The National Programme on Conservation and Utilization of Microbial Genetic Resources and Invertebrates of Agricultural Importance

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COLLECTIONS OF VIRUSES AND PHYTOPLASMAS


VURV-V SVOR SPCH UPOC-VIR-FLO CAPM VIRUBRA CFVS


Collection of Plant Pathogenic Viruses (VURV–V), VÚRV, v. v. i., Praha

Cataloging of viral isolates has started with viruses kept in Crop Research Institute Prague in the eightieths of the last century. The collection has expanded as the number of isolates and solved projects has grown.

• Characterization of collection
In the collection, there are maintained economically important viruses of cereals, vegetables, fruit trees, hop and grapevine. The collection contains 91 different strains and isolates of plant viruses and three isolates of European stone fruit yellows phytoplasma (ESFY). The total number of the collection items changes as new isolates are collected from the nature.
The collection contains Agropyron mosaic virus, Alfalfa mosaic virus, Apple chlorotic leaf spot virus, Apple mosaic virus, Apple stem grooving virus, Apple stem pitting virus, Arabis mosaic virus, Barley yellow dwarf virus, Bean common mosaic virus, Beet chlorosis virus, Beet yellows virus, Broad bean wilt virus-1, Broad bean wilt virus-2, Brome mosaic virus, Cauliflower mosaic virus, Cherry leaf roll virus, Cocksfoot streak virus, Cucumber mosaic virus, European mountain ash ringspot-associated virus, Grapevine fleck virus, Grapevine leafroll-associated virus 1-A, Grapevine leafroll-associated virus 1-B, Grapevine rupestris vein feathering virus, Grapevine virus A, Grapevine virus B, Hop mosaic virus, Lettuce mosaic virus, Lolium latent virus, Myrobalan latent ring spot virus, Oat necrotic mottle virus, Pea enation mosaic virus, Pepper mild mottle virus, Pleione flower breaking virus, Plum pox virus, Potato potyvirus Y, Prunus necrotic ringspot virus, Rupestris stem pitting associated virus, Ryegrass mosaic virus, Squash mosaic virus, Strawberry latent ring spot virus, Tobacco ringspot virus, Tomato aspermy virus, Tomato mosaic virus, Turnip mosaic virus, Turnip yellow mosaic virus, Watermelon mosaic virus 2, Wheat dwarf virus, Wheat streak mosaic virus a Zucchini yellow mosaic virus.

• Activities, services
Individual isolates of the collection are used by an expert diagnosis as comparative standards. They are used for research work e.g. for testing resistant plants and offered to other institutions. The isolates are gradually characterized by the use of molecular biological methods.

• Methods of culture maintenance
Plant viruses are stored dried in host plant leaves at 2-6 °C. Some of them are preserved frozen in leaves at -70 °C, by freeze-drying and freezing in liquid nitrogen. Viruses which lose their infectiousness by drying or freezing are maintained and regularly inoculated to new indicator plants mechanically or by the use of insects like aphids (Myzus persicae, Rhopalosiphum padi) or a leafhopper (Psammotettix alienus). Infected plants are maintained in isolated greenhouse compartments or on woody plants in a screen house.

• System of quality
Individual isolates of the collection are inoculated to new host plants separately in isolated greenhouse compartments at a different terms to avoid any mixing or replacement of the collection items. The worker responsible for a consign virus probes the virus presence in a plant by evaluating symptoms on indicator plants, ELISA, RT-PCR and an electron microscopy. Plants infected by any quarantine virus can be maintained only in a special isolated quarantine greenhouse. This viruses can not be exchanged between institutions.

• Membership
The collection workers are members of European Plant Protection Organization, Panel on Fruit Tree Viruses, International Council for the Study of Virus and Virus-like Diseases of the Grapevine, Temperate Fruit Virus Working Group, International Society for Horticultural Science (ISHS), International Working Group on Legume and Vegetable Viruses, Small Fruit Virus Working Group, Plum Pox Working Group, European Foundation for Plant Pathology, International Foundation for Science - Stockholm, International Society for Horticultural Science, European Association for Research on Plant Breeding (EUCARPIA), Julius Kühn-Institut (JKI) Bundesforschungsinstitut für Kulturpflanzen, Quedlinburg.

