal Netherlands Academy of Arts and Sciences; 2 = Dutch Ministry of Agriculture (FES); 3 = National Research Foundation, South Africa; 4 = Mushroom Research Centre, and Royal Jubilee Grant, Thailand; 5 = Fundação para a Ciência e a Tecnologia, Portugal; 6 = EU, FP7; 7 = Tree Pathology Corporate Programme, South Africa; 8 = China Scholarship Council; 9 = The Brazilian federal Agency, CAPES; 10 = Chinese Academy of Science; 11 = Department of Primary Industries, Australia.
16 2.3 Research environment and embedding
The research is embedded in the Experimental Plant Sciences research school. Prof. dr P.W. Crous is affiliated as professor to the departments of plant pathology at the universities of Stellenbosch, Free State and Pretoria (South Africa), Wageningen and Utrecht (Netherlands) and Melbourne (Australia). This research programme is partly funded by the Odo van Vloten Foundation, and the Dutch Fund for Economic Improvement (FES), which coordinated a programme on plant health (quarantine), and also one on building the tree of life, and QBOL (FP-7; DNA Barcoding of plant pathogenic fungi). We have excellent collaboration with the Dutch National Plant Protection Organisation in Wageningen; sometimes in the context of joint projects and other times in the form of contract research. Research staff from the evolutionary phytopathology programme coordinate (along with staff from the Collection) the general “CBS course in Mycology” (a two-week course, run in February each year), as well as an undergraduate course at Wageningen university “Magical mushrooms, slayers and sex: how moulds mould society” (PHP-50306), which is taught during September–October each year (including a two-week-long practical). Several members of staff at CBS also provide guest lectures during this course. The research programme is frequently host to international guest students, researchers or interns spending time in the group to acquire specific skills under the supervision and training of the group’s staff. Significant scientific interaction also occurs via email in the form of advice and assistance with, for example identification of morphology or improvements of alignments or phylogenies, with students and researchers from all over the world. PhD students from the programme are enrolled in the Experimental Plant Sciences research school (EPS), which was recently accredited by the KNAW, and received the rating of “excellent”. EPS has regular meetings, yearly PhD days, newsletters, and offer several training courses which the PhD students can attend to get the credits required to obtain a certificate at completion of their PhD study.
Institutional and national collaboration
The Evolutionary Phytopathology programme has strong links with the Collection & Bioinformatics programme, as well as with the Origins of Pathogenicity in Clinical Fungi programme in CBS. Within the Netherlands, we are closely linked to Plant Research International (Dothideomycete research, genomics, and SPIN programme with Musa diseases in Indonesia), and the laboratory of phytopathology at the department of Plant Sciences of University of Wageningen. Given the nature of research and the joint curation of the fungal database of Q-bank, we also closely collaborate with the Dutch National Plant Protection Organisation. We also coordinated the fungal component of the Dutch Programme for Plant Health (FES), along with other groups in Leiden, Amsterdam and Wageningen.
International collaboration
Internationally, the programme is strongly linked to the departments of plant pathology at the universities of Stellenbosch (grapevine and fruit tree research) and the University of Pretoria (Forestry and Agricultural Biotechnology Institute, diseases of plantation forestry) in South Africa. Fungal pathogens of indigenous Australian crops are also being researched in collaboration with the Royal Botanical Gardens of Sydney (Sydney), Department of Primary Industries (Queensland), and Department of Plant Pathology (Melbourne). We have strong links to the Departments of Plant Pathology in Thailand (Chiang Mai, Chiang Rai), Brazil (Viçosa, São Paulo), Iran (Tehran) and China (Beijing Forestry University, Chinese Academy of Sciences, and North-West A&F University), where we also co-supervise students. We also co-supervise PhD students from universities in Portugal (Instituto Superior de Agronomia de Lisboa) and Italy (University of Florence, Catania) on grapevine diseases, and have informal research ties with universities in Slovenia
Part 2 - Evolutionary Phytopathology Programme – P.W. Crous 17 (Agricultural Institute of Slovenia) and Germany (University of Halle). As European partner we are also actively involved with an American consortium to elucidate the Fungal Tree of Life at genome level (F- 1000 project in collaboration with JGI, California).
2.4 Quality and scientific relevance
During the past few years we have significantly advanced the field in depositing thousands of ex-type sequences of plant pathogenic fungi in GenBank: the vast majority of these originate from our group and are also indicated as such in the comment field. A significant number of entries in the Q-bank fungal database is based on sequences generated in our programme and are associated with CBS strains. We have also embraced genomics, and started to generate whole genome sequences for many of the pathogens we work on, in an attempt to resolve various research questions relevant to society and industry.
Several highlights from this period (2008–2013) are listed below:
• Fungal taxonomists routinely encounter problems when dealing with asexual fungal species due to poly- and paraphyletic generic phylogenies, and unclear species boundaries. These problems are aptly illustrated in the genus Phoma. This phytopathologically significant fungal genus was subdivided into nine sections which are mainly based on a single or just a few morphological characters. However, this subdivision is ambiguous as several of the section-specific characters can occur within a single species. In addition, many sexual genera have been linked to Phoma. In this study we delineated the generic boundaries, and came to a generic circumscription which is more correct from an evolutionary point of view by means of multilocus sequence typing. A total of 324 strains were included in the analyses of which most belonged to Phoma taxa, with 54 related to pleosporalean fungi. In total, 206 taxa were investigated, of which 159 are known to have affinities to Phoma. The phylogenetic analysis revealed that the current Boeremaean subdivision is incorrect from an evolutionary point of view, revealing the genus to be highly polyphyletic. Phoma species are distributed in six distinct clades within the Pleosporales, and appear to reside in different families. The majority of the species, however, including the generic type, clustered in a recently established family, Didymellaceae. Based on the sequence data obtained, the Didymellaceae segregate into at least 18 distinct clusters, of which many can be associated with several specific taxonomic characters. Four of these clusters were defined well enough by means of phylogeny and morphology, so that the associated taxa could be transferred to separate genera. Additionally, this study addresses the taxonomic description of eight species and two varieties that are novel to science, and the recombination of 61 additional taxa. • The genus Colletotrichum includes a number of plant pathogens of major importance, causing anthracnose diseases of a wide variety of woody and herbaceous plants. Fruit production is especially affected, both high-value crops in temperate markets such as strawberry, mango, citrus and avocado, and staple crops such as banana. Furthermore, numerous novel taxa were introduced in the C. acutatum (treating 31 taxa, and introducing 21 novel species), C. boninense (treating 17 taxa, and introducing 12 novel species), and C. gloeosporioides (treating numerous taxa of which 22 are accepted, and introducing 9 novel taxa, as well as one novel subspecies) species complexes. Although some species appear to have preferences to specific hosts or geographical regions, others are plurivorous and are present in multiple regions. The future for Colletotrichum biology will thus have to rely on consensus classification and robust online identification tools. In support of these goals, a Subcommission on Colletotrichum has been formed under the auspices of the International Commission on Taxonomy of Fungi, which will administer a carefully curated barcode database for sequence-based identification of species within the BioloMICS web environment (currently part of Q-bank).
18 • The genera Alternaria, Cercospora, Phoma, Pseudocercospora and Septoria are linked to devastating diseases of staple food crops globally, several of them also representing important quarantine organisms. Furthermore, from these studies we concluded that the application of European and American names to Asian and African plant pathogens, and vice versa, was often incorrect. For all taxa investigated multi-gene DNA data were deposited in GenBank and other databases to expedite future identification of these plant pathogenic fungi. No single locus was found to be the ideal DNA barcode gene for these taxa, and species identification will have to be based on a combination of two or more gene loci with or without the addition of morphological characters. • The Botryosphaeriales contain numerous plant pathogenic fungi of quarantine and economic importance, e.g. Phyllosticta spp. associated with Citrus Black Spot disease. By employing a multi- gene phylogenetic analysis on 129 isolates, 12 new Phyllosticta species were introduced, while epitype and neotype specimens were designated for a further seven species. One species of interest is P. citrimaxima associated with tan spot of Citrus maxima fruit in Thailand, which adds a fifth species to the citrus black spot complex. Other than the Phyllostictaceae, a further five families are now recognised in the Botryosphaeriales. Furthermore, molecular clock dating on radiations within the Botryosphaeriales, based on estimated mutation rates of the SSU rDNA locus, suggests that the order originated in the Cretaceous period around 103 (45–188) mya, with most of the diversification in the Tertiary period. We also provided an account of all genera and species in the Botryosphaeriaceae known from culture, including the morphological features and descriptions of 17 genera and 110 species. Keys to the genera and species are also provided, along with definitive DNA barcodes for the species in each genus.
Key publications
Aveskamp MM, Woudenberg JHC, de Gruyter J, Turco E, Groenewald JZ, Crous PW (2009). Development of taxon-specific SCAR markers based on actin sequences and DAF: a case study in thePhoma exigua species complex. Molecular Plant Pathology 10: 403–414. Arzanlou M, Crous PW, Zwiers L-H (2010). Evolutionary dynamics of mating-type loci of Mycosphaerella spp. occurring on banana. Eukaryotic Cell 9: 164–172. Aveskamp M, de Gruyter H, Woudenberg J, Verkley G, Crous PW (2010). Highlights of the Didymellaceae: A polyphasic approach to characterise Phoma and related pleosporalean genera. Studies in Mycology 65: 1–60. Damm U, Cannon PF, Woudenberg JHC, Crous PW (2012). The Colletotrichum acutatum species complex. Studies in Mycology 73: 37–113. Crous PW, Braun U, Hunter GC, Wingfield MJ, Verkley GJM, Shin H-D, Nakashima C, Groenewald JZ (2013). Phylogenetic lineages in Pseudocercospora. Studies in Mycology 75: 37–114. Phillips AJL, Alves A, Abdollahzadeh J, Slippers B, Wingfield MJ, Groenewald JZ, Crous PW (2013). The Botryosphaeriaceae: genera and species known from culture. Studies in Mycology 76: 51–167. Wikee S, Lombard L, Nakashima C, Motohashi K, Chukeatirote E, Cheewangkoon R, McKenzie EHC, Hyde KD, Crous PW (2013). A phylogenetic re-evaluation of Phyllosticta (Botryosphaeriales). Studies in Mycology 76: 1–29.
2.5 Output
Evolutionary Phytopathology has had a substantial output for the period (2008–2013), and has published 210 papers in six years.
Part 2 - Evolutionary Phytopathology Programme – P.W. Crous 19 Table 2.2. Scientific publications. 2008 2009 2010 2011 2012 2013 Total Academic publications Refereed journals 22 38 27 37 42 36 202 Book Chapters & – 1 5 1 – 1 8 Proceedings Total 22 39 32 38 42 37 210 Monographs & books 1 2 4 – 2 2 11 Ph.D. theses 1 – 2 – 2 2 7 Popular publications 2 2 2 2 2 4 14 and products
As can be seen from this table, the research output of the group has remained relatively stable, though there has been a marked increase in impact factor. Numbers of books and PhD students have also remained relatively constant.
2.6 Earning capacity
Our research programme has been successful in acquiring funds from a wide variety of national and international programmes and institutions. Nationally we have obtained funds from FES (Dutch gas reserves), in two major projects, namely “Plant Health”, and “Making the tree of Life Work”, which were supplemented with funds from the Academy to barcode the CBS culture collection. We also obtained a grant in the SPIN collaborative programme between the Academy and Indonesia, linked to Panama disease of banana in Indonesia (starting 2014), and Q-bank (Dutch government). Internationally we have been successful in obtaining EU funding, namely CETAF-EDIT (linked to DNA barcoding), and grants linked to plant health, namely QBOL (FP-7), and are involved with a COST-action dealing with sustainable control of grapevine trunk diseases. We were also successful in obtaining a personal grant, namely a JSTP-NWO grant with China (Q&Q fungi), aiming to supplement Q-bank with more data linked to quarantine fungi. We have firm links with Australia, Brazil, China, Thailand and South Africa, and regularly obtain funds in these countries for collaborative projects related to plant health.
2.7 Academic reputation
Awards
2010 - Elected Corresponding Member of the Royal Academy for Overseas Sciences, Belgium 2011 - Founders’ Award from the European Mycological Association 2012 - Honorary Membership of the Mycological Society of America 2013 - Elected Fellow of the Southern African Society for Plant Pathology
Appointments
University of Pretoria (extraordinary professor) University of Stellenbosch (extraordinary professor) University of the Free State (extraordinary professor) University of Utrecht (professor) University of Wageningen (professor) University of Melbourne (professor) International Mycological Association (President, 2006–2010; Past President, 2011–2014).
20 Editorships in academic journals and book series
Australasian Plant Pathology CBS Biodiversity Series CBS Laboratory Manual Series IMA Fungus (ME) Journal of Plant Pathology Persoonia (EiC) Studies in Mycology (ME)
Memberships in Scientific Boards
Centre of Excellence in Tree Health Biotechnology, South Africa (2005–) Christine Buisman Stichting (2003–) Consortium of European Taxonomic Facilities (2003–) International Commission for the Taxonomy of Fungi (Dothideomycete subcommission) (2010–) International Mycological Association (executive committee 2002–). Johanna Westerdijk Stichting (2003–) Odo van Vloten Stichting (2003–) Willie Commelin Scholten Stichting (2003–)
Selection of invited lectures
Crous PW (2008). Calibrating Europe’s Biodiversity using DNA Barcodes (ECBOL). EuroBioForum, 19 September, Strasburg, France. (keynote) Crous PW (2008). Fungal Diversity: The future of research. XII International Congress of Mycology. IUMS Congress, 5–9 Augustus, Istanbul, Turkey. (keynote) Crous PW (2010). Fungal Biodiversity: separating good from evil. Scientific Spring Meeting of the Dutch Society for Microbiology, 19 April 2010, Papendal, Arnhem, The Netherlands. (keynote) Crous PW (2013). Names of plant pathogenic fungi: starting over. Doidge Lecture, at the biennial meeting of the Southern African Society for Plant Pathology. (20–24 January 2013, Bela Bela, Limpopo Province, South Africa). (keynote) Crous PW (2013). From Latin to barcode: advances and instabilities in the application of names to plant pathogenic fungi. (46th Congress of the Brazilian Plant Pathology Society, 20–25 October 2013, Ouro Preto, Brazil). (keynote)
Bibliometric analysis
• Number of peer reviewed publications: 462 (Web of Science, March 2014) • H-index: 47 (Web of Science, March 2014); 65 (Google Scholar, March 2014) • Total number of citations: 9672 (Web of Science, March 2014); 16802 (Google Scholar, March 2014)
2.8 Societal relevance: quality, impact and valorisation
A major aim of our programme is to make our data available to end-users and society in general. We therefore give preference to publishing in open access journals, and also ensure that our relevant published DNA barcodes, which are essential to rapidly and accurately identify the various plant pathogenic species, are also placed in Q-bank, of which we co-curate the fungal database. Furthermore, via MycoBank, we also
Part 2 - Evolutionary Phytopathology Programme – P.W. Crous 21 develop specialized polyphasic identification keys (in collaboration with the Bioinformatics software), so that end-users can easily identify their plant pathogens. Practical applications linked to our work in the past few years have been the elucidation of the Sigatoka disease complex of banana, and clarifying the distribution and rapid detection of these pathogens, as well as their mating types; clarifying the movement of Petri disease organisms from grapevines to alternate woody hosts; providing evidence for sexual reproduction in Cercospora beticola populations on sugar beet; developing SCAR markers to detect varieties of Phoma exigua of quarantine importance; clarifying the Colletotrichum species complexes occurring on peppers, strawberries and vegetables; identification and rapid detection of Phyllosticta spp. that can cause Citrus Black Spot disease of Citrus globally; clarification of the genera and species associated with flyspeck and sooty blotch disease of apples; resolving the Zymoseptoria and Parastagonospora species respectively associated with septoria leaf blotch and glume blotch of cereals; Stagonosporiopsis ray blight disease of ornamentals, and monographing major genera such as Botryosphaeria, Calonectria, and Cladosporium. Of all the ex-type sequences linked to fungal species in GenBank, the by far largest number has been generated in our programme. Since 1985, we have also published more novel fungal species than any other group in the world, and almost all have associated DNA barcodes. Importantly, novel species are linked to type cultures and where possible, existing names are linked to neo- or epitype cultures in cases where type material is absent, thus tying down the application of the name for future research. Astonishingly, ex-type cultures are not available for many important plant pathogens and related species. We are also actively involved in streamlining fungal systematics, and organised the “Best Gene” for fungi symposium, which resulted in the ITS being chosen as barcode locus for fungi. We are now actively pursuing the “Genera of Fungi” project, which will again see DNA barcodes fixed to type species of fungal genera, thereby stabilizing the genetic application of names. We also provide DNA barcodes to Life Technology, who again uses these barcodes on their DNA chip for rapid species identification. We see the data that we generate as the biggest product, and this is made available to the research community via different online portals and databases.
2.9 Viability
Our group was originally instrumental in obtaining the FES grant dealing with plant health (DNA barcoding plant pathogenic fungi, and setting up an associated online database in collaboration with the Bioinformatics Programme). This project directly lead to the successful EU programme QBOL, which focused on plant health in a quarantine context, and in which we coordinated the fungal component. Via the SPIN collaborative programme with Wageningen University (PRI), we also share a PhD student working on Panama disease of banana (starting 2014). We have several international collaborative projects dealing with plant health, and plant pathogens in general. Our JSTP-NWO project with China (Q&Q fungi) will again pave the way for more funding related to plant health. Given the projected growth of the world’s population in the coming years, food security, and especially trade via the Netherlands, remains an important focus of our programme. The group has 4 postdocs, and 5 PhD students (internally) with numerous students that visit for periods of 3-12 mo, as well as sabbatical visitors from Brazil, Europe, Japan, and South Africa. Students have traditionally acquired positions in industry (NL) and universities (South Africa, Brazil, Thailand, China, Germany).
2.10 Strategy
The Evolutionary Phytopathology programme presently represents one of the largest and most diverse groups in Europe working on the characterisation and detection of plant pathogenic fungi. By incorporating a genomics aspect (in collaboration with JGI, Plant Research International and Wageningen Phytopathology), we have stimulated a lot of new ideas and approaches to tackling well-known problems in plant pathogenic fungi. This approach has led to considerable success nationally and internationally, often
22 leading to larger, joint projects with different international laboratories. Due to our unique expertise within the group, and because we are a fungal biodiversity institute, we often get involved with international students and groups working on diverse topics in phytomycology. However, we think that we have found a coherent manner in tackling this difficulty, as we have similar phylogenetic and mating type questions that we can also formulate for these groups of fungal pathogens, which again leads to good collaboration in several networks (e.g. see high research output). This approach has led to considerable national and international interest in our group, and will in coming years lead to more externally-funded projects. The research approach taken in our group has proven to be in high demand nationally and internationally (links with quarantine services and departments of plant pathology). This has resulted in the fact that we have been successful in obtaining funding, training students, and also publishing in the top journals of plant pathology and mycology. By incorporating a genomics component to our programme, we hope that this will create the opportunity to break through to more high impact journals outside the traditional field, and also obtain more prestigious research grants (NWO, STW). Threats to the group are related to the relatively small research staff, which at times makes it difficult to manage all the projects. Preparation for and upload of data to curated databases is time-consuming and this process goes at the cost of research time. With an increased output of such data the amount of time necessary to upload the data to curated databases also increases. Although the data itself has a societal use for plant protection officials and other colleagues, the process of preparation, uploading and curation cannot be considered or accounted for as “research time” in a strict sense. It is also unclear how we will manage all the genomic data that will become available in the coming years, and this type of research will have to be expanded. The practical application of the research results in the form of kits for rapid pathogen detection in soil and agricultural produce needs to be further developed (which could turn this threat into an opportunity).
Part 2 - Evolutionary Phytopathology Programme – P.W. Crous 23 3. Origins of Pathogenicity in Clinical Fungi Programme
Programme leader Prof. dr G.S. de Hoog
3.1a Objective(s) and research area
Invasive Fungal Infections (IFI) are gaining importance in the clinical laboratory. Not only patients with AIDS or leukemia and transplant recipients constitute large high-risk hospital populations, but emerging infections are observed in individuals suffering from chronic diabetes or under antibiotic or corticosteroid use. The 5–10% of the healthy adult population suffering from nail and skin mycoses are generally disregarded, while they may form the source for disseminated infections. At a global scale, thousands of identifications of fungal clinical samples are performed each day. Emerging fungal pathogens with high mortality and morbidity include both Ascomycetes like e.g. the black yeasts and Fusarium, and Zygomycetes like Rhizopus. Availability of reliable diagnostic tools based on well-circumscribed taxonomic entities is of prime importance. Understanding pathogenicity, virulence and antifungal resistance predict clinical course and adequate therapy. Recent developments in human immunology have led to understanding of enigmatic severe, mutilating, sometimes even fatal black yeast infections in seemingly healthy individuals: several of these patients appeared to have rare genetic malfunctions in their immune system. Although originally described for Candida, a mutation in the signalling pathway between Dectin-1 receptor and macrophage appeared to be present in patients with hitherto unexplained black yeast infections.
Our objectives are:
1. to reconstruct the phylogeny of species under study, and resolve diversities at and below the species level in order to define species as units of evolution; 2. to reveal the natural ecology and routes of transmission of pathogenic and opportunistic fungi, and to understand the fungal contribution to such infections; 3. to provide tools to recognize the entities in clinical practice, varying from simple genomic alternatives to next generation proteomics; 4. to reveal factors contributing to virulence by comparative genomics, in the assumption that predisposed opportunists are connected by latent homology and virulence factors may otherwise function in ecology as diverse types of extremotolerance.
3.1b Forward look
Our research group focuses on fungal biodiversity in the clinical area, and therefore focuses on less- known species outside the major agents in Candida and Aspergillus. In recent years enormous progress has been made in our understanding of fungal pathology Kingdom-wide. A broad diversity of molecular and proteomic techniques is available to unravel species complexes into narrow-defined siblings which often appear to differ in their behaviour towards host and environment. Entities are circumscribed much more precisely than in the past, using multilocus phylogeny supplemented with phenotypic, ecologically relevant parameters eventually supplemented with mating studies, which in concert should explain evolution and clinical course of fungi causing infection. Comparative genomics reveals traits that have expanded or contracted and provide insight into genes playing a role during evolution. Subsequently diagnostic tools can be developed, not only by the use of an array of molecular techniques, but also by proteomics which has become in reach due to extremely powerful mass spectrometric instrumentation. The latter techniques not only concern peptide analysis with MALDI-TOF, but also whole-protein analysis with LC-
24 tandem-mass spectrometry. At present our research team covers large areas of the fungal Kingdom where medically relevant fungi are located, ranging from Mucorales to Sordariomycetes, Dothideomycetes and Chaetothyriomycetes. Our main areas of research will be multilocus phylogeny, comparative genomics, and next generation proteomics using LC-tandem-MS.
3.2 Composition
Table 3.1. Research staff at programme level, 2008–2013. 2008 2009 2010 2011 2012 2013 Funding* Research staff Prof. dr G.S. de Hoog 1.0 1.0 1.0 1.0 1.0 1.0 1 Postdoc Dr A.D. van Diepeningen – – – 1.0 1.0 1.0 1 Dr C. Gueidan 1.0 1.0 – – – – 1 Dr G. Walther 1.0 1.0 – – – – 2 Dr J. Freeke – – – – – 1.0 3 PhD M. Sudhadham 1.0 1.0 – – – – 4 M.H.L. Tata 1.0 – – – – – 5 H. Badali 1.0 1.0 1.0 – – – 6 M.J. Najafzadeh 1.0 1.0 1.0 – – – 6 S. Abdallah Ahmed – – – 1.0 1.0 1.0 7, 14 S. Dolatabadi – – – 1.0 1.0 1.0 6, 14 K. Samerpitak – – – 1.0 1.0 1.0 8, 14 K. Dukik – – – – – 1.0 3 A. Al-Hatmi – – – – – 1.0 9 B. Brankovics – – – – – 1.0 10 L. Moreno – – – – – 1.0 11 M. Chen – – – – – 1.0 12, 14 Y. Zhang – – – – – 1.0 13, 14 Laboratory A.H.G. Gerrits van den Ende 1.0 1.0 1.0 1.0 1.0 1.0 1 assistant Technician K.F. Luijsterburg 1.0 1.0 1.0 1.0 1.0 1.0 1
*1 = Royal Netherlands Academy of Arts and Sciences; 2 = Odo van Vloten fonds; 3 = ThermoFisher Co.; 4 = Wetenschappelijk Onderzoek van de Tropen WOTRO; 5 = NUFFIC; 6 = Ministry of Health and Education, Iran; 7 = University of Khartoum, Sudan; 8 = Khon Kaen University, Thailand; 9 = Ministry of Health, Oman; 10 = NWO JSTP; 11 = Brazilian Federal Agency CAPES; 12 = Second Military Medical University Shanghai, China; 13 = Xinjiang Medical University, Urumqi, China; 14 = IBED, University of Amsterdam.
3.3 Research environment and embedding
Institutional and national collaboration
Our team participates in weekly CBS seminars and organizes group seminars every week. Members of our team participate in the international CBS Course Medical Mycology which is held every year in The Netherlands or elsewhere, particularly in China. We also co-organized an international course in veterinary
Part 3 - Origins of Pathogenicity in Clinical Fungi Programme – G.S. de Hoog 25 mycology in Nijmegen, The Netherlands, and are in the process of preparing a course on mycotoxins jointly with partners from Wageningen, Belgium and Slovakia. Most members of the group are member of the Netherlands Society for Human and Animal Mycology, which also provides podia for presentations by students. We host the yearly KNPV Fusarium day for Dutch and Belgian researchers. There are strong research links with the University of Wageningen on Luminex detection of clinical and mycotoxigenic Fusarium spp., and with hospitals in Nijmegen on epidemiological methods and antifungal susceptibility testing. Immunology of black yeasts is a new research line with Radboud University. With Erasmus University in Rotterdam we work on mycetomes and on aspects of human immune disorders. Sybren de Hoog is affiliated as professor to the Institute of Biodiversity and Ecosystem Dynamics of the University of Amsterdam, which is a funding agent of part of our programme.
International collaboration
Sybren de Hoog is professor at the Peking University Health Science Center, Research Center for Medical Mycology, Beijing, China, at the Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China, at the Shanghai Institute of Medical Mycology, Changzheng Hospital, Second Military Medical University, Shanghai, China, at the Basic Pathology Department, Federal University of Paraná State, Curitiba, Paraná, Brazil, and at the Biological Sciences Department, Faculty of Science, King Abdulaziz University, Jeddah, Saudi Arabia. Our research group receives regular guests from abroad, partly in the framework of the above collaboration agreements, as a result of intensive cooperation in ISHAM Working groups (below), and through individual contacts. Several of our Chinese guests were supported by the China Exchange Programme (CEP) of the Netherlands Academy of Sciences (KNAW) in cooperation with the Chinese government. Our team convenes the following ISHAM Working groups which each count 30–200 members: Fusarium (Anne van Diepeningen), Black Yeasts and Chromoblastomycosis [Sybren de Hoog with Peying Feng (China), Liyan Xi (China) and Roxana Vitale (Argentina)], Barcoding of Medical Fungi (Sybren de Hoog with Wieland Meyer (Australia)], Nomenclature of Medical Fungi [Sybren de Hoog with Gerhard Haase (Germany), Tom Walsh (USA), Vishnu Chaturvedi (USA), Wieland Meyer (Australia) and Michaela Lackner (Austria)]. We also organize regular workshops as part of the CBS Spring Symposia, such as on Mucorales (Utrecht 2010 and 2013), Barcoding (Utrecht 2013 and 2014), Black yeasts (Curitiba 2011, Guangzhou 2013, Tehran 2015), Dermatophytes (Utrecht 2015), and Fusarium (Coimbatore 2014 and Utrecht 2015). Sybren de Hoog gave keynote and opening lectures in several of these workshops, as well as in those on Sporotrichosis (Brazil 2013) and Paracoccidiodomycosis (Brazil 2014). He also presented an opening lecture and was awarded the European research prize at the Trends in Medical Mycology congress in Copenhagen (2013). In addition, an extended network has been built around the Atlas of Clinical Fungi, with contributors from CBS and from Spain, Hungary, China, Argentina, the UK and South Africa. A Chinese translation of the book is largely finished, a Russian version makes good progress, and an Arabic version has been initiated.
3.4 Quality and scientific relevance
Much of our work is devoted to diagnostics of clinically relevant fungi, for which we try to reconstruct a reliable taxonomic system, taking evolutionary processes as well as phylogenetic outcomes into account. We combine molecular and phenotypic data with ecological features, and try to explain behavioural trends through comparative genomics. It is becoming evident that ecology and pathology are fine-tuned, and often are remarkably specific for taxonomic entities. Within each entity ‒ species or cluster of species ‒ trends of evolution can be revealed, enabling understanding of evolutionary host shifts and changes from general vitality factors to dedicated virulence factors, allowing an evolution where fitness is increased by the use of a human host at any time in the life cycle of the fungus.
