Natasa Mehle

• PhD
• Research Associate at National Institute of Biology

In July 2024, a pooled leaf sample (D760/24) was collected from several plants of three watermelon cultivars (Citrullus lanatus cvs. Crimson Sweet, Asahi Miyako Hybrid F1 and Top Gun) grown in an open field (approx. 0.5ha) in Dombrava, Slovenia. The plants which were included in the pooled sample showed virus-like symptoms, such as leaf mosaic, wil...

Nanopore sequencing has proven to be a useful tool for the generic detection of plant viruses, especially in laboratories working with small number of samples. In this chapter, we describe the steps prior to library preparation as well as the library preparation itself, which we found provides comparable results to Illumina sequencing.

Viticulture is a traditional branch of agriculture in the Czech Republic. Grapevines (Vitis vinifera L.) are cultivated on more than 18,000 hectares in the wine-growing regions of Bohemia and South Moravia. South Moravia alone accounts for more than 90 % of the total wine-growing area in the country. Grapevine yellows are a complex of diseases asso...

Flavescence dorée (FD) phytoplasma from 16SrV-C and -D subgroups cause severe damage to grapevines throughout Europe. This phytoplasma is transmitted from grapevine to grapevine by the sap-sucking leafhopper Scaphoideus titanus. European black alder and clematis serve as perennial plant reservoirs for 16SrV-C phytoplasma strains, and their host ran...

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...

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...

p>High-throughput sequencing (HTS) is a powerful tool that enables the simultaneous detection and potential identification of any organisms present in a sample. The growing interest in the application of HTS technologies for routine diagnostics in plant health laboratories is triggering the development of guidelines on how to prepare laboratories f...

High-throughput sequencing (HTS) technologies have the potential to become one of the most significant advances in molecular diagnostics. Their use by researchers to detect and characterize plant pathogens and pests has been growing steadily for more than a decade and they are now envisioned as a routine diagnostic test to be deployed by plant pest...

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...

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...

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...

As the causal agent of the grapevine yellows disease Bois noir, ‘Candidatus Phytoplasma solani' has a major economic impact on grapevines. To improve the control of Bois noir, it is critical to understand the very complex epidemiological cycles that involve the multiple “Ca. P. solani” host plants and insect vectors, of which Hyalesthes obsoletus i...

Tobamovirus species represent a threat to solanaceous crops worldwide, due to their extreme stability and because they are seed borne. In particular, recent outbreaks of tomato brown rugose fruit virus in tomato and pepper crops led to the establishment of prompt control measures, and the need for reliable diagnosis was urged. Another member of the...

High-throughput sequencing (HTS) technologies have the potential to become one of the most significant advances in molecular diagnostics. Their use by researchers to detect and characterize plant pathogens and pests has been growing steadily for more than a decade and they are now envisioned as a routine diagnostic test to be deployed by plant pest...

Tobamovirus species represent a threat to solanaceous crops worldwide, due to their extreme stability and being seed-borne. In particular, recent outbreaks of tomato brown rugose fruit virus in tomato and pepper crops led to the establishment of prompt control measures, and the need for reliable diagnosis was urged. Another member of the genus, tom...

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...

A search for phytoplasma‐associated diseases was conducted for the first time in the main grapevine‐growing localities of the Dukagjini plain in Kosovo. A total of 144 samples were collected from grapevine cultivars displaying leaf yellowing, reddening, discolouration and irregular wood ripening, and analysed using nested and quantitative PCR assay...

High-throughput sequencing (HTS) technologies and bioinformatic analyses are of growing interest to be used as a routine diagnostic tool in the field of plant viruses. The reliability of HTS workflows from sample preparation to data analysis and results interpretation for plant virus detection and identification must be evaluated (verified and vali...

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...

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...

Phytoplasmas of the 16SrIII group are wide spread, and have a broad plant host range. Among these, ‘Candidatus phytoplasma pruni’ (‘Ca. P. pruni’; phytoplasmas of 16SrIII subgroup A) can cause serious diseases in Prunus species and ‘Ca. P. pruni’-related strains can infect other plant species, including grapevines. In this study, a new real-time PC...

In recent years, pepino mosaic virus (PepMV) has rapidly evolved from an emerging virus to an endemic pathogen, as it causes significant loses to tomato crops worldwide. At present, the main control strategy for prevention of PepMV disease in tomato production remains based on strict hygiene measures. To prevent damage caused by PepMV, cross-protec...

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...

In July 2017, a survey was conducted in a fig collection plot at Locorotondo (south of Italy) to investigate the possible presence of phytoplasmas in plants showing yellowing, deformed leaves, short internodes, mottling and mosaic. Samples were collected from symptomatic plants and tested by nested PCR assays using universal and specific primers to...

Hazelnut (Corylus avellana) is cultivated on 118 ha and ranks eighth in Slovenian fruit growing production, representing 2.8% of the total area of fruit plantations in the country. However, decline of some of the trees appeared in 2012 in two plantations located in eastern Slovenia. Together these orchards cover 5 ha, with around 1600 trees planted...

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...

Izvleček Bresti (Ulmus glabra Huds., U. minor Mill., U. laevis Pallas) so avtohtone drevesne vrste naših nižinskih in montanskih območij, kjer gradijo dobovo-jesenovo-brestove loge (npr. Querco roboris-Ulmetum laevis in druge). Bresti povečujejo estetsko vrednost gozda in so cenjeni zaradi kvalitetnega lesa. Predstavljajo približno 0,1% lesne zalo...

Izvleček Bresti (Ulmus glabra Huds., U. minor Mill., U. laevis Pallas) so avtohtone drevesne vrste naših nižinskih in montanskih območij, kjer gradijo dobovo-jesenovo-brestove loge (npr. Querco roboris-Ulmetum laevis in druge). Bresti povečujejo estetsko vrednost gozda in so cenjeni zaradi kvalitetnega lesa. Predstavljajo približno 0,1% lesne zalog...

Digital PCR-based methods, such as droplet digital PCR, are one of the best tools for determination of absolute nucleic-acid copy numbers. These techniques avoid the need for reference materials with known target concentrations. Compared to real-time PCR, they provide higher accuracy of quantification at low target concentrations, and have higher r...

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...

Phytoplasmas from the 16Sr-X apple proliferation (AP) group are quarantine species in Europe and causal agents of the most important diseases of fruit trees within the family Rosaceae, namely apple proliferation, European stone fruit yellows and pear decline. In this study, a detailed insight into the molecular diversity of isolates of two phytopla...

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...

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...

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...

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...

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...

Field elm (Ulmus minor Mill.) and wych elm (U. glabra Huds.) notably contribute to the biological diversity in lowland and montane forest ecosystems in Slovenia where natural presence of these species in forests constitute 0.1% of wood stock. In southwestern Slovenia (Beka, Kastelec, and Gabrovica) from May to October 2016, field elm trees exhibite...

This test performance study (TPS) was carried out on DNA samples from grapevine, clematis, fungi and bacteria to compare and validate loop-mediated isothermal amplification (LAMP) tests for detection of Grapevine flavescence dorée phytoplasma and 'Candidatus Phytoplasma solani' (Grapevine Bois noir phytoplasma). Two LAMP tests, for Grapevine flaves...

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...

Grapevine yellows diseases occur in most viticultural regions worldwide and they are associated with at least 24 different phytoplasmas. Their epidemiology is often different, and it can be strongly linked to the environment, particularly to factors such as the presence of alternative plant hosts and the biology of the insect vector (s). Sometimes...

Research into grapevine yellows diseases and their control relies on detection and identification of the phytoplasmas associated with them. Detection methods for phytoplasmas can be divided into four main categories: biological tests, microscopy techniques, and immunological and molecular approaches. The suitability of each of these methods for dif...

Many aspects of interactions between grapevine yellows phytoplasmas (GYP) and their grapevine hosts remain unclear. However, based on the available data, it appears that damage caused by GYP to their hosts is greater than might be expected from their relatively low titre in the phloem and their uneven distribution throughout the plant. Moreover, it...

Phytoplasmas were discovered almost 50 years ago and initially they were named mycoplasma-like organisms. These cell-wall lacking bacteria and members of the class Mollicutes inhabit plant phloem sieve elements and are transmitted and spread primarily by leafhoppers, plant hoppers and psyllids that feed on infected plants phloem. Phytoplasmas inter...

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...

Pepino mosaic virus (PepMV) has caused great concern in the greenhouse tomato industry after it was found causing a new disease in tomato in 1999. The objective of this paper is to investigate alternative hosts and compare important biological characteristics of the three PepMV strains occurring in Europe when tested under different environmental c...

In most cases, plant pest diagnostic methods are interpreted as providing qualitative data. However, although qualitative, it is still important to identify all the sources and components of uncertainty in the diagnostic process and to ensure that appropriate measures are taken to reduce the uncertainty and that the form of reporting the result doe...

A European quarantine organism ‘Candidatus Phytoplasma pyri’ causing devastating pear decline disease has been reported to affect pear trees in several European countries. In this study a multilocus sequence analysis was successfully used to gain detailed insight into the molecular diversity of thirty closely related ‘Candidatus Phytoplasma pyri’ i...

The first droplet-digital-PCR-based absolute quantification of “flavescence dorée” phytoplasma, agent of a quarantine phytoplasma yellows in grapevine was performed. Quantitative PCR that targets the secY gene was transferred to droplet digital PCR and used for absolute quantification of “flavescence dorée” phytoplasma, without the need for calibra...

Table S1 Hypergeometric distribution calculations for determination of the probability of FDp detection.

Table S4 Homogenization buffer selection and determination of the minimal number of berries needed for positive result.

Table S2 Comparison of ELISA buffer and Lysis buffer homogenization efficiency by qPCR.

Table S3 Checklist of MIQE précis with details on parameters associated with LAMP 23S rRNA assay.

In Europe the most devastating phytoplasma associated with grapevine yellows (GY) diseases is a quarantine pest flavescence dorée (FDp) from the 16SrV taxonomic group. The on-site detection of FDp with an affordable device would contribute to faster and more efficient decisions on the control measures for FDp. Therefore, a real-time isothermal LAMP...

Hydroponic systems and intensive irrigation are used widely in horticulture, and thus have the potential for rapid spread of water-transmissible plant pathogens. Numerous plant viruses have been reported in aqueous environments, although information on their survival and transmission is minimal, due mainly to the lack of effective detection methods...

This paper reports the development of a single tube, real-time, reverse transcription loop-mediated isothermal amplifi-cation (RT-LAMP) assay for detecting Potato spindle tuber viroid (PSTVd), one of the quarantine pathogens of potato in Europe and North America. The method enables detection of a broad range of PSTVd isolates, and is about 10 times...

The method described here is a rapid, total DNA extraction procedure applicable to a large number of plant samples requiring pathogen detection. The procedure combines a simple and quick homogenization step of crude extracts with DNA extraction based upon the binding of DNA to magnetic beads. DNA is purified in an automated process in which the mag...

The real-time PCR detection system for grapevine yellows phytoplasmas described here is composed of two assays for group-specific detection of flavescence dorée (FD) and bois noir (BN) phytoplasmas and a universal phytoplasma assay. It uses hydrolysis minor groove binder probes (TaqMan-MGB). The addition of an assay for amplification of plant DNA c...

In this chapter, we describe a real-time PCR detection system for fast, reliable, specific, and sensitive detection and discrimination of ‘Candidatus Phytoplasma mali’, ‘Ca. P. prunorum’, and ‘Ca. P. pyri’ from the 16SrX (apple proliferation-AP) group. These phytoplasmas are causal agents of fruit tree diseases within the Rosaceae family, namely ap...

Potato spindle tuber viroid (PSTVd) is the causal agent of a number of agriculturally important diseases. It is a single-stranded, circular and unencapsidated RNA molecule with only 356-360 nucleotides and no coding capacity. Because of its peculiar structural features, it is very stable ex vivo and it is easily transmitted mechanically by contamin...

The presence of plant viruses outside their plant host or insect vectors has not been studied intensively. This is due, in part, to the lack of effective detection methods that would enable their detection in difficult matrixes and in low titres, and support the search for unknown viruses. Recently, new and sensitive methods for detecting viruses h...

Many thrips are pests of commercial crops due to the damage they cause by feeding on developing flowers or vegetables. Thrips may also serve as vectors for plant diseases, such as tospoviruses. Their small size and predisposition towards enclosed places makes them difficult to detect by phytosanitary inspection. In this review, several methods avai...

Fruit species are affected by severe diseases associated with phytoplasmas. European stone fruit yellows (ESFY) phytoplasma, ('Candidatus Phytoplasma prunorum'), causes important disorders and decline of trees in many cultivated Prunus species, such as apricot (P. armeniaca), Japanese plum (P. salicina) and peach (P. persica). ESFY phytoplasma is t...

The 'flavescence dorée' (FD) phytoplasma is associated with the most devastating grapevine yellows disease 'flavescence dorée'. It belongs to the ribosomal group16SrV in which a high 16SrDNA identity among subgroup members exists. To investigate the genetic diversity of this phytoplasma in Slovenia a large analysis of strains in known and possible...

Phytoplasmas, as fastidious wall-less mollicutes, colonize phloem tissue of many plant species, including grapevine and fruit trees. They are transmitted between plants by vegetative propagation and by sap-sucking insect vectors, which enable spread of the diseases. In Slovenia in the period 2001-2010, 3,189 plant and 109 insect samples were collec...

In 2009 in Slovenia, an uncultivable cell-wall-less pathogenic bacterium of the class Mollicutes -a phytoplasma belonging to the 16SrV (Elm yellows) group was found in the mosaic leafhopper (Orientus ishidae) [Hemiptera, Cicadomorpha: Cicadellidae]. Molecular analyses showed that the phytoplasma isolates from O. ishidae resembled those of flavescen...

We report here on the development of combination of assays for fast, reliable, specific and sensitive detection and discrimination of 'Candidatus Phytoplasma mali', 'Ca. P. prunorum' and 'Ca. P. pyri' from the 16Sr-X (apple proliferation - AP) group. These phytoplasmas are causal agents of diseases of fruit trees within the family Rosaceae, namely...

Assays based on real-time PCR (TaqMan) that can detect a number of viroids in the genus Pospiviroid have been developed and evaluated. The assays are designed for detecting viroids from tomato leaf material but detection from other solanaceous hosts of these viroids has been confirmed. These methods have been validated by nine laboratories and comp...

In the period 2000-2008 more than 1300 fruit trees from different regions of Slovenia were tested for the quarantine phytoplasmas Apple proliferation (AP), Pear decline (PD), and European stone fruit yellows (ESFY). The majority of samples were collected within systematic official surveys, which was conducted for assessing the presence of these phy...

Over the last decade a new virus disease caused by Pepino mosaic virus (PepMV) has been threatening the tomato industry worldwide. Reliable detection is vitally important to aid disease control. Methods must be both sensitive and capable of detecting the range of distinct genotypes that have been identified. The development of five new reverse tran...

Over the last decade a new virus disease caused by Pepino mosaic virus (PepMV) has been threatening the tomato industry worldwide. Reliable detection is vitally important to aid disease control. Methods must be both sensitive and capable of detecting the range of distinct genotypes that have been identified. The development of five new reverse tran...

Stone fruit species are affected by severe disease caused by European stone fruit yellows phytoplasma (ESFY; 'Candidatