Microbiology

Biotechnological Hub of the NIB (BTH-NIB)

The purpose of the investment project BTH-NIB is the assurance of the appropriate infrastructural conditions for the use of research and developmental opportunities in the fields of operation of the NIB.

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Microbiology

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Research tools and methods

Focusing on molecular detection methods quantitative real-time PCR (qPCR) has been a mainstay in our laboratory for many years. Now, we increasingly combine it with digital PCR (dPCR), DNA barcoding and new generation sequencing (NGS) technologies to further improve our detection capabilities. Part of our research is devoted to development and implementation of molecular methods that are easy to perform, fast and can be used on site (LAMP). All novel methods are complemented with classical microbiological methods, serology (IF and ELISA), molecular approaches (PCR, repetitive PCR) and transmission electron microscopy (TEM).

Besides detection, we are experts in extraction and concentration of nucleic acids from different plant, food, feed and environmental samples. We have improved and developed methods for concentration purification or removal of viruses in aqueous solutions exploiting monolithic chromatography.

 

Bacteria

Our bacterial studies are focused on efficient detection of plant pathogenic bacteria through development of novel methods, validation approaches, study of the microbe diversity and epidemiological aspects relevant to their detection. Our real-time PCR assays are widely used and have been included in the diagnostic protocols of the European and Mediterranean Plant Protection Organization. Among these are an assay for detection of a slow growing grapevine pathogen, Xylophilus ampelinus, the first real-time PCR to be included in the EPPO guidelines and real-time PCR for the detection of the fire blight pathogen, Erwinia amylovora, in symptomatic and latently infected samples. We are especially interested in improving the reliability and the analytical sensitivity of Ralstonia solanacearum and Erwinia amylovora detection through the use of digital PCR format of real-time PCR assay. Currently we focus on Xylella fastidiosa and other bacteria of Xanthomonadaceae family in seeds. Recently isothermal on site LAMP tests were developed for Ralstonia solanacearum and human and avian pathogenic E. coli. Moreover, we are analyzing the genomes of putative new Dickeya sp. causing soft rots of orchids, the genome of an efficient lytic phage against them and their interactions. One of the aims of the research is also to test the potential of bacteriophages to be used as bioagents against bacterial plant diseases.

 

Phytoplasma

Our current work on phytoplasmas has been focused on their epidemiology, we have been analyzing their occurrence and strain variability in their host plants and insect vectors. Research has been focused on phytoplasma species from the apple proliferation group infecting fruit trees (i.e. ‘Candidatus Phytoplasma mali’, ‘Ca. P. pyri’ and ‘Ca. P. prunorum’) and phytoplasmas associated with grapevine yellows (i.e. ‘Ca. P. solani’, causing Bois Noir disease and phytoplasma FDp, a causal agent of Flavescence dorée). We developed several methods for the detection of the phytoplasmas. Our real time PCR assays, which are widely used, including DNA isolation based on magnetic beads have the following characteristics: high specificity and sensitivity, and high and rapid throughput. Real time PCR was transferred to droplet digital PCR and used for absolute quantification of FDp, without the need for calibration curves. To enable faster and on site detection of phytoplasmas isothermal LAMP assays were developed. Real time PCR and LAMP for detection of FDp have been included in the diagnostic protocols of the European and Mediterranean Plant Protection Organization.

 

Viruses

Our virology studies are focused mainly on plant viruses (PVY, PepMV, GFLV…) that represent pests to economically important plants, such as potato, tomato and grapevine. We study distribution of viruses in plants, their movement inside plants (GFP PVY) and changes that are caused by the infection, virus genetic diversity and its biological significance. We also research the diversity of plant virus populations. Particularly, we are interested into the within plant population structure of viruses and its dynamics (using NGS for detection and potato virus Y as our model virus). We are also using NGS to search for viruses in environmental (e.g. effluent waters) samples. Within last few years we have also focused on waterborne viruses (plant – PepMV and human – RoV, NoV, SaV, AsV) that use environmental waters as a transportation between hosts. We have developed and improved methods for waterborne virus concentration, detection, quantification and removal.