26 The genus Fusarium contains at least seven species complexes and several single species that are of clinical relevance. In leukemic patients Fusarium strains cause disseminated infections with a high mortality, while in other immunosuppressed patients the species generally leads to deep localized infections. In otherwise healthy individuals the genus is involved in onychomycosis, cutaneous infections, and keratitis. Especially Fusarium keratitis occurs frequently: in dry tropical areas more than 100,000 new cases are seen every year. Furthermore, many members of the genus Fusarium are able to produce mycotoxins, which can have a large impact on human metabolism and immune system. Fusarium ability to form biofilms enable it to thrive in the drinking water system as potential source of infection as well as enhance its pathogenicity in the human body. Multi-gene and full genome data of the genus are generated now being used for the development of new diagnostic tools and epidemiological studies, while also proteomic diagnostic tools are evolved. The order Chaetothyriales contains many black fungi with an infective potency, sometimes even causing disseminated infections in seemingly healthy patients. The agents of the severely mutilating disease chromoblastomycosis are restricted to this order. Pathogenicity is a polyphyletic latent homology within the order, expressed resp. absent in closely related species, and thus pathogenicity is unlikely to play an evolutionary role over larger phylogenetic entities. Environmental parameters such as oligotrophism, thermotolerance and hydrocarbon assimilation are more likely as evolutionary determinants. Aromatic hydrocarbons play a role in the ecology of ants, and indeed numerous undescribed black yeast-like fungi are consistently associated with these insects. Research has been started into exploitation of black fungi in bioremediation of oil-polluted soils. Truly extremophilic black fungi on rock are mainly found in the Dothideomycetes, where numerous new species and genera are being described. The groups are linked by expression of meristematic growth under conditions of stress, i.e. on rock and in tissue, respectively. Comparative genomics may reveal the factors involved, and explain the marked clinical differences between chromoblastomycosis and phaeohyphomycosis. Next generation proteomics will be developed to detect the genes concerned in vivo. The Onygenales is the major order of fungal pathogens, comprising the dermatophytes and the classical systemic agents (Coccidioides, Histoplasma, etc.). The large group of the dermatophytes have mainly been studied by ITS, and another level of precision with multilocus analysis is now needed to properly classify the anthropophilic species. The systemic pathogens are found in two other clades, and several phylogenetic species seem to be host-specific on particular mammals and geographically restricted. Some of the species have a relatively sloppy space, also being able to cause infection on non-optimal hosts such as humans. As some clades seem to consist exclusively of pathogens, a very interesting question will concern the origin of pathogenicity, which may be linked to known evolutionary ages of their mammal hosts. Comparative genomics in dermatophytes will focus on keratinolysis, where the transition from geophilic to anthropophilic specialization will be one of the intriguing questions. The opportunistic genus Scedosporium, the main species S. apiospermum and S. boydii consist of a number of separate lineages and is interpreted as a complex undergoing sympatric evolution with incomplete lineage sorting. Overall clinical spectra and antifungal profiles of the two species are very similar; differences in (multi-)resistance are mostly linked to individual strains and not to taxonomic entities. In order to understand the transmission of virulence and resistance over lineages, the enigmatic processes of sexuality within the genus have to be studied. The genus Ochroconis (Sympoventuriaceae, Venturiales) is revised and currently contains 13 species for which the phylogenetic position has been determined using multilocus sequencing. Phylogenetic distances of all markers analyzed are exceptionally large, both between and within species. A new genus Verruconis is proposed for the neurotropic opportunist Ochroconis gallopava. The first genomes will soon become available in the framework of our cooperation with the Broad institute, allowing comparative genomics with these fungi and the chaetothyrialean black yeast which are ecologically remarkably similar. In Sporothrix, the human infection known as sporotrichosis has an entirely different background than supposed some years ago. One species Sporothrix brasiliensis is transmitted by cats and shows limited
Part 3 - Origins of Pathogenicity in Clinical Fungi Programme – G.S. de Hoog 27 geographic distribution, suggesting slow vectors of dispersal. In contrast, S. globosa populations are dispersed on a global scale. The latter species originates from plant material, similar to S. schenckii. In both species the type of plant material was relatively variable, but nevertheless Sporothrix infections consistently in the form of outbreaks, sometimes with thousands of cases within a short timeframe. A hypothesis was therefore put forward that highly specific conditions in the plant material are required to promote the growth of Sporothrix, leading to outbreaks when these conditions are met. Fermented, self- heated plant debris may stimulate the thermodependent yeast-like invasive form of the fungus, which facilitates infections in mammals. The unique zoo- and sapronoses caused by Sporothrix species, as well as the unique host-shift from plant material to cats will reveal fascinating evolutionary mechanisms as soon as the genomes will become available, sequenced by partners in Brazil. Our study demonstrates that Madurella species are nested within the Chaetomiaceae, a family of fungi that mainly inhabit animal dung, enriched soil, and indoor environments. We hypothesize that cattle dung, ubiquitously present in rural East Africa, plays a significant role in the ecology of Madurella. If animal dung is an essential factor in inoculation by Madurella, prophylactic strategies may involve restructuring of villages to reduce the frequency of contact with etiologic agents of mycetoma. We also noted that the Chaetomiaceae are much more frequently involved in human infection than previously supposed, and the species possess a hidden clinical potential which needs to be explored. The order Mucorales comprises predominantly fast-growing saprotrophic fungi, some of which are used for the fermentation of foodstuffs but that are also known to cause infections in patients with severe immune or metabolic impairments. A first inventory of biodiversity inMucorales has been made involving 203 taxa. The analysis revealed that conventional phenotypic classifications of the Mucoraceae were highly artificial. The ITS region turned out to be an appropriate barcoding marker in Mucorales and can be sequenced directly in most strains leading to unambiguous recognition of molecularly well-delimited species. However, many taxa proved to be confused or undescribed. Novel molecular methods are being adapted to recognition of clinically relevant species. Some of these use powerful isothermic DNA amplification. In model studies the methods are compared with e.g. quantitative real-time PCR (qPCR), PCR-based reverse line blot (PCR-RLB), or AFLP data. We aim to recommend simple, rapid, and cost-effective methods for the identification of recently discovered or re- established species that are unknown in the routine laboratory or are indistinguishable with conventional techniques. On the other hand, a new field to be explored is the use of next generation proteomics for the rapid detection of species and to reveal their essential properties expressed in the host. To this aim we apply a prototype tandem-MS instrument with expertise from our industrial research partner. In addition to development of applicable technologies for identification, we aim to provide therapeutic data in the form of in vitro activities of panels of antifungal drugs against well-defined fungal species and varieties. This is done in close cooperation with partners in Nijmegen and Buenos Aires.
Key publications
Seyedmousavi S, Guillot J, Hoog GS de (2013). Black fungi, emerging opportunists in animals – a review. Clinical Microbiology Reviews 26: 19–35. Hoog GS de, Abdalla Ahmed S, Najafzadeh MJ, Sutton DA, Saradeghi Keisari M, Fahal AH, Eberhardt U, Verkley GJ, Xin L, Stielow B, Sande WWJ van de (2013). Possible new routes of infection of human eumycetoma. PLOS Neglected Tropocal Diseases 7: e2229. Samerpitak K, Choi HJ, Gerrits van den Ende AHG, Machouart M, Gueidan G, Hoog GS de (2014). Taxonomy of Ochroconis and Scolecobasidium, members of the novel order Ochroconiales. Fungal Diversity (online). Selbmann L, Onofri S, Zucconi G, Fonseca L, Isola D, Hoog GS de, Gueidan C (2013). Mountain tips as reservoirs for new rock-fungal entities: Saxomyces gen. nov. and four new species from the Alps. Fungal Diversity DOI 10.1007/s13225-013-0234-9.
28 Davari M, Babai-Ahari A, Arzanlou M, Sabbagh SK, Waalwijk C, Lee TAJ van der, Wei SH, Gerrits van den Ende AHG, Hoog GS de, Diepeningen AD van (2013) Geographic differences in trichothecene chemotypes of the FHB-causing ‒ Fusarium graminearum s.str. in the Northwest and North of Iran. World Mycotoxin Journal 6: 137–150. Dolatabadi S, Walther G, Gerrits van den Ende AHG, Hoog GS de (2014). Diversity and delimitation of Rhizopus microsporus. Fungal Diversity 64: 145–163. Li DM, Hoog GS de (2009). Cerebral phaeohyphomycosis - a cure at what lengths? Lancet Infectious Diseases 9: 376–383.
3.5 Output
Our programme has had a substantial output for the period (2008–2013), and has published 194 papers in six years.
Table 3.2. Scientific publications. 2008 2009 2010 2011 2012 2013 Total Academic publications Refereed journals 22 27 22 41 27 48 187 Book Chapters & 2* 1* – 1* 1* 2* 7 Proceedings Total 24 28 22 42 28 50 194 Monographs & books – 1 – – – 1 2 Ph.D. theses 2 1 2 1 – – 6 Popular publications – – – – – – – and products
*) Edited special issues in Medical Mycology (2x), Fungal Biology, Mycopathology, Mycoses, Studies in Mycology and Persoonia (3 planned for 2014: in Mycopathology, Fungal Diversity and Mycoses, respectively)
3.6 Earning capacity
The research group of Medical and Extremophilic Fungi has been successful in acquiring funds from a variety of national and international programmes and institutions. For our collaboration with medical centres and research institutes in China we obtained a KNAW-China Exchange Programme (CEP) grant in 2011 (11CDP009) and in 2013 a new project started on mycotoxigenic fungi, funded via a personal grant JSTP- NWO (833.13.006). Our PhD students are supported by grants from their own universities, supplemented with grants from IBED, University of Amsterdam, where they will have their promotion. A large collaboration with ThermoFisher Scientific Co. started in 2013, supporting a PhD student and a Postdoc researcher, and running costs. In 2014 the project will be extended with two more Postdoc researchers. In 2012 a project with free equipment was funded by BioMérieux. A grant was obtained from the King Abdulaziz University in Saudi Arabia in view of bioremediation studies. Smaller contributions we acquired from various sources, mostly earmarked for international activities such as working groups, websites and workshops. Through our network we are able to keep our costs at moderate levels. Examples are a black yeast genome sequencing agreement with Broad Institute (http://www.broadinstitute.org/annotation/genome/Black_Yeasts/MultiHome. html), agreements on antifungal testing and other methods with Radboud and Canisius in Nijmegen, The Netherlands, and proteomic studies in the framework of our contract with ThermoFisher Scientific.
Part 3 - Origins of Pathogenicity in Clinical Fungi Programme – G.S. de Hoog 29 3.7 Academic reputation
Awards
International Society for Human and Animal Mycology (Past President, honorary member). European Confederation of Medical Mycology (co-founder). Netherlands Society Medical Mycology (co-founder, scientific secretary). Pan-African Medical Mycology Society (co-founder). Eduard Drouhet Medal of the European Confederation of Medical Mycology.
Appointments
University of Amsterdam, Institute for Biodiversity and Ecosystem Dynamics IBED (professor). Peking University Health Science Center, Research Center for Medical Mycology, Beijing, China (extraordinary professor). Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China (honorary professor). Shanghai Institute of Medical Mycology, Changzheng Hospital, Second Military Medical University, Shanghai, China (extraordinary professor). Basic Pathology Department, Federal University of Paraná State, Curitiba, Paraná, Brazil (professor). Biological Sciences Department, Faculty of Science (North Jeddah), King Abdulaziz University, Jeddah, Saudi Arabia (professor).
Editorships in academic journals and book series
Mycoses Mycopathologia Mycological Progress Guest editor of special issues of Medical Mycology, Mycoses, Mycopathologia, Fungal Biology, Fungal Diversity, Fungal Biology Reviews, and Persoonia
Memberships in Scientific Boards
ISHAM Working Groups on Black Yeasts and Chromoblastomycosis (convener Sybren de Hoog) ISHAM Working Group on Fusarium (convener Anne van Diepeningen) ISHAM Working Group Barcoding Medical Fungi (convener Sybren de Hoog) ISHAM Working Group Nomenclature of Medical Fungi (convener Sybren de Hoog)
Selection of invited lectures
Plenary opening lecture at Trends in Medical Mycology, Copenhagen, Denmark, October 2013. Keynote lecture at Sporothrix meeting, Rio de Janeiro, Brazil, 2013. Opening lecture at Iranian Congress of Mycology, Sari, Iran, 2011. Keynote at Microbiological congress “Power of fungi and mycotoxins” in Trogir, Croatia, 19-22 October, 2011. Opening lecture at Fourth meeting on Black Yeasts and Chromoblastomycosis “Hidden Danger, Bright Promise”, Curitiba, Brazil, 1−4 December, 2011.
30 Bibliometric analysis
• Number of peer reviewed publications: 126 (Web of Science, March 2014) • H-index: 35 (Web of Science, March 2014); 51 (Google Scholar, March 2014) • Total number of citations: 4343 (Web of Science, March 2014); 14675 (Google Scholar, March 2014)
3.8 Societal relevance: quality, impact and valorisation
It is our general aim to transmit our data to the user in the clinical laboratory and in industry in a way that is practical, useful and understandable. Phylogenetic, epidemiologic and other biodiversity data are published in accessible journals for dedicated audiences, and are followed by two types of studies. One concerns the development of diagnostic methods that can be used in routine to recognize the entities that are relevant to the respective areas. To this aim, methods like RCA (rolling circle amplification), LAMP (loop-mediated isothermal amplification), MALDI-TOF and others have been designed for clinically relevant species. With the help of an industrial partner, next generation proteomics using mass spectrometry are being developed as tools for recognition and monitoring. Our global activities in the various ISHAM Working Groups, for FungiScope, and for the San Antonio Reference Lab (USA) stimulate identification of strains that otherwise would have gone unnoticed.
3.9 Viability
Over the past peer review period the number of postdocs and PhD students in the group have tripled. A constant source of income is generated by the different universities where Sybren de Hoog has been appointed as professor, particularly Amsterdam, Jeddah and Curitiba. Experienced guest workers from China were supported by the KNAW China Desk. We have significant links with industry. A project with three postdocs and one PhD student, and including instrumentation and running costs is provided by ThermoFisher Co., while we also concluded a project for BioMérieux. A JSTP-NWO project supports a further PhD student. We are regularly invited to join national and international projects.
3.10 Strategy
Our most important focus is on less-current fungal species involved in medical mycology. Originally our position was particularly strong in black fungi where we are globally leading, but now a significant part of our output concerns other fungi, such as Fusarium, Sporothrix, Scedosporium, Madurella, dermatophytes, systemic Onygenales, and Mucorales. The networks are expanded by the yearly organization of workshops, mostly in Utrecht, on topics such as black fungi, Mucorales, dermatophytes and Fusarium. New concepts on ecology and pathology have been developed in recent years. As an example, infection routes and host shifts were studied in Sporothrix, which species occur in very large outbreaks of sometimes thousands of cases. We explained the outbreaks by assuming formation of a yeast-like form similar to the known tissue phase in humans under particular conditions of self-heated rots of plant material. Our very successful workshops led to several special journal issues, such as Medical Mycology, Fungal Biology, Fungal Diversity, Persoonia, Mycoses and Mycopathologia. The next Barcoding meeting will be a special issue of Fungal Biology Reviews. An important development is our contract with the Broad Institute at Harvard (USA) to sequence a large series of genomes of black fungi, for which a dedicated website has been made at Broad. In addition, relevant genomes are sequences at the Genome Institute in Brasilia, Brazil, and in centres in China and Saudi Arabia. Exchange of living material on a global basis has however severely been jeopardized by increasingly strict regulations of biosafety, and unjustified fear for bioterrorism.
Part 3 - Origins of Pathogenicity in Clinical Fungi Programme – G.S. de Hoog 31 In addition, the following projects have been submitted or are in preparation:
(1) Three-year continuation of research agreement with ThermoFisher Scientific, covering three postdocs and one PhD student, free next generation MS equipment and running costs. (2) Bioremediation of hydrocarbon-polluted soil by black fungi, funded by King Abdulaziz University, Jeddah, Saudi Arabia (3) Genomics of black fungi, to be submitted at National Research Foundation of China. (4) Role of black yeast biofilms in galvanic cells, submitted at Science Foundation of Poland. (5) Blackening of intestine castings, to be submitted at International Casting Society. (6) Role of hydrocarbon-assimilating black fungi in biofilters, to be submitted at Stichting Technische Wetenschappen. (7) Diagnostic tools for mycetoma, to be submitted at EU Horizon 2020. (8) Network on Zygomycetes biodiversity, to be submitted at Leibniz Competition SAW, Germany. (9) Immunology of black fungi, to be submitted at Radboud University, Nijmegen and industrial partners. (10) Biodegradation of extracted plant material, to be submitted at industrial partner, China. (11) Guest workers are expected with grants from CAPES (Brazil), Erasmus, and Chinese funding.
Our team will be one of the two core organizing groups to make a bid for the congress of the International Society for Human and Animal Mycology (ISHAM) in Amsterdam 2018; if we are elected (in 2015), about 1000–1500 participants are expected.
32 4. Yeast and Basidiomycete Programme
Programme leader Dr Teun Boekhout
4.1a Objectives(s) and research area
Yeasts are among the best studied eukaryotes on earth due to some very well studied species, such as Saccharomyces cerevisae and Schizosaccharomyces pombe. Research in the Yeast and Basidiomycete (Y&B) programme focuses on pathogenic basidiomycetous yeasts, such as Cryptococcus neoformans and C. gattii, and skin inhabiting Malassezia spp. C. neoformans is one of the major fungal pathogens causing over a million new infections per year with approximately 600,000 casualties per year. C. gattii, a sibling species of C. neoformans, is of emerging importance as it infects immunocompetent humans and animals and is causing some major ongoing outbreaks. Moreover, the species is also emerging in Europe. Malassezia yeasts are carried by all humans and are a major component of the human skin microbiome. These yeasts are involved in a number of skin diseases, such as pityriasis versicolor, seborrheic dermatitits, atopic dermatitis etc., but also can cause sepsis, especially in neonates that receive parenteral nutrition. Our research projects aim to understand the relationship between pathogen biodiversity and virulence related properties of these clinically important species. In-depth studies will focus on the relationship between evolution (incl. biodiversity, phylogeography, speciation, taxonomy) and pathogen traits (i.e. virulence and susceptibility to antifungals). In this medical mycological oriented research program we also aim to develop innovative diagnostics for the clinic using comparative genomics, extensive validation studies and testing in clinics. Unification of the phylogeny and taxonomy of all yeasts within the frame of the Fungal Tree of Life (TOL) is a second major research topic. Our involvement as editor in the standard reference work “The Yeasts, a Taxonomic Study” [5th edition, 2011, 1532 cites Google Scholar Jan. 29 2014] gives us a good position to realize this work. With colleagues from State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China, we performed a multigene phylogenetic analysis of all currently accepted basidiomycetous yeast species (BYe-TOL project). This has already resulted in the recognition of two new classes, Monillielomycetes and Malasseziomycetes, in Ustilaginomycotina. Also in the Pucciniomycotina and Agaricomycotina major changes in the phylogenetic relationships, and, consequently, their taxonomy are foreseen. In the context of the KNAW-supported barcoding project of the CBS collection (Dr Benjamin Stielow) and in close cooperation with Dr Marizeth Groenewald, curator of the CBS yeast collection, we initiated a multigene-based phylogenetic study of all ascomycetous yeasts. Although, still ongoing, we foresee a major impact on our understanding of the phylogenetic relationships among these yeasts, yeast-like taxa and other fungi. One of the main results will be a completely revised taxonomy of yeasts. Comparative genomics is increasingly important to understand the proper phylogenetic relationships among yeasts [a field already explored by us quite some time ago] and to understand their traits (i.e. life styles). We are PI of the CNRS supported European network iGenolevures (France, 2014–2018) that is the successor of the highly successful French Genolevures network. Groups from France, Spain (Gabaldon), Portugal and The Netherlands participate in the network. Species discovery is also part of our research efforts. Due to our long term expertise we are frequently consulted about questions related to species distinction. Ecological aspects of yeast and basidiomycete fungi are addressed in a number of collaborative projects (see below).
4.1b Forward look
Medical important yeasts will continue to be a main cause of disease with high morbidity and mortality, and thus cause a great burden on the society. Moreover, the work we have been doing on this terrain turned
Part 4 - Yeast Research Programme – T. Boekhout 33 out to be quite fruitful, e.g. judged by number of citations. In the research on Cryptococcus we plan to investigate more in detail which phenotypic trait is involved in hyper- and hypovirulence and this will be performed in close collaboration with Prof. Dr Theo Geijtenbeek (AMC, Amsterdam). This collaboration will be extended to other clinically important fungi, such as Aspergillus, Fonsecaea, Candida, etc. Note that our previous PhD student Ferry Hagen (graduation 2011) submitted an application for a VENI grant (NWO) in which he proposes to study the emergence of virulence related traits during the evolutionary history of C. gattii, a species that causes some major outbreaks, through genomics-based coalescence analysis (collaboration with Center of Mathematics and Informatics, CWI, Amsterdam, Dr Leo van Iersel, Prof. Dr Gunnar Klau en Prof. Dr Leen Stougie). Malassezia yeasts, especially comparative genomics, understanding of infraspecific structure of M. furfur with hybrids, role in the skin microbiome, and the apparent preference of some genotypes to cause invasive disease will continue to get our attention. The importance of Malassezia yeasts is currently highlighted by many research groups, especially focussing on skin microbiome risk in disease and (comparative) genomics. We believe that its involvement in the human skin microbiome may offer good possibilities for obtaining grants (e.g. with Theo Geijtenbeek and Sonja Gringhuis (AMC) as well for H2020 funding). Development of efficient detection tools using comparative genomics, wet lab testing and clinical validation will be continued. Presently we work on this supported by a grant from Qatar Foundation in collaboration with Dr Toni Gabaldon and the postdoc Dr Salvador Capella (Centro de Regulacion Genomica, Barcelona, Spain) and Dr Saad Taj-Aldeen and colleagues at Hamad Medical Corporation in Doha, Qatar. Importantly, the pipe line will have a universal character and will be able “to digest” genomes of pro- and eukaryote pathogens, and probably even viruses. Resistance markers for e.g. azoles and echinocandins will also be included. This project is running smoothly and aims to develop a probe finder bioinformatics pipeline that may become the basis of an SME. We plan to submit a proposal for an Innovative Training Networks (ITN in Horizon2020) in April 2014 on the further development of diagnostics in clinical mycology. Finally, it is important for the development of our research agenda to know if, and when, the two medically oriented research groups in CBS will merge. This merger will provide more critical mass that we consider important for the further development of medical mycology in CBS. In the area of yeast biodiversity we will finish our multigene phylogenetic analyses of all presently described species (ca. 2000) and the results will be published in a number of papers. Probably even more important is that the results will form the foundation for the establishment of a new taxonomy of yeasts, based on 1. Phylogenetic evidence; 2. Monophyly, and 3. One fungus = One name. We expect that this [more] natural system of classification will have predictive value that may be useful in screens, such as those for compounds, enzymes etc. The new taxonomy will be discussed first with the Yeast Taxonomy Committee of International Commission of Yeasts (ICY, IUMS) in order to reach maximum consensus. The new taxonomy may likely be published in a volume of Studies in Mycology. Thereafter this will be the base for the new edition of “The Yeasts, a Taxonomic Study”. It was decided to leave Elsevier for this project and to start a new collaboration with Trunity (USA, http://www.trunity.com/) to make a web-based monograph that is updatable in time. Moreover, the number of topics to be included will be expanded, e.g. next to the species descriptions, it will also cover Food and Spoilage yeasts, Pathogen yeasts, Agricultural important yeasts, Biotech aspects, Molecular Biology, Laboratory Protocols, and Tutorials for students. This web-based product may be further developed in collaboration with commissioners/experts in the various fields from ICY, many of which are also involved in teaching fundamental and applied aspects of yeast biology. Species discovery will be ongoing and mainly done in collaboration with ecologically oriented researchers and in our ecologically oriented projects. One of them is te ongoing Qatar Foundation sponsored multi partner project “Mapping the Microbial Diversity in the Arabian Gulf”. Comparative genomics will be increasingly important in the field of yeast biodiversity. We will continue to collaborate in appropriate European networks, such as iGenolevures (funded by the French funding body CNRS). In the Horizon2020
34 program (EU) and elsewhere (China?) we will also look for funding opportunities for significant grants on this topic. Besides, we will invest some of our own resources to see what can be done in CBS using IonTorrent technology. Results from comparative genomics of yeasts will be strongly beneficial to society as screens for compounds, enzymes, etc, can firstly be performedin silico, and subsequently in vivo, using targeted species/strain sets. Ecologically focused projects will be mainly executed in collaborations in which we take responsibility for yeast [and sometimes also filamentous fungal] diversity. We expect that two students who worked [and work] in the Colombian Amazon rain forests will graduate in 2014. Importantly, in the future this ecologically oriented work may result in obtaining a better understanding of the global emergence of C. gattii since we postulated that it originated from native trees in pristine Amazon rain forests in Northern Brazil [Hagen et al. 2013, PLos ONE 8: e71148. doi:10.1371/journal.pone.0071148]. More recently we got involved in an assessment of diversity of marine microbes around Qatar [funded by Qatar Foundation] that will comprise culturing studies and metagenomics analyses of pro- and eukaryotic microorganisms. Besides GIS mapping, analysis of ecological important parameters will be done by Qatarese partners and [oil] industry. For filamentous fungi we will collaborate with various CBS colleagues (Jos Houbraken, Anne van Diepeningen) and other experts (e.g. Irina Druzhinina, Vienna). We expect several new species to be identified [in fact the first novel Aspergillus spec. is already identified]. New PhD students are expecting in two research projects funded by Qatar Foundation. Unexpectedly, we noted that some fungi from the anoxic zone In the Arabian Sea were able to grow anoxically. One of these isolates was recently described as a new species in the basidiomycete genus Tritirachium (Pucciniomycotina). Morphological and ultrastructural differences were noted between oxic and anoxic growth. In a controlled growth experiment with another anoxically growing fungus a link between anoxic growth and nitrogen metabolism was observed (Stief et al., BMC Microbiology accepted, highly accessed: 2000 in 3 weeks). It may well be possible that fungal growth under anoxic condition is also important for fungi involved in deep seated infections. We will try to set up some pilot experiments to see if this hypothesis makes sense, and, if so, look for funding opportunities. This will be relevant to immunological interactions. Within CBS I expect that the smooth collaboration with the curator of the Yeast Collection (Dr M. Groenewald) will be continued or even intensified. During the last couple of years we together started some ambitious projects on yeast diversity and classification that will result in some landmark publications with a high impact on the field [not necessarily meaning that they will be published in journals with a high impact factor though].
4.2 Composition
Table 4.1. Research staff at programme level, 2008–2013. 2008 2009 2010 2011 2012 2013 Funding* Research staff Dr T. Boekhout 1.0 1.0 1.0 1.0 1.0 1.0 1 Postdoc Dr R. Boesten 1.0 1.0 1.0 1.0 – – 2 Dr S. Capella Gutierrez – – – – 0.3 1.0 7 PhD F. Hagen 1.0 1.0 1.0 1.0 – – 3 K. Khayhan 1.0 1.0 1.0 1.0 1.0 1.0 4 R. Quintilla Mateo – – – 1.0 1.0 1.0 5 A. Vasco Palacios – – 1.0 1.0 1.0 1.0 6 H. Mohamed – – – – – 0.3 7 Technician B. Theelen 1.0 1.0 1.0 1.0 1.0 1.0 1
Part 4 - Yeast Research Programme – T. Boekhout 35 *1 = Royal Netherlands Academy of Arts and Sciences; 2 = EuroTransbio (EU); 3 = Odo van Vloten Foundation; 4 = Naresuan University Grant, Phayao, Thailand; 5 = EU Initial Training Network; 6 = NUFFIC, with additional grants from Schlumberger Foundation (Paris) and International Foundation of Science (Stockholm); 7 = Qatar Foundation.
4.3 Research environment and embedding
Institutional and national collaboration
Within CBS the Yeast and Basidiomycete research group closely interact with the curator of the yeast collection, Dr M. Groenewald. She and Dr T. Boekhout are involved in a number of joint projects, such as the Cornucopia project, the new edition of “The Yeasts, a Taxonomic Study”, Assembling the Tree of Life (TOL) of yeasts, Barcoding of the yeast collection, and several smaller projects such as species descriptions, and various manuscripts. We believe that this close interaction between the yeast collection and - research is beneficial for both. In addition, this collaboration also results in a bigger size of the Y&B research group. Note that the barcoding and TOL projects are also performed in collaboration with the team of Dr. Benjamin Stielow (Collection Department). A multigene-based phylogenetic study of all ascomycetous yeasts (ca. 1500 spp.) is developed in the context of the KNAW-supported barcoding project (Dr Benjamin Stielow, Dr Marizeth Groenewald). Although, still ongoing, we foresee a major impact on our understanding of the phylogenetic relationships among yeasts, yeast-like taxa and other fungi, as well as a completely revised taxonomy of these yeasts. This new taxonomy will be developed with experts of the various groups of yeasts in order to reach a consensus taxonomy that will stand for a significant amount of time. In some of the ecologically oriented biodiversity projects the filamentous fungi encountered are being studied by various experts at CBS (e.g. Drs A. van Diepeningen, J. Houbraken) or abroad (e.g. Dr I. Druzhinina, Vienna, Austria). At the national level, collaborations exists with Department of Medical Microbiology, University Medical Centre of Utrecht (Dr E. Boel, diagnostics of fungal infections); Department of Experimental Medicine, Academic Medical Centre, University Amsterdam (host-pathogen interactions, Prof. dr T. Geijtenbeek and Dr S. Gringhuis); Department of Clinical Microbiology, Canisius Wilhelmina Hospital (CWZ) in Nijmegen (diagnostics and Cryptococcus biology, Dr F. Hagen), Departments of Cryo-electronmicroscopy of Utrecht University (electronmicroscopy, Dr W.M. Müller); Mathematics and informatics VU Amsterdam (Prof. dr L. Stougie) and Centre for Mathematics and Informatics (CWI), Amsterdam (Prof. dr G. Klau, Dr L. van Iersel) to develop and test algorithms to detect reticulate evolution. TB has a zero appointment at the Department of Infectious Diseases at University Medical Centre of Utrecht (UMCU), and it may well be that he will be appointed an associate professorhsip at IBED (University of Amsterdam) soon.