Catalogue

Application:
• Application of strains
The isolates of the collection are used as positive standards by diagnosis of cereal, vegetable, fruit tree and grapevine viruses. They are no substitutable at this function by the correct expert diagnosis.
They serve as a source of infectious material for testing possibly resistant plants, evaluating the immunity against plant viruses and for diagnosis by the comparison of symptoms on indicator plants. The viral isolates are used for solving of a research work.
Samples of the isolates of the collection are available to other research and diagnostic institutions of the Czech Republic.

• Customers
- Czech University of Life Sciences Prague,
- Mendel University in Brno, ZF Lednice,
- Research and Breeding Institute of Pomology Holovousy Ltd. s.r.o.,
- SEMO Smržice,
- research workers of CRI

Contact:
Crop Research Institute Prague
Drnovská 507
161 06 Praha 6 – Ruzyně
https://www.vurv.cz/

Staff:
Ing. Jana Brožová, Ph.D.(curator)
e-mail: brozova@vurv.cz, tel: 233022388
Ing. Jan Ripl
e-mail: ripl@vurv.cz, tel. 233 022 468

Collection of Ornamental Plant Viruses , (VÚKOZ, v. v. i., Průhonice)

The research on virus diseases of ornamental plants has a long tradition at the Silva Tarouca research institute for landscape and ornamental gardening at Průhonice. Altogether thirty seven viruses were isolated and identified here. The majority of these viruses has been maintained in leaf material dried over calcium chloride. Economic important viruses in ornamental crops were chosen of that collection and included in 1997 into the Czech National Programme on Protection of Genetic Resources of Economic Significant Microorganisms and Tiny Animals and their utilization in reference diagnostic in 1997.

• Characterization of collection
At present twenty six viruses represented by one hundrerd and three isolates are maintained in the collection.
Apple chlorotic mosaic virus (ACLSV) 1
Arabis mosaic virus (ArMV) 2
Calibrachoa mottle virus (CbMV) 1
Chrysanthemum virus B (CVB) 3
Cucumber mosaic virus (CMV) 8
Cymbidium mosaic virus (CyMV) 2
Dahlia mmosaic virus (DMV) 1
Dasheen mosaic virus (DsMV) 2
Hydrangea ring spot virus (HdRSV) 2
Impatiens necrotic spot virus (INSV 5
Odontoglossum ring spot virus (ORSV) 2
Pelargonium flower break virus (PFBV) 5
Petunia asteriod mosaic virus (PetAMV) 5
Plum pox virus (PPV) 1
Poplar mosaic virus (PopMV) 4
Potato virus Y (PVY 3
Scophularia mottle virus (ScrMV) 3
Tobacco mosaic virus (TMV) 12
Tobacco necrosis virus (TNV 9
Tobacco streak virus (TSV) 8
Tomato aspermy virus (TAV) 5
Tomato bushy stunt virus (ToBSV) 2
Tomato mosaic virus (ToMV) 2
Tomato spotted wilt virus (TSWV) 8
Tulip breaking virus (TBV) 3
Tulip virus X (TVX) 4

Particular isolates are characterized by transmitting of infective sap from the original host to indicator plants. The plant species used for biological tests by sap inoculation are: Capsicum annum, Chenopodium quinoa, Chenopodium amaranticolor, Datura stramonium, Mimulus sp., Nicotiana benthamiana, Nicotina debney, Nicotiana glutinosa, Nicotiana megalosiphon, Nicotina occidentalis, Nicotiana rustica, Nicotiana tabacum ‘Samsum’, ‘Xanthi’, ‘White Burely’ and Petunia hybrida. Serological tests ELISA have been used for further identification of the isolate. The antisera originate from commercial firms Leowe Biochemica - Germany, DSMZ – Germany, Bioreba – Switzerland, B Q Support – Netherlands, Neogen – Scotland or from research institutes in the Czech Republic, Germany, Netherlands and UK. Elecrone microscopy (EM) has been used for checking whether the isolate is not a mixture of two or more viruses. In some cases immunoelectron microscopy (EM) was used for proving the diagnostic of the virus (see fig. 1. - ?). Tobacco streak virus was identified also by the transcription-polymerase chain reaction (RT-PCR).