Bacterial, virus and viroid collections

Reference and other control materials are essential in diagnosis and in research work. In the field of plant pathology certified reference materials of known composition, the concentration of the target organism and other known characteristics are not available. Most of the controls that we use in our work we therefore prepare ourselves e.g. positive controls of DNA extraction that contain defined low concentration of the target organism in the background plant material. Controls are used to check and confirm the proper performance of the tests or the experiments (their specificity, sensitivity and robustness) and in the development and validation of methods. For this purpose, we maintain a working collection of bacteria on the Microbank system (Pro-Lab Diagnostics) since 1990, following the World Federation for Culture Collections guidelines. The collection of phytoplasmas that cannot be grown in culture media, and virus isolates most relevant to our work are maintained in tissue culture or in infected plants in the quarantine greenhouse, while other viruses and viroids are stored frozen. The collection of viruses, viroids and phytoplasmas contains 359 isolates and more than 2000 isolates of bacteria including (i) the type isolates of plant pathogens (reference isolates), (ii) isolates, which can cross-react in the detection test, (iii) isolates, representing microflora of the host plants, and (iv) isolates harmful organisms, which were isolated from the samples and identified at National institute of biology. The last group is indispensable in the introduction and development of new methods. Isolates present in Slovenia may in fact differ from the isolates used in the development of reagents and consequently such tests are not always suitable for the analysis of the local samples. The Slovenian isolates were already used to improve existing methods and the development of methods of source-tracking and for phylogenetic analysis of plant pathogens.

Selected references

Research papers:

 

  • DULAR, Matevž, GRIESSLER BULC, Tjaša, GUTIÉRREZ-AGUIRRE, Ion, HEATH, Ester, KOSJEK, Tina, KRIVOGRAD-KLEMENČIČ, Aleksandra, ODER, Martina, PETKOVŠEK, Martin, RAČKI, Nejc, RAVNIKAR, Maja, ŠARC, Andrej, ŠIROK, Brane, ZUPANC, Mojca, ŽITNIK, Miha, KOMPARE, Boris. Use of hydrodynamic cavitation in (waste)water treatment. Ultrasonics Sonochemistry, ISSN 1350-4177, 2016, vol. 29, str. 577-588, doi: 10.1016/j.ultsonch.2015.10.010.
  • RAČKI, Nejc, KRAMBERGER, Petra, STEYER, Andrej, GAŠPERŠIČ, Jernej, ŠTRANCAR, Aleš, RAVNIKAR, Maja, GUTIÉRREZ-AGUIRRE, Ion. Methacrylate monolith chromatography as a tool for waterborne virus removal. Journal of chromatography. A, ISSN 0021-9673, 2015, vol. 1381, str. 118-124, doi: 10.1016/j.chroma.2015.01.003.
  • RUŠČIĆ, Jelena, GUTIÉRREZ-AGUIRRE, Ion, TUŠEK-ŽNIDARIČ, Magda, KOLUNDŽIJA, S., SLANA, A., BARUT, Miloš, RAVNIKAR, Maja, KRAJAČIĆ, Mladen. A new application of monolithic supports: the separation of viruses from one another. Journal of chromatography. A, ISSN 0021-9673, 2015, vol. 1388, str. 69-78, doi: 10.1016/j.chroma.2015.01.097.
  • KUTNJAK, Denis, RUPAR, Matevž, GUTIÉRREZ-AGUIRRE, Ion, CURK, Tomaž, KREUZE, Jan F., RAVNIKAR, Maja. Deep sequencing of virus derived small interfering RNAs and RNA from viral particles shows highly similar mutational landscape of a plant virus population. Journal of virology, ISSN 0022-538X, 2015, vol. 89, no. 9, str. 4760-4769, doi: 10.1128/JVI.03685-14.
  • RUPAR, Matevž, FAUREZ, Florence, TRIBODET, Michel, GUTIÉRREZ-AGUIRRE, Ion, DELAUNAY, Agnes, GLAIS, Laurent, KRIŽNIK, Maja, DOBNIK, David, GRUDEN, Kristina, JACQUOT, Emmanuel, RAVNIKAR, Maja. Fluorescently tagged Potato virus Y: a versatile tool for functional analysis of plant-virus interactions. Molecular plant-microbe interactions, ISSN 0894-0282, 2015, vol. 28, no. 7, str. 739-750, doi: 10.1094/MPMI-07-14-0218-TA.
  • KOGOVŠEK, Polona, HODGETTS, Jennifer, HALL, J., PREZELJ, Nina, NIKOLIĆ, Petra, MEHLE, Nataša, LENARČIČ, Rok, ROTTER, Ana, DICKINSON, M., BOONHAM, Neil, DERMASTIA, Marina, RAVNIKAR, Maja. LAMP assay and rapid sample preparation method for on-site detection of flavescence dorée phytoplasma in grapevine. Plant Pathology, ISSN 0032-0862, 2015, vol. 64, no. 2, str. 286-296. http://onlinelibrary.wiley.com/doi/10.1111/ppa.12266/abstract;jsessionid=7850B082A5DCFD15057B22A9A431AFE1.f03t02, doi: 10.1111/ppa.12266.
  • RAČKI, Nejc, MORISSET, Dany, GUTIÉRREZ-AGUIRRE, Ion, RAVNIKAR, Maja. One-step RT-droplet digital PCR: a breakthrough in the quantification of waterborne RNA viruses. Analytical and bioanalytical chemistry, ISSN 1618-2642, 2014, vol. 406, issue 3, str. 661-667. doi: 10.1007/s00216-013-7476-y.
  • DREO, Tanja, PIRC, Manca, RAMŠAK, Živa, PAVŠIČ, Jernej, MILAVEC, Mojca, ŽEL, Jana, GRUDEN, Kristina. Optimising droplet digital PCR analysis approaches for detection and quantification of bacteria: a case study of fire blight and potato brown rot. Analytical and bioanalytical chemistry, ISSN 1618-2642, 2014, vol. 406, issue 26, str. 6513-6528, doi: 10.1007/s00216-014-8084-1.
  • GUTIÉRREZ-AGUIRRE, Ion, HODNIK, Vesna, GLAIS, Laurent, RUPAR, Matevž, JACQUOT, Emmanuel, ANDERLUH, Gregor, RAVNIKAR, Maja. Surface plasmon resonance for monitoring the interaction of Potato virus Y with monoclonal antibodies. Analytical biochemistry, ISSN 0003-2697, 2014, vol. 447, str. 74-81. doi: 10.1016/j.ab.2013.10.032.
  • MEHLE, Nataša, GUTIÉRREZ-AGUIRRE, Ion, PREZELJ, Nina, DELIĆ, Duška, VIDIC, Urška, RAVNIKAR, Maja. Survival and transmission of Potato virus Y, Pepino mosaic virus, and Potato spindle tuber viroid in water. Applied and environmental microbiology, ISSN 0099-2240, 2014, vol. 80, no. 4, str. 1455-1462. doi: 10.1128/AEM.03349-13.
  • MEHLE, Nataša, DREO, Tanja, JEFFRIES, C., RAVNIKAR, Maja. Descriptive assessment of uncertainties of qualitative real-time PCR for detection of plant pathogens and quality performance monitoring. Bulletin OEPP, ISSN 0250-8052, 2014, vol. 44, no. 3, str. 502-509, doi: 10.1111/epp.12166.
  • BÜHLMANN, Andreas, DREO, Tanja, REZZONICO, Fabio, POTHIER, Joël F., SMITS, T., RAVNIKAR, Maja, FREY, J., DUFFY, Brion. Phylogeography and population structure of the biologically invasive phytopathogen Erwinia amylovora inferred using minisatellites. Environmental microbiology, ISSN 1462-2912. [Print ed.], 2014, vol. 16, issue 7, str. 2112-2125. http://dx.doi.org/10.1111/1462-2920.12289, doi: 10.1111/1462-2920.12289.
  • RAČKI, Nejc, DREO, Tanja, GUTIÉRREZ-AGUIRRE, Ion, BLEJEC, Andrej, RAVNIKAR, Maja. Reverse transcriptase droplet digital PCR shows high resilience to PCR inhibitors from plant, soil and water samples. Plant methods, ISSN 1746-4811, Dec. 2014, vol. 10, str. 1-20, doi: 10.1186/s13007-014-0042-6.
  • LENARČIČ, Rok, MORISSET, Dany, PIRC, Manca, LLOP, Pablo, RAVNIKAR, Maja, DREO, Tanja. Loop-mediated isothermal amplification of specific endoglucanase gene sequence for detection of the bacterial wilt pathogen Ralstonia solanacearum. PloS one, ISSN 1932-6203, Apr. 2014, vol. 9, iss. 4, str. 1-8, ilustr., doi: 10.1371/journal.pone.0096027.
  • KUTNJAK, Denis, SILVESTRE, Rocio, CUELLAR, Wilmer J., PEREZ, Wilmer, MÜLLER, Giovanna, RAVNIKAR, Maja, KREUZE, Jan F. Complete genome sequences of new divergent potato virus X isolates and discrimination between strains in a mixed infection using small RNAs sequencing approach. Virus Research, ISSN 0168-1702. [Print ed.], 2014, vol. 191, str. 45-50. http://dx.doi.org/10.1016/j.virusres.2014.07.012, doi: 10.1016/j.virusres.2014.07.012.
  • STEYER, Andrej, GUTIÉRREZ-AGUIRRE, Ion, KOLENC, Marko, KOREN, Simon, KUTNJAK, Denis, POKORN, Marko, POLJŠAK-PRIJATELJ, Mateja, RAČKI, Nejc, RAVNIKAR, Maja, SAGADIN, Martin, FRATNIK STEYER, Adela, TOPLAK, Nataša. High similarity of novel orthoreovirus detected in a child hospitalized with acute gastroenteritis to mammalian orthoreoviruses found in bats in Europe. Journal of clinical microbiology, ISSN 0095-1137, 2013, vol. 51, no. 11, str. 3818-3825, doi: 10.1128/JCM.01531-13.
  • PREZELJ, Nina, NIKOLIĆ, Petra, GRUDEN, Kristina, RAVNIKAR, Maja, DERMASTIA, Marina. Spatiotemporal distribution of flavescence dorée phytoplasma in grapevine. Plant Pathology, ISSN 0032-0862, 2013, vol. 62, no. 4, str. 760-766. http://onlinelibrary.wiley.com/doi/10.1111/j.1365-3059.2012.02693.x/pdf, doi: 10.1111/j.1365-3059.2012.02693.x.
  • LENARČIČ, Rok, MORISSET, Dany, MEHLE, Nataša, RAVNIKAR, Maja. Fast real-time detection of Potato spindle tuber viroid by RT-LAMP. Plant Pathology, ISSN 0032-0862, 2013, vol. 62, issue 5, str. 1147-1156, doi: 10.1111/ppa.12017.
  • RUPAR, Matevž, RAVNIKAR, Maja, TUŠEK-ŽNIDARIČ, Magda, KRAMBERGER, Petra, GLAIS, Laurent, GUTIÉRREZ-AGUIRRE, Ion. Fast purification of the filamentous Potato virus Y using monolithic chromatographic supports. Journal of chromatography. A, ISSN 0021-9673, 2013, vol. 1272, str. 33-40, doi: 10.1016/j.chroma.2012.11.058.
  • DREO, Tanja, PIRC, Manca, RAVNIKAR, Maja. Real-time PCR, a method fit for detection and quantification of Erwinia amylovora. Trees, ISSN 0931-1890, 2012, vol. 26, no. 1, str. 165-178. http://dx.doi.org/10.1007/s00468-011-0654-7, doi: 10.1007/s00468-011-0654-7.
  • KOGOVŠEK, Polona, KLADNIK, Aleš, MLAKAR, Jana, TUŠEK-ŽNIDARIČ, Magda, DERMASTIA, Marina, RAVNIKAR, Maja, POMPE NOVAK, Maruša. Distribution of Potato virus Y in potato plant organs, tissues and cells. Phytopathology, ISSN 0031-949X. [Print ed.], 2011, vol. 101, no. 11, str. 1292-1300. http://dx.doi.org/10.1094/PHYTO-01-11-0020, doi: 10.1094/PHYTO-01-11-0020.
  • GUTIÉRREZ-AGUIRRE, Ion, STEYER, Andrej, BANJAC, Marko, KRAMBERGER, Petra, POLJŠAK-PRIJATELJ, Mateja, RAVNIKAR, Maja. On-site reverse transcription-quantitative polymerase chain reaction detection of rotaviruses concentrated from environmental water samples using methacrylate monolithic supports. In: GIESE, R. (edit.). Selected Papers of the 4th Summer School of Monolith Technology for Biochromatography, Bioconversion and Phase State Synthesis, Portorož, Slovenia, 30 May-02 June 2010, (Journal of Chromatography A, ISSN 0021-9673, Vol. 1218, no 17 (2011)). [S. l.]: Elsevier, cop. 2011, vol. 1218, no. 17, str. 2368-2373. doi: 10.1016/j.chroma.2010.10.048.
  • HANSSEN, Inge M., RAVNIKAR, Maja, et al. Seed transmission of Pepino mosaic virus in tomato. European journal of plant pathology, ISSN 0929-1873, 2010, vol. 126, no. 2, str. 145-152. http://dx.doi.org/10.1007/s10658-009-9528-x, doi: 10.1007/s10658-009-9528-x.
  • NIKOLIĆ, Petra, MEHLE, Nataša, GRUDEN, Kristina, RAVNIKAR, Maja, DERMASTIA, Marina. A panel of real-time PCR assays for specific detection of three phytoplasmas from the apple proliferation group. Molecular and cellular probes, ISSN 0890-8508, 2010, vol. 24, issue 5, str. 303-309. http://dx.doi.org/10.1016/j.mcp.2010.06.005, doi: 10.1016/j.mcp.2010.06.005.
  • HANSSEN, Inge M., GUTIÉRREZ-AGUIRRE, Ion, PAELEMAN, A., GOEN, K., WITTEMANS, L., LIEVENS, B., VANACHTER, A. C. R. C., RAVNIKAR, Maja, THOMMA, B. P. H. J. Cross-protection or enhanced symptom display in greenhouse tomato co-infected with different Pepino mosaic virus isolates. Plant Pathology, ISSN 0032-0862, 2010, vol. 59, no. 1, str. 13-21. http://dx.doi.org/10.1111/j.1365-3059.2009.02190.x, doi: 10.1111/j.1365-3059.2009.02190.x.

Review papers and book chapters:
    • ERJAVEC, Jana, KOS, Janko, RAVNIKAR, Maja, DREO, Tanja, SABOTIČ, Jerica. Proteins of higher fungi - from forest to application. Trends in biotechnology, ISSN 0167-7799. [Print ed.], 2012, vol. 30, issue 5, str. 259-273. http://dx.doi.org/10.1016/j.tibtech.2012.01.004, doi: 10.1016/j.tibtech.2012.01.004.
    • MEHLE, Nataša, RAVNIKAR, Maja. Plant viruses in aqueous environment: survival, water mediated transmission and detection. Water research, ISSN 0043-1354. [Print ed.], 2012, vol. 46, iss. 16, str. 4902-4917. http://dx.doi.org/10.1016/j.watres.2012.07.027, doi: 10.1016/j.watres.2012.07.027.
    • GUTIÉRREZ-AGUIRRE, Ion, RAČKI, Nejc, DREO, Tanja, RAVNIKAR, Maja. Droplet digital PCR for absolute quantification of pathogens. In: LACOMME, Christophe (edit.). Plant pathology: techniques and protocols, (Methods in molecular biology, ISSN 1064-3745, 1302). 2nd ed. New York: Humana Press, cop. 2015, str. 331-347, doi: 10.1007/978-1-4939-2620-6_24.
    • MEHLE, Nataša, NIKOLIĆ, Petra, RUPAR, Matevž, BOBEN, Jana, RAVNIKAR, Maja, DERMASTIA, Marina. Automated DNA extraction for large numbers of plant samples. In: DICKINSON, Matthew (edit.), HODGETTS, Jennifer (edit.). Phytoplasma: methods and protocols, (Methods in Molecular Biology, ISSN 1064-3745, vol. 938), (Springer Protocols). New York: Humana Press, 2013, str. 139-145. http://www.springer.com/us/book/9781627030885#
    • MEHLE, Nataša, PREZELJ, Nina, HREN, Matjaž, BOBEN, Jana, GRUDEN, Kristina, RAVNIKAR, Maja, DERMASTIA, Marina. A real-time PCR detection system for the bois noir and flavescence dorée phytoplasmas and quantification of the target DNA. In: DICKINSON, Matthew (edit.), HODGETTS, Jennifer (edit.). Phytoplasma: methods and protocols, (Methods in Molecular Biology, ISSN 1064-3745, vol. 938), (Springer Protocols). New York: Humana Press, 2013, str. 253-268. http://www.springer.com/us/book/9781627030885#
    • PIRC, Manca, RAVNIKAR, Maja, DREO, Tanja. Assesing DNA extraction methods for real-time PCR detection of quarantine plant pathogenic bacteria. In: ZHOU, Chunxu (edit.), LING, Xia (edit.). DNA binding and DNA extraction: methods, applications and limitations, (DNA and RNA). New York: Nova Biomedical, cop. 2011, str. 205-225. https://www.novapublishers.com/catalog/product_info.php?products_id=28239

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