International collaboration
The Y&B research group has established several collaborations that are important for its mission. TB has an appointment as visiting professor at Chinese Second Military Hospital, Shanghai, China, and he is a research associate at the State Key of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China. In the medical mycological oriented yeast research we aim to develop innovative diagnostics for clinical use by combining comparative genomics and wet lab validation and testing. This work was funded by EU [EuroTransbio] and presently by Qatar Foundation and is executed in collaboration with Dr Toni Gabaldon from the Centre de Regulacion Genomica (GCR, a top class institute in the field of genomics) in Barcelona and Hamad Medical Corporation, Doha, Qatar (Dr S. Taj-Aldeen). Comparative genomics is increasingly important to understand the proper phylogenetic relationships among yeasts, to understand
36 fungal traits (i.e. life styles), but also for diagnostics development. We are PI of the CNRS supported network iGenolevures (France, 2014–2018) that is the successor of the highly successful Genolevures network. Groups from France, Spain (Gabaldon), Portugal and Netherlands participate in iGenolevures. Extensive international collaborations were realized within two working groups of International Society for Human and Animal Mycology (ISHAM). The first is the ISHAM working group on “Typing of Cryptococcus and cryptococosis”, and the second is the working group on Malassezia. Within Europe, a collaborative network was initiated to study the presence and distribution of C. gattii in the Mediterranean region after our successful collaborative work in the context of the European Confederation of Medical Mycology (ECMM) on the emergence of C. gattii in Europe (Hagen et al. 2012, EID 18: 1618 that was discussed as a top 10 paper in Medical Mycology at both ICAAC 2013, USA and TIMM6, Copenhagen). Extensive international collaborations exist in the cryptococcal research field. Just to name J. Kwon- Chung (NIH, USA), J. Heitman (Durham, USA), A. Litsintseva (Durham, USA), J. Kronstad (Vancouver, Canada), W. Meyer (Sydney, Australia), I. Polacheck (Jerusalem, Israel), R. May (Birmingham, UK), M. Fischer (Imperial College, London, UK), F. Dromer & G. Janbon (Institut Pasteur, Paris, France), R. Wahyuningsih (Jakarta, Indonesia), A. Botha (Stellenbosch, S. Africa), and Kantarawee Khayhan (Phayao University, Phayao, Thailand [Naresuan University University PhD grant 2008–2012]. From May 15- 19 2014 we organize the 9th International Congress on Cryptococcus and cryptococcosis (ICCC9) in Amsterdam, The Netherlands. The second is the ISHAM working group on Malassezia (Drs A. Velegraki, G. Gaitanis, Greece; Drs C. Cafarchia, R. Iatti, D. Otranto, Italy, a.o.), but we also participate in the Malassezia genome consortium with participation from Procter & Gamble (Cincinnati, USA, C. Saunders & colleagues), T. Dawson (Singapore), J. Heitman (USA), J. Kronstad (Canada), A. Scheynius, Karolinska Institute, Sweden, a.o.). One of our aims is to bring these two consortia in Malassezia research together and develop a unifying research agenda, e.g. on human skin microbiome biology. With colleagues from the Key State Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China, we performed a multigene phylogenetic analysis of all currently accepted basidiomycetous yeast species (BYe-TOL project, ca. 700 spp.). This has already resulted in the recognition of two new classes, Monillielomycetes and Malasseziomycetes, in Ustilaginomycotina (Wang et al. submitted). Also in the Pucciniomycotina and Agaricomycotina major changes in the phylogenetic relationships of yeasts, and, consequently, their taxonomy are foreseen. Teun Boekhout is Chief Editor for the coming edition of “The Yeasts, a Taxonomic Study” [editors F.- Y. Bai, China; H.M. Daniel, Belgium; M. Groenewald, CBS]. As indicated, we decided to leave Elsevier as a publisher for this project as this multinational is not able to cope with the demands for a flexible and updatable web-based product. Therefore, we start working with Trunity, a US-based electronic publisher. Thus the “new edition” of “The Yeasts” will be web-based, updatable, with a flexible format that allows addition of new chapters or changing the order of chapters, and will contribute to the development of tutorials for teaching and student curricula. The scope of the “book” will be expanded to cover more aspects of yeast biotechnology, food microbiology, clinical microbiology, and other applied aspects of yeast biology. In the Cornucopia Initial Training Network (ITN, EU) functional aspects of yeast biodiversity are being studied by a number of PhD students and postdocs at various academic and industry labs in Europe. Aspects studied are “extreme” yeasts, probiotic yeasts, wine yeasts, brewing yeasts, volatile production etc. Raquel Quintilla Mateo, the PhD student at CBS, focuses on understanding the genetic basis of “extreme” yeasts. This work is done in close collaboration with M. Groenewald (CBS Yeast collection) and Prof. J. Thevelein (KU Leuven, Leuven, Belgium). The 4th year of her PhD thesis will be spent in Leuven. Presently, we are involved in two research project funded by Qatar Foundation that are realized in close collaboration with researchers from Qatar (Dr. Saad Taj-Aldeen, Hamad Medical Corporation and Dr. Rashmi Fotedar, Biotechnology Center, Ministry of Environment), and also include participants from Germany (Kaiserslautern, T. Stoeck) and Spain (Barcelona, T. Gabaldon).
Part 4 - Yeast Research Programme – T. Boekhout 37 4.4 Quality and scientific relevance
Key publications
Arendrup MC, Boekhout T, Akova M, Cornely OA, Lorthlaro O, ESCMID/EFISG study group (2013). ESMID/ECMM Guideline diagnosis and management of emerging yeast infections. Clinical Microbiology and Infection (doi: 10.1111/1469-0691.12360) Hagen F, Ceresini PC, Polacheck I, Ma H, Nieuwerburgh F van, Gabaldón T, Kagan S, Pursall ER, Sionov E, Falk R, Hoogveld HL, Iersel LJJ van, Klau GW, Kelk SM, Stougie L, Bartlett KH, Castañeda E, Lazera M, Meyer W, Deforce D, Meis JF, May RC, Klaassen CHW, Boekhout T (2013). Ancient dispersal of the human fungal pathogen Cryptococcus gattii from the Amazon rainforest. PLos ONE 8: e71148. doi:10.1371/journal.pone.0071148. [blog http://latinamericanscience.org/2013/08/human- fungal-outbreaks-traced-to-brazil-rainforest/] Hagen F, Colom MF, Dromer F, Swinne D, Tintelnot K, Iatta R, Montagna MT, Torres-Rodriguez JM, Viviani MA, Velegraki A, Gari-Toussaint M, Burggraaf A, Kamermans A, Sweere J, Meis JF, Klaassen CHW, Boekhout T (2012). Emergence of autochthonous and dormant Cryptococcus gattii infections in Europe: a Multi-Locus Sequence Typing study. Emerging Infectious Diseases 18: 1618–16124. [TIMM6 & ICAAC 2013 Top 10 papers in Mycology] Gringhuis SI, Kaptein TM, Wevers BA, Theelen B, Vlist M van der, Boekhout T, Geijtenbeek TBH (2012). Dectin-1 is an extracellular danger sensor for induction and processing of IL-1β through activation of a Malt1-ASC-caspase-8 complex. Nature Immunology 13: 246–255. [News and Views Nature Immunology 13: 211–212] Kurtzman CP, Fell JW, Boekhout T (Eds) (2011). The Yeasts, a Taxonomic Study, 5th ed., 3 Vol., Elsevier, Amsterdam, 2080 pp. Boekhout T, Guého E, Mayser P, Velegraki A (Eds) (2010). Malassezia and the skin. Science and Clinical Practice. Springer Verlag, Berlin, 319 pp. Driel KGA van, Peer AF van, Grijpstra J, Wösten HAB, Verkleij A, Müller WH, Boekhout T (2008). The septal pore cap protein SPC18 isolated from the basidiomycetous fungus Rhizoctonia solani also resides in pore-plugs. Eukaryotic Cell 7: 1865–1873 [featured in Mycological Research News Nov. 2008].
4.5 Output
Our group has had a substantial output for the period (2008–2013), and has published 85 papers and two major reference works in six years.
Table 4.2. Scientific publications. 2008 2009 2010 2011 2012 2013 Total Academic publications Refereed journals 11 14 12 15 19 14 85 Book Chapters & – – 4 32 1 1 38 Proceedings Total 11 14 16 47 20 15 123 Monographs & books – – 1 1 – – 2 Ph.D. theses 1 – – 1 1 – 3 Popular publications – 1 – – – – 1 and products
38 4.6 Earning capacity
Our research is funded by various organizations, such as EU (ITN, 7th framework), Qatar National Research Fund (part of Qatar Foundation), KNAW (NL-China collaborative grant on TOL of basidiomycetous yeasts, Barcoding of ascomycetous yeasts, including a multigene study of all accepted species), National Research Foundation of China (TOL of basidiomycetous yeasts), NUFFIC), Schlumberger Foundation, International Foundation of Science (all supporting research on ectomycorrhiza in Colombia Amazon rain forest), and Naresuan University Thailand (University grant for a PhD student). Last year we also participated in a proposal LIFELINK that was submitted to the NWO Roadmap scheme together with partners from NIOZ, IBED and NATURALIS. Development and continuation of projects will strongly depend on the ability to obtain future funding. Due to the rather difficult funding situation in The Netherlands, we are already considering alternative funding sources. Therefore, ongoing collaborations in Europe (Horizon2020), China (with various options for joint grants) and Qatar (with funding opportunities form Qatar Foundation) will become more important. Besides we will continue to get funds from Netherlands Organization for Science (NWO, ALW and STW). We already participate in a proposal for an Innovative Training Network (ITN) on translational research of clinically important yeasts that will be submitted April 2014; similarly we will resubmit a proposal with Prof. Theo Geijtenbeek (AMC, Amsterdam) to NWO on host-pathogen interactions of fungal [yeast] infections that was not funded in an earlier round. We also consider funding for human skin mycobiome projects together with partners from the Malassezia consortia in which we participate, e.g. H2020 or private funds. Together with Prof. Theo Geijtenbeek and Dr Sonja Grinhuis (Center for Experimental and Molecular Medicine, AMC, Amsterdam) we will consider a top grant on host-pathogen mechanisms probably related to the skin microbiome and interactome. Importantly, it is important to find a balance between performing research, writing proposals, writing manuscripts, supervising students, and, probably the most important, thinking about interesting new research avenues.
4.7 Academic reputation
Appointments
Visiting professor Shanghai Key Laboratory of Molecular Medical Mycology, Shanghai, China (2012–) Research Fellow Institute of Microbiology, Chinese Academy of Sciences (IM-CAS), Beijing, China (2013–)
Editorships in academic journals and book series
Editor FEMS Yeast Research (2006–2012) Editor Fungal Biology (2006–) Associate Editor Medical Mycology (2009–) Editor Malassezia and the skin (eds. T. Boekhout, E. Guého, P. Mayser, A. Velegraki, Springer, Berlin) (2010) Editor 5th edition The Yeasts, a Taxonomic Study (3 Vol., 2080 pp., eds C.P. Kurtzman, J.W. Fell & T. Boekhout, Elsevier, Amsterdam) (2011) Editor The Yeasts, a Taxonomic Study (6th edition / ebook, eds T. Boekhout, F.Y. Bai, M. Groenewald & MH.M. Daniel) (2012–)
Manuscripts are reviewed for the following journals: Antimicrob. Agents Chemother.; Antonie van Leeuwenhoek; Appl. Env. Microbiol.; Appl. Microb. Biotechnol.; Biodivers. Conserv.; Biol. Conserv.; Biol. J. Linnean Soc.; Biophys. Biochim. Acta; BMC Microbiology; BMC Evol. Biol.; Clin. Infect. Dis.; Clin. Microbiol. Infect.; Clin. Microbiol. Rev.; Eukar. Cell.; Eur. J. Clin. Microbiol. Infect. Dis.; FEMS Yeast Res.;
Part 4 - Yeast Research Programme – T. Boekhout 39 FEMS Microb. Ecol.; Flora Neotropica; Fungal Genet. Biol.; Future Medicine; Int. J. Infect. Dis.; Genome Biol. Evol.; Indonesia Microbiol.; Int. J. Syst. Evol. Microbiol.; J. Clin. Microbiol.; J. Gen. Appl. Microbiol.; J. Industr. Microbiol. Biotechnol.; J. Infect. Dis.; J. Invest. Dermatol.; J. Med. Microbiol., J. Microbiol. Meth.; Memórias do Instituto Oswaldo Cruz; Mol. Genet. Genomics.; mBIO; Med. Mycol.; Mycologia; Mycol. Res.; Plant Biology; Plant Biosystems; PLOS One; PLOS Genetics; PLOS Pathogens; Rev. Inst. Med. Trop. Sao Paulo; Yeast.
Memberships in Scientific Boards
Member Safety and Security Commission KNVM (2006–) Vice-chair of ESF-EMBO conference on Comparative Genomics of Eukaryotic Microorganisms (San Feliu, Spain) (2007–) Commissioner International Commission of Yeasts (ICY, IUMS) (2007–) Member ISHAM Global Panel of Opinion Leaders (2008–) Member Scientific Advisory Boardth 7 International Conference on Cryptococcus and cryptococcosis, Nagasaki, Japan (2008) Member Scientific Advisory Boardth 17 Congress of the International Society for Human and Animal Mycology, Tokyo, Japan (2009) Vice-chair EMBO conference on Comparative Genomics of Eukaryotic Microorganisms, San Feliu, Spain (see Fischer G et al. 2009 Meeting Report. Genome Biol. 10:318; http://genomebiology.com/2009/10/12/318) (2009) Chair EMBO conference on Comparative Genomics of Eukaryotic Microorganisms, San Feliu, Spain (see de Mendoza and Ruiz-Trillo Meeting Report. EvoDevo 2011, 2:22; http://www.evodevojournal.com/ content/2/1/22) (2011) Member Scientific Advisory Boardth 18 Congress of the International Society for Human and Animal Mycology, Berlin, Germany (2012) Member Scientific Advisory Board 13th International Congress on Yeasts, Madison, Wisconsin, USA (2012) Vice-chair Mycology / Eukaryotic Microbiology Division, International Union of Microbiological Societies (IUMS) (2012–2014) Member of European Society of Clinical Microbiology and Infectious Diseases (ESCMID) and European Confederation of Medical Mycology (ECMM) working group of rare yeast infections (2012–) Member International Commission on Taxonomy of Yeasts (ICY, IUMS) (2013)
Selection of invited lectures
Boekhout T (2013). Yeast Diversity United (Keynote). 13th Int. Congr. Culture Collections WFCC, Beijing, China. Boekhout T, Posteraro B (2013). MALDI-TOF MS, a robust tool for fungal identification and beyond). th6 Trends in Medical Mycology (Meet the Expert session), Copenhagen. Boekhout T (2012). Yeast Diversity United: Exploring Yeast Biodiversity (Keynote). 13th International Congress on Yeasts, Madison, USA. Boekhout T (2012). Possibilities and limitations of non-conventional yeasts to understand evolution of disease potential. EMBO Conferences Experimental Approaches in Ecology and Evolution using Yeasts, Heidelberg, Germany. Boekhout T (2012) Ethics of Scientific Publishing: a Continuing Story. 18th Congress of the International Society for Human and Animal Mycology, June 11–15, 2012, Berlin, Germany
40 Bibliometric analysis
• Number of peer reviewed publications: 230 (Web of Science, March 2014) • H-index: 36 (Web of Science, March 2014); 47 (Google Scholar, March 2014) • Total number of citations: 4731 (Web of Science, March 2014); 10051 (Google Scholar, March 2014)
4.8 Societal relevance: quality, impact and valorisation
Our work on clinically important yeasts bridges fundamental and applied clinical research. Patterns of biodiversity are compared with those on susceptibility to antifungals and virulence features, and thus helps to understand the urgency of treatment. Our involvement in the joint working group on rare yeast infections of the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) and the European Confederation of Medical Mycology (ECMM) resulted in a guidelines paper (CMI) how to diagnose and treat infections of such rare yeast infections. Similarly we organize in collaboration with Drs Edwin Boel en Anneloes Vlek (UMCU, Utrecht) a multicentre investigation on the reliability of MALDI-TOF MS diagnostics of yeasts that included 14 centres from across Europe. From this work some clear recommendations appeared how to further optimize maldi-based diagnostics. Currently, in collaboration with Dr. Edwin Boel (UMCU) and Dr. Jos Houbraken (CBS), we are optimizing maldi-based diagnostics of filamentous fungi, most notable Aspergillus fumigatus and related species. With some interns from laboratory schools (mainly MLO) we are expanding the CBS in-house maldi database with a broad variety of yeast species that are relevant for the clinic, food industry and other parts of society. This database will be used in the future to assist in identifications for third parties, e.g. hospitals. Our aims to develop novel diagnostsic for fungal infectious agents based on comparative genomics of fungal genomes, yielded promising results. It may well be that this will lay the foundation for a start up company with the partners involved (CBS, CRG Barcelona, HMC Doha) as well as [some] patents. We also foresee that [part of] the clinical centres in The Netherlands and abroad need help in their fungal diagnostics and quality control. We need to consider how this can be best realized, e.g. as an expert centre dedicated to this market. The barcoding database, the maldi database, and our novel diagnostics and probe generating pipeline may contribute to this. We also expect that our multigene phylogeny studies on all currently accepted yeast species will lay the basis for a new classification of yeasts. This will be highly important as some clinically AND industrially important genera, such as Cryptococcus and Candida, are highly polyphyletic in the current concepts [actually they are almost meaningless]. Despite the notion that clinicians may not like the name changes at first, after they realize that the new taxonomy that will be based on solid phylogenetic analysis and a consensus approach among both taxonomists, the applied scientists, clinicians and other end users will see the future advantages, e.g. to better understand disease as well as resistance in some groups of yeasts. Moreover, limiting the above mentioned polyphyletic genera Candida and Cryptococcus to smaller monophyletic lineages that contain some of the most important yeast pathogens, may also contribute to the more readily societal and regulatory acceptance of species hitherto classified in those genera and that are useful for [agro]industrial purposes. Furthermore, we are regularly consulted by industry (a.o. DSM, Heineken) for questions related to yeasts, e.g. biosafety, typing of strains. Moreover, we are consultant for the Skin and Hair Division of Procter & Gamble (Cincinnati, USA) and collaborate with Bruker Daltonik GmbH (Bremen, Germany) on the further development of Maldi-tof-based diagnostics of fungi. We are regularly consulted by the Dutch Commision on Genetic Modification (COGEM) to advice on species and strains that are considered to be genetically engineered. Finally, we occasionally organize teaching course on medical yeasts (e.g. Hamad Medical Corporation, Doha, 16–18 February 2011; Medellín, Colombia November 2014), and take part in the Medical Mycology Course of Institut Pasteur, Paris (2012, 2014), various CBS courses, and we present a teaching module on Medical Mycology in the 2nd year course on “Treatment of Infectious Diseases”, University Medical
Part 4 - Yeast Research Programme – T. Boekhout 41 Centre Utrecht. In the future it is likely that we will be involved in teaching mycology at University of Amsterdam. We also supervise many trainees from laboratory schools (MLO and HLO level) who get training in laboratory practise.
4.9 Viability
The Yeast & Basidiomycete research group comprises one researcher (TB) and one technician (Bart Theelen) permanent staff. Thus finding external funding is important for survival. Within CBS, TB collaborates nicely with M. Groenewald, curator of the CBS yeast collection, which enlarges the scope of the research group. As an example we name the ongoing multigene phylogenetic studies of all ascomycetous yeasts and the barcoding of the CBS yeast collection. MG is also one of the editors for the future web-based edition of “The Yeasts, a Taxonomic Study”. One may, however, ask how important the study of yeast diversity is and what it contributes to the society? Noting that the reference work “The Yeasts, a Taxonomic Study” (5th edition, 2011) is very well cited one may conclude that studies to uncover yeast diversity and to present this diversity in a modern multigene/phylogenomic context are important. Indeed several clinically and technologically relevant species have been described. Yet, the funding capability of this type of work by regular funding bodies in our country is almost zero. During the period that covers this peer review we were able to generate money for projects in the field of molecular phylogeny with our colleagues from State Key Laboratory of Mycology, Chinese Academy of Sciences, Beijing, China. Similarly for the clinical yeast field we obtained a grant from Qatar Foundation, and the same is true for an environmental project on microbes in the sea near Qatar. The first project will set the standard for a new taxonomy that will be widely used [however, probably only after some lag phase] and the two other projects may contribute to uncover unknown fungal [incl. yeasts] biodiversity. In the coming period comparative genomics will become more and more important. Presently we are gearing up to realize collaborations in this area in The Netherlands [e.g. CWI via a VENI proposal of Dr. Ferry Hagen], Europe [via the French Genolevures network and a proposal for an Innovative Training Network (ITN)] as well as via our Chinese colleagues. Ongoing collaborations with Prof. Dr. Toni Gabaldon (Bio-informatics, Centro de Regulacion Genomica, Barcelona, Spain) also yield possibilities for future EU programs. So far, big Dutch companies [e.g. Heineken, DSM] showed only non-financial interest in comparative genomics of yeasts, but at the Europena level this may be somewhat different [e.g. Carlsberg in Denmark seems to have an interest in comparative genomics of yeasts and we are collaborating on a yet rather small scale] . The anticipated merger of the Yeast & Basidiomycete Research group with the Origin of Clinical Fungi program in a new Medical Mycology Research program may challenge the continuity of yeast research at CBS. Given that the taxonomic diversity covered by CBS has already significantly eroded in recent years the future of yeast research in CBS needs serious consideration. As long as TB will stay as a PI at CBS he will continue to pursue his research agenda in the yeast domain, but his may change thereafter.
4.10 Strategy
The Y&B research group at CBS focusses at scientifically interesting but feasible research topics that contribute significantly to the development of the field. Funding of such projects will be challenging as almost everywhere on the globe the funding climate is getting worse. Strategically, we collaborate with researchers from Qatar that gives us access to funds from Qatar Foundation (currently 2 projects funded) and China that gives us access to international collaborative grants from this country (currently 1 grant). Beyond this we need to actively search for grants in Netherlands (e.g. NWO), Europe (Horizon2020), private funds, etc. Private funds also need to be considered as was done by A. Vasco-Palacios (Colombian PhD student) to support her work on ectomycorrhiza in Colombia Amazonia, e.g. by Schlumberger Foundation, 3 successive grants.
42 We plan to continue our collaboration with Bruker that allowed us to develop in-house databases for MALDI-TOF MS diagnostics of yeasts and filamentous fungi. Bruker placed a BioTyper machine at CBS in exchange for 100 isolates per year. This collaboration turned out to be successful with a number of joint publications and some being prepared. Furthermore, this collaboration is also successful for the CBS collection as it resulted in a significant order of strains to be used in validation (FDA) purposes. Such collaborations with industry may also result in joint grant applications. Bruker, for instance, will be involved in the H2020 ITN application (April 2014). In cryptococcal research we investigated the evolutionary history of some major outbreaks caused by C. gattii, and this turned out to be a highly successful strategy resulting in a high number of citations. Similarly, the recent discovery of the importance of Malassezia spp. in the skin microbiome will boost research in this field and our long standing interest and collaborations will certainly be beneficial for our involvement. We imagine that skin microbiome studies aiming at mycobiota, constitutes a research item with good potential for funding, especially if the host interactions are considered. Host-pathogen interactions with Cryptococcus and Malassezia and some other pathogens with the human host will be continued to be studied in collaboration with Prof. T. Geijtenbeek (Experimental and Molecular Medicine, AMC, Amsterdam). This collaboration already yielded some papers in journals with a high impact factor (e.g. Nature Immunology, PLOS Pathogens). We aim to get some grants (NWO, TOP grant) to develop this field further. Our former PhD student F. Hagen applied for a VENI grant from NWO to understand the evolutionary history of the Cryptococcus gattii complex and evolution of virulence. For our future work on “The Yeasts, a Taxonomic Study” we refer to the text in the sections above. Here we will be looking for possibilities to get grants in the area of eLearning to utilize the content for the development of eLearning tools as well as improve presentation tools at the web, e.g. for dynamic phylogenetic trees and with cross referencing to many other databases on e.g. literature, treatment of yeast infections, and this work may include also entertaining movies of fermentations, action of medical doctors to diagnose infections etc. Briefly, we anticipate: 1. to continue our research onCryptococcus , focusing on understanding of host- pathogen interactions related to hypo- and hypervirulence; 2. To direct research of Malassezia to understand their role in the healthy and diseased skin microbiome, as well as invasiveness; 3. Use multigene and whole genomes for (phylo)genomics studies to understand yeast diversity, phylogeny and improve the taxonomy; 4. To further develop the electronic version of “The Yeasts, a Taxonomic Study”.
Part 4 - Yeast Research Programme – T. Boekhout 43 5. Applied and Industrial Mycology Programme
Programme Leader Prof. dr dr h c R.A. Samson
5.1a Objectives(s) and research area
Filamentous fungi are playing an important role in our daily life, for instance as parts of food products or as food spoilers. In addition, they also occur inside human dwellings in air and on walls as so-called indoor fungi. These genera are overrepresented as spoilage agents of food and beverages, as producers of toxic compounds and as fungi in human dwellings. They even occasionally occur as human and animal pathogens. On the other hand, many species of these genera are very important industrial microorganisms in food fermentation and biotechnology. Finally, the organisms are avid producers of a wide variety of (putative) bioactive compounds. The area of the research group is to study the biodiversity, phylogeny and cell biology of fungi with special relation to food and indoor mycology, with an emphasis on the genera Penicillium, Aspergillus, Paecilomyces and Talaromyces. The mission of the program is to reach a deeper understanding of applied and fundamental aspects of fungi in their relation to food-association and indoor situations. These include a novel polyphasic classification of the subgroups of the order Eurotiales, and the development of tools for a practical barcoding system of these groups. To understand the biology of the important fungi in these applied fields, the biology of the fungal cell is studied, in particular on the topic of cell stress. These studies include extreme stress resistant ascospores, the sensitivity of fungal conidia for antifungal compounds and the reaction of growing fungi on drops in relative humidity. The research of the group has always been intertwined with numerous (smaller) projects with external parties (industrial companies and governmental institutions) that request expertise with problems related to food spoilage, indoor environments and industrial applications. This is illustrated by the fact that several large fundamental or applied research projects have been directly initiated as a result of these smaller projects.
5.1b Forward look
The group harbours a unique collection of fungi, many of them belonging to the Eurotiales, related to industrial and indoor situations. For the next five years our group will continue to work on the polyphasic taxonomy of these fungi and expand the collection with newly obtained strains. An on-line platform will be developed combining phenotypic and sequence data of Penicillium, Aspergillus and Talaromyces species and linking this database with the accepted species names in these genera (via MycoBank). The research collection is used for innovative projects with industry, including the development of tools for detection and identification of indoor and food spoilage fungi and as panels of selected relevant fungi to test inactivation of fungi in indoor- and food situations. Furthermore, the research collection will be probed on the production of novel natural compounds in collaborations with different institutes and universities. The group will expand the know-how on this topic and remains to generate substantial outcome from small or medium projects with industrial partners and (governmental) institutions as well as identification services. The group also aims at the funding of a number of more fundamental research projects in applied mycology. In the next years the group will become a part of the new research group “Industrial Mycology”, but will continue its research on the established themes taxonomy, food mycology and indoor mycology, and with the generation of income.
44 5.2 Composition
Table 5.1. Research staff at programme level 2008–2013. 2008 2009 2010 2011 2012 2013 Funding* Research staff Prof. dr R.A. Samson 1.0 1.0 1.0 0.6 0.6 0.6 1 Dr J. Dijksterhuis 1.0 1.0 1.0 1.0 1.0 1.0 1 Dr J. Houbraken – – – – – 1.0 1 Post-doc Dr J. Varga 1.0 1.0 – – – – 1 Dr R. van Leeuwen – 1.0 1.0 1.0 1.0 1.0 4 Dr C. Visagie – – – – 1.0 1.0 3 PhD R. van Leeuwen 1.0 – – – – – 4 T.T. Wyatt 0.1 1.0 1.0 1.0 1.0 0.3 4 F. Segers – – – 1.0 1.0 1.0 4 E. van Nieuwenhuijzen – – – 0.8 0.8 0.8 4 N. Yilmaz – – 1.0 1.0 1.0 1.0 3 Technicians T. van Doorn 1.0 1.0 1.0 1.0 1.0 1.0 2, 4 J. Houbraken 1.0 1.0 1.0 1.0 1.0 – 1 M. Meijer 1.0 1.0 1.0 1.0 1.0 1.0 1, 2, 3 R. Jacobs – – – – 1.0 – A. Verkennis – – – – 0.4 1.0 2, 5
*1 = Royal Netherlands Academy of Arts and Sciences; 2 = Applied projects. 3 = Sloan Indoor barcoding project; 4 = STW project; 5 = ZonMW project.
5.3 Research environment and embedding
This research group studies the biodiversity, phylogeny and cell biology of fungi with special relation to food and indoor mycology, with an emphasis on the order Eurotiales. The mission of the programme is to attain a better understanding of applied and fundamental aspects of fungi related to food-association and indoor situations. The research group continued to work on the axis APPLICATION (applied projects; contact with companies and institutions) - TAXONOMY (continuing to be the standard for recognition of food- related fungi; exploring the basis of variation of these fungi) - FUNDAMENTAL (bringing applied problems into the realm of fundamental research project to address the question why certain food-related fungi have developed certain survival strategies). This axis enables the group to do basic research with an eye on possible valorisation of the data. The group tries to find the balance between applied and fundamental research. However, the group members still spend approximately 50 % of their time on research for applied projects of third parties, and teaching commercial courses. The demand of third parties for applied research remains to have a great impact concerning the time which can be spent on fundamental research.
Institutional and (inter)national collaboration
In Utrecht, the research group has a strong cooperation with the Departments of Microbiology (6 common publications in 2013) and Membrane Biochemistry and Biophysics (2 common publications) of the
Part 5 - Applied and Industrial Mycology Programme – R.A. Samson 45 University of Utrecht. With each departments 2 joint research projects were completed and/or running between 2008 and 2013. Firm cooperation is also started with the department of Molecular Biophysics. Recently research co-operations are started with the Hubrecht Institute (KNAW), the department of Pharmaceutical Sciences, and Hogeschool Utrecht. Links also exist with the University Medical Centre (UMC). Nationally, long term connections exist with the laboratory for Biophysics of Wageningen University (4 common publications), with the group Food Microbiology, and with Plant Research International. Longstanding cooperation is also existing with the Technical University of Eindhoven (1 common publications, 2 joint research projects). Further cooperation exists with the Radboud University University Nijmegen Medical Centre (4 common publications). Recently, a link has been established with the University of Amsterdam within a STW project on bacterial cell division and including the search for novel antibiotics. Dr Dijksterhuis is member of the user’s committee of this project. Importantly, well established contact exists with research laboratories of companies, as DSM Food Specialties in Delft (3 common publications) and Unilever Research Laboratories in Vlaardingen. Both co-operations include secured projects, but also cooperation within STW projects (users committee) which has led to peer-reviewed publications. Another project with Ecofide (Weesp) has led to one publication. This shows that co-operations with industry lead to scientific publications. Internationally, many co-operations exist with laboratories including: The Swedish University of Agricultural Sciences (SLU), Uppsala, Sweden; the Applied Mycology Group, Cranfield University UK; Queens University, Belfast, UK; University of Nottingham, Nottingham, UK; DTU, Copenhagen Denmark; Helmholtz Centre for Infection Research, Braunschweig, Germany; Leibniz-Institute for Natural Product Research and Infection Biology, Jena, Germany; CIML Immunology, Marseille, France; University of Wien, Austria; University of Szeged, Hungary; Institute of Sciences of Food Production, Bari, Italia; Agriculture and Agri-Food Canada, Ottawa, Canada; ARS-USDA, Peoria, USA; Davidson College, Davidson, USA; National Academy of Agricultural Science, RDA, Suwon, Korea; The University of Sydney, New South Wales, Australia. Dr Samson holds an adjunct professorship at the Faculty of Food Science and Technology and Plant Pathology of the Kasetsart University in Bangkok. Since January 2009 he is also visiting professor at Instituto de Tecnologia Quimica e Biologica of the Universidade Nova de Lisboa in Portugal.
5.4 Quality and scientific relevance
The genera Penicillium and Aspergillus have been studied at CBS since the 1940’s, and this tradition has been continued by Rob Samson and his group. In the late 1990’s, molecular techniques were successfully implemented in these studies. In 1986, collaboration was initiated with Prof. Jens Frisvad (Lyngby) using extrolites data as taxonomic characters, and this has now resulted in a polyphasic taxonomic approach towards species recognition in Penicillium and Aspergillus. The initiative of organizing international forums in Penicillium and Aspergillus and publication of four important papers were instrumental in the leadership of CBS in these taxonomic fields. Since Penicillium and Aspergillus are important in food and indoor mycology, the research in these two applied fields was a logical consequence. Since the initiation of research on these topics, this group has gained a significant international reputation. The aim of the taxonomic research is to complete and publish the monographs of Aspergillus and Penicillium using the polyphasic approach. With Dr Janos Varga (Szeged, Hungary) who held a post doc position from 2006 until 2009, a monographic treatment of Aspergillus will be envisaged, while the treatment of Penicillium will follow later. Studies on the significance ofAspergillus and Penicillium as food spoilers and mycotoxin producers will be published in separate papers, but these data were compiled into two books on Food- borne fungi. For indoor mycology biodiversity studies, a DNA barcoding initiative was started in 2007, which will be continued, while the biology of indoor moulds on building materials is studied together with Prof. Olaf Adan from the Technical University of Eindhoven.
46 Drs Houbraken and Samson have completed a key study that reveals the taxonomic relationships within the family Trichocomaceae. This study shows that the genera Penicillium and Aspergillus are closely related and other genera including Talaromyces, Paecilomyces and Thermoascus are more distant. Furthermore, a new sectional classification scheme was proposed in Penicillium and the genus Talaromyces was re- defined following the single name nomenclatural rules. Biodiversity studies in the aforementioned genera have been conducted, resulting in the discovery of new species and new insights. Dr Dijksterhuis has been project leader of a STW project on the mode of action of natamycin, a polyene antifungal compound used in the cheese and sausage industry. This project included a firm collaboration with the Department of Membrane Biochemistry and Biophysics (Dr Breukink, Prof de Kruijff), Utrecht University. The project was completed in 2008 and produced 8 publications of which 2 were joint and led to 2 PhD theses (one of Richard van Leeuwen, CBS, in June 2009). The surprise was that this polyene had a completely different mode of action to the known polyenes, including a novel mechanism of inhibition on transporter molecules. The project also included an extensive micro-array analysis of conidial germination and the effect of natamycin on conidia of A. niger. Dr. Dijksterhuis also led a project on the molecular mechanisms of heat resistance which started in 2008 in cooperation with Dr Lugones and Prof Wösten of the Department of Microbiology (University of Utrecht) which had one post-doc and PhD student. The project has been completed and attracted the attention of different companies in order to identify novel compounds that stabilize whole cell preparations or biomolecules. Ascospores of fungi belonging to the genus Neosartorya (Aspergillus) are extraordinary stress resistant and accumulate large amounts of trehalose-based oligosaccharides of which a number have never been described in nature before. The project has already led to 1 publication, and 4 more are expected in 2014 as is the defence of a PhD thesis by Timon Wyatt (January 2014). A third project on the behaviour of fungal cells during humidity changes was started in 2011 in cooperation with the Departments of Molecular Biology and Biophysics in Utrecht and the Technical University Eindhoven. We used advanced fluorescent techniques in order to study these changes and one PhD student is involved (Frank Segers). Very recently, a novel ZonMW project in cooperation with different groups in Utrecht and the Hubrecht Institute has started in order to identify novel antibiotic compounds. Drs Dijksterhuis and Houbraken are currently involved in a number of confidential projects that are initiated by companies, which have a strong bias to innovation. Occasionally, projects done with companies lead to peer reviewed publications, for example, a study on the sensitivity of non-target water fungi for antifungals used in agriculture and the discovery of a heat resistant sexual state in Paecilomyces variotii. The electron microscopical facilities of the Institute generate unique micrographs of many fungal species that occurred in 11 publications during the period of review and expected to do so in 8 manuscripts in 2014 en 2015. A selection of these pictures has been shown in an exhibition (visited by 80.000 people) entitled: “Beauty and Science” held in the prestigious museum Boijmans van Beuningen, Rotterdam, in 2011. The research staff teaches the course Introduction to Food and Airborne fungi (one-week course, run in October each year in Utrecht, and in June in Ottawa). Every year, two short courses are given in Stuttgart (Germany) on the same subject and this course was also taught in Edirne, Turkey. Furthermore, tailor-made courses are organised for industries. They also participate on a regular basis in two VLAG courses at Wageningen University courses namely “Management of Microbiological Hazards in Foods” and “Genetics and physiology of food-associated micro-organisms”. VLAG is one of the seven graduate schools of the University of Wageningen. The group Applied and Industrial Mycology frequently has visiting students, researchers or interns spending time in the group to acquire specific skills under the supervision and training of the group’s staff. From 2008 on, Dr Dijksterhuis has been involved in the organisation of the annual meeting of the Royal Dutch Society of Microbiology held at the CBS on the last Friday of November. The meetings have a fixed theme each year and, on average, two international speakers are invited. The meetings are well visited (80–120 visitors) and very positively evaluated and seen as an important meeting platform for mycology in the Netherlands.
Part 5 - Applied and Industrial Mycology Programme – R.A. Samson 47 Several highlights from the period 2008–2013 are listed below: • Polyphasic taxonomic studies on Aspergillus and Penicillium elucidated species complexes but also demonstrated the great species diversity in both genera. Publications on Aspergillus sections Nigri, Circumdati, Fumigati and other groups have been consulted by many applied researchers in view of the aspects of pathogenicity and toxicity. Drs Samson, Houbraken and Visagie were instrumental in setting up the list of names of the Trichocomaceae in view of the One Fungus One Name concept following the recent changes in the Code of Nomenclature. • The preparation and publication of the full colour book Food and airborne borne fungi was archived with the collaboration with Prof. Jens C. Frisvad and other Danish colleagues. This book complies the most common species in food and indoor environments and is used for the courses and many other training events (inter)nationally. • Much time was spent collecting and barcoding indoor moulds in view of IMBOL a project supported by the US Sloan Foundation in collaboration with Dr Keith Seifert (Agriculture and Agri-Food Canada, Ottawa). Two books on indoor moulds were published and edited with Drs Flannigan, Miller and Adan. • A Studies in Mycology issue was published in 2013 entitled “Development in Aspergillus” in cooperation with Prof Han Wösten, Molecular Microbiology, Utrecht. • A STW project resulted in a joint paper in PNAS on a novel mode of action of the polyene natamycine.
Key publications
Amend AS, Seifert KA, Samson RA, Bruns TD (2010) Indoor fungal composition is geographically patterned and more diverse in temperate zones than in the tropics. PNAS 107: 13748–13753 Samson RA, Houbraken J, Frisvad JC, Thrane U, Andersen B (2010). Food and Indoor Fungi. CBS Laboratory Manual series 2, 390 pp. Samson RA, Varga J, Frisvad JC (eds) (2011). Taxonomic studies on the genus Aspergillus. Studies in Mycology 69: 1–97. Samson RA, Yilmaz N, Houbraken J, Spierenburg H, Seifert KA, Peterson SW, Varga J, Frisvad JC (2011). Phylogeny and nomenclature of the genus Talaromyces and taxa accommodated in Penicillium subgenus Biverticillium. Studies in Mycology 70: 159–183. Welscher YM te, Leeuwen MR van, Kruijff B de, Dijksterhuis J, Breukink E (2012) Polyene antibiotic that inhibits membrane transport proteins. Proceedings of the National Academy of Sciences of the USA 109: 11156–11159. Dijksterhuis J, Wösten HAB (2013) Development of Aspergillus niger. Studies in Mycology 74, 85 pp. Hong S-B, Lee M, Kim D-H, Varga J, Frisvad JC, et al. (2013) Aspergillus luchuensis, an industrially important black Aspergillus in East Asia. PLoS ONE 8(5):e63769. doi:10.1371/journal.pone.0063769
5.5 Output
Table 5.2. Scientific publications. 2008 2009 2010 2011 2012 2013 Total Academic publications Refereed journals 13 12 15 25 11 25 101 Book Chapters & 7 2 – 7 – 5 21 Proceedings Total 20 14 15 32 11 30 122 Monographs & books 1 2 2 4 – – 9
Ph.D. theses 1 1 – 1 – 1 4
48 Table 5.2. (Continued). 2008 2009 2010 2011 2012 2013 Total Popular publications – 1 1 2 1 1 3 9 and products
5.6 Earning capacity
During the period the group received funding form the Sloan Foundation for DNA barcoding. This grant was extended a second time in 2011. Another grant was received in the frame of Dutch government in collaboration with the Dutch industry PURAC. A small grant was obtained from the Korean programme Biogreen for collaboration in the studies of Aspergillus from Korea and the microbiological surveys of meju a traditional Korean fermented food. In the period of review 3 STW projects are awarded to our group (one as a groupleader) and one ZonMW project.
Applied projects
From the beginning of his CBS career Rob Samson started to receive an increasing number of requests for identification of food spoilage or indoor fungi and also request for consultancy about problems within the food industry and also with respect to indoor mycology. In Fig. 5.1 an overview of the number of projects in the period of 2008–2013 is illustrated which varies from 147 to 178 per year. Most of these projects deal with food spoilage and problems in indoor environments. Although many of the projects are routine cases, some of them have an added value becauseJaar obtainedNumber isolates of projects are usedTurnover in biodiversity studies and the indoor barcoding initiative. The turnover generated2013 is between 128- and158 181155559 k€ per annum. Aantal identificatiesFrom 2010 on, the coordination,Omzet administration2012 and identification178 of128683 strains sent to CBS for identification 82 117373 2011 149 127524 was resumed as an270 activity67025 in the group (M.2010 Meijer). Per year 148244 to142961 357 isolates were identified within 112–155 projects. 244The combined81000 turnover of2009 identifications and147 applied181132 projects during 2013 was 273 k€. The projects are done357 in cooperation24600 with a wide2008 variety of companies152 167620 and institutions including food- and indoor mycology.
Applied projects and iden7fica7ons 155559 117373
272932 2008
2009
2010 Iden0fica0ons
Applied projects 2011
2012
2013 Fig. 5.1. An overview of the turnover 0 50000 100000 150000 200000 generated from applied projects and Turnover in Euro (€) identifications during the last 6 years.
Part 5 - Applied and Industrial Mycology Programme – R.A. Samson 49 5.7 Academic reputation
Awards Prof. dr R.A. Samson
2008 - Honorary Membership of the Mycological Society of America 2009 - Honorary doctorate Swedish University of Agricultural Sciences Uppsala 2010 - Honorary Membership of the Hungarian Society of Microbiology
Appointments Prof. dr R.A. Samson
Secretary General of the International Union of Microbiological Societies
Editorships in Academic Journals and book series
Prof. dr R.A. Samson CBS Biodiversity Series (EiC) CBS Laboratory Manual Series (EiC) Studies in Mycology (EiC) Persoonia Dr J. Dijksterhuis FEMS Microbiological Letters
Memberships in Scientific Boards
Prof. dr R.A. Samson International Union of Microbiological Societies International Commission for the Taxonomy of Fungi International Commission of Penicillium and Aspergillus International Commission of Food Mycology International Commission of Indoor Mycology Dr J. Dijksterhuis Section Mycology of the Dutch Society for Microbiology (NVvM) Section Scanning Electron Microscopy of the Dutch Society for Microscopy (KREM) Dr J. Houbraken International Commission of Penicillium and Aspergillus International Commission of Food Mycology
Other Proofs of Academic Reputation
Drs Samson and Dijksterhuis regularly perform in the corona (committee) for the defence of PhD theses in- and outside NL, and have performed as co-promoters.
Selection of invited lectures
Samson RA (2009) Living with Fungi. Keynote. Honorary doctorate award Swedish University of Agricultural Sciences, Uppsala, October 3, 2009. Samson RA (2010) Global warming and the change in the airborne mycotoxin-producing mycobiota. The World Mycotoxin Forum - 6th Conference. 8–10 November 2010 – Noordwijkerhout. Samson RA (2010) New taxonomic and identification schemes for Aspergilli causing human and animal
50 health hazards. Keynote Honorary Member of the Hungarian Society for Microbiology. October 12, 2010. Dijksterhuis J (2011) Fungal spores as survival vehicles in space and time. Spring Conference 2011 of the Society for General Microbiology. 11–14 April 2011, Harrogate UK. Samson RA (2013) Fungal biodiversity and the unlimited use of microbial resources. Keynote 11th Congress of Indonesian Society For Microbiology. Pontianak, Indonesia.
Bibliometric analysis
• Number of peer reviewed publications: 98 (Web of Science, March 2014) • H-index: 25 (Web of Science, March 2014); 62 (Google Scholar, March 2014) • Total number of citations: 1922 (Web of Science, March 2014); 15234 (Google Scholar, March 2014)
5.8 Societal relevance: quality, impact and valorisation
Yearly enormous amounts (1.3 M t) of food are lost worldwide and for an important part this is the result of food spoilage by fungi. This immediately provides our group an important societal importance. This is clearly illustrated by the large number of requests from different companies and institutions for applied research projects. The average number of projects remains high throughout the last 5 yr. This generates income for the institute, but more importantly, contacts are made and strengthened with companies via these projects and information is generated on current food spoilage problems. It is easy to see that these projects also have an economical and societal impact of a different magnitude. If fungal growth is identified and successfully removed or stopped as a result of our projects, this will be of great importance for food safety. With the growing worldwide demand on food, this topic is of increasing importance during the next decades. By reducing food losses caused by fungi, an increase of amount of edible food can be realized without an increase of the area of land needed to produce crops. There is an ongoing societal interest into indoor air quality and health aspects, which are partially linked to the presence of indoor fungi. A considerable part of our projects is related to this subject and for example deal with air quality in musea, deterioration of archives, water damages and requests of governmental health institutions and individuals. An increasing number of confidential projects are conducted on request of different companies that are of a strong innovative nature. We regard this as a unique example of knowledge transfer which is highly relevant for society and seen by the KNAW as an example of how scientific research and society can interact. These projects occur in both areas of food- and indoor mycology. Our group has started to build up expertise in the area of the identification of natural compounds produced by fungi and this is another aspect very relevant for society. These include the search of novel antibiotics, not only against multi-resistant bacteria, but also against other pathogens (e.g. eukaryotes). Fungi also are known to produce compounds that can be used as novel drugs and our search (may) address(es) other compounds with functions outside the medical area.
5.9 Viability
Our group is a world-leader in taxonomy of food- and indoor associated fungi, with the emphasis on the order Eurotiales including the genera Penicillium, Aspergillus, Paecilomyces and Talaromyces. We will continue studying the taxonomy of these genera and this will result in various taxonomic publications. Some of these publications will primarily focus on the taxonomic aspects while others will combine new species discovery with additional properties such as enzyme production and unique starter culture development. An extended and unique collection of food- and indoor related fungi has built up during different decades and will be used for various (applied) research topics. Contacts within many companies and
Part 5 - Applied and Industrial Mycology Programme – R.A. Samson 51 institutions by means of smaller projects provide a unique platform that leads to larger applied and fundamental research projects (e.g. STW). It also results in the generation of turnover for the institute, but the societal effect is expected to generate profits of a higher magnitude. The group combines knowledge transfer and innovation. Several current projects with companies are confidential and very innovative. These requests of companies are currently more numerous than in the past; aspects of patents are currently being evaluated. The group has a high publication rate for its size and many of the manuscripts are surprisingly fundamental and in high impact journals. Publications in (applied) journals have attracted attention from companies and as such resulted in new (applied) projects. The group has realised funding by STW (three projects) and ZonMW. The group has built up a strong research connection with the department of Microbiology of Utrecht University leading to two promotions and 6 joint publications in 2013. These projects will generate more publications in the next years and novel applications will be submitted. We will also evaluate how we can play a role in the Horizon 2020 framework of the EU. The group organizes courses (e.g. Utrecht, the Netherlands; Ottawa, Canada) on recognition of food fungi that have traditionally captured interest worldwide from either companies or universities. Furthermore, tailor-made courses are given for food production companies. The cryo electron microscopy facility, which is manned by our group, will generate the highest number of publications in its existence during current and next years.
5.10 Strategy
The potential of the collection of food- and indoor fungi is enormous with respect to comparative (genomic research) studies in collaboration with numerous institutions. We would like to increase our knowledge in the area of comparative genomics between closely related food fungi. We envision that this approach may open new insights in the specificity of food fungi and lead to possible ways to battle food spoilage. Within this framework, the identification and taxonomy of food spoilage fungi remains vital. As fungal species are surprisingly specific in food situations, wrong identification frustrates the process of knowledge acquisition. Unravelling the taxonomic relationships within the order Eurotiales, that harbours many food fungi, remains an important focal point for the group. For this will we evaluate possibilities to submit projects for the Horizon 2020 EU framework that also will address the topic of food safety. We remain active in this area as several companies have requested confidential projects to be realized in 2014. We aim to use our connections with companies and institutions for novel research applications for STW. In this process we also evaluate the possibilities to collaborate in the writing of patents. In 2013 we have already built up considerable expertise in this process together with STW. The large number of requests for applied research requires expertise on different topics, and has led to different areas of competence within the group. This might be interpreted as a lack of focus. For instance, the topic of indoor fungi remains a second important research theme in our group as a result of many requests of clients. A large STW project on fungal growth and humidity changes is currently active. Up to now, research was never done on the behaviour of growing fungal hyphae during changes in relative humidity. In 2014, we expect the first results of monitoring fungal growth under varying relative humidity and expect new insights, and new research leads. Fungal growth on indoor surfaces is occurring in a substantial number of dwellings worldwide and linked to health aspects. Here, knowledge transfer between institutes and companies may lead to novel ways to prevent indoor fungal growth. During the period of review, in a number of cases certain groups of fungal species from the collection or freshly isolated from a relevant location were requested. For example, to test if a certain compound (antibiotic, sanitizer or else) is able to kill survival structures of fungi, or developing tests to study the growth of certain strains/species on newly formulated (food) products. Composing panels of fungi for
52 testing purposes could be a typical example of knowledge transfer as often irrelevant fungal species are used in such protocols. A novel aspect of research is to explore the collection for putative (bio) active or other compounds produced by fungi and of use in medical or other applications. As is stated by the KNAW, this is an important novel area of research for our institute. In 2013, a project in cooperation with different groups of Utrecht University and the Hubrecht Institute was started and hundreds of extracts of fungi were collected and tested on antibiotic capacity. We would like to initiate other collaborations in this regard, for instance within Utrecht University and also with the University of Amsterdam. Dr Dijksterhuis is member of the users committee of a STW-project (VICI to Dr Leendert Hamoen) in which the search for novel antibiotics is connected to the cell biology of Bacillus subtilis. The group has a decade long interaction with Prof. Jens Frisvad, DTU, Denmark, who is a world leader on the production of fungal metabolites, and recently cooperation has been initiated with Prof. Marc Stadler, HZI, Germany. During the period of review contact has also been made with Prof. Tielens (Erasmus University, Utrecht University) in order to find novel antibiotics against trypanosomes. If these initiatives will take shape in the future, the expertise of technician(s), as those that are now funded by the ZonMW project is of vital importance and prolongation of their input is necessary. The fungal spore is a vehicle that is important for the distribution of (spoilage) fungi and often causes the problems observed in food- and indoor situations. Research on fungal spore biology including recognition, germination and stress resistance has been strong at our group for the last years and during the period of review has been the topic of 14 publications. In 2014 and 2015, at least 4 new publications are expected to be written on the subject. Spore biology also remains to be a topic of interest for different companies that we cooperate with and will be the subject of novel applications for funding. In the next years the activities of our group are brought under the umbrella of the group “Fungal Physiology” led by Prof. Ronald de Vries. We expect to be able to maintain our activities as described above, but actively aim to be a constructive and a cooperative partner and hope to create synergy for the new group. In fact, there is already a lot of communication between both groups and “joint” publications are already in preparation.
Part 5 - Applied and Industrial Mycology Programme – R.A. Samson 53 6. Fungal Physiology Programme
Programme leader Prof. dr R.P. de Vries
6.1a Objectives(s) and research area
The global move to a biobased economy requires significant research input to make this economically feasible. Plant biomass is a major resource for this change as it is the largest renewable resource on earth. The use of plant biomass as a resource for industry and society involves two main areas: depolymerisation of the biomass to obtain starting compounds and conversion of these compounds in end products. Fungi play a major role in both processes. They are the most efficient organisms in decomposing plant biomass and produce a large array of enzymes that can be used for depolymerisation of plant biomass. This ability is accompanied by a highly diverse carbon metabolism involving many pathways for conversions of plant biomass monomers. In addition, Saccharomyces cerevisiae is the most widely used yeast for metabolic conversions into high-value product such as ethanol. However, major hurdles still need to be taken to make plant biomass an economically sustainable resource for society. The depolymerisation efficiency of currently available enzyme cocktails is too low on most lignocellulose substrates and the production costs of the enzymes is too high. In addition, S. cerevisiae only has a limited ability for conversion of plant-biomass based monomers and will need to be engineered by introducing additional pathways and transporters. Considering the relevance of these issues, much research across the globe is devoted to solving these problems. Our programme distinguishes itself from other programmes in that it addresses the issue from a fungal biodiversity and evolution angle. By performing fundamental studies aimed at understanding the differences between fungi with respect to plant biomass utilization and the underlying mechanism, we aim to generate a better understanding of this topic that can then be used in more applied studies. This is carried out by a combination of in-depth studies in a few selected model organisms with large comparative studies across the fungal tree of life.
6.1b Forward look
The programme started in 2009 with a group-leader, a technician and a PhD student supplemented with two postdocs and two PhD students at the Utrecht University group of Prof. de Vries, and now consists of a group- leader, three technicians, seven postdocs (one bioinformatician) and 11 PhD students (as of February 2014), demonstrating the relevance of the field. Funding is obtained from various sources, such as NWO (including one VENI and one VICI grant), STW, BeBasic, EU FP7, Brazil, China and direct funding from several companies. Despite the increase in size all research still circles around the central question: how do fungi use natural carbon source, in particular plant biomass. This ensures strong interactions and synergy between the group members. The main model organism is Aspergillus niger, which has a long history in academia and industry related to plant biomass degradation. This species and its distant relative Aspergillus nidulans are the main organisms for in depth studies into the mechanism underlying plant biomass utilization by fungi. These studies have also lead to a JGI CSP2011 project, which I coordinate and that involves generating genome sequences for 10 additional Aspergilli and a comparative analysis between all available Eurotiales genomes. The consortium for this project involves approximately 60 groups worldwide. Other organisms that are used for more in depth studies are the ascomycetes Aspergillus vadensis (a non- proteolytic close relative of A. niger), Magnaporthe oryzae (plant pathogen), Myceliophthora thermophile and heterothallica (thermophiles), Podospora anserine (dung-fungus) and Trichoderma reesei (the main industrial cellulose producer), and the basidiomycetes Agaricus bisporus (the commercially grown white- button mushroom) and Dichomitus squalens (a white rot fungus). These are the reference for a large study in comparative growth profiling which has now included nearly 350 fungal species and for which the data
54 is available through a public database (www.fung-growth.org). This data has been used in more than 20 fungal genome annotations studies, many of which have been published in high-impact journals and was strongly facilitates by the fungal collection at CBS. The research in the group consists of a combination of physiology, molecular biology, biochemistry, genetics, (comparative) genomics, transcriptomics and proteomics, and phylogeny and involves many (inter-)national collaborations. This multidisciplinary approach have made us one of the leading groups in the field of plant biomass utilization by fungi. Considering the emphasis on a biobased economy in both Dutch funding and the Horizon 2020 program of the EU, our research program should have no problems participating in calls in the coming years.
6.2 Composition
Table 6.1. Research staff at programme level, 2009–2013. 2009 2010 2011 2012 2013 Funding* Research staff Prof. dr R.P. de Vries 0.7 1.0 1.0 1.0 1.0 1 Postdoc Dr J. van den Brink 0.5 1.0 1.0 1.0 1.0 1 Dr Isabelle Benoit 1.0 1.0 1.0 1.0 1.0 2, 3 Dr M. Zhou – – 0.4 1.0 1.0 3 Dr O. Bouzid 1.0 1.0 1.0 0.3 – 2 Dr D. Falkowski – – – – 1.0 4 Dr M. Nicolau de Almeida – – – – 1.0 4 PhD S. Klaubauf – 1.0 1.0 1.0 1.0 1 A. Pathyshakuliyeva – 1.0 1.0 1.0 1.0 5 H. Culleton – – 0.3 0.3 0.3 6 S. Budak – – 0.3 0.3 0.3 7 G. Piccolo Maitan-Alfenas – – – 1.0 1.0 4 D. Robl – – – – 0.7 4 E. Battaglia 1.0 1.0 – – – 2 B.S. Gruben 1.0 1.0 1.0 – – 2 J. Kowalczyk – – – – 1.0 3 C. Khosravi – – – – 1.0 3 T. Benocci – – – – 1.0 3 A. Liwanag – – – – 1.0 8 S. Casado Lopez – – – – 1.0 3, 6 Technician A. Wiebenga 1.0 1.0 1.0 1.0 1.0 1 E. Majoor – – 1.0 1.0 1.0 3 I. Allijn – – 0.2 0.2 – 6 A. Vugts – – – 0.3 0.7 6 A. Vivas-Duarte Vermeulen – – – 0.6 0.8 3 S. Misiedjan – – – – 0.3 1 H. Beenen – – – 1.0 1.0 9
*1 = Royal Netherlands Academy of Arts and Sciences; 2 = through Utrecht University; 3 = Dutch Organsisation for Scientific Reseacrh (NWO); 4 = Science without borders program, Brazil; 5 = Dutch Technology Foundation STW; 6 = Funded by/collaboration with companies; 7 = PhD grant, Saudi Arabia; 8 = EU FP7; 9 = WUR.
Part 6 - Fungal Physiology Programme – R.P. de Vries 55 6.3 Research environment and embedding
Prof. de Vries has an associate affiliation with the Department of Biology, Utrecht University, which will be converted to a professorship by March 2014. All PhD students from the group will therefore graduate at Utrecht University and the group has access to all facilities of the Department of Biology of Utrecht University. The group participates in various national (BeBasic, Wageningen Lignin Network) and international (Eurofung, FP& collaborative project, FP7 Marie Curie ITN project) networks, which provide additional collaborations, feedback and infrastructure for the research in the group. Prof. de Vries and other members of the group participate in courses at Utrecht University and Wageningen University, as well as several MSc and PhD courses in other countries. In addition, Prof. de Vries is co-supervisor for PhD students in Brazil, Sweden and Finland. Supervision of these students is largely by e-mail and skype, but also includes visits to their labs and of them to CBS. The strategic collaboration with University of Helsinki (see below) ensures the required expertise in basidiomycete fungi and currently involves two EU projects, two PhD projects ay University of Helsinki and one PhD project at CBS. Bi-weekly skype meetings and 6-8 meetings in person per year ensure a high level of interaction and knowledge exchange. The research programme is regularly hosts international guests (postdocs, PhD students and Erasmus students) for internships or as part of collaborative projects.
Institutional and national collaboration
The Fungal Physiology programme has strong links with the Applied and Industrial Mycology Programme of Rob Samson based on a common interest in the genus Aspergillus and related fungi. These two programs will merge in 2015 when Rob Samson will retire. It also has strong ties to the Collection & Bioinformatics programme through their comparative studies across the fungal kingdom, which is facilitated by the collection, and through the set-up of the Fung-Growth database, which is supported by the Bioinformatics group. One of the PhD students of the programme, Sylvia Klaubauf, is also strongly linked to the Evolutionary Phytopathology Programme, as her project includes a revision of the Pyricularia genus, which includes the main rice pathogen Magnaporthe oryzae. This part of her PhD project is supervised by Prof. Pedro Crous. Within the Netherlands, there are strong collaborations with Utrecht University. As mentioned, Prof. de Vries has an affiliation to the Department of Biology, but we also have intensive collaborations with the Department of Chemistry (Prof Heck – Proteomics; Prof. Weckhuijsen – lignin degradation). Strong ties are also established with Wageningen University, in particular with respect to sugar analysis (Prof. Schols, Food Chemistry), proteomics and metabolite analysis (Dr Vervoort, Biochemistry), lignin degradation (Dr Mooibroek, Food and Biobased Research), mushroom production (Dr Sonnenberg, Plant Research International), and fungal physiology (Dr Debets, Genetics). In addition, there are several ongoing collaborations with industry based in The Netherlands (DSM, Dupont-Genencor, Dyadic, CNC, Walkro, Mushroom Factory, ServiceXS, Proteonic).
