Maja Ravnikar

• PhD Prof.
• Head of Department at National Institute of Biology

The presence of bacteria and viruses in freshwater represents a global health risk. The substantial spatial and temporal variability of microbes leads to difficulties in quantifying the risks associated with their presence in freshwater. Fine particles, including bacteria and viruses are transported and accumulated into shallow streambed (i.e., ben...

High-throughput sequencing (HTS) and sequence mining tools revolutionized virus detection and discovery in recent years and implementing them with classical plant virology techniques results to a powerful approach to characterize viruses. An example of a virus discovered through HTS is Solanum nigrum ilarvirus 1 (SnIV1) (Bromoviridae), which was re...

Plant viruses pose a significant threat to agriculture. Several are stable outside their hosts, can enter water bodies and remain infective for prolonged periods of time. Even though the quality of irrigation water is of increasing importance in the context of plant health, the presence of plant viruses in irrigation waters is understudied. In this...

Tomato brown rugose fruit virus (ToBRFV) has recently emerged as a major disease of tomatoes and peppers. ToBRFV is a seed- and contact-transmitted virus. In Slovenia, ToBRFV RNA was detected in samples of wastewater, river, and water used to irrigate plants. Even though the source of detected RNA could not be clearly established, this raised the q...

The advances in high-throughput sequencing (HTS) technologies and bioinformatic tools have provided new opportunities for virus and viroid discovery and diagnostics. Hence, new sequences of viral origin are being discovered and published at a previously unseen rate. Therefore, a collective effort was undertaken to write and propose a framework for...

Background In agroecosystems, viruses are well known to influence crop health and some cause phytosanitary and economic problems, but their diversity in non-crop plants and role outside the disease perspective is less known. Extensive virome explorations that include both crop and diverse weed plants are therefore needed to better understand roles...

High-throughput sequencing (HTS) and sequence mining tools revolutionized virus detection and discovery in recent years and implementing them with classical plant virology techniques results to a powerful approach to characterize viruses. An example of a virus discovered through HTS is Solanum nigrum ilarvirus 1 (SnIV1) (family Bromoviridae ), whic...

As a result of the COVID-19 pandemic, many new materials and masks came onto the market. To determine their suitability, several standards specify which properties to test, including bacterial filtration efficiency (BFE), while none describe how to determine viral filtration efficiency (VFE), a property that is particularly important in times of pa...

The fungus Phyllosticta citricarpa (McAlpine) Van der Aa is a quarantine pathogen in the EU and is of high economic importance in many parts of the world where favourable climate conditions drive the development of citrus black spot (CBS) disease. Disease symptoms include necrotic lesions on leaves and fruits. Low disease pressure can reduce crop m...

The SARS-CoV-2 pandemic has accelerated the development of virus concentration and molecular-based virus detection methods, monitoring systems and overall approach to epidemiology. Early into the pandemic, wastewater-based epidemiology started to be employed as a tool for tracking the virus transmission dynamics in a given area. The complexity of w...

The recent COVID-19 pandemic highlighted the importance of rapid and reliable pathogen detection. In the field of plant health accurate and timely detection is a corner stone for successful pest control. Plant pests can be diagnosed with a variety of tests, which may be developed by commercial companies or research institutions. The reliability of...

There are two types of interlaboratory comparison studies: proficiency tests, which aim at monitoring the proficiency of laboratories, and test performance studies (TPS), which aim at evaluating the performance of (a) specific test(s) and whether it (they) is (are) fit for purpose. This booklet covers only TPS organisation. A TPS is usually organis...

The organisation of a test performance study (TPS) involves different steps that are mostly sequential, but some may be conducted simultaneously. This chapter details the following: the steps regarding the selection of the tests to be validated; the selection of the laboratories to participate in the TPS; the preparation of the materials and the di...

Ensuring the reliability of diagnostic activities is an essential cornerstone of plant health strategies to reduce the risk of entry and spread of plant pests in a region and ultimately their impacts. Diagnostic tests should be validated to ensure that they are fit for purpose. Validation is usually done by diagnostic laboratories, although compani...

p>Appropriate statistical analysis of the validation data for diagnostic tests facilitates the evaluation of the performance criteria and increases the confidence in the conclusions drawn from these data. A comprehensive approach to analysing and reporting data from validation studies and inter-laboratory comparisons such as test performance studie...