• Preservation
Isolates of the majority of viruses are maintained for the long-term in infected leaves dried over calcium chlorate (CaCl2) at the temperature 4° C. Leaves taken of the infected plant cut in small pieces are placed into a big Petri dish containing crystallic CaCl2 at the bottom covered with nylon net. Approximately after 20 days the dried sample is placed to a test tube containing CaCl2 at the bottom covered with a piece of cellulose wool. The test tubes are corked up and stored at 4° C. This method can be used without any problems especially for viruses characterized by natural mechanical transmission.
Viruses transmitted under natural conditions by vectors are stored also in infected living plants in an isolation glasshouse. If an insect transmitted virus was isolated from a bulb or a cormel plant these originally infected bulbs and cormels are grown further for having the possibility of the virus reisolation. Living plants infected by Tomato spotted wilt virus or Impatiens necrotic spot virus are grown in an isolation chambre according quarantine regulations.

Application:
The collection of viruses and antisera is used in the diagnostic of virus infections in ornamental plant species. It is available to the State plant protection service for reference diagnostic.The virus isolates are used as positive controls in testing the breeding material of Impatiens, Petunia, Dahlia and Tulipa appointed for further propagation and export. The collection is used in checking imported ornamental plant material which is further propagated in this country for the enlargement of ornamental plant species assortment. It is important also in monitoring the possible occurrence of quaratine viruses of the genus Tospovirus and other viruses having a broad spectrum of host plant species. The rsolvers are members of the working group on virus diseases of ornamental plants at the International Society for Horticultural Science (ISHS). The members of this working group exchange virus isolates, offer antisera and information on results of the experiments on viruses of ornamental plants. The isolates are available after application to other institutions in the Czech Republic and in abroad.

Contact:
Silva Tarouca research institute for landscape and ornamental gardening
Květnové nám. 391
252 43 Průhonice, Czech Republic
phone: +420 296528294, fax: +420 267750440

Staff: Curator: Ing. et Ing. Dita Šetinová
phone: 296528368
setinova@vukoz.cz

Collection of Hop Pathogens, (ChI, s. r. o., Žatec)

The collection was established in 2003. The main attention is paid to hop pathogens - viruses and viroids.

• Characterization of collection
8 virus isolates
1 fungus isolate

• Methods of culture maintenance
infected hop plant, CaCl2, lyophilization

• Control
Each year monitoring is performed pathogens control by ELISA and RT-PCR, the same time there is a continuous obtaining of new isolates
Isolates: ApMV, ArMV, HLV, HMV, HLVd

Application:
Diagnosis of pathogens - ELISA, PCR, phytopathological – virology

Utilization of strains
positive controls for testing of nursery material
providing of strains for research

Users
The Central Institute for Supervising and Testing in Agriculture (CISTA)
The Czech University of Life Sciences Prague (CULS), Institute of Plant Molecular Biology - The Biology Centre of the Czech Academy of Sciences, České Budějovice

Contakt:
Hop Research Institute Zatec

Staff:
Ing. Petr Svoboda, CSc., curator
svoboda@chizatec.cz
+420 415 732 121
fax +420 415 732 150
www.chizatec.cz





Collection of Phytopathogenic Microorganisms UPOC, (PřF UP v Olomouci)

The collection of fungal microorganisms pathogenic to plants has been formed by Prof. Lebeda from 1977, during his work in Plant Breeding Station of Sempra company in Smržice. Following his employment (from 1994) at the Dept. Botany, Faculty of Science, Palacký University in Olomouc the collection was enlarged by isolates of cyanobacteria and algae (administered by Dr. Hašler and managed by prof. Poulíčková) and collection of viruses and phytoplasmas (managed by Prof. Navrátil at Department of Cell Biology and Genetics). The UPOC collection has expanded by expeditions organized yearly to collect new samples.

• Characteristic of collection (strains), number of cultures

The national database include
177 isolates of 15 species of phytopathogenic fungi and fungi-like organisms
31 strains of 31 species of cyanobacteria (12) and algae (7)
5 isolates of 4 species of phytoplasmas and 28 isolates of 7 virus species
others are part of working collection.