International collaboration
Four types of international collaborations are commonly running in the Fungal Physiology programme: 1. Strategic collaborations Some of the range of fungi, aspects of plant biomass utilisation and methodologies that are being used by the programme require expert advice that is not present within CBS. Also, some groups in other countries address some of the topics that are also addressed in the Fungal Physiology programme.
56 In these cases strategic collaborations are initiated to ensure a long-term collaboration and efficient exchange of knowledge, methodology and sometimes people. Current strategic collaborations are: a. Plant biomass utilization by basidiomycetes: Dr Kristiina Hildén and Dr Miia Mäkelä, University of Helsinki, Finland b. Plant biomass utilization by mycorrhizae: Prof. Francis Martin, INRA-Nancy, France c. Proteomics of fungi during growth on plant biomass: Prof. Adrian Tsang, Concordia University, Canada d. Aspergillus niger regulators involved in plant biomass utilization: Prof. David Archer, University of Nottingham, UK e. Plant biomass utilization by Trichoderma reesei: Dr Bernhard Seiboth, Technical University of Vienna, Austria f. Characterisation of plant polysaccharide degrading enzymes: University of Vicosa, Brazil 2. Research projects Currently, two EU FP7 projects are running in the programme (a collaborative project and a Marie Curie ITN network), as well as four other international projects funded by other sources involving 1 PhD student at CBS and 4 PhD students in Finland (2), Sweden (1), Brazil (1) and UK (1). 3. Genome annotation projects The Fungal Physiology programme participates/has participated in more than 25 genome annotation projects and coordinates one of these projects. These typically involve between 20 and 60 labs from around the world. 4. Incidental collaborations We regularly collaborate with a variety of other labs for specific short-term studies resulting in shared publications. These are usually based on our specific expertise.
6.4 Quality and scientific relevance
The establishment of the Fungal Physiology programme at CBS has enabled me to broaden my expertise on plant polysaccharide degradation to a larger set of fungi and further develop aspects as fungal diversity and evolution and comparative genomics. This has resulted in a strong recognized position as international leader in this field which has fostered many new collaborations. The quality of the research is also reflected in obtaining a VICI grant by Prof. de Vries and a VENI grant by Dr van den Brink, which has provided further momentum for the programme. Some highlights from this first period at CBS (2009–2013) are listed below: • The diversity of fungi with respect to carbon source utilisation is much higher than was previously assumed, even between closely related species. This has been evaluated mainly through various genome annotation projects, resulting in papers in Science (2), Nature Biotechnology (2) and PNAS (2). • Regulatory systems involved in plant biomass utilization by fungi are less conserved than would be expected based on sequence homology, even within the genus Aspergillus. We not only discovered that the set of target genes of the regulators differ from species to species, but also identified a regulator involved in galactose metabolism that is unique to Aspergillus nidulans. • During mushroom formation of Agaricus bisporus, the mycelium growing in compost is responsible for obtaining carbon sources and transporting them to the developing fruiting body. This transport is often considered an aspecific osmotic process. We discovered that this transport is in fact specific to hexose-related carbon sources, while others (e.g. pentoses) are not transported but converted in the mycelium. • The zygomycete Rhizopus oryzae is a primary coloniser of plant biomass consuming mainly the ‘easy’ components before it is replaced by other fungi. However, it also degrades biomass of ascomycete and basidiomycete fungi, indicating a dual life style as both a primary saprobe of plant
Part 6 - Fungal Physiology Programme – R.P. de Vries 57 biomass and a pathogen of other fungi. • Analysis of mixed cultivations of fungi and of fungi and bacteria (Dr Benoit) has revealed both positive and negative interactions and demonstrated the presence of bacterial biofilms coating fungal hyphae.
Key publications
Genome related: Morin et al. (2012). The genome sequence of the Button Mushroom Agaricus bisporus reveals mechanisms governing adaptation to a humic-rich ecological niche. PNAS 109: 17501–17506. Floudas et al. (2012). The Paleozoic origin of enzymatic lignin decomposition reconstructed from 31 fungal genomes. Science 336: 1715–1719. Eastwood et al. (2011). The plant cell wall-decomposing machinery underlies the functional diversity of forest fungi. Science 333: 762–765. Berka et al. (2011). Comparative genomic analysis of the thermophilic biomass-degrading fungi Myceliophthora thermophila and Thielavia terrestris. Nature Biotechnol 29: 922–927. Other: Patyshakuliyeva A, Jurak E, Kohler A, Baker A, Battaglia E, de Bruijn W, Burton KS, Challen M, Coutinho PM, Eastwood D, Gruben BS, Mäkelä MR, Martin F, Nadal M, van den Brink J, Wiebenga A, Zhou M, Henrissat B, Kabel M, Gruppen H, de Vries RP (2013). Carbohydrate utilization and metabolism is highly differentiated in Agaricus bisporus. BMC Genomics 14: 663. Meijer M, Houbraken JAMP, Dalhuijsen S, Samson RA, de Vries RP (2011). Growth and hydrolase profiles can be used as characteristics to distinguish Aspergillus niger and other black Aspergilli. Studies in Mycology 69: 19–30. Christensen U, Gruben BS, Madrid S, Hansen SF, Mulder H, Nikolaev I, de Vries RP (2011). Unique regulatory mechanism for D-galactose utilization in Aspergillus nidulans. Applied and Environmental Microbiology 77: 7084–7087.
6.5 Output
Fungal Physiology has had a substantial output for the period (2009–2013), and has published 63 papers in five years.
Table 6.2. Scientific publications. 2008 2009 2010 2011 2012 2013 Total Academic publications Refereed journals – 6 3 16 14 10 49 Book Chapters & – 3 3 4 1 3 14 Proceedings Total – 9 6 20 15 13 63 Monographs & books – – – 1 – – 2 Ph.D. theses – – – 1 1 – 2 Popular publications – – – – 7 2 9 and products
Considering the recent increase in the numbers of PhD students and Postdocs in the Programme it can be expected that the number of publications will increase significantly over the next years.
58 6.6 Earning capacity
The Fungal Physiology programme has been highly successful in acquiring funds from a variety of national and international programmes and institutions. Since the start of the programme in 2009 we have obtained from national funding one STW project, one NWO-JSTP project (collaboration with China), one NGI-Zenith project, one Senter-Novem project, one NWO-VENI grant, one NWO-VICI grant, two VanGogh collaboration grants and one BeBasic grant, and are partner in an EMBO long-term fellowship grant with the Department of Chemistry, Utrecht University. From international funding we have obtained three FP7 projects (NEMO, Optibiocat, Subicat) and one COST network, one DOE-JGI CSP2011 project (USA), four Science without borders grants (Brazil) and one sandwich PhD grant (Saudi-Arabia). Finally, we obtained direct funding from industry for five projects (DSM, Dupont-Genencor, WeissBioTech, Megazyme, Mushroom Factory).
6.7 Academic reputation
Awards
2013 - VICI grant from NWO 2005 - VIDI grant from NWO
Appointments
Utrecht University (Professor, per 18 March 2014)
Editorships/Editorial board membership in academic journals
Editor for Research in Microbiology Editor for two special issues of Fungal Genetics & Biology Editorial Board member for Applied and Environmental Microbiology Editorial Board member for Letters in Biotechnology
Memberships in Scientific Boards
Fungal Program of the Joint Genome Institute of the US-DOE Aspergillus Genome Resource Policy Committee
Selection of invited lectures de Vries RP (2013). Fungal strategies for plant biomass degradation. XI International Fungal Biology Conference, Karlsruhe, Germany. de Vries RP (2013). Degradation of biomass of terrestrial plants and algae by fungi. Seagriculture - exploring the seaweed chain, Texel, The Netherlands. (keynote) de Vries RP (2012). Physiological and molecular aspects of the utilisation of complex substrates by fungi. The International Union of Biological Sciences (IUBS) 31st General Assembly (GA) on biological sciences and bioindustry. Suzhou, China de Vries RP (2012). Fungal biodiversity with respect to carbon utilisation: Taxonomic relationships versus parallel evolution. Workshop on Taxonomy, Bremen, Germany de Vries RP (2012). Differences in the utilisation of complex substrates within the genus Aspergillus. 9th Symposium of the Kluyver Centre for Genomics of Industrial Fermentation, Noordwijkerhout, The Netherlands
Part 6 - Fungal Physiology Programme – R.P. de Vries 59 Bibliometric analysis
• Number of peer reviewed publications: 96 (Web of Science, March 2014) • H-index: 32 (Web of Science, March 2014); 42 (Google Scholar, March 2014) • Total number of citations: 5464 (Google Scholar), 3142 (Web of Science, March 2014)
6.8 Societal relevance: quality, impact and valorisation
The transition to a biobased economy requires significant research inputs, both from academia as well as the private sector. The research of the Fungal Physiology programme links perfectly to the aims of using plant (and algal) biomass as a renewable resource for society that may largely replace fossil fuels. Our aim to understand fungal biodiversity with respect to plant biomass utilization and its underlying mechanisms provides the basic knowledge needed to further improve existing applications and design new applications. The research in the group is set-up in such a way that largely fundamental research is complemented with more applied projects (often to public-private partnerships) and direct collaborations with industry. The research focus on understanding the utilization of plant biomass by fungi provides direct links to various applications: • Identification and/or development of better enzyme mixtures for plant biomass saccharification for the production of biofuels, biochemicals and biomaterials • Improving enzyme production in filamentous fungi, resulting in lowered production costs • Improving substrate utilisation in the cultivation of edible mushrooms resulting in higher mushroom productivity per amount of compost • Opening up fungal biodiversity as a source of enzymes and metabolites for the biobased economy In addition, the results related to fungal biodiversity and evolution also have an impact on ecology as fungi play an important role in many ecosystems. Understanding their ability to consume plant biomass (the most common substrate in many of these biotopes) aids in the understanding of the role of fungi in these ecosystems.
6.9 Viability
Of the staff indicated in Table 6.1 only the group leader and one technician are on KNAW funding, while all other staff is employed on competitive grants. The increase in the group size over the last five years clearly demonstrates the programmes potential to obtain external funding. This is largely due to the international reputation of the programme as well as to the unique combination of having a strong group in plant biomass utilization by fungi together with direct access to the largest public fungal culture collection. In addition, the main topic of the group (plant biomass utilization by fungi) fits perfectly in the global move to a biobased economy, as well as in the themes of the Dutch Topsector themes and the new EU Horizon 2020 programme. As these themes are not likely to change in the foreseeable future, this also means that the viability of the Fungal Physiology programme will continue to be strong.
6.10 Strategy
Over the last five years, the Fungal Physiology programme has developed into one of the leading groups worldwide in its field. While the programme initially focussed mainly on molecular and regulatory aspects of plant biomass utilization by fungi, this has evolved to address the complete process and its underlying mechanisms. Also the recent expansion of the group with a bioinformatician and its increasing participation in large scale (comparative) genomic and transcriptomic projects have not only resulted in significant deepening of our understanding of the topic, but has also fostered many new collaborations with groups from around the world.
60 Recently awarded grants (VICI, EU Optibiocat) have added an additional bioinformatician as well as an enzymologist to the programme, strengthening those skills. The combination of skills, scientific expertise and access to the CBS collection has raised significant interests from both academic groups and industries in collaboration with the programme, resulting in several new initiatives for STW and Horizon2020 proposals. The main threat to the group is the balance between staff on permanent (2) and temporary (currently around 20) contracts, which puts significant pressure on continuation, supervision and support ofthe various projects in the groups. Crucial skills for the group (e.g. bioinformatics, enzymology) are based on temporal staff that under the Dutch law cannot be extended indefinitely, resulting in a loss of expertise in the near future that will be essential to be replaced, but will likely again be depending on external funding. The professorship at Utrecht University for Prof. de Vries which is expected to be finalised by March 2014 may partially solve this problem as it would enable us to continue contracts of temporal staff for an additional period at Utrecht University. However, it is an issue that requires careful monitoring to prevent a sudden loss of crucial expertise. An additional issue to address is the changing requirements to stay at the forefront of our field. This includes state of the art equipment to perform both molecular biological and biochemical research. While the former is well established at CBS, the latter requires further development. To solve this, granting schemes that enable the acquisition of state of the art equipment will be addressed.
Part 6 - Fungal Physiology Programme – R.P. de Vries 61 7. Bioinformatics
Programme leader Dr V. Robert
7.1a Objectives(s) and policy
Culture collections such as the Centraalbureau voor Schimmelcultures (CBS, The Netherlands) need to manage large numbers of strains (the records) and characteristics (the fields). Until a few years ago, collected data were mainly used for taxonomic purposes. Most of the time, these were not disclosed to users outside the institute. Only the strain’s species name was available together with a few additional data such as depositor, substrate, and strain origin, in printed catalogues. Several years ago, CBS decided to tackle this problem and to create databases that would contain all possible sources of information at the strain and species levels. This task was far from trivial since it involved integration of many different sources of information [e.g., administrative data, bibliography, geography, pictures, nomenclature, morphology, physiology, biochemistry, molecular data (electrophoresis results, sequences, …)], as well as hyperlinks to other web-based repositories. From the beginning, it was apparent that conventional searching tools in commercially available databases did not fit CBS’ needs. Therefore, new software called BioloMICS was created, capable of searching, identifying, classifying, and analyzing all available data in a polyphasic way. This software is under continuous development and is the central focus of the bioinformatics groups since it is currently managing all the data (scientific and administrative) of the CBS and many associated projects. In 2004, we also introduced the MycoBank concept. MycoBank is an on-line database aimed as a service to the mycological and scientific community by documenting mycological nomenclatural novelties (new names and combinations) and associated data, for example descriptions and illustrations. The nomenclatural novelties receive unique MycoBank numbers that can be cited in the publication where the nomenclatural novelty is introduced. In 2009, due to the ever-increasing needs in terms of data storage, developments of specific algorithms and research projects requiring specific programming, it was decided to establish a group in Bioinformatics. The main focus of the group is to provide a number of services to the other CBS groups (mainly the collection). The bioinformatics group also develops its own research projects, either alone or in collaboration with other research groups worldwide. The group on bioinformatics is a hybrid group essentially consisting of a group, three collaborators based at CBS and a series of external collaborators working on a number of projects that are described below. The group is more service than researched oriented. We are developing a number of tools needed for the daily management of the collection (databases, websites, software, IT infrastructure, etc). We are also helping researchers (internal and external) to analyse their data. In addition to databases and software developments, our group is also managing the most important aspects of the IT infrastructure of the CBS and associated projects since mid-2009. We are currently managing 56 virtual servers and a complete IT infrastructure hosted in a datacenter (firewalls, switches, SANs, servers, etc). We are currently also managing 92 websites and a large number of web services, all hosted on our servers.
7.1b Forward look
The bioinformatics group was recently established and yet it is holding a central position within CBS since all data and websites are managed by our software and IT infrastructure. Many scientific analyses made by CBS researchers are done in collaboration with our group. We have been successfully included in several international projects since the creation of the group and continue to be involved in the writing of new ones.
62 Needless to say that bioinformatics is one of the key components of existing research projects and its importance will inevitably increase exponentially in the future in correlation with the explosion of scientific data.
Several aspects will have to be addressed in the future:
1. Maintenance and development of existing databases 2. Developments of the next generation databases (MongoDB) and associated software for large data stores 3. Maintenance of the data management software 4. Maintenance and development (for NGS data) of the LIMS software 5. Maintenance of the stock management software 6. Developments of new algorithms for data analyzes 7. Next generation websites and web services 8. Research projects and publications 9. Communication and teaching 10. Funding
Our service-based tasks will certainly increase in the future but our research activities need to be increased. This will not be possible with the limited number of people currently active in our group. This means that internal and external funding will have to be found in order to achieve such ambitious goals.
7.2 Composition
Table 7.1. Staff at programme level, 2009–2013. 2009 2010 2011 2012 2013 Funding* Group leader Dr V. Robert 1.0 1.0 1.0 1.0 1.0 1 Researchers Dr L. Vaas – – – 1.0 1.0 2 Dr C. Brouwer – – – – 0.3 11 Developers Dr D. Vu 1.0 1.0 1.0 1.0 1.0 1 Dr S. Szoke 1.0 1.0 1.0 1.0 1.0 3–10 A. Ammor Ben Hadj – 0.5 1.0 1.0 1.0 3–10 B. Jabas 0.5 0.5 0.5 0.5 0.5 3–10 N. van de Wiele – – – 1.0 1.0 2, 3, 5, 8 O. Chouchen 1.0 1.0 1.0 1.0 1.0 3–10 O. Bensghaier – – – – 0.5 2, 5 M. Triki – – – – 0.5 2, 5 A. Dridi – – – 0.5 0.5 5 S. Ben Daoud 0.01 0.01 0.01 0.01 0.01 3–10 M. Jaidane 0.2 – – 0.4 0 10 F. Borges Dos Santos – – – 0.8 0.3 11 IT Staff E. Blom – – 0.2 0.2 0.2 3–10
*1 = Royal Netherlands Academy of Arts and Sciences (KNAW); 2 = NH&MRC, Australian grant; 3 = EMbaRC, EU FP7; 4 = QBOL, EU FP7; 5 = Dutch Ministry of Science (FES Barcoding Informatics); 6 = Indoor fungi project, Sloan Foundation Project; 7 = Q-bank project, EU FP7; 8 = i4Life project, EU FP7; 9 = Q-collect project, EU FP7; 10 = private funding BioAware and Biorganize; 11 = voluntary basis/stage.
Part 7 - Bioinformatics Programme – V. Robert 63 7.3 Research environment and embedding
7.3.1 QBOL project
The Quarantine Barcoding Of Life project (QBOL) aimed at the development of accurate identification tools for plant pathogens and pests, which is vital to support European Plant Health Policies. For this project Council Directive 2000/29/EC has listed some 275 organisms for which protective measures against introduction into and their spread within the Community needed to be taken. Those threats are now greater than ever because of the increases in the volumes, commodity types and origins of trade, the introduction of new crops, the continued expansion of the EU and the impact of climate change. Currently identifying pathogens (in particular new emerging diseases) requires a staff with specialized skills in all disciplines (mycology, bacteriology, etc.); which is only possible within big centralized laboratory facilities. Taxonomy, phytopathology and other fields which are vital for sustaining sound public policy on phytosanitary issues are threatened with extinction. Modern molecular identification/detection techniques may tackle the decline in skills since they often require much less specialist skills to perform, are more amenable for routine purposes and can be used for a whole range of different target organisms. In the last few years, DNA barcoding has arisen as a robust and standardized approach to species identification. QBOL wanted to make DNA barcoding available for plant health diagnostics and to focus on strengthening the link between traditional and molecular taxonomy as a sustainable diagnostic resource. Informative genes from selected species of the EU Directive and EPPO lists have been sequenced from vouchered specimens. The sequences, together with taxonomic features, have been included in a new internet-based database system created by the CBS bioinformatics group. A validation procedure on developed protocols and the database have been undertaken across worldwide partners to ensure robustness of procedures for use in a distributed network of laboratories across Europe. The multinational QBOL consortium has a long-term vision of developing a sustainable diagnostic resource to enable ‘DNA-barcode identification’ ultimately for all quarantine plant pests or pathogens of statutory importance. The aim is to continue to develop a DNA barcode database containing relevant reference DNA/RNA sequences of quarantine and regulated plant pathogens linked to appropriate taxonomic metadata. In addition to develop the tools and methods to enable the identification of all quarantine and regulated plant pests and pathogens worldwide by any plant diagnostic center, national reference organization and authority freely using the internet, to enable the implementation of Council Directive 2000/29/EC. This project started in 2009 and finished in 2012. It received a very good appreciation rate by the EU and has been prolonged with the Q-collect project (EU FP7 2013-2015) that just started at the end of 2013 and that will link more data and resources to the databases created within the QBOL and Q-bank projects.
7.3.2 Q-bank
The Q-bank project (www.q-bank.eu) started a numbers of years ago and it was initially called the FES Plant Health project. It was supported by the Dutch government and the basic idea behind it (at least the informatics part that is interesting to us here) was to create software to store any biological data associated with reference collections. Our BioloMICS software was selected as a basis and was further developed according to the needs of the participants of the project. At the end of the FES project in 2008, the QBOL project mentioned above allowed us to continue software developments that were initiated by the FES project. At the end of the QBOL project and before the start of the Q-collect project (not described here since it has just been started), a transitional period was again funded by the Dutch government in order to maintain databases and continue software developments. Our group is again responsible for the developments made for Q-bank partners. We are also hosting their databases, websites and providing
64 support to them. In 2015, following the recommendations of the EU commission, the intention is to migrate Q-bank under the auspices of EPPO in order to obtain permanent support from the EU.
7.3.3 NCB - Netherlands Centre for Biodiversity - project: European BOLD mirror and Bioinformatics infrastructure
The FES/NCB project is a large and ambitious project subsidized by the Dutch government and aims at providing collection and molecular (DNA barcodes) information to the general public on collections available from NCB Naturalis (Plants and Animals mainly) and CBS-KNAW (Fungi). Such data should be fully and freely available for third parties via databases accessible through one or several websites. While parts I and II of the project are dealing with the production of DNA barcodes, part III has the following general objectives as defined by:
1. Create the needed infrastructure for the storage, analyses and publication of DNA barcodes. This includes hardware and software components. 2. Make agreements with internationally recognized authorities, consortia and networks at European (ECBOL) and International (iBOL) level to host a Barcoding of Life mirror or similar system. 3. Establish a help desk, back office and network to answer questions related to the created infrastructure. 4. Create the necessary conditions to ensure the continuity of the infrastructure after the end of the project.
The project started in 2010 and will end early 2015.
For this project, a huge amount of time was invested in the creation of software tools that were again integrated in our BioloMICS software. The most important component in terms of time and money invested was certainly the Laboratory Information Management System (LIMS). It took us several years to develop it and to make it completely integrated within the other aspects of our collection management software. The LIMS module consists of eight (sub-)modules including data storage, sample tracking, complex query building, generation of reports, automatic importation of trace files, trace file edition, sequence identification and automated annotation of sequences that are able to manage data produced by DNA barcoding workflows from the creation of DNA extractions to the identification of the obtained sequences. This system is now used by several laboratories including CBS-KNAW to manage all the aspects of large DNA barcoding projects.
7.3.4 iBOL project: European BOLD mirror
The International Barcode of Life Project (iBOL) is building a DNA-based identification system for all species of eukaryotes. It will achieve this goal through a five-year project, leading to an increasingly comprehensive library of DNA barcodes, short sequence reads from standardized genomic region(s) that enable species discrimination. The International Barcode of Life Project entered its ramp-up phase on July 1, 2009 and saw formal activation 15 months later. Activities over this interval have focused on satisfying the five conditions necessary for iBOL to achieve its research objectives – building an international research alliance and the administrative system to support it, sourcing specimens for analysis, establishing core sequencing facilities, establishing an informatics platform for data curation and analysis, and building a research program which evaluates the societal impacts of the overall iBOL research effort. The balance of this summary provides a brief overview on each of these areas. CBS-KNAW is an active member of iBOL and our bioinformatics group was asked to co-chair the BOLD mirrors work package. Our tasks were to create the European BOLD mirror website and to provide basic
Part 7 - Bioinformatics Programme – V. Robert 65 search functionalities as well as pairwise and polyphasic identifications using several loci. We were also asked to create synchronization web services with Canada (BOLD), China (CAS) and Australia (ALA). For our group, the project started in 2010 when the NCB project (mentioned above) allowed us to start the developments. All the tasks mentioned above have been achieved and the EUBOLD mirror website is now active since November 2013 and accessible at www.eubold.org.
7.3.5 I4Life
Indexing for Life (i4Life) was a European e-Infrastructure project, co-funded by the European Commission’s Seventh Framework Programme for Research and Technological Development. The project was launched at the University of Reading on 1st November 2010. The i4Life is a continuation and expansion of currently existing 4D4Life project. i4Life had one principal goal – to provide tools for the comparison and harmonization of the various species catalogues used by six global biodiversity programmes using the Catalogue of Life as a yardstick. This goal was achieved by establishing of a virtual research community that will interlink and harmonize the taxonomic catalogues presently used by each of the global partners (EOL, GBIF, EMBL, IUCN, BOLD, etc.) and to create an enhanced list of the entire set of organisms. Partners to this project are major global programmes exploring the full extent of life on earth, a central conceptual scientific axis in human knowledge of global biodiversity. They were: GBIF for distribution modeling, the ENA project at EMBL-EBI and the Barcode of Life Initiatives (CBOL and EUBOLD represented by CBS bioinformatics group) for molecular diversity, IUCN Red Lists for conservation assessment, the Encyclopedia of Life with its life desks and the Species 2000 Catalogue of Life taxonomic framework. During this i4Life project, several software were created, like cross-mapping tools that compare list of species names between different databases to point discrepancies and incoherencies. Many web services for automated data exchange between partners have been established as well. As creator and owner of the EUBOLD database and website, the role of our team was to provide the flow of data to and from the Catalog of Life (CoL) system. This was successfully done. Since we are also in charge of the MycoBank database and since the CoL wanted to create structural links to and from MycoBank, the system was extended and CoL is now using MycoBank as preferential partner for fungal names. This project started in 2010 and ended at the end of 2013. It gained the highest possible grade and was rated as “excellent” by the EU reviewers.
7.3.6 MycoBank
MycoBank was officially launched in 2004 as an online repository with the primary aim to register all fungal taxonomic novelties published (including new names and combinations), and make this available in an open access database to the mycological community. One of the major constraints experienced by mycologists was that many newly published fungal names were not accessible to researchers in developing countries or simply overlooked, because they were published in obscure sources. Due to the large number of names published each year in a range of publications, MycoBank curators were not always able to verify and include all of them in the database. To address this issue, we approached a large number of journal editors that published taxonomic novelties, and suggested that they request authors to deposit nomenclatural data, descriptions and illustrations in MycoBank, as good practice. This equates MycoBank as a phenotypic equivalent of GenBank, the main database for genotypic data. Authors would receive a unique identifier to link the registration to the name (equivalent to a GenBank accession number for data sequence), and would simultaneously be assured that no homonyms were published, as the search engine would inform authors if the name was already occupied (www. mycobank.org). Registration was seen as a two-step process; upon acceptance of the article, authors deposit their taxonomic novelties, provide the MB numbers in the protolog, and upon publication, notify MycoBank to ensure that the taxonomic novelty could be released to the community with date, volume and page numbers.
66 The system was so popular among mycologists, that proposals to make the deposit of the key elements mandatory for the valid publication of new scientific names of fungi, at all ranks, were prepared, and debated at the 9th International Mycological Congress in Edinburgh in 2010. These were put before the Nomenclature Section of the 18th International Botanical Congress in Melbourne in July 2011, and incorporated into the International Code of Nomenclature for algae, fungi, and plants. MycoBank can do much more than complete the basic requirements of the Code, but the only mandatory elements are the: name; rank; authorship; bibliographic details of the anticipated place of publication; diagnosis (or description) for names of novel taxa (which from 1 January 2012 can be in English or Latin); full bibliographic details of the basionym or replaced name for new combinations, names at new ranks, or replacement name; and for names of novel taxa also details of the name-bearing type and the institution or other place in which it is permanently preserved. Although MycoBank was initially set up by the CBS-KNAW staff in close collaboration with Index Fungorum, in 2009 it was decided that the ownership of the MycoBank system, database and website should be transferred to the International Mycological Association (IMA). In 2010 a new version of the MycoBank website was launched, based on the BioloMICS software. The advantage of the latter software is that the structure of the database could evolve according to the needs identified by the end-users and the curators of MycoBank. The new BioloMICS-based version of MycoBank has been regularly updated and improved since then. We present here all the major developments achieved during the past four years, as well as some usage statistics of the MycoBank system. In the last section, we will briefly describe how we see the database evolving in coming years.
Infrastructure. The latest version of the MycoBank software was released in April 2012, allowing curators to create new tables and fields according to the natural evolution of their increasing needs and the one of the end-users, without the intervention of any software developers. This is essential when new types of data and the associated analytical tools will be incorporated into the system. In order to keep MycoBank users aware of the latest news and improvements related to the database and software, a “News”, a “Frequently Asked Questions” and a ”Help” section were created.
Queries. The new software interface was created in order to improve flexibility for queries. Basic (www. mycobank.org/Biolomics.aspx?Table=Mycobank) and advanced queries (www.mycobank.org/Biolomics. aspx?Table=Mycobank_Advanced) are now possible. Advanced users can build complex Boolean queries by combining AND, OR and NOT together with brackets.
Name registration. The interface for the registration of the scientific names of new taxa, and new names, has also been redesigned and simplified, with fewer required steps than the previous version.
Type registration. In the summer of 2013, a new typification registration system was added to MycoBank. Mycologists can now log in to the system, and choose to register a type specimen for an existing taxon. It means that mycologists can now get “MBT” numbers (MycoBank Typification numbers) for the designation of lectotypes, epitypes and neotypes.
Multi-lingual system. The original software was English based but has been modified to allow multiple languages to be displayed. Arabic, Chinese, Dutch, French, German, Portuguese, Spanish and Thai are now available as well.
Forum. Since the Amsterdam declaration on fungal nomenclature, and the introduction of the new Code, mycologists have several new challenges to face reaching consensus with regard to the “one fungus one name” nomenclature. Two years ago, when discussions were initiated, we felt that there was a need to create a discussion forum to exchange ideas about dual nomenclature, and the name that should be retained. Hence, the Forum option was created and a large number of topics and discussions were initiated.
Part 7 - Bioinformatics Programme – V. Robert 67 Annotations and remote curation. The annotation system was created to allow users (after registration open to anyone) to post comments, suggest corrections or propose new data associated with already deposited taxa.
Web services and central system for registration of fungal names. Many users and websites are interested to obtain data in batch and incorporate this in their own databases. Since MycoBank is a public database used by many other repositories it was important to provide a number of web services that can be consumed by third party machines. We therefore created several dynamic web services that can easily be changed or adapted if needed. The system was released in June 2013, and is going to be used for the synchronization between MycoBank, Index Fungorum and Fungal Names website.
Links to third parties. Many other websites are rich resources that can be associated with fungal names available in the MycoBank system.
Identification services. MycoBank is not only a repository of data associated with fungal names and vouchers, but also offers unique online pairwise sequence identification services (www.mycobank.org/ biolomicssequences.aspx) against curated databases such as Q-bank, CBS collections websites, Fungal Barcoding, EUBOLD system, ISHAM ITS Database, UNITE or NCBI/Genbank databases. Users interested in identifying unknown sequences can compare them against all the wanted reference databases at the same time or separately and results are gathered centrally and proposed as a unique matching list. Other more advanced identification services are also possible using a combination of morphological, physiological and/or molecular data.
Table 7.2. Top 10 countries using MycoBank. Rank Country Percentage of total users 1 USA 13.65 2 France 6.31 3 Germany 6.03 4 Spain 4.87 5 Italy 4.20 6 Brazil 3.80 7 Russia 3.40 8 India 3.32 9 Canada 3.22 10 China 3.03
Statistics. In total 254,120 unique visitors have visited the English version of MycoBank between April 2012 and 3 December 2013. In December 2012 we launched several language versions of the website, French (3992 unique visitors), Arabic (2466), Chinese (1953), Dutch (1079), German (1828), Portuguese (2141) and Spanish (2207). Recently, a Thai language version has been introduced. On an average day, 1872 unique users visit one of the MycoBank portals, while the average visit duration is between 6–10 min per user. The MycoBank user-base is truly global: 13.65 % of the users are located in the USA, but people from 205 countries have used MycoBank since April 2012. Table 7.2 lists the top 10 countries using of MycoBank around the world. Researchers depositing new scientific names in MycoBank, interested in forum discussions or willing to annotate taxon records have to be registered in MycoBank. Presently 5680 profiles have been registered
68
Fig. 7.1. Decennial evolution of the number of described species between 1759 and 2009. since MycoBank was initiated in 2004. During the period between 1985 and 2012, 8031 different taxonomists published at least one new fungal species. The average number of authors was 1.86/species. The first 50 authors contributed to 22.9 % of the new species. The first 100 authors contributed to 32.1 % of the new species. The first 1000 authors contributed to 74.3 % of the new species and 6077 authors published between 1 and 5 new species only.
Future. MycoBank is one of the three repositories that fill an important requirement in terms of the registration of scientific names now required by the Code. While it is increasingly becoming a rich source of knowledge at species, genus, family and higher levels, the databases of the International Nucleotide Sequence Database Collaboration (INSDC), a consortium consisting of NCBI, EMBL and DDBJ, serves as the international repository for molecular sequences. The task of linking MycoBank entries based on reference material (specimens and strains) to INSDC sequences, often only known from environmental sequences, is a real challenge. It incorporates subjective taxonomic interpretations with many species described and circumscribed on the basis of non-molecular criteria (morphology, physiology, ecology, etc). Voucher data annotated consistently in all databases will possibly remain the most effective way to link species names and their associated molecular data. The links between species (and subsequently to higher taxonomic ranks) and sequences can only be done via strains and specimens. The biological repositories of fungal voucher data, culture collections and herbaria (listed in Index Herbariorum) are of major importance by housing reference material, information and strains. Other initiatives such as the barcoding of life (BOLD systems, EUBOLD, China BOLD, etc.) or the UNITE database are providing useful links between reference material and barcoding sequences. Some projects are dealing with the establishment of reference databases in specific fields such as medical mycology. The Straininfo and WDCM databases are gathering strain data from culture collections and are providing links to INSDC databases, but their scope is limited to cultivable strains and it is now commonly accepted that most of the diversity is not present in culture collections or museums but is simply unknown. There still remain many research collections around the world with useful information of unique strains or specimens, but these are often unavailable to third parties. MycoBank also maintains an ex-type strain and specimen database that is linked to species descriptions, which is in the process of being linking to INSDC-based sequences, in order to objectify or at least to provide a molecular background to species circumscriptions. Hence, some prominent mycologists and institutions are actively working on this matter and are preparing workshops, guidelines and tools to better fill the gap of linking sequences to species. One of the ways to solve this problem is to suggest MycoBank depositors of new species to provide molecular data, in addition to strains or specimens, or links to these data during the registration process.
Part 7 - Bioinformatics Programme – V. Robert 69 Linking species data via molecular data using strains and specimens is important, but will not solve all problems or opportunities induced by the usage of modern technologies. Next Generation Sequencing (NGS) methods or high throughput screening technologies already allow us to obtain large datasets that would not be accessible using traditional sampling, isolation and collecting methods. New species are traditionally based on the isolation of one, or ideally several specimens that are studied and deposited in reference collections. With NGS it is possible to obtain millions of sequences from a single soil sample in a few hours and get an idea about the relative abundance of the taxa present. It is also possible and relatively easy to monitor the changes in ecosystems or hosts over time. The known diversity constitutes only a small fraction of the real fungal biodiversity. Given the drastic reduction of taxonomists and financial support attributed to systematics, it is unlikely that traditional taxonomic approaches will ever allow us to get a near complete idea of the scope of microbial diversity. Therefore, ignoring the impact of new technologies such NGS for the discovery of existing diversity would be a major mistake. Currently, there are no mechanisms allowing researchers to record, share and describe new taxa on the basis of such new technologies, other than the recently proposed system of UNITE. Although there are a number of issues associated with these new technologies in terms of data quality, reproducibility and quantity, there is no definitive reason to ignore them. Hence, MycoBank, in collaboration with INSDC, UNITE and other DNA Barcoding initiatives (in its broad definition) will propose mechanisms and tools to record non-specimen based descriptions for candidates species. We are currently working on tools for the semi-automated curation of large datasets, for fast and accurate assignments to species or candidate species. Given the amount of data to be handled and analyzed, new technologies need to be developed. This can only be accomplished through the collaboration of several groups of experts ranging from ecologists, taxonomists, molecular researchers, bioinformaticians, informaticians, mathematicians, database specialists to technologists focused in molecular or information technologies and hardware devices such as CPUs, GPUs or FPGAs.
9.3.7 The quest for a general and reliable fungal DNA barcode (Sloan foundation and EU-Embarc projects)
DNA sequences are key elements for both identification and classification of living organisms. Mainly for historical reasons, a limited number of genes are currently used for this purpose. From a mathematical point of view, any DNA segment, at any location, even outside of coding regions and even if they do not align, could be used as long as PCR primers could be designed to amplify them. Thanks to the support of the Sloan foundation and EU-Embarc projects, two methods have been used to search genomic data for the most efficient DNA segments that can be used for identification and classification. While our bioinformatics group was heavily involved in the preparation of the paper on fungal ITS barcoding (Schoch et al. PNAS, 2012), it remained obvious that alternative regions to the ITS should be investigated. A number of researchers have done it and found that some regions are certainly performing as well as, or better in some clades than ITS. However, the selection of such alternative regions was usually based on rather subjective decisions. When the first large molecular phylogenetic studies were completed, it was obvious that many clades were poorly supported statistically when only one or two genes were used. Recently, many authors explored possibilities for analyzing several genes to obtain the “true phylogeny”. Some suggested that a few genes randomly selected were sufficient to obtain the “true phylogeny”. Others found that excellent classifications and identifications could be obtained using single loci present in most known fungal clades. In a first approach, our group tried to find the “Ideal Locus”. Such locusa would provide a phylogeny as close as possible to the one putatively expected from the whole genome phylogeny and would distinguish distantly and closely related species. With the “Ideal Locus” method, we used all available fungal genomes and classified all protein coding segments in euKaryote Orthologous Groups (KOGs) of proteins. As many of these genes were not found in all species, it was necessary to be selective and keep only those found in all species of interest. At this point one or several copies of the same KOG and for each species/
70 genome were obtained. Another selection step was introduced to reduce the number of copies to one by keeping the copy that maximized the similarity with the other copies of the same KOG in the other analyzed genomes. Multiple alignments for each KOG were produced using one KOG copy per genome. The resulting multiple alignments were transformed into distance matrices. A reference matrix (i.e. the “true matrix”) was created by concatenating all multiple alignments of the different KOGs to build a large multiple alignment representing the KOGs present in all of the selected genomes. All single gene distance matrices were compared to the reference matrix using the Pearson correlation algorithm (Mantel test) and ranked according to how well they fitted with the ideal or reference phylogeny. From there, the best possible genes for phylogenetic analyses on the group of interest were selected, from which potential barcoding candidates were then selected. Results based on 25 genomes and 531 KOGs in common showed that one third of the genes (29.8 %) produced a phylogeny that was highly correlated with the reference matrix. Seventy per cent of the gene’s matrices had a correlation higher than 0.70 with the reference matrix. Only a few genes (25) showed no, or a very low correlation with the reference matrix. Neither the length (Pearson’s coefficient of correlation = 0.28) nor the evolutionary rates (Pearson’s coefficient of correlation = 0.018) of the KOGs were related to, or could explain, the level of correlation with the reference matrix. The loci showing the highest level of correlation with the reference matrix are presented in Table 7.3. Their predicted functions were obtained from the NCBI website. The best possible KOG (1234) had a correlation of 0.986 with the reference matrix (i.e. the “true phylogeny”). A large number of loci showed such high correlation levels with the reference matrix and could therefore be used to reconstruct a robust phylogeny from very diverse taxa. However, for routine identification it was not possible to find “universal primers” that could be used for PCR amplification of phylogenetically diverse groups.
Table 7.3. List of the first 20 KOGs showing the highest correlation with the super matrix and therefore being the best candidate genes for phylogeny reconstruction and potentially for barcoding. Rank KOG # Correlation Length Predicted function level (source: ftp://ftp.ncbi.nih.gov/pub/COG/KOG/kog) 1 KOG 1234 0.987 435 ABC (ATP binding cassette) 1 protein 2 KOG 0724 0.982 435 Zuotin and related molecular chaperones (DnaJ superfamily), contains DNA-binding domains 3 KOG 2472 0.981 587 Phenylalanyl-tRNA synthetase beta subunit 4 KOG 0714 0.980 309 Molecular chaperone (DnaJ superfamily) 5 KOG 2002 0.979 513 TPR-containing nuclear phosphoprotein that regulates K(+) uptake 6 KOG 3844 0.978 366 Predicted component of NuA3 histone acetyltransferase complex 7 KOG 2369 0.977 449 Lecithin:cholesterol acyltransferase (LCAT)/Acyl- ceramide synthase 8 KOG 0363 0.976 523 Chaperonin complex component, TCP-1 beta subunit (CCT2) 9 KOG 0362 0.976 522 Chaperonin complex component, TCP-1 theta subunit (CCT8) 10 KOG 1450 0.976 382 Predicted Rho GTPase-activating protein 11 KOG 1439 0.975 444 RAB proteins geranylgeranyltransferase component A (RAB escort protein) 12 KOG 1113 0.975 248 cAMP-dependent protein kinase types I and II, regulatory subunit
Part 7 - Bioinformatics Programme – V. Robert 71 Table 7.3. (Continued). Rank KOG # Correlation Length Predicted function level (source: ftp://ftp.ncbi.nih.gov/pub/COG/KOG/kog) 13 KOG 1156 0.974 422 N-terminal acetyltransferase 14 KOG 0863 0.973 218 20S proteasome, regulatory subunit alpha type PSMA1/ PRE5 15 KOG 1533 0.973 277 Predicted GTPase 16 KOG 0340 0.973 421 ATP-dependent RNA helicase 17 KOG 1273 0.973 295 WD40 repeat protein 18 KOG 1980 0.973 546 Uncharacterized conserved protein 19 KOG 1068 0.972 222 Exosomal 3’-5’ exoribonuclease complex, subunit Rrp41 and related exoribonucleases 20 KOG 1485 0.972 201 Mitochondrial Fe2+ transporter MMT1 and related transporters (cation diffusion facilitator superfamily)
In a second approach, called Best Pair of Primers (BPP) method, we mined 77 Fungal genomes to find short conserved regions (coding or not) that could be used as forward and reverse primers. The variable regions between these two primers were analyzed and subsequently the ability of the amplified regions to be used as reliable phylogenetic representatives and/or as potential barcode candidates for identification was assessed. More candidates are currently under investigations and seem to be extremely promising as secondary or even primary barcodes for fungi. All the results mentioned above have been used to launch a large scale study including a large number of mycology research laboratories and collections such as CBS, CDC Atlanta, Pasteur Institute Paris, University of Perugia, University of Sydney, Agriculture and Agri-Food Canada (AAFC), DSMZ University of Athens, etc. Preliminary results are extremely promising and some primers pairs are even working better than common ITS primers. A series of publication is currently under preparation.
7.3.8 DNA barcoding of pathogenic fungi as the basis for the development of novel standardized diagnostic tools (NH & MRC Australian grant)
This ongoing project combines the unique expertise of three leading international research teams in the field of molecular phylogeny/diagnostic/mycoses (University of Sydney, Australia), bioinformatics (CBS, Netherlands) and fungal taxonomy (University of Adelaide, Australia) to tackle a major medical mycology/ quarantine problem - the lack of an easy and universally standardized identification system for fungal pathogens. Pathogenic yeast and filamentous fungi are increasingly implicated as cause of life-threatening diseases, however, the current lack of reliable and fast identification tools delays significantly a timely and appropriate treatment of mycoses. We use whole genome comparisons to: (i) identify the most appropriate genetic locus/loci capable to generate species-specific genetic signatures to be used as DNA barcodes, (ii) design primers and develop a highly standardized method to obtain unique DNA barcodes, (iii) generate unique DNA barcodes for all human/animal pathogenic fungi, and (iv) establish a globally accessible online database. This enables faster diagnoses of pathogenic fungi in the clinical and quarantine setting, and permits information driven therapeutic choices and better possibilities to safeguard Australia from emerging infectious diseases threats. This application aligns with the NH&MRC Strategic Plan initiatives of “Building a Better Health System” and “Emerging Infectious Disease Threats” by addressing a major global one-health issue. Every fungal pathogen is characterized by a unique genetic signature, which specifies its phylogenetic position in the “Tree of Life”. The unique genetic signature(s) of a microorganism can be identified
72 by comparative genomics. The identified locus/loci will form the basis for the development ofnew identification tools, which will revolutionize the way, how fungal disease agents will be identified in a clinical or quarantine setting in the future.
7.3.9 Stock management
The CBS collection is certainly the largest and the most important fungal culture collection in the world and we have a large client base. More than 20 years ago CBS initiated the development of software to manage all the aspects related to the management of the collection (strains data, stock, ordering, invoicing, website, etc.). CBS was a pioneer in this field. In 1999, the yeast collection migrated to the BioloMICS software to store all scientific data and publish them online. In 2008, it was decided to migrate the fungal collection to the same system and we have unified both databases into a single new one. This wasa major task but only scientific data were stored in BioloMICS. In 2011, it was decided that the rest of the procedures, data and tools associated with stock management, orders, invoices and delivery of strains should also be moved from the old “vooraad” system to BioloMICS. Due to the complexity and the large number of procedures involved, the migration from the old system to BioloMICS was a huge challenge and it took almost two years to achieve this goal. We have now a system that integrates all the aspects of the administrative and scientific management of all data within CBS. This is a huge step forward since many procedures have been simplified and the integration of data allows us to save a lot of time. Also, the new system is much more transparent than the old system that was a “black box” in some cases. The definitive migration was done in January 2014 and technicians are now getting used to the new way of working which involves less work to them since all the orders placed via the website are filled in by the clients. A large number of specific scripts have been written to make their work easier and faster. This is therefore a major step forward for the collection and the CBS as well.
7.4 Future projects and scientific relevance
As can be seen above, the staff of the bioinformatics groups was involved in many projects and we have supported many researchers in their endeavors. Some led to publications listed below while others have never been published. While it is difficult to predict what will happen in the future when talking about informatics and bioinformatics, there are some projects that we are currently preparing. Some of them are described below.
7.4.1 Next Generation Sequencing and data management pipelines
The huge potential of Next Generation Sequencing applied to metagenomics studies has given biologists an unprecedented ability to obtain data describing the complexity of the biosphere. This productivity is however unbalanced in comparison with data processing, biological interpretation and, above all, real impact in the various components of society. This situation clearly outlines the risk of a wonderful underutilized tool. At current stage, while some bioinformatics tools are available to handle and analyse relatively small datasets at a pace that is too slow outside the research arena. Many of the existing tools were created for first generation sequencing or small datasets and are not efficient at all for the “big data” revolution. There is therefore an unbalanced situation between data production, the primary bioinformatics processing and the final biological interpretation. This is largely due to the fact that processing and interpretations look for among data relations, which are normally the square number of the items to be compared. Another problem of the current situation is that in most instances metagenomics data are left in some database with various levels of interpretation and are normally not used to expand the biological knowledge beyond the limits of the specific study for which they were produced. This leads
Part 7 - Bioinformatics Programme – V. Robert 73 to an excess of metagenomic information without links to environmental and functional states. A third limit of the current approach is that most of the biological data are not centrally available on a common platform under a standard format making them ready for cross-studies comparisons that would include additional metadata as well, such as climate, geographic, time and many other characteristics that might be of interest. Many studies have demonstrated the huge potential of Next Generation Sequencing (NGS) methods in terms of new species discovery, environment or patients monitoring, ecological studies, etc. There is no doubt that NGS will become the major tools for species identification and routine diagnostic. While read lengths are still quite short for most existing systems ranging between 50 bp and 800 bp, they are likely to improve soon. This will enable easier, faster and more reliable contig assembly and subsequent matching against reference databases. Data generation is no longer a bottleneck, the storage, speed of analysis and interpretation of DNA sequence data are becoming huge challenges. Also, the integration or the use of data originating from diverse datasets and a variety of data providers are serious issues that need to be addressed. Poor sequence record annotations and species name assignments are known problems that should be addressed soon and would allow for the creation of reference databases used for routine diagnostics based on NGS. Samples with huge amounts of short DNA fragments need to be analysed and compared against reference databases in an efficient and fast way. One of the challenges that we need to address is data upload from client’s computers to central or distributed data storage and analysis services. Another one is the efficient parallelization of analyses using cloud or grid solutions. The reliability and up-time of storage and analyses facilities is another important problem that need to be addressed if one wants to use it for routine diagnostics. Finally, the management, reporting and visualization of the analyses results are among the last issues, but not the least challenging. To allow large implementation and adoption of NGS for routine industrial use or health diagnostics and monitoring, better and faster alignment algorithms, software and infrastructures are needed.
We propose to create metagenomics pipelines that will be able to:
1. Accept metagenome samples containing up to 106 sequences reads 2. Simultaneous analysis of large amounts of metagenome samples analysis requests (up to 1000 samples/day) 3. Automated integration of new discoveries in reference databases (self-learning or automated database improvement) 4. Linking metagenomes samples to other reference databases (for example bibliographical, proteomics, climate or ecological databases) 5. Advanced reporting system including statistics and graphical reports. Metagenomics samples analysis results will include: a. Number of unique sequence reads b. Number of known species, genera, etc. c. Number of unknown sequences d. Evolution of samples in monitoring or epidemiological scenarios e. Geographical comparisons and contextualization
7.4.3 StrainsBook, the universal organism’s data system
Biodiversity researchers are collecting large amounts of strains or specimens around the world and characterize them with a lot of metadata. Except for the researchers working in large museums or culture collections, most of them keep their data in files on their local drives and computers and have no ways to share their data and make them publically available. Museums and official culture collections around the world are holding and accepting only a small fraction of the actual diversity that is published
74 in scientific journals (around 0.01 %, source Erko Stackebrand in MIRRI project). Many strains/ specimens data are lost or not maintained after publication. This is a huge problem in science that needs to be addressed. The StrainsBook system will be available for free to researchers working in many fields such as:
1. Biodiversity studies (on any organism from microbes to higher plants or animals) 2. Agricultural and plant protections studies 3. Medical studies and epidemiology 4. Any studies working with specimens or living organisms
Since the StrainsBook system will be able to cope with all types of data and organisms, it will be used by a very large panel of researchers and people. Therefore, it will be used in many scenarios such as biodiversity management and conservation programs anywhere in the world, quarantine and invasive organisms fight, epidemiology or medical diagnostic. It will be beneficial to researchers in third world countries since they don’t have the needed financial and technical resources to tackle this problem.
Our proposal is to create a complete pipeline for the researchers working with strains or specimens and that need a system to manage their collections from the field to the laboratory and to provide tools to analyse and share their findings with the rest of the scientific community. The new pipeline will include:
1. A mobile application (IOS, Android and Windows Mobile) to collected based samples information (pictures, GIS coordinates, time and miscellaneous metadata) directly on the field. 2. A desktop application that will synchronize with the mobile app and where a complete and extensible set of data will be recordable such as administration, bibliographical, geographical, ecological, chemical, physiological, medical, molecular, links to other specialized repositories (e.g. GenBank, GBIF, etc.) and many more. This application will not only allow to store data but also to perform advanced queries, polyphasic identifications and classifications based on any combination of characteristics. 3. Data will be stored in a local light weight database. 4. Data will be shareable using the central StrainsBook facility that will be accessible, searchable by anyone or by a selected list of co-workers. 5. The StrainsBook website will include user-friendly basic and advanced searching facilities, distribution maps, online polyphasic identifications and web services to download data. 6. A publication tool will also be available to produce e-books or to export data in basic data exchange formats.
The system will be completely free (free mobile and desktop applications) and the StrainsBook system will be hosted and maintained by CBS-KNAW. This idea and project has been submitted to the JRS Biodiversity Foundation as a grant proposal.
7.5 Output
49 scientific papers, 92 websites, 15 software versions and manuals were published in 6 years.147 presentations and courses have been given during the same period. Details are provided below.
Key publications
Robert V, Casadevall A (2009). Vertebrate endothermy restricts most fungi as potential pathogens. Journal of Infectious Diseases 200: 1623–1626. O’Donnell K, Sutton DA, Rinaldi MG, Sarver BAJ, Balajee SA, et al. (2010). Internet-Accessible DNA
Part 7 - Bioinformatics Programme – V. Robert 75 Sequence Database for Identifying Fusaria from Human and Animal Infections. Journal of Clinical Microbiology 48: 3708–3718. Robert V, Szöke S, Eberhardt U, Cardinali G, Meyer W, et al. (2011). The quest for a general and reliable fungal DNA barcode. The Open Applied Informatics Journal 5: 55–71. Desnos-Ollivier M, Robert V, Raoux-Barbot D, Groenewald M, Dromer F (2012). Antifungal susceptibility profiles of 1698 yeast type strains uncovering potential emerging human pathogens. PLoS ONE 7: e32278. doi:10.1371/journal.pone.0032278 Schoch CL, Seifert KA, Huhndorf S, Robert V, Spouge JL, et al. (2012). The internal transcribed spacer as a universal DNA barcode marker for Fungi. PNAS 109: 6241–6246 O’Donnell K, Humber RA, Geiser DM, Kang S, Park B, Robert V, et al. (2012). Phylogenetic diversity of insecticolous fusaria inferred from multilocus DNA sequence data and their molecular identification via FUSARIUM-ID and Fusarium MLST. Mycologia 104: 427–445. Osmundson TW, Robert VA, Schoch CL, Baker LJ, Smith A, et al. (2013). Filling Gaps in Biodiversity Knowledge for Macrofungi: Contributions and Assessment of an Herbarium Collection DNA Barcode Sequencing Project. PLoS ONE 8(4): e62419. doi:10.1371/journal.pone.0062419
Software (partial list)
Robert V, Szoke S, Jabas B, Chouchen O, Vu D, Stegehuis G (2009). BioloMICS Software. Version 8. Robert V, Vu D (2009). Sequencing LIMS systems. Version 1.0 Beta. Robert V, Szoke S, Jabas J, Vu D, Chouchen O, Amor A (2011). BioloMICS software. Version 8.9. Robert V, Szoke S, Jabas J, Vu D, Chouchen O, Amor A (2011). BioloMICS software help document. 352 pp. Robert V, Szoke S, Jabas B, Vu D, van de Wiele N, Borges dos Santos F, Amor A, Dridi A, Chouchen O (2012). BioloMICS Windows Software. Version 9. Robert V, Amor A, Jabas B (2012). Mycobank Software Version 2.0. Van de Wiele N, Borges dos Santos F, Szoke S, Jabas B, Robert V (2012). BioloMICS manual. 325 pages. Robert V, Amor A, Jabas B (2012). MycoBank webservices. Version 1. Robert V, Szoke S, Jabas B, Vu D, van de Wiele N, et al. (2013). BioloMICS Windows Software. Version 10. Robert V, Amor A, Jabas B, Vu D, van de Wiele N (2013). EUBOLD website and associated database. Version 1.
Table 7.4. Scientific publications. 2008 2009 2010 2011 2012 2013 Total Academic publications Refereed journals 4 2 4 14 9 14 47 Book Chapters & – – – – 1 1 2 Proceedings Total 4 2 4 14 10 15 49 Monographs & books – – 1 – – – 1 Ph.D. theses – – – – – – – Popular publications – 3 – 4 4 4 15 and products (software)
7.6 Earning capacity
Our group only has one permanent staff member and the rest of the revenues depend completely on soft money obtained from grants. The most important ones have already been mentioned above. Another major source of revenues comes from the sales of the BioloMICS software and associated services. All this is
76 done by BioAware, a spinoff company of the CBS-KNAW, created in 2000. Thanks to this structure, the group has been relatively stable during the last few years. Recent improvements of the software have made it more and more popular and we hope to further increase our revenue in the coming years.
7.7 Academic reputation
Memberships in Scientific Boards
DNA Barcodes, Versita (2011–, Editorial advisory board member) iBOL mirrors (2011–, co-chair) iBOL scientific advisory board (2009-2013, scientific advisor) World Federation of Culture Collections CODATA (2012–, advisor)
Editorships, reviewer in academic journals and book series
Antonie van Leeuwenhoek BMC bioinformatics DNA Barcodes FEMS Yeast Research PLOS One
Other appointments
Lecturer, annual course on medical mycology of Institut Pasteur, Paris (2004–) CEO BioAware, Hannut, Belgium CEO Biorganize, Sousse, Tunisia
Selection of invited lectures (among 147 lectures given between 2008 and 2013)
Robert V. EUBOLD System & BioloMICS software. 4th International Barcoding of Life meeting, Adelaide, Australia, November 28th 2011. Robert V. Fungal barcoding database, innovative concepts. MSA meeting, University of Yale, USA, July 18th 2012. Robert V. MycoBank and Nomenclatural databases as working tools for taxonomists: opportunities and challenges. Nomenclatural Meeting, Beijing, China, August 10th 2012. Robert V. Should we be working with specimens, strains, genomes, single sequences or continue with species? Chair and invited speaker, Workshop on DNA Barcoding at EMBL-EBI, Cambridge, UK, September 24th 2012. Robert V. BioloMICS software, the ultimate tool for the management of culture collections. US Culture Collection Network, National Center for Marine Algae and Microbiota, East Boothbay Harbor, Maine, USA, September 26th 2013.
7.8 Societal relevance: quality, impact and valorisation
The fact that our group has been involved in many research projects (some are listed above) and plays a central role in the management, analysis and publication of data in our institute and outside as well, already shows that the role of our team is crucial to the success of these projects. Users paying or freely accessing our software and websites is another proof of the success of our work. We have approximately 200 unique daily users working with our BioloMICS desktop software while for
Part 7 - Bioinformatics Programme – V. Robert 77 MycoBank and the CBS collection websites only we have a total of 2200 unique daily visitors. There is no doubt that the increased reputation of the CBS-KNAW is due to its researchers but also to its databases, websites and associated software. The visibility of our institution is now heavily dependent of electronic publications, websites, databases and software.
7.9 Viability
As already mentioned above, our group is relatively small and has not received enough financial support from the institute but on the other hand we have been able to raise money via grants and sales of the software and associated services. That we like it or not future research and technologies will generate more and more data that will have to be managed by software, stored in efficient databases and published in a diversity of ways. Our group will play the central role in this but we will certainly need to increase resources allocated to bioinformatics. Our team is currently under high pressure due the large number of projects, software, websites, databases and IT infrastructure to maintain or develop. Too much pressure is of course counterproductive and a better balance between available resources (staff and money) and workload, need to be established to insure high productivity levels. Bioinformaticians, information technology staffs and pure informaticians are rare and highly qualified resources. Although our staff is quite stable since the creation of the group and it is crucial to keep this stability since the experience and the programming skills required to work with large software such as BioloMICS cannot be acquired in a short period of time. It takes at least two years for an already experienced programmer to know all the aspects of the system. In addition, our informaticians need to understand the “language” of biologists, which is not always an easy task. Stability of the group is therefore essential to its viability.
7.10 Strategy
Our strategy for the future is to capitalize on our strengths, the BioloMICS software and on our current experienced staff to develop tools that are highly needed in our field. Some of them have already been developed in section 9.4. After many years of software developments focusing on the management, storage and publication of biological data, we are also going to further develop new algorithms and statistical tools to analyse and visualize large datasets using cloud and grid-based technologies. CBS-KNAW is “seated” on a data goldmine that has not been used to its full potential until now. Our group will certainly use it to produce high impact or interesting papers such as the one published in Journal of Infectious Diseases by Robert and Casadevall in 2009 entitled: Vertebrate endothermy restricts most fungi as potential pathogens. This paper received a lot of general media attention in 2009 even if it was based on simple fungal temperature data collected by CBS curators during a century of work. Given the huge amount and complexity of data to be analysed in the future, our groups needs to increase its collaboration within the Netherlands and Internationally. We are already collaborating with for example, the NCBI, the EMBL-EBI, Naturalis, the Institut Pasteur, the CDC in Atlanta, the University of Perugia, the University of Sydney and the Chinese Academy of Sciences, but we need to extend these collaborations with future common projects.
78 8. Collection
Programme leader Dr G.J.M. Verkley
8.1a Objectives(s) and policy
The CBS Collection of Fungi holds close to 80.000 strains, and is currently the largest public service culture collection in the world. The main objective of the Collection programme is to preserve living strains of all culturable members of the Kingdom Fungi and Oomycota. The Collection grows rapidly, with some 3000 strains being annually deposited. The CBS Bacterial Collection (NCCB) consists of another 10.000 strains, including a unique Plasmid and Phage Collection. CBS offers an unchallenged and globally unique reference collection for mycological research. Each year the collection distributes between 5000–6300 strains to scientists in over 60 different countries (for detailed figures see Table 1.8). The high quality of CBS strains is ensured by the practice of having identities and typical features authenticated by specialists within CBS and elsewhere. Researchers within CBS are provided with another 10000–15000 CBS strains annually for their projects, which results in an improved identification and classification of these CBS strains. Since 2009, all new deposits in CBS are also authenticated by DNA sequencing of the ITS and LSU ribosomal DNA. In fact, for all holdings ITS and LSU sequences are being generated in the barcoding project “Making the Tree-of-Life work”, funded by the Dutch government (FES, 2009–2015) and the KNAW “Stimuleringsfonds”. By constantly adding DNA- and other scientific data to the strain records, the Collection is able to keep information up to date and increase the value of the holdings to the scientific community. Users can access this information through the CBS database portal to browse the collections and associated data, and their up-to-date names in MycoBank. By collaborative preservation research in the EMbaRC project (2009–2012), preservation methods are further optimized and new ones tested and implemented. Due to all these activities CBS is well on track for becoming a “Biological Resource Centre” (BRC), as it already fulfils most key requirements set out in the ambitious OECD Best Practise Guidelines for BRCs.
8.1b Forward look
Apart from DNA barcoding, the collection programme plans to implement other (more) sensitive identification tools to authenticate new accessions and newly created stocks. MALDI-TOF MS is especially useful to authenticate yeasts (and most bacteria), and when proven reliable enough in future, it can also be used for certain filamentous fungi. For genome studies, and also for the ambitious novel product discovery line CBS will develop within the industrial mycology programme, CBS has the best possible starting position with a diverse collection at hand to sample from. Identification and authentication of the strains and stocks is key to develop economic and target-driven screening strategies. International collaboration between culture collections remains essential. Microbial genetic resources are an important basis for successful bio-economic development, and collections keeping these resources need to better meet the needs of the industrial and institutional players that drive the bio-economy. European microbial culture collections, including CBS, have therefore initiated the Microbial Resources Research Infrastructure (MIRRI), which will actively involve such players. Collections can also develop coordinated approaches to answer to new legislation on access and benefit sharing that will enter into force together with the Nagoya Protocol. Ongoing concern about bioterrorism has strongly increased the interest of politicians in culture collections. Attention is being paid to the organisms that are maintained and curated, and to the way the distribution of these organisms is managed, with special focus on the security measurements instituted to prevent undesired use. Biosecurity risk management will be designed and implemented in CBS. It will
Part 8 - Collection – G. Verkley 79 include security measures and procedures for the institute (CBS) itself and its personal, students and guests with the aim to minimize risk of loss, theft, misuse or intentional release from the CBS facilities of pathogens and toxin-producing organisms. The yeast curator will be appointed in the near future as biosecurity risk manager and have the day to day responsibility for the biosecurity risk management. Biosecurity risk management practices will involve the integration of biorisk management throughout CBS activities and seek its continuous improvement. This will be done by assigning adequate resources and responsibilities in order to guarantee compliance with legal requirements and assure proper communication to staff and relevant third parties. Issues that will be addressed are assigning biosecurity risk groups levels to biological agents in CBS, physical security of storage and working areas, security management of personnel and visitors, an incident response plan, staff training and developing a biosecurity-conscious culture, security of data and information, material control and accountability, supply of material and transport security within and outside CBS.
8.2 Composition
Table 8.1. Staff at programme level, 2008–2013. 2008 2009 2010 2011 2012 2013 Funding* Research staff Dr G.J.M. Verkley 1.0 1.0 1.0 1.0 1.0 1.0 1 Dr M. Groenewald 0.8 0.8 0.8 0.8 0.8 0.8 1 Dr A.W.A.M. de Cock 0.6 0.6 0.6 0.6 0.6 0.6 1 Dr J.A. Stalpers 1.0 1.0 1.0 1.0 – – 1 Ir G.J. Stegehuis 0.8 0.8 0.8 0.8 0.8/– – 1 Postdoc Dr U. Eberhardt 1.0 1.0 1.0 1.0/– – – 2 Dr B. Stielow – – – –/1.0 1.0 1.0 2, 3 Technicians W. Epping 0.6 0.6 0.6 0.6 0.6 0.6 1 (Collections) M. Figge 0.8 0.8 0.8 0.8 0.8 0.8 1 D. Vos-Kleyn 0.5 0.5 0.5 0.79 0.5 0.5 1 R. Verwoerd-Kuyt 0.8 0.8 0.8 0.8 0.8 0.8 1 T. Merkx 0.737 0.737 0.737 0.737 0.737 0.737 1 E. Mul 0.6 0.6 0.6 0.6 0.6 0.6 1 A. de Nooijer 0.5 0.5 0.5 0.5 0.5 0.5 1 M. Setropawiro 0.9 0.9 0.9 0.9 0.9 0.9 1 T. Snippe 0.8 0.8 0.8 0.8 0.8 0.8 1 H. Stasia – – 1.0 1.0 1.0 1.0 1 IJ. Vlug 1.0 1.0 1.0 1.0 1.0 1.0 1 F. Woode – – 1.0 1.0 1.0 1.0 5 Technicians K. Dukik 1.0 1.0 1.0 1.0 1.0/– – 2, 4 (DNA M. Wouters – 1.0 1.0 1.0 1.0/– – 2 barcoding) S. Roos – – – 1.0 1.0/– – 2 R. Renfurm 1.0 1.0 1.0 1.0 1.0 1.0/– 2, 5 Dr M. de Vries – – – 1.0 1.0 1.0 5 D. Smits – – – – 1.0 1.0 5 G. Omer – – – – 0.8 0.8 5
80 Table 8.1. Staff at programme level, 2008–2013 (Continued). 2008 2009 2010 2011 2012 2013 Funding* Staff for F. Claus 1.0 1.0 1.0 1.0 1.0 1.0 1 service/sales M. van der Merwe – – – – –/0.632 0.632/– 1 T. van den Berg 0.8 0.8 0.8 0.8 0.8 – 1 A. Herder – – 0.6/– 0.6 0.6 0.6/– 1
*1 = Royal Netherlands Academy of Arts and Sciences (KNAW); 2 = KNAW Stimulerings fund for DNA Barcoding & DNA Bank; 3 = Odo van Vloten Foundation, CBS; 4 = EMbaRC, FP7; 5 = Dutch Ministry of Science (FES Barcoding).
In April 2011 Joost Stalpers retired from his position as head curator of the collection, and Gerard Verkley was appointed as his successor. After a temporary appointment, Marizeth Groenewald obtained a permanent position as the curator of the yeast collection in January 2013. Database manager Gerrit Stegehuis left CBS in 2011 and this position was taken over by Duong Vu, who became a member of the bioinformatics programme, on which the collection now depends for technical support and development of new modules in the BioloMICS databases. In the technical staff, Hanslin Stasia was appointed on a permanent position in 2013.
8.3 Scientific environment and international collaborations
The CBS Collection regularly exchanges strains with other culture collections, such as MUCL, NRRL, and ATCC. We also accept complete or parts of research collections built up by internationally reputed specialist who no longer can maintain these collections; for example, the Alternaria collection of the late Dr Emory Simmons has been incorporated in 2013 and the ML yeast collection of the retired Dr Jack Fell was taken up in the CBS collection in 2010. In collaboration with the evolutionary phytopathology programme, the collection staff organizes the annual CBS Fungal Biodiversity Course. This course runs in February for two weeks. The collection participated in five EU projects during the evaluation period. The EDIT project (European Distributed Institute of Taxonomy, 2006–2011, http://www.e-taxonomy.eu/), a FP6 Network of Excellence, for which NL-TAF was coordinating workpackage 3 (WP3) on integration of the infrastructure basis for leading taxonomic facilities in Europe. The main task of CBS in WP3 was to coordinate DNA Barcoding, for which it established the European Consortium for the Barcode of Life (www.ecbol.org). SYNTHESYS (I) (www.synthesys.info, I, 2004–2009) is another FP6 programme, comprising 20 European natural history museums and botanic gardens, aiming to create an integrated European infrastructure for researchers in the natural sciences. SYNTHESYS I set standards for collection management and databases, and aimed to raise scientists’ awareness of best practice by offering improved training and workshop opportunities, and guidelines for the care, storage and conservation of collections. The project also provided new policies on emerging technologies for storing collections, such as DNA samples or tissue banks. On SYNTHESYS grants, many scientists visited the Collections and other groups in CBS. To SYNTHESYS II (2009–2013) CBS was not a full partner (no visits), but it did contribute to research activities. Within a joint consortium between several research institutions in the Netherlands and United Kingdom, the first entire genomes from insect, plant and fungal museum specimens (some of them more than 100 years of age) were sequenced as a proof-of-principle concept with state-of-the-art next generation DNA sequencing methodology. CBS was also partner in the Global Biological Resources Centre Network (GBRCN) Demonstration Project (2008–2011), an OECD initiative that was funded by the German government and anticipated
Part 8 - Collection – G. Verkley 81 as an important step towards establishing a sustainable global network for BRCs. In the European Consortium of Microbial Resource Centers (EMbaRC) project, funded under FP7, CBS and eleven other microbial culture collections collaborated in coordinating activities and research from 2009–2012. J. Stalpers initially led the Task JRA 2.2, and later G. Verkley took over. Joint research activities in EMbaRC aimed the harmonization of methods and procedures for culture preservation and validation, improved coordination of quality control and delivery to users. Other contributions of the CBS collection staff focused on the discovery of new gene sections for molecular taxonomy and identification by in silico comparison of entire fungal genomes using in-house developed software. After an extensive testing period using in silico predicted targets by CBS (U. Eberhardt, B. Stielow) and various other labs, the results will soon be published. The research activities included work on challenging groups like the coniothyrium-like fungi (G. Verkley) and candida yeasts (M. Groenewald), aiming to resolve their taxonomy and phylogeny. EMbaRC ended in August 2012. CBS also participates in the Microbial Resource Research Infrastructure (MIRRI) project, which aims to build one pan-European infrastructure for microbial collections that will more effectively facilitate access throughout Europe to high-quality microbial cultures, their derivatives and associated data and services, for research, development and applications. After its acceptance on the European Strategy Forum on Research Infrastructures (ESFRI) road-map, MIRRI obtained funding from the European Commission and on Nov 1st, 2012 it entered a three-year preparatory phase. In this phase partners will focus on governance and structure, and technical, legal, and financial issues to build the network. This will establish the links across the distributed research infrastructure (RI) and between the RI microbiological resource centre (MRC) community, its users, policy makers and potential funders. G. Verkley leads Workpackage 9, which focuses on the preparation of a legal operational framework for the RI and addresses Access and Benefit Sharing (ABS), intellectual property and biosecurity issues. Throughout the evaluation period CBS staff has been actively serving on boards of two important organisations for culture collections, viz., the World Federation of Culture Collections and the European Culture Collections Organisation (ECCO). At the ECCO Annual Meetings and at the WFCC ICCC12 and 13 (held every three-years), in Florianapolis, Brasil and Beijing, China, respectively, many important issues were discussed and several contributions were made by CBS in the programme, concerning taxonomy of fungi and yeasts, law and regulations (especially Nagoya Protocol on ABS, CBD) and biosecurity. In June 2011, the CBS Collection hosted the 30th ECCO Annual Meeting. Over seventy curators and other scientists from 22 countries gathered in Utrecht for a series of sessions dedicated to research and important issues for culture collections, including ABS and collaboration with industrial partners. CBS Collection staff successfully collaborated within EMbaRC and GBRCN Demonstration Project to design a Code of Conduct for Biosecurity for European Microbial BRCs. During a workshop held 1–2 September, 2011, in Utrecht, the concept was discussed by 15 specialists representing most partners of EMbarC. The final text and procedural document were published in 2013 (Rohde, Smith & Stalpers, IJSEM 63, 2013, 2374–2382). This code considers the regulatory and working environment in public service culture collections, defines the responsibilities and provides practical advice in the implementation of best practice. Furthermore, it was introduced to the 7th Review Conference to the BTWC, United Nations, Geneva, Dec. 2011, where it was well-received.
8.4 CBS Holdings
During the evaluation period the collection of fungi and yeasts has been growing with up to 3500 strains per year [2143 (2008), 2295 (2009), 3328 (2010), 3487 (2011), 2586 (2012), 3080 (2013)]. The numbers of strains in the public CBS and NCCB-collection per main category are presented in Table 8.2. Practically all accepted strains can be cryopreserved. Most sporulating fungal strains and bacteria can also be freeze- dried successfully. A relatively small number of strains are recalcitrant to cryopreservation and cannot be freeze-dried. They are still kept on agar, and maintaining this agar collection is extremely laborious
82 and (therefore) expensive. In the past decade CBS has been able to considerably reduce the number of strains in the agar collection, due to improved preservation methods for certain groups of fungi for which cryopreservation was considered to be unreliable over longer periods. Currently about 2500 strains are (also) kept on agar.
The CBS collection currently contains 9432 ex-type strains of yeasts and filamentous fungi. An ex-type strain fixes the undisputed application of a fungal name, while other strains that have been authenticated by a specialist can also serve as reference material for the identification of a fungal taxon. These strains are becoming increasingly important since the numbers of trained taxonomists around the world are constantly decreasing. Moreover, type and authentic strains are the preferred material to generate DNA barcodes and whole genome sequences from.
Table 8.2. Numbers of strains in the public collection per main group (CBS and NCCB collections, including unreleased strains). Organism Number of accessions (per 22-01-2014) Filamentous fungi 65725 Yeasts 9405 Oomycota 1709 Bacteria 8618 Actinomycetes 1283 Plasmids 562 Total 87302
DNA Bank The primary purpose of the DNA-Bank is to store good quality genomic DNA extracts of CBS strains for later use. The focus is at this moment on DNA of type strains and highly pathogenic strains. Central storage is more economic and will make the DNA more easily accessible, avoiding unnecessary repetition of DNA extraction from the same cultures. The DNA-Bank is safe-guarding genomic DNA for future use, even after the strain might be lost accidentally. It has to be therefore designed also as a facility for long-term storage. In the future the DNA-Bank may also incorporate extracts of fungi that cannot be cultivated, and DNA that has been isolated from herbarium specimens. However, there is no structural funding available for this to date. CBS provides DNA samples to customers on request. Researchers may prefer to receive DNA of highly pathogenic microorganisms instead of living cultures, for which the labs may not be equipped to ensure safe handling. In addition, costs of transport by courier following international quarantine and biosafety regulations, can be largely avoided if DNA is shipped instead of live material. Initially CBS obtained funds from KNAW to sequence all (ex-)type strains, and built up a collection of 2500 DNA-extracts that are stored for long-term in the dynamic gas-phase of a liquid nitrogen container, at a temperature constantly below -170 °C. This is relatively expensive compared to other DNA storage methods, but the advantage for CBS is that it is compatible with the system already in place for cryopreservation of strains. In 2009, when funding was obtained from FES and KNAW to barcode all holdings, a high-throughput barcoding pipeline was set up and extraction methods optimized for it. These extracts were mainly stored in mechanical freezers at -80 °C. At present the collection has generated at least one extracts for 50 000 strains (59 000 extractions including repeats). Each DNA-Bank accession obtains a unique identifier (CBS D-XXXXX), independent of the unique identifiers connected to the source. For long-term storage, a sample of DNA in buffer is maintained for each accession.
Part 8 - Collection – G. Verkley 83 8.5 Barcoding the CBS Collection of Fungi and Yeasts (2010–2015)
The CBS barcoding project “Making the Tree-of-Life work” is funded through a grant of the Dutch Ministry of Science and Education, supplemented with funds from the KNAW Stimuleringfonds to cover costs of barcoding the entire CBS collection. Choice of barcode regions – By time the project started no region of the genome had been formally accepted by the Consortium for the Barcode of Life (CBOL) as DNA-barcode for the kingdom Fungi. The DNA sequence markers most generally used by mycologists to identify fungal species are the internal transcribed spacer region (ITS) and the 5’ primed end of the large ribosomal subunit (LSU, D1/D2 region). One important advantage of these two regions is that they can normally be amplified and Sanger sequenced with standard non-degenerate primers from a wide variety of fungal taxa. For this reason, it was decided to choose both regions as “barcode” at the start of this project. An important step forward was made when the mycological community involved in barcoding gathered for a meeting of the Fungal Working Group (FWG) of CBOL on the 16–17th of April 2011, in Amsterdam. The participants provided sequences of three regions of the ribosomal DNA (ITS, LSU and SSU) and one protein coding gene, the RNA polymerase II (RPB 1/RPB 2) for selected fungal groups and compared the results. As expected, the FWG concluded that the ITS was the best suitable region as barcode, because of the relatively high success rate in PCR and sequencing and an average of 71 % identification success for all fungi (more info on http://www.fungalbarcoding.org/). A formal proposal to approve the ITS as the first official barcode region was subsequently accepted at the Fourth International Barcode of Life Conference, 28 Nov – 3 Dec 2011, Adelaide, Australia. Although no second region was proposed by the FWG to serve as secondary barcode, sequencing of the LSU will be continued in CBS because this region has additional value for the assignment of species to genera when reference data of related organisms are scarce or absent. DNA-extraction – At the beginning of the project DNA was usually extracted from actively growing cultures, after a variable period of incubation on a suitable medium for growth. In order to reduce the time needed for DNA extraction, a method was developed that avoids this cultivation step, by directly extracting the contents from straws with the cryopreserved fungal material (Liquid nitrogen, LN) and from glass ampoules with freeze-dried fungal spores. Apart from saving time and expensive media, this new procedure also minimizes the risk of spontaneous contaminations. Furthermore, the extraction protocols were repeatedly modified and improved, while initial mechanical disruption of the fungal cells remains a critical step for DNA extracts. DNA extraction methods used thus far for barcode production include one based on the BIO101 kit (FastDNA; Q.BIOgene), the Mobio Microbial DNA isolation kit and a combination of a commercial lysis buffer (Prepman, Applied Biosystems) and a DNA purification kit for genomic DNA (Jetquick, Genomed) or its high throughput version employing glass fiber plates (Pall). The DNA purification protocol based on 96 well fiber plates has been recently further optimized by reducing time-intensive working steps (fewer hands), by application of a small semi-automated multichannel robot (Integra Vioflo 96). Pipetting of entire 96 well plates saves labour time, improves accuracy of dispensed liquids and almost entirely avoids handling errors at one of the key steps in the pipeline. PCR and sequencing – Much time has been invested in optimizing the protocols for PCR and sequencing. One of the crucial bottlenecks in terms of time investment (labour costs), for a high throughput sequencing pipeline are PCR redoes based on negative results. A highly efficient and cost effective PCR protocol has been implemented into the routine work flow, giving now an average PCR success rate of 90–95 % (previously 65–70%). Also, investments made into new state-of-the-art PCR cyclers (SensoQuest) and more effective enzymes have paid off. In 2012, the pipeline was equipped with a state-of-the-art Hamilton MicroLab Star robot that has reduced considerably the time spent on manual work in the molecular lab. As a result, the number of strains extracted and sequenced in the workflow has increased dramatically. Currently, the pipeline uses an in-house Applied Biosystems/ Hitachi 3730xl sequencer (Sanger technology) that will be available for use until the end of this project.
84 Table 8.3 provides information regarding the current progress status of this project. Researchers in the evolutionary phytopathology and applied and industrial mycology programme also provide barcode sequences of CBS strains.
Table 8.3. Progress status of the DNA Barcing project per Jan 2014. DNA extracts 61.617 Total number of ITS sequences (contigs) 49.950 Total number of LSU sequences (contigs) 46.439 Total number of other gene loci (contigs) 28.990 Contigs 125.379 (250 K single files) Total number of ITS sequences from types >9000 Total number of LSU sequences from types >9000
Sequence validation. After sequences are edited and contigued by technicians, they are validated by curators, to assess the authenticity of the sequence. The validation process is a bottle-neck as the curators team is small and validation can only be done by manual BLAST and comparing results. Failed sequences are redone once. As validation proceeds a more high-quality reference sequences become available in the CBS sequence database for comparison, making validation more accurate and a bit easier. Currently (22- 1-2013) ITS sequences of 18 972 strains have been validated, and LSU of 19 471 strains (total 38 443 sequences validated). The CBS database already contains numerous validated sequences of taxa or even entire groups of fungi that are barely or not at all present in GenBank at this time. A subset of the validated sequences is already available for blasting by users of the CBS website tool, although they cannot see or download these sequences at this time. As more sequences will be validated and/or published in the near future more of the CBS sequence data will be released in various databases.
8.6 Research by collection staff
Curators and post-docs in the collection have to dedicate 90 % of their time to quality management, barcoding, databases and services. There is thus limited time to conduct research. Mostly this is done in close collaboration with scientists in the other CBS programmes. Gerard Verkley recently completed several years of work on septoria-like coelomycetes in collaboration with the evolutionary phytopathology programme, and continued working on taxonomy and phylogeny of coniothyrium-like fungi, the asexual morphs of Pleosporales and other Dothideomycetes (Ascomycota) which are of considerable importance to society, destructive as plant pathogens, of clininal relevance, or beneficial as effective biological control agents or bioremediators. Soils are particularly rich in these fungi, and the bulk of isolates deposited in CBS over decades are still unidentified. Only a small number of species have been formally described from soil, and a recently completed study of strains clustering in Montagnulaceae yielded two novel genera and nine new species. He will continue to work on coniothyrium-like fungi grouping in other clades. The sequences obtained in the barcoding project have proven very useful to select strains for morphotaxonomic analyses and sequencing of additional genes to resolve phylogeny. Marizeth Groenewald’s main research focus is on the taxonomy and phylogeny of ascomycete yeasts. Several papers have been published together with collaborators describing novel species and clarify the taxonomic status of existing species. One of the most interesting studies was that on Geotrichum species where the study showed that within one strain the ITS region, the barcode of fungi, can have different copies with different sequences, making it impossible to use this region for good species identification. This study also helped to explain some contradictory results from the barcoding project of strains that
Part 8 - Collection – G. Verkley 85 belong to Geotrichum and close relatives and even species in other genera. Good collaboration was built up the past years between the Yeast Collection and the Yeast and Basidiomycetous research group. One of the main research focuses involves the use of the MALDI-TOF MS system to create and maintain an elaborate yeast MSP database that can successfully be used for rapid and correct identification of important yeasts in the medical field and food industry. The results obtained from the MALDI-TOF MS system nicely correlate with the barcoding data and it can even distinguish between species where D1/D2 and ITS fail. Several papers have been published on this and this work will continue in order to set up a quality control system that can be used for the daily validation of the strains in the yeast collection. Benjamin Stielow joined the CBS barcoding project in 2011, as young post-doctoral researcher coming from Germany to manage and further develop the barcoding project. He is interested in applied molecular taxonomy of various fungi, using state-of-the-art methodology and genomic techniques. He intensively collaborates with various staff from CBS, universities and scientific institutions around the world, with the aim to enable selected researchers to use the massive amounts of data that are generated in the barcoding project to advance our understanding of genetic diversity within the kingdom fungi and publish the results. Beside the barcoding pipeline, he has setup a next-generation sequencing (ion torrent) facility at the CBS, together with Michel de Vries, to further strengthen the possibilities and to develop novel state-of-the- art research tools for CBS researchers. Benjamin Stielow will resign as group leader from the barcoding project in July 2014, and will continue within an industry funded project at the CBS, combining advanced genomics with high resolution tandem mass-spectrometry in a systems biology framework. Arthur de Cock. When Gerrit Stegehuis left CBS in 2011, Arthur de Cock’s appointment (0.6 fte) came 100% dedicated to curation of MycoBank. In spite of this, he has continued to publish on Oomycetes, time permitting.
Key publications
Groenewald M, Boekhout T, Neuvéglise C, Gaillardin C, van Dijck PWM, Wyss M (2013). Yarrowia lipolytica – Safety assessment of an oleaginous yeast with a great industrial potential. Critical Reviews in Microbiology doi:10.3109/1040841X.2013.770386. Groenewald M, Coutinho T, Smith MTh, van der Walt JP (2012). Re-classification and mating type behavior of Geotrichum bryndzae, Geotrichum phurueaensis, Geotrichum silvicola and Geotrichum vulgare. International Journal of Systematic and Evolutionary Microbiology 62: 3072–3080. Staats M, Erkens RHJ, Vossenberg van de B, Wieringa JJ, Kraaijeveld K, Stielow B, Geml J, Richardson JE, Bakker FT (2013). Genomic treasure troves: complete genome sequencing of herbarium and insect museum specimens. PLOS One 8(7): e69189. Verkley GJM, Quaedvlieg W, Shin H-D, Crous PW (2013). A new approach to species delimitation in Septoria. Studies in Mycology 75: 213–305. Verkley GJM, Dukik K, Renfurm R, Göker M, Stielow JB (2013). Novel genera and species of Coniothyrium-like fungi in the Montagnulaceae (Ascomycota). Persoonia 32: 25–51. Wagner L, Stielow B, Hoffmann K, Petkovits T, Papp T, Vagvoelgyi C, de Hoog GS, Verkley G, Voigt K (2013). A comprehensive molecular phylogeny of the Mortierellales (Mortierellomycotina) based on nuclear ribosomal DNA. Persoonia 30: 77–93.
8.7 Quality Management and collection safety
CBS has built up a good reputation as supplier of high quality cultures and customer-friendly services. For the collection holdings and these services, CBS successfully implemented a quality management system (QMS) according to ISO 9001:2000 in 2007 (now 9001:2008 due to an upgrade) and has been working under this system continuously ever since. On November 1, 2013, the collection was re-certified for another three-year period. The ISO certificate covers the following activities:
86 • Accession, preservation and maintenance of micro-organisms • Sales of cultures and derivatives (DNA) • Patent deposits, Safe deposits and Maintenance deposits • Information services A quality manual (“Kwaliteitshandboek”) describing these processes and the supporting activities is available to all CBS employees via intranet (in Dutch). This manual provides flow-charts and step-by- step descriptions of main procedures and protocols. In the introduction the quality policy statements and objectives are explained, and the assignments of responsibilities to staff for the QMS, QMS structure and planning, components of the improvement cycle, viz., internal audits, management review, customer satisfaction, process performance indicators and product conformity data, human resources aspects such as competence and awareness, training, and data on equipment.
Several safety measures are in place to secure the continuity and preservation quality of the CBS holdings:
Physical safety - restricted controlled access. The building-complex of CBS and the Hubrecht Institute can only be entered by authorized personnel using electronic e-key cards. During visiting hours, guests are registered by reception personnel. The CBS main area is only accessible for authorized e-key holders. Within the CBS main area, several areas and labs are only accessible for those whom are granted access through authorised staff. These areas include all storage facilities for the CBS Collection, viz. LN-storage room, lyophilisation lab and storage room for freeze-dried samples, agar storage and oil collection storage rooms, transfer rooms with safety cabinets, quarantine room. All entries are automatically registered. Recently, the Dutch government has subsidized measurements to physically strengthen the area where the collections are stored and state-of-the-art personalized security entry-control are being installed. The restricted access regime also assures that Dutch regulations for biosafety and recommendations for biosecurity are met. Building Control System & Monitoring of vital equipment. Vital culture storage equipment such as LN-storage containers and several mechanical freezers are connected to an electronic Building Control System (GBS). The temperature and several other parameters of the LN-storage vessels are internally logged, and can be downloaded and viewed with special software at any time. In case of malfunctioning an alarm is transferred to the technical staff at all times, and in case of power failure CBS can rely on an emergency power aggregate. Quality control and backup of the preserved materials. Before a strain is accepted for accession in the collection and first stock will be made, the identity, general condition, and purity are checked phenotypically. DNA barcode sequences are generated from material used to create first stock (in LN), and compared to reference sequences. When new stock of a strain is created the identity, growth, and purity are also checked before and after preservation. CBS preserves practically all cultures in metabolically inactive condition, so that they are preserved as much as possible in the pristine original condition in which they were received. The most important preservation methods are cryo-preservation and freeze-drying (lyophilisation), for which cutting-edge equipment is installed. Almost all strains are cryo-preserved and stored in liquid nitrogen containers. CBS uses Taylor-Wharton 24 K gas-phase containers, in which the strains are stored in a dynamic gas phase maintained by a constant flow of cold gas from above, resulting in temperatures that are constantly below -180 °C even at the top of the containers. Most yeasts and bacterial strains, and about 60 % of the non-yeast fungal strains are also preserved in a freeze-dried state. CBS uses two state-of-the-art freeze-dryers (Christ-Epsilon 2-8D and 2-10D). All crucial parameters are recorded during the freeze-drying process, and stored for later reference. A backup collection is situated at another location in the Netherlands, where one cryo-vial of each strain is stored in the same type of storage system as in Utrecht. With very few exceptions, all CBS strains are cryo-preserved in the back-up collection. Frozen samples of new accessions are brought to the backup location once per year. Of the complete stock of lyophilized strains, one ampoule per strain is also stored
Part 8 - Collection – G. Verkley 87 in the Hubrecht building (‘fire-collection’ back-up). Also, of all new filamentous fungi an actively growing agar slant under mineral oil is stored (keeps 10–15 years, no maintenance).
8.8 Scientific output
Taking into consideration the time available for collection staff to conduct research (ca. 10 %), the collection has had a substantial output for the period (2008–2013).
Table 6.2. Scientific publications. 2008 2009 2010 2011 2012 2013 Total Academic publications Refereed journals 8 9 12 17 18 23 87 Book Chapters & – 1 2 – 2 – 5 Proceedings Total 8 10 14 17 20 23 92 Monographs & books 1 – – – – 1 2 Ph.D. theses – – – 1 – – 1 Popular publications – 3 2 – – – 5 and products
8.9 Earning capacity
Together with collaborating collections, we have been successful in obtaining EU funding for EMbaRC and MIRRI. Our services, e.g. supply of living cultures, deposit of patent strains and safe deposits, and identifications (bacterial strains) have contributed significantly to the CBS annual budget (Table 1.8) for re-investment into the institute and collection.
8.10 Academic reputation
Editorships in academic journals and book series
CBS Lab Manual series (Vol 1.) Studies in Mycology (Vol. 75)
Memberships in Scientific Boards
Foundation NL-BIF (2011–) World Federation of Culture Collections (2010–2013, secretary) European Culture Collections Organisation (2009–2012, collection officer)
Selection of invited lectures
Verkley GJM (2012). European Collections partner to the Microbial Resource Research Infrastructure (MIRRI) develop common approaches to answer to the Nagoya Protocol. NBRC 10th Anniversary Symposium: Impact of the Nagoya Protocol on management of Biological Resource Centers, Dec. 6, 2012, Tokyo, Japan. Verkley GJM, Quaedvlieg W, Shin H-D, Crous PW (2013). A new approach to species delimitation in Septoria. ICCC13, BRCs in the era of microbial genomics and diversity driven innovation of
88 biotechnology, Sep. 23, 2013, Beijing, China. Verkley GJM (2013). Implementation of the Nagoya protocol in Europe – update on developments and on activities by the Microbial Resource Research Infrastructure (MIRRI). ICCC13, Sep. 25, 2013, Beijing, China.
8.11 Societal relevance: quality, impact and valorisation
The collection has a strong reputation as a general fungal biodiversity collection and reference collection for pathogenic fungi and yeasts, with over 5500 strains of clinical relevance today, multiple isolates of all quarantine regulated fungi in the EU, and numerous other plant pathogenic fungi. It is also unique for having the only complete set of yeast type-strains, and for having representatives of practically all fungal lineages that can be kept in axenic culture. Statistics on usage of the holdings are provided in Chapter 1 (Table 1.8). The fast growing number of type-strains in CBS illustrates the scientific relevance of the collection, now 9500 and almost doubled since 2008. This increase is not only due to new deposits and exchanges of types, but is for a major part due to the many taxonomists who designate CBS strains as (epi-)types of novel or existing fungal names. Furthermore, CBS strains are increasingly selected for genome sequencing projects in the framework of global initiatives, such as the F-1000 genomes project (JGI), while pi’s of genome sequencing projects more often deposit key strains in CBS to safeguard their availability in future, or such strains are actively acquired by CBS curators. In the commercial sector CBS strains are also seen as a reliable source for building libraries and databases with validated data. Bruker uses CBS yeast strains to supplement and update their MSP library of their commercial MALDI-TOF-MS system. This is also true for Life Tech that uses the sequences obtained from CBS strains and validated by CBS curators and expert staff to update the sequence library that is used together with their commercial MicroSeq™ kit.
8.12 Viability
In the Netherlands, the major biological collections holding millions of plant and animal specimens recently merged into the Naturalis Biodiversity Centre (NCB). As part of a major grant to build the NCB, 5 Mln budget was reserved for the project “Making the Tree-of-Life work” (FES, financed through the Ministry of Economic Affairs) to barcode specimens. As the only independent collection, CBS was allowed to participate, underpinning the confidence scientific partners and funders have in the quality of CBS research and the relevance the CBS collection. Over the years the CBS collection has grown steadily, but in the last 10 years faster than before. As full partner in a continuous series of projects funded by the EU Framework Programmes 6 and 7, the collection has also gained visibility in Brussels. If the latest of these, the current ESFRI project MIRRI, makes it to the construction phase in 2015, this will open new opportunities for CBS to participate in new project grants that would be financed in part by the EC and by EU member states that decide to become member of a European research infrastructure consortium (ERIC) for MIRRI and support it financially.
8.13 Strategy
The development towards becoming a BRC is proceeding successfully. By incorporating DNA sequencing into the normal procedures for assessing the identity and authenticity of new and older holdings, the collection has made an important step forward. The funds for barcoding the collection have made it possible to hire temporary appointed technicians and postdoc who are trained in molecular techniques, but the current permanent technical staff is insufficiently skilled to take over after the projects ends. Current staff will have to be trained and for newly hired technicians skills in culture preservation and
Part 8 - Collection – G. Verkley 89 molecular work will be equally important (including managing the molecular pipeline). The molecular pipeline needs to be kept state-of-the-art in the interest of all CBS programmes, and requires input from staff of all programmes. The entry into force of the Nagoya Protocol to the CBD, which is expected before end of 2014, may have a negative impact on numbers of strains deposited in and ordered from culture collections world- wide. The collection is closely following the development of the EU Regulations for Access and Benefit Sharing, which will become active shortly after, and the head curator is leading the workpackage in MIRRI that is also dealing with these issues. Whatever the outcome of the EU negotiations, it seems unavoidable that the collections in Europe will be faced with additional administrative burdens. CBS and other holders of genetic resources in Netherlands will try to convince the Dutch authorities that they need additional support (e.g., to hire more administrative staff), but the chances of getting it seem small now. Developing proposals for Best Practise that minimize the burdens for collections are a way forward, but have to be acceptable for the EC as well. CBS has always complied fully with all applicable national and international laws for biosafety and biosecurity (export control), and will soon also implement a biosecurity risk management according to OECD standards. These measures will further increase trust in the CBS as a responsible keeper and provider of hazardous microorganisms, with authorities and users of genetic resources alike. Curators’ duties of managing the collections, entering data, and DNA-based validation of strains are putting increasing pressure on the already very limited time they have to do research (now ca. 10%). The effects of the Nagoya Protocol are difficult to estimate, but it seems that with a curators team of only two persons, which is a low number (relative to size of the collection) compared to other collections in Europe, it could become increasingly difficult to develop and assure the international scientific profile of the curators, which is of importance for the reputation of the CBS collection as is the quality of the strains kept.
90
Bibliometric analysis of the Centralbureau voor Schimmelcultures Fungal Biodiversity Center (CBS)
Rodrigo Costas-Comesaña, PhD
Centre for Science and Technology Studies (CWTS) Leiden University PO Box 9555, 2300 RB Leiden
The Netherlands
January 20, 2014 Final version
Part 9 - Appendix – Bibliometric analyses 91
ii
92 Contents
Contents iii
1 Introduction 4
2 2.1BibliometricDatabase structure indicators 55
2.2 Indicators of output 5
2.3 Indicators of impact 6
2.4 Indicators of scientific collaboration 9
3 3.1DataCoverage collection of WoS publications 1111
4 Overall results 13
5 Discussion and conclusions 17
Appendix I. Calculation of field-normalized indicators 18
Part 9 - Appendix – Bibliometric analyses 93 1 Introduction
Centraabureau
voorThis studySchimmelcultures provides insight Fungal into Biodiversity the publication Center outp ut and citation impact of the (hereafter referred to as ‘CBS’) covered in the Web of Science database.
The present study analyses publications that appeared between 2006 and 2011 and the citation impact of these publications in the period 2006-2012. Publications counts are also provided until 2013. The impact, as measured by citations, is compared to worldwide reference values. The study is based on a quantitative analysis of scientific articles published in journals and serials processed for the Web of Science (WoS) versions of the Science Citation Index and associated citation indices: the Science Citation Index (SCI), the Social Sciences Citation Index (SSCI), and the Arts & Humanities Citation Index (A&HCI).
The structure of this report is as follows. Chapter 2 gives a general introduction to the methodology and an overview of the bibliometric indicators that were calculated in the study. Chapter 3 describes the data collection and gives an overview of the internal coverage by unit of analysis. Chapters 4 reports the main results of the CBS. Chapter 5 presents a brief discussion and main conclusions related with this study.
iv
94 2 Bibliometric indicators
In this chapter, we discuss the methods underlying the bibliometric analyses presented in this report. 2.1 Database structure
At CWTS, we calculate our indicators based on our in-house version of the Web of Science (WoS) database of Thomson Reuters. WoS is a bibliographic database that covers the publications of about 12,000 journals in the sciences, the social sciences, and the arts and humanities. Each journal in WoS is assigned to one or more subject categories. We note that our in-house version of the WoS database includes a number of improvements over the original WoS database. Most importantly, our database uses a more advanced citation matching algorithm and an extensive system for address unification. Our database also supports a hierarchically organized field classification system on top of the WoS subject categories. To determine the appropriateness of our indicators for assessing a particular research entity, we often look at the internal WoS coverage of the entity. The internal WoS coverage of an entity is defined as the proportion of the references in its oeuvre that points to publications covered by WoS (see Chapter 3). The lower the internal WoS coverage of an entity's output, the more careful one should be in the interpretation of our indicators. The rest of this chapter provides an in-depth discussion of the main bibliometric indicators that we use in this report. Overview of CWTS bibliometric indicators.
Indicator Dimension Definition
P Output Total number of publications of a unit covered by WoS. Int. coverage Output Internal coverage. Proxy of the share of the oeuvre of a unit that is covered by WoS. Measured by the proportion of the cited references in the WoS- covered publications of a unit that point to other WoS-covered publications. MCS Impact Average number of citations of the publications of a unit (excluding author self citations). TCS Impact Total number of citations. MNCS Impact Average normalized number of citations.
MNJS Journal impact Average normalized citation impact of the journals in which a unit has published. PP(top 10%) Impact Proportion of publications that belong to the top10% of their field. P(top 10%) Impact Total number of publications that belong to the top10% of their field. PP(uncited) Impact Proportion of uncited publications. 2.2 Indicators of output
To measure the total publication output of a unit, we use a very simple indicator. This is the number of publications indicator, denoted by P. This indicator is calculated by counting the total
Part 9 - Appendix – Bibliometric analyses 95 number of publications of a research unit. Only publications of the document types article and review are taken into account. 2.3 Indicators of impact
A number of indicators are available for measuring the average scientific impact of the publications of a unit. These indicators are all based on the idea of counting the number of times the publications of a unit have been cited. Citations can be counted using either a fixed-length citation window or a variable-length citation window. In the case of a fixed-length citation window, only citations received within a fixed time period (e.g., four years) after the appearance of a publication are counted. In the case of a variable-length citation window, all citations received by a publication up to a fixed point in time are counted, which means that older publications have a longer citation window than more recent publications. An advantage of a variable-length window over a fixed-length window is that a variable-length window usually yields higher citation counts, which may be expected to lead to more reliable impact measurements. In this study, we have used a variable length window, so all citations received by all publications up to 2012 are counted, for the whole publication period 2008-2011. In the calculation of our impact indicators, we disregard author self citations. We classify a citation as an author self citation if the citing publication and the cited publication have at least one author name (i.e., last name and initials) in common. We disregard author self citations because they have a different nature than ordinary citations. Many self citations are given for good reasons, in particular to indicate how different publications of a researcher build on each other. However, sometimes self citations can serve as a mechanism for self-promotion rather than as a mechanism for indicating relevant related work. This is why we consider it preferable to exclude self citations from the calculation of our impact indicators. By disregarding self citations, the sensitivity of our impact indicators to manipulation is reduced. Disregarding self citations means that our impact indicators focus on measuring the impact of the work of a researcher on other members of the scientific community. The impact of the work of a researcher on his own work is ignored.
As we have mentioned previously, each journal in WoS is assigned to one or more subject categories. These subject categories can be interpreted as scientific fields. ThereNature areProceedings about 250 subjectof the Nationalcategories Academy in WoS. Publications of Sciences in multidisScienceciplinary journals such as , , and were individually allocated, as much as possible, to subject fields on the basis of their references. The reassignment was done proportionally to the number of references pointing to a subject category. It is important to highlight that the impact indicators are calculated based on this assignment. Each publication in
6
96 WoS has a document type. The most frequently occurring document types are article, book review, correction, editorial material, letter, meeting abstract, news item, and review. In the calculation of bibliometric indicators, we only take into account publications of the document types article and review. Publications of other document types usually do not make a significant scientific contribution.
Our most straightforward impact indicator is the mean citation score indicator, denoted by MCS. This indicator equals the average number of citations per publication. Only citations within the relevant citation window are counted, and author self citations are excluded. Also, only citations to publications of the document types article and review are taken into account.
A major shortcoming of the MCS indicator is that it cannot be used to make comparisons between scientific fields. This is because different fields have very different citation characteristics. For instance, using a three-year fixed-length citation window, the average number of citations of a publication of the document type article equals 2.0 in mathematics and 19.6 in cell biology. It clearly makes no sense to make comparisons between these two fields using the MCS indicator. Furthermore, when a variable-length citation window is used, the MCS indicator also cannot be used to make comparisons between publications of different ages. In the case of a variable-length citation window, the MCS indicator favors older publications over more recent ones because older publications tend to have higher citation counts.
Our mean normalized citation score indicator, denoted by MNCS, provides a more sophisticated alternative to the MCS indicator. The MNCS indicator is similar to the MCS indicator except that it performs a normalization that aims to correct for differences in citation characteristics between publications from different scientific fields and between publications of different ages. To calculate the MNCS indicator for a unit, we first calculate the normalized citation score of each publication of the unit. The normalized citation score of a publication equals the ratio of the actual and the expected number of citations of the publication, where the expected number of citations is defined as the average number of citations of all publications of the document types article and review that belong to the same field and that have the same publication year. The field (or the fields) to which a publication belongs is determined by the WoS subject categories of the journal in which the publication has appeared. The MNCS indicator is obtained by averaging the normalized citation scores of all publications of a unit. If a unit has an MNCS indicator of one, this means that on average the actual number of citations of the publications of the unit equals the expected number of citations. In other words, on average the publications of the unit have been cited equally frequently as publications that are similar in terms of field and
Part 9 - Appendix – Bibliometric analyses 97 publication year. An MNCS indicator of, for instance, two means that on average the publications of a unit have been cited twice as frequently as would be expected based on their field and publication year. We refer to Appendix I for an example of the calculation of the MNCS indicator.
The MNCS indicator can be influenced considerably by a single highly cited publication, which can lead to improper conclusions regarding research performance. Therefore, we have another important impact indicator in addition to the MNCS indicator. This is the proportion of top 10% publications indicator, denoted by PP(top 10%). For each publication of a research group, this indicator determines whether based on its number of citations the publication belongs to the top 10% of all publications in the same field (i.e., the same WoS subject category) and the same publication year. The PP(top 10%) indicator equals the proportion of the publications of a research group that belong to the top 10%. If a research group has a PP(top 10%) indicator of 10%, this means that the actual number of top 10% publications of the group equals the expected number. A PP(top 10%) indicator of, for instance, 20% means that a group has twice as many top 10% publications as expected. Of course, the choice to focus on top 10% publications is somewhat arbitrary. Instead of the PP(top 10%) indicator, we can also calculate for instance a PP(top 1%), PP(top 5%), or PP(top 20%) indicator. In this study, however, we use the PP(top 10%) indicator. On the one hand this indicator has a clear focus on high impact publications, while on the other hand the indicator is more stable than for instance the MNCS indicator (see Appendix I for an illustration of the calculation on the indicator).
To assess the impact of the publications of a unit, our general recommendation is to rely on a combination of the MNCS indicator and the PP(top 10%) indicator. The MCS indicator does not correct for field differences and should therefore be used only for comparisons of groups that are active in the same field. An important weakness of the MNCS indicator is its strong sensitivity to publications with a very large number of citations. If a unit has one very highly cited publication, this is usually sufficient for a high score on the MNCS indicator, even if the other publications of the group have received only a small number of citations. Because of this, the MNCS indicator may sometimes seem to significantly overestimate the actual scientific impact of the publications of a unit. The PP(top 10%) indicator is much less sensitive to publications with a very large number of citations, and it therefore does not suffer from the same problem as the MNCS indicator. A disadvantage of the PP(top 10%) indicator is the artificial dichotomy it creates between publications that belong to the top 10% and publications that do not belong to the top 10%. A publication whose number of citations is just below the top 10% threshold does not contribute to the PP(top 10%) indicator, while a publication with one or two additional citations does contribute to the indicator. Because the MNCS indicator and the
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98 PP(top 10%) indicator have more or less opposite strengths and weaknesses, the indicators can be considered complementary to each other. This is why we recommend taking into account both indicators when assessing the impact of a unit’s publications.
It is important to emphasize that the correction for field differences that is performed by the MNCS and PP(top 10%) indicators is only a partial correction. As already mentioned, these indicators are based on the field definitions provided by the WoS subject categories. It is clear that, unlike these subject categories, fields in reality do not have well-defined boundaries. The boundaries of fields tend to be fuzzy, fields may be partly overlapping, and fields may consist of multiple subfields that each have their own characteristics. From the point of view of citation analysis, the most important shortcoming of the WoS subject categories seems to be their heterogeneity in terms of citation characteristics. Many subject categories consist of research areas that differ substantially in their density of citations. For instance, within a single subject category, the average number of citations per publication may be 50% larger in one research area than in another. The MNCS and PP(top 10%) indicators do not correct for this within- subject-category heterogeneity. This can be a problem especially when using these indicators at lower levels of aggregation, for instance at the level of departments or individuals.
We use the mean normalized journal score indicator, denoted by MNJS, to measure the impact of the journals in which a unit has published. To calculate the MNJS indicator for a unit, we first calculate the normalized journal score of each publication of the group. The normalized journal score of a publication equals the ratio of on the one hand the average number of citations of all publications published in the same journal and the same year and on the other hand the average number of citations of all publications published in the same field (i.e., the same WoS subject category) and the same year. The MNJS indicator is obtained by averaging the normalized journal scores of all publications of a unit. The MNJS indicator is closely related to the MNCS indicator. The only difference is that instead of the actual number of citations of a publication the MNJS indicator uses the average number of citations of all publications published in a particular journal. The interpretation of the MNJS indicator is analogous to the interpretation of the MNCS indicator. If a unit has an MNJS indicator of one, this means that on average the group has published in journals that are cited equally frequent as would be expected based on their field. An MNJS indicator of, for instance, two means that on average a group has published in journals that are cited twice as frequently as would be expected based on their field. 2.4 Indicators of scientific collaboration
Part 9 - Appendix – Bibliometric analyses 99 Indicators of scientific collaboration are based on an analysis of the addresses listed in the publications produced by a unit. We first identified publications authored by a single institution (‘no collaboration’). We then identified publications that have been produced by institutions from different countries (‘international collaboration’) and publications that have been produced by institutions from the same country (i.e. ‘national collaboration’). These types of collaboration are mutually exclusive. Publications involving both national and international collaboration are classified as international collaboration.
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100 3 Data collection
The analysis presented in this report focuses on publications from the period 2006–2013, although citation analysis will refer only to publications between 2006-2011 with citations up to 2012. This different time scope is caused by the fact that the year 2013 is not yet fully processed in the CWTS CI-database (the full load of the year 2013 is expected to happen around the first quarter of 2014).
The data collection follows the so-called ‘address based approach’. Thus, all publications having at least one author affiliated with the CBS are identified from the address list of the publications. All name variations of the CBS were taken into account and in general terms, we have followed the same methodology for the address data collection as presented in the CWTS Leiden Ranking (see www.leidenranking.com/methodology/datacollection). Also variants of the KNAW (both in Dutch and in English) have been considered in this case. Other keywords such as ‘Fungal’ or ‘Schimmel’ were used in combination with wildcard characters. All variants have been checked manually so the correctness of the data is granted.
Double occurrences of publications were excluded within the CBS, in a way that a publication co-authored by several authors belonging to the CBS is counted only once. Based on the information contained in the affiliation field of the WoS bibliographic records, publications were collected from the period 2006-2013 were extracted from our database. 3.1 Coverage of WoS publications
To gain insight in the WoS coverage of the publications included in the study, we analyzed the references of these publications. To this end, references included in the publications produced by the CBS during 2006-2011 were matched with our WoS database. In this way, we can estimate the importance of WoS publications for the CBS by determining to what extent CBS authors themselves cite WoS publications, and to what extent they refer to other non-WoS publications. Based on our database, we can trace references dated between 1980 and 2011. The column ‘Internal coverage’ in Table 1 indicates that the internal coverage of the WoS- covered publications of the CBS. As it can be seen, this coverage is quite high with almost 78% of the references provided by the CBS publications also pointing to other publications covered in the Web of Science, thus suggesting that this study is adequate to characterize the WoS-covered publications of the CBS. Another point however is the fact that CBS, as a taxonomic institution,
Part 9 - Appendix – Bibliometric analyses 101 also publishes a lot outside the Web of Science, and this is something that also needs to be taken into account for the consideration of the results of this report.
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102 4 Overall results
In this chapter, the results of the performance analysis are reported. Section 4.1 shows the overall results. Table 1 includes the overall bibliometric indicators for the CBS for the periods 2006-2011 (i.e. publications from 2006 to 2011). In terms of the citation analysis, we have used a variable length window: all citations received by all publications up to 2012 are counted. This means that for publications from each of the publication years (2006–2011), citations are counted up to and including 2012. For example, a five year citation window is considered for papers published in 2008 since the publication year is considered the first year to yield citations. Similarly, papers published in 2009 have a four year citation window. At the same time we also include the number of publications until the year 2013 in order to present a more complete picture on the WoS-covered output of the CBS, although no citation metrics are provided for these years as the short citation windows would be quite unreliable. Table 1. P (2006- P (2006- PP P top PP(top Internal PP PP(int. Period Organization Main indicators2013) for2011) the CBS TCS (full MCS output (uncited) 2006-2013 MNCS and MNJS citation 10% analysis10%) 2006-2011/12)coverage (collab.) collab.) 2006- 2011/12 CBS 719 526 6281 11.94 11.8% 1.82 1.46 106 20.1% 77.9% 96.6% 89.5%
2006/12 CBS 85 1713 20.15 2.4% 1.73 1.27 11 13.0% 72.7% 92.9% 84.7%
2007/12 CBS 96 2149 22.39 4.2% 2.54 1.65 23 24.4% 76.1% 94.8% 86.5%
2008/12 CBS 85 924 10.87 7.1% 1.45 1.11 13 15.1% 76.8% 100.0% 92.9%
2009/12 CBS 86 775 9.01 8.1% 1.95 1.53 22 25.4% 78.3% 98.8% 94.2%
2010/12 CBS 73 407 5.58 12.3% 1.55 1.56 14 18.8% 83.3% 97.3% 89.0%
2011/12 CBS 101 313 3.10 33.7% 1.58 1.62 23 22.5% 80.6% 96.0% 90.1%
2012 CBS 113
2013 CBS 80
The indicator of output for the period 2006-2013 (P (2006-2013) shows that 719 publications can be attributed to the CBS. Focusing on the period subject of analysis P (2006-2011), the impact of 526 publications can be analyzed. These publications have received a total of 6281 citations (excluding self-citations), which means that CBS papers receive on average almost 12 citations. Still, almost 12% of CBS publication in the period 2006-2011 did not receive any citation.
Field normalized indicators show that CBS publications are cited 82% above the international level (set by the reference value 1) with an MNCS value of 1.82. CBS authors also publish in high impact journals in their field(s) as the MNJS value is also above 1 (1.42). Regarding top cited publications, around 106 publications of the CBS qualify as being among the top 10% most cited of their different fields, thus with 20% of highly cited publications it can be
Part 9 - Appendix – Bibliometric analyses 103 said that overall the CBS has produced double the number of highly cited publications than expected (i.e. 10%). In the two final columns of the table we also present the share of publications produced by the CBS in both national or international. In general it is remarkable the high degree of collaboration, with nearly 97% of CBS publications in collaboration (both national or international) and almost 90% of the publications in international collaboration.
Figure 1 shows the trend analysis of the number of publications produced by the CBS. As shown, the number of publications per year is normally around 90 publications, although from the period 2007 to 2010 it is noticeable a decrease in the number of WoS-covered publications. After that year there is a substantial increase in the number of publications with its highest peak in 2012. The decrease in the year 2013 is not informative as this year is not yet fully covered in the WoS database. The number of highly cited publications shows a relatively stable pattern over time, although in table 1 it is possible to see how the number of top CBS publications oscillates normally between 11 and 23 (i.e. from one year to another almost doubling the number of highly cited papers). Figure 1. Trend analysis of the CBS publications and top 10% publications (2006-2013)
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60 Publications P top 10%
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0 2006 2007 2008 2009 2010 2011 2012 2013
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104 Figure 2 shows the trend analysis of the PP (top 10%) indicator (i.e. the share of highly cited publications of the CBS). The most remarkable aspect of figure 2 is the fact that the CBS has presented a share of top 10% highly cited publications above 10%. The trend is in general terms increasing, but there are two peaks in 2007 and 2009 when almost ¼ of the CBS publications where among the most cited publications worldwide in their field(s). Figure 2. Trend analysis of PP(top 10%) indicator of the CBS (2006-2011/12) PP(top 10%)
30.0%
25.0%
20.0%
15.0%
10.0%
5.0%
0.0% 2006 2007 2008 2009 2010 2011
Figure 3 shows the trend analysis of other two field-normalized indicators, namely the MNCS and the MNJS. As in the previous figure, the most remarkable aspect of figure 3 is the fact that the CBS exhibits values of MNCS and MNJS above 1 during the whole period of the study. The impact of the journals (MNJS) presents a quite stable pattern over time with values generally above 1.50. The MNCS value however shows a more decreasing pattern. The main reason for this decreasing patterns it hat it finds its highest value in 2007 (reaching 2.50!) but then decreasing to values around 1.50. An indicative event of this decrease is the fact that the MNCS score reaches a value slightly below the MNJS value in 2011.
Part 9 - Appendix – Bibliometric analyses 105 Figure 3.
Trend analysis of PP(top 10%) indicator of the CBS (2006-2011/12)
3.00
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1.50 MNCS MNJS 1.00
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106 5 Discussion and conclusions
The Centraalbureau voor Schimmelcultures (CBS) Fungal Biodiversity Centre - an institute of the Royal Netherlands Academy of Arts and Sciences (KNAW) situated in Utrecht presents a total output of 719 publications (article and reviews) covered in the Web of Science during the period 2006-2013. The internal coverage of the CBS is quite high (around 78%), indicating that this bibliometric analysis is quite adequate for the WoS-covered publications, although it is important to highlight that other publications not covered in the WoS are not included in this study. The field-normalized impact indicators of the unit (focusing on the 2006-2011 production) show a high performance level of the unit, with field normalized indicators (i.e. PP (top 10%), MNCS and MNJS) much higher than the average international level. The trend analysis shows an increase in the number of WoS-covered publications, particularly after 2010. The production of highly cited publications is quite stable around values of 10 or 20 top papers per year. The same goes for the share of highly cited publications over time, with a slight increase over the period. The impact level of the publication journals is also high and quite stable over time. It is noticeable however a decrease in the overall field-normalized impact (i.e. the MNCS value) particularly during the last years, although with high levels of performance during the whole period. As a general conclusion, the CBS bibliometric performance analysis shows a strong performance, with an increasing pattern in WoS-covered production and a stable and high impact performance over time.
Part 9 - Appendix – Bibliometric analyses 107 Appendix I. Calculation of field-normalized indicators
To illustrate the calculation of the MNCS indicator, we consider a hypothetical research group that has only five publications. Table A1 provides some bibliometric data for these five publications. For each publication, the table shows the scientific field to which the publication belongs, the year in which the publication appeared, and the actual and the expected number of citations of the publication. (For the moment, the last column of the table can be ignored.) As can be seen in the table, publications 1 and 2 have the same expected number of citations. This is because these two publications belong to the same field and have the same publication year. Publication 5 also belongs to the same field. However, this publication has a more recent publication year, and it therefore has a smaller expected number of citations. It can further be seen that publications 3 and 4 have the same publication year. The fact that publication 4 has a larger expected number of citations than publication 3 indicates that publication 4 belongs to a field with a higher citation density than the field in which publication 3 was published.
The MNCS indicator equals the average of the ratios of actual and expected citation scores of the five publications. Based on Table 1, we obtain
Hence, on average the publications of our hypothetical research group have been cited more than twice as frequently as would be expected based on their field and publication year. Table A1. Publication Field Year Actual Expected Top 10% Bibliometric data for the publications of a hypothecitationstical research citations group threshold
1 Surgery 2007 7 6.13 15 2 Surgery 2007 37 6.13 15 3 Clinical neurology 2008 4 5.66 13 4 Hematology 2008 23 9.10 21 5 Surgery 2009 0 1.80 5
To illustrate the calculation of the PP(top 10%) indicator, we use the same example as we did for the MNCS indicator. Table A1 shows the bibliometric data for the five publications of the hypothetical research group that we consider. The last column of the table indicates for each publication the minimum number of citations needed to belong to the top 10% of all
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108 1 publications in the same field and the same publication year. Of the five publications, there are two (i.e., publications 2 and 4) whose number of citations is above the top 10% threshold. These two publications are top 10% publications. It follows that the PP(top 10%) indicator equals
In other words, top 10% publications are four times overrepresented in the set of publications of our hypothetical research group.
1 If the number of citations of a publication is exactly equal to the top 10% threshold, the publication is partly classified as a top 10% publication and partly classified as a non-top-10% publication. This is done in order to ensure that for each combination of a field and a publication year we end up with exactly 10% top 10% publications.
Part 9 - Appendix – Bibliometric analyses 109 PEER REVIEW CBS-KNAW 2008–2013