As a result of the pandemic COVID -19 many new materials and masks came on the market. To determine their suitability, several standards specify which properties to test, including bacterial filtration efficiency (BFE), while none describe how to determine viral filtration efficiency (VFE), a property that is particularly important in times of pand...

As a result of the pandemic COVID -19 many new materials and masks came on the market. To determine their suitability, several standards specify which properties to test, including bacterial filtration efficiency (BFE), while none describe how to determine viral filtration efficiency (VFE), a property that is particularly important in times of pand...

Background In agroecosystems, viruses are well known to influence crop health and a few cause phytosanitary and economic problems, but their diversity in non-crop plants and role outside the disease perspective is less known. An extensive virome exploration that includes both crop and diverse weed plants is therefore needed to better understand rol...

Viruses influence plants in agroecosystems, where their pathogenic nature in crops is primarily studied. Within the same systems, their diversity in non-crop plants and role outside the disease perspective is less known. To better understand their diversity and ecology, we performed an extensive virome exploration focusing on tomatoes and diverse w...

The concept of environmental DNA (eDNA) utilizes nucleic acids of organisms directly from the environment. Recent breakthrough studies have successfully detected a wide spectrum of prokaryotic and eukaryotic eDNA from a variety of environments, ranging from ancient to modern, and from terrestrial to aquatic. With their diversity and ubiquity in nat...

High-throughput sequencing (HTS) has become an important tool for plant virus detection and discovery. Nanopore sequencing has been rapidly developing in the recent years and offers new possibilities for fast diagnostic applications of HTS. With this in mind, a study was completed, comparing the most established HTS platform (MiSeq benchtop sequenc...

Pepper (Capsicum annuum) and Tomato (Solanum lycopersicum) plants showing virus-like disease symptoms were collected in 2017, 2019, and 2020, in different parts of Slovenia (Supplementary Figure 1). Total RNA was extracted from leaf tissue of individual samples using RNeasy Plant Mini kit (Qiagen) and pooled in four composite samples as follows: 2...

Waterborne plant viruses can destroy entire crops, leading not only to high financial losses but also to food shortages. Potato virus Y (PVY) is the most important potato viral pathogen that can also affect other valuable crops. Recently, it has been confirmed that this virus is capable of infecting host plants via water, emphasizing the relevance...

Increasing research demonstrates the potential of donor-derived cell-free DNA (dd-cfDNA) as a biomarker for monitoring the health of various solid organ transplants. Several methods have been proposed for cfDNA analysis, including real-time PCR, digital PCR, and next generation sequencing-based approaches. We sought to revise the droplet digital PC...

Using high-throughput sequencing, we investigated viruses associated with tomatoes, and weeds surrounding tomato cultivation areas in Slovenia, where 436 samples were collected from 2019 to 2020. We pooled the extracted ribosomal RNA depleted total RNAs and sequence in total 69 sample pools using Illumina platform, and assembled viral genomes from...

Phytoviromics studies, or analysis of all viral sequences from plant samples, were intensified by a recent increase in accessibility of high throughput sequencing. This opened possibilities for novel discoveries, and wider understanding of the role of plant viruses across ecological scales. Here, we report the detection of tomato matilda virus (TMa...

Viruses cause a big fraction of economically important diseases in major crops, including tomato. In the past decade (2011–2020), many emerging or re-emerging tomato-infecting viruses were reported worldwide. In this period, 45 novel viral species were identified in tomato, 14 of which were discovered using high-throughput sequencing (HTS). In this...

Rivers transport pathogenic microorganisms (including fecal indicator bacteria and human enteric viruses) from point and non-point sources over long distances, posing a direct risk for human health. Yet, pathogens in surface waters can be deposited and transitorily immobilized and accumulated together with other fine particles in streambed sediment...

In this study, we investigated the virome of tomato, and of a variety of weed species surounding tomato production areas in Slovenia and Serbia. A total of 475 symptomatic or asymptomatic tomato, and weed samples were collected in selected tomato growing areas, during a two-year survey (2019-2020). Total RNA was extracted from each sample and were...

Tomato production worldwide is affected by numerous plant virus species. The early and accurate detection of viruses is a critical step for disease control. However, the simultaneous detection of the most known tomato viruses can be difficult because of the high number and diversity of tomato-infecting viruses. Here, we have identified four new vir...

p>VALITEST is an EU-funded project built to improve the reliability of diagnostic tests performed in plant health laboratories across the European and Mediterranean region. The project is undertaken by a consortium of 16 partners composed of research institutions, private companies (such as diagnostic kit providers), national plant protection organ...