• Activities/research activities of Department of Botany
Genetic variability of plant pathogenic fungi;
Biodiversity and genetic variability of host-pathogen interactions in crop and wild plant pathosystems;
Mechanisms of resistance in host-pathogen interactions;
Plant breeding (classical and biotechnological approaches) in disease resistance;
Tests of plant pathogen for fungicide resistance;
Biology, ecology and genetics of cyanobacteria and algae
Epidemiology, biology and genetics of phytoplasmas and viruses

• Services
providing samples for non-commercial purposes
consultancy and determination
methods of culture maintenance see methodology (in Czech language)
quality systems, internal norms, correct laboratory practice
according to requirements of the National programme

• Involvement to national and international organizations/platforms
IBEB (International Bremia Evaluation Board)

• international cooperation

interchange of isolates, developing methods for storage, cultivation and testing of host plants resistance

• catalogue - on www VURV Praha

Utilization of collection:

• Utilization of strains
teaching, research, screening of plant resistance, reference strains for phytosanitary diagnostics, State phytosanitary administration and Central Checking and Testing Agricultural Institute, consultancy activities and postgraduate teaching of professional staff of water management institutions/authorities

• Users, clients
students, teachers, scientists, specialists of State Phytosanitary Administration, breeding companies and water authorities, …

Microorganisms in UPOC collection

Contact:

Institution/place
Palacký University in Olomouc
Faculty of Science
Department of Botany
Šlechtitelů 11
783 71 Olomouc-Holice

Staff, contacts:

• head/responsible person
Prof. Ing. Aleš Lebeda, DrSc. (curator)

• others
Doc. RNDr. Michaela Sedlářová, Ph.D.
Mgr. Pavla Šiková
RNDr. Barbora Mieslerová, Ph.D.
RNDr. Božena Sedláková, Ph.D.
RNDr. Petr Hašler, Ph.D.
Prof. RNDr. Milan Navrátil, CSc.

• contact: e-mail, telephon, fax: 585634824,
ales.lebeda@upol.cz
585634800
michaela.sedlarova@upol.cz
585634809
fax 585634824

Collection of Animal Pathogenic Microorganisms (CAPM),
(VÚVeL, v. v. i., Brno)

The Collection of Animal Pathogenic Microorganisms (CAPM) was founded in 1962. At present the CAPM is a part of the Department of Bacteriology at the Veterinary Research Institute in Brno.

• Characterization of collection
The major activities of the CAPM involve acquisition, preservation and distribution of microorganisms - animal viruses and zoopathogenic bacteria useful for veterinary practice, applied microbiology and research. The CAPM holds more than 300 strains of viruses and almost 600 strains of bacteria which are listed in printed catalogues.

• Activities, services
The main function of the CAPM is to collect, maintain, preserve and supply pure cultures of microorganisms. The CAPM accept for deposit viral and bacterial strains which are subject to patent applications on the national level.

• Other services:
Safe deposit of cultures
Freeze-drying of cultures
Consultation

• Preservation
Cultures are preserved by freeze-drying or freezing in liquid nitrogen or kept at -80 °C.

• System of quality
Laboratory works are carried out in compliance with the approved methodology (“Standard procedure for preservation, storing, characterization and documentation of genetic resources”), and the principles of Good Laboratory Practice (GLP) are being observed in the workplace.

• Membership
The CAPM is a member of the World Federation for Culture Collections - WFCC , the European Culture Collections' Organisation - ECCO and the Federation of Czech and Slovak Culture Collections - FCCM .

Application:
• Application of strains
Viral and bacterial strains are predominantly used for research, diagnostic and educational purposes. Besides agriculture, education and health care, they can be also employed in biotechnology and in industry.

• Customers
CAPM cultures are supplied to laboratories, schools and educational institutions both in Czech Republic and other countries.

Contact:

Veterinary Research Institute
Hudcova 70, 621 00 Brno
Czech Republic
Telephone 533 332 131
Fax 541 211 229
WWW www.vri.cz

Staff
MVDr. Markéta Reichelová - Head, curator (bacteria)
reichelova@vri.cz

Other staff:
Mgr. Hana Malenovská - curator (viruses)
malenovska@vri.cz


CO2 incubator


Card index


Lyophilizer


Inverse microscope


Bovine respirovirus 3, strain CAPM V-32


Cowpox virus, strain CAPM V-158


Rotavirus A, strain CAPM V-334

Collection of Potato Viruses (VIRUBRA),
(VÚBHB, s. r. o., Havlíčkův Brod)

The collections of potato pathogenic viruses and viroids were in Potato Research Institute in Havličkuv Brod founded and maintained since seventies of the last century. Methods of long-term conservation and stabilization of viruses (procedures of freez-drying and drying over strong desicants) were solved in research projects dealing with viruses occurring on potato in CR. Afterwards experimental utilization of tissue culture technique for maintenance of isolates in in vitro conditions was devised. The originál collection included altogether 231 isolates of six main potato viruses.