Water scarcity is one of the greatest threats for human survival and quality of life, and this is increasingly contributing to the risk of human, animal and plant infections due to waterborne viruses. Viruses are transmitted through polluted water, where they can survive and cause infections even at low concentrations. Plant viruses from the genus...

Viral contamination is a major concern for biological products. Therefore, virus testing of raw materials and cells is essential for the safety of the final product. We used high-throughput sequencing to detect viral-like sequences in selected CHO cell lines. Our aim was to test various approaches of sample preparation, to establish a pipeline for...

Virusi v vodnih okoljih so bili v zadnjem desetletju intenzivno preučevani, vendar pa virusi v jamskih vodah ostajajo slabo opisani. Cilj raziskave je bila karakterizacija viroma vode vzorčene v različnih jamah, v katerih živijo tudi predstavniki vrste Proteus anguinus oziroma človeške ribice. Predstavniki dvoživk, ki živijo v tako občutljivih vodn...

Aquatic viruses have been extensively studied over the past decade, yet aspects of virus communities in cave waters remain poorly described. Our goal was to characterize viromes of cave water sampled in oligotrophic environments where Proteus anguinus, also known as olm or European cave salamander is present. Due to their dependence on, in many cas...

Potato virus Y (PVY), the type member of the genus Potyvirus (family Potyviridae), is the most widespread virus affecting potato and is included in the top five most economically detrimental plant viruses. Recently, the structure of the PVY virion has been determined by cryo-electron microscopy, which has opened the doors to functional studies that...

The concept of environmental DNA (eDNA) utilizes nucleic acids of organisms directly from the environment. Recent breakthrough studies have successfully detected a wide spectrum of prokaryotic and eukaryotic eDNA from a variety of environments, ranging from ancient to modern, and from terrestrial to aquatic. These numerous sources promise to establ...

Ralstonia solanaceraum is the quarantine plant pathogenic bacterium that causes bacterial wilt in over 200 host plants, which include economically important crops such as potato, tomato, tobacco, banana, and ginger. Alternative biological methods of disease control that can be used in integrated pest management are extensively studied. In search of...

Viruses represent one of the most important threats to agriculture. Several viral families include highly stable pathogens, which remain infective and can be transported long distances in water. The diversity of plant viruses in wastewater remains understudied; however, their potential impact is increasing with the increased irrigation usage of rec...

While one of the biggest problems we are facing today is water scarcity, enormous quantities of water are still being used in irrigation. If contaminated, this water can act as an effective pathway for the spread of disease-causing agents, like viruses. Here, we present a novel, environmentally friendly method known as cold atmospheric plasma for i...

One of the main challenges in the gene therapy viral vector development is to establish an optimized process for its large scale production. This requires optimization for upstream and downstream processes as well as methods that enable the step-by step analytical characterization of the virus, the results of which inform the iterative refinement o...

Potato virus Y (PVY) is among the most economically important plant pathogens. Using cryoelectron microscopy, we determined the near-atomic structure of PVY’s flexuous virions, revealing a previously unknown lumenal interplay between extended carboxyl-terminal regions of the coat protein units and viral RNA. RNA–coat protein interactions are crucia...

Infections with pathogenic Escherichia coli can lead to different animal- and human-associated diseases. E. coli infections are common in intensive poultry farming, and important economic losses can be expected during infections with avian pathogenic E. coli (APEC) strains followed by colibacillosis. Loop-mediated isothermal amplification (LAMP) as...

High-throughput sequencing has dramatically broadened the possibilities for plant virus research and diagnostics, enabling discovery of new or obscure viruses, and virus strains and rapid sequencing of their genomes. In this research, we employed high-throughput sequencing to discover a new virus infecting tomato, Henbane mosaic virus (Potyvirus, P...

Molluscum contagiosum virus (MCV) is the sole member of the Molluscipoxvirus genus and the causative agent of molluscum contagiosum (MC), a common skin disease. Although it is an important and frequent human pathogen, its genetic landscape and evolutionary history remain largely unknown. In this study, ten novel complete MCV genome sequences of the...

The use of acoustic cavitation for water and wastewater treatment (cleaning) is a well known procedure. Yet, the use of hydrodynamic cavitation as a sole technique or in combination with other techniques such as ultrasound has only recently been suggested and employed. In the first part of this paper a general overview of techniques that employ hy...

Recent advances in high-throughput sequencing technologies and bioinformatics have generated huge new opportunities for discovering and diagnosing plant viruses and viroids. Plant virology has undoubtedly benefited from these new methodologies, but at the same time, faces now substantial bottlenecks, namely the biological characterization of the ne...

With 113.4 ha, hazelnut occupies the 7th place among fruit species and represents 2.7% of the area of intensive fruit orchards in Slovenia. In two plantations located in eastern Slovenia that together cover 5 ha with around 1,200 trees planted 12 to 15 years ago, decline of some of the trees appeared in 2012. By October 2017, up to 15% of these tre...

en High‐throughput sequencing (HTS) technologies have revolutionized plant pest research and are now raising interest for plant pest diagnostics, with plant virus diagnostics at the forefront of development. However, the application of HTS in plant pest diagnostics raises important challenges that plant health regulators will have to address. Adapt...

RNA viruses have a great potential for high genetic variability and rapid evolution that is generated by mutation and recombination under selection pressure. This is also the case of Potato virus Y (PVY), which comprises a high diversity of different recombinant and non-recombinant strains. Consequently, it is hard to develop reverse transcription...

Members of the genus Dickeya are plant pathogens that mostly cause soft‐rot diseases. Diversity within the genus has led to the recent description of three novel species (i.e. a mainly clonal D. solani of economic consequence in potatoes, D. aquatica from water samples and D. fangzhongdai isolated from necrotic pear trees in China). However, multil...

Viruses exist in aquatic media and many of them use this media as transmission route. Next-generation sequencing (NGS) technologies have opened new doors in virus research, allowing also to reveal a hidden diversity of viral species in aquatic environments. Not surprisingly, many of the newly discovered viruses are found in environmental fresh and...

The localization of cysteine proteinase PLCP-2 was investigated in potato plants (Solanum tuberosum L.) cultivar Désirée by electron microscopy. Healthy and PVY^NTN infected potato plants were grown in vitro on media with or without a supplement of jasmonic acid. We had already shown that PLCP-2 is present in leaves, stems, tips of shoots...

Plant diagnostic laboratories are facing increasing demands for accreditation of their testing activities from both private and public organizations. For diagnostics, validating tests for the intended use is one of the key elements of accreditation. Thus far, validation has mainly focused on tests with a fixed scope, i.e. a test for organism X in m...

This is the first surveillance study using methacrylate monolithic supports to concentrate environmental coastal water samples, prior to molecular target detection by RT-qPCR. Rotaviruses (RoV) and Noroviruses (NoV) were monitored in a polluted area at the Bay of Koper (Gulf of Trieste, Northern Adriatic Sea) and at a nearby bathing area and mussel...

Viruses represent the most abundant and diverse of the biological entities in environmental waters, including the seas and probably also freshwater systems. They are important players in ecological networks in waters and influence global biochemical cycling and community composition dynamics. Among the many diverse viruses from terrestrial environm...

Electrophoresis 2017, 38, 2827–2836. DOI: 10.1002/elps.201700152 The cover picture shows the first chromatographic separation of mixed viruses (Virus 1, 2, 3) and virus strains (Virus 2a, 2b) from each other by using methacrylate monoliths and weak anion exchanger, as described in the review “Application of monolithic chromatographic supports in vi...

Next generation sequencing (NGS) technologies are becoming routinely employed in different fields of virus research. Different sequencing platforms and sample preparation approaches, in the laboratories worldwide, contributed to a revolution in detection and discovery of plant viruses and viroids. In this work, we are presenting the comparison of t...

Bacterial soft rots are a serious limitation to the production of orchids and others horticultural plants. Here, we report on the characterization of causative bacteria isolated from symptomatic Phalaenopsis orchids from a commercial production site. The most commonly isolated bacteria were identified as Dickeya spp. Partial sequencing of 16S rDNA,...

Soft rot pathogenic bacteria from the genus Dickeya cause severe economic losses in orchid nurseries worldwide, and there is no effective control currently available. In the last decade, the genus Dickeya has undergone multiple changes as multiple new taxa have been described, and just recently a new putative Dickeya species was reported. This stud...

As obligate parasites, plant viruses, require in order to survive, to be transmitted to another plant. Experimentally, viruses such as Potato virus Y (PVY) can be transmitted by mechanical means such as wounding and grafting. In its natural environment, PVY transmission is mediated by sap-feeding aphid vector, or vegetatively through propagated org...

RNA viruses are one of the fastest-evolving biological entities. Within their hosts, they exist as genetically diverse populations (i.e., viral mutant swarms), which are sculpted by different evolutionary mechanisms, such as mutation, natural selection, and genetic drift, and also the interactions between genetic variants within the mutant swarms....

The aim of this study was to accurately quantify the impact of hydrodynamic cavitation on the infectivity of bacteriophage MS2, a norovirus surrogate, and to develop a small scale reactor for testing the effect of hydrodynamic cavitation on human enteric viruses, which cannot be easily prepared in large quantities. For this purpose, 3 mL scale and...

Pollution of coastal waters is a major global problem, since these areas are often recipients of wastewaters and are used for recreation and for aquaculture. Microbial quality of waters has been traditionally assessed by monitoring faecal coliform bacteria (FC), however, several studies showed that the quantification of FC is insufficient to protec...

Key properties of monolithic chromatographic supports, make them suitable for separation and/or concentration of large biomolecules, especially virus particles and viral genomes. One by one, the studies that have been completed so far, contributed to the knowledge that monolith chromatography has hardly any limitation to be applied in virus researc...

Plant viral diseases are currently a major economic problem in agriculture throughout the world. The objective of the COST Action is to coordinate and raise the European capacity to apply Next Generation Sequencing (NGS) technologies for the study and diagnosis of viral diseases in vegetatively propagated plants, seeds and seedlings. NGS enables ra...

Waterborne and seed-borne Pepino mosaic virus (PepMV) and Potato spindle tuber viroid (PSTVd) pose serious threats to tomato production due to seed transmission and mechanical transmission, coupled with their long-term stability outside the host plant. Therefore, rapid and sensitive diagnostic procedures are needed to prevent the spread of these qu...

Recent advances in high-throughput sequencing technologies and bioinformatics have generated huge new opportunities for discovering and diagnosing plant viruses and viroids. Plant virology has undoubtedly benefited from these new methodologies, but at the same time, faces now substantial bottlenecks, namely the biological characterization of the ne...

As obligate parasites, plant viruses, require in order to survive, to be transmitted to another plant. Experimentally, viruses such as Potato virus Y (PVY) can be transmitted by mechanical means such as wounding and grafting. In its natural environment, PVY transmission is mediated by sap-feeding aphid vector, or vegetatively through propagated org...

A fluorescence-based real-time loop-mediated isothermal amplification (LAMP) assay for ‘Candidatus Phytoplasama solani’ (Bois noir phytoplasma; BNp) detection was developed and optimised for rapid laboratory and on-site BNp detection. This assay is highly specific, rapid and as sensitive as qPCR. It was validated according to European and Mediterra...

Currently, around 50% of the world's population lives in towns and cities within 100 km of the coast. Monitoring of viruses that are frequently present in contaminated coastal environments, such as rotavirus (RoV) and norovirus (NoV), which are also the major cause of human viral gastroenteritis, is essential to ensure the safe use of these water b...

The physical, chemical, and biological gradients in interfaces between fresh and marine waters often correspond to enhanced rates of biogeochemical processes and have an impacts on ecosystem responses. Seasonal changes in temperature, salinity, inorganic and organic nutrient concentrations, in relation to the microbial dynamic and community composi...

The use of acoustic cavitation for water and wastewater treatment (cleaning) is a well known procedure. Yet, the use of hydrodynamic cavitation as a sole technique or in combination with other techniques such as ultrasound has only recently been suggested and employed. In the first part of this paper a general overview of techniques that employ hyd...

The genus Dickeya contains bacteria causing soft rot of economically important crops and ornamental plants. Here, we report the draft genome sequences of two Dickeya sp. isolates from rotted leaves of Phalaenopsis orchids.

In total, 150 protein extracts from 94 different basidiomycete and ascomycete wild mushroom species were tested for antibacterial activity against the quarantine plant-pathogen bacterium Ralstonia solanacearum. In in vitro microtiter plate assays, 15 extracts with moderate to high antibacterial activities were identified: 11 completely inhibited ba...

Little is known about the natural occurrence and genetic variability of nepovirus large satellite RNA (satRNA). In this study, we screened 71 Grapevine fanleaf virus (GFLV) isolates mainly from Slovenia, but also from other countries in Europe and the USA, for the presence of satRNA, using a newly developed RT-PCR assay. GFLV satRNA (satGFLV) was d...