• Characterization of collection
The majority of isolates are currently maintained in aseptic in vitro conditions and regularly restored using nodal segment of plantlets. Original potato cultivars infected with particular virus or viroid isolate are for long term storage maintained in vitro in microtubers. The plant segments are kept on semi-solid agar media and special environmental conditions and chemical components are utilized. Before transmission of isolates into in vitro conditions as well as at regular intervals during their maintenance the isolates are checked using serological, biological and selected molecular diagnostic methods, including sequincing of their nucleic acid.
At present 544 isolates of main potato viruses, devided into seven sub-sets are mainatained.
Ptato leafroll virus (PLRV) - 61 isolates in vitro on originál potato cultivars
Potato virus Y (PVY) – 12 isolates on tobacco plants and 100 isolates on potato cultivars
Potato virus A (PVA) – 5 isolates on tobacco plants and 25 isolates on potato cultivars
Potato virus M (PVM) – 43 isolates on potato cultivars
Potato virus X (PVX) – 27 isolates on potato cultivars
Potato virus S (PVS) – 262 isolates on potato cultivars
Potato virus V (PVV) – 1 isolate on potato cultivar
Potato Mop-Top virus (PMTV) – 5 isolates on potato cultivars
Potato Aucuba mosaic virus (PAMV) – 2 isolates on potato cultivars
Potato Rough Dwarf virus (PRDV) – 1 isolate on potato cultivar
Tobacco rattle virus (TRV) – 1 isolate on potato cultivar
Potato Spindle Tuber Viroid (PSTVd) – 2 potato isolates on potato cultivars and 7 isolates from ornamental plants.

• Activities, services
Individual isolates of the collection are used in a number of solved research projects, and are on request supply to all the laboratories of large scale testing as the positive controls for ELISA and further laboratory diagnostic tests.

• Methods of culture maintenance
In in vitro conditions the isolates are kept in aseptic conditions on semi-solid artificial medium and depending on initial host plants they are restored using nodal segment of plantlets. For long term storage the technique of micro tubers has been incorporated. Testing of individual isolates has been performed and repeated in regular intervals and positive isolates are re-introduced into collection.

Contact:
Potato Research Institute Havlíčkův Brod L.t.d.
Dobrovského 2366
58001 Havlíčkův Brod

Staff:
Ing. Martin Kmoch, Ph.D. (curator)
e-mail: kmoch@vubhb.cz
tel.: 569 466 231
webové stránky: www.vubhb.cz

Collection of Fruit Trees Viruses (CFVS),
(VŠÚO Holovousy, s. r. o.)

In collection are preserved the most incident viruses and phytoplasmas of fruit arts (apple – ApMV, ACLSV, ASGV, ASPV, CPm; pear - ACLSV, ASGV, ASPV, CPp; plum – ACLSV, PPV, PDV, PNRSV; sweet and sour cherry - ACLSV, PDV, PNRSV), some small fruits viruses (raspberry - CLRV, RBDV)

• Activities/research activities, services
care of collection – cutting, fertilizing, watering at plants cultivated in glasshouse
regularly passage on fresh medium at elements cultivated in culture in vitro

• Preservation methods
a part of collection is preserved in cultivation containers with standard soil substrate in collection-glasshouse
a part of collection is preserved in tissue culture in vitro in Erlenmeyer flasks on agar medium in cultivation box

• Quality system
quality system is not available, the work is regulated with manual of safety work and work manual in own laboratory

• Registration in international and national organizations (WFCC, ECCO, FCCM), other international activities the collection is not registred in international system, the international activities are not accomplished

Utilization of collection:
• Utilization of strains
positive controls for testing of nursery material
providing of stems for research intention

• Users, clients
Laboratory of Research and Breeding Institute of Pomology Holovousy Ltd.
Laboratory of Central Institute for Supervising and Testing in Agriculture Brno

Contakt:
Institution/place
VŠÚO Holovousy, s.r.o.

Staff
Mgr. Lucie Valentová (curator)
e-mail: lucie.valentova@vsuo.cz, tel: +420 491 848 221
Web sites: www.vsuo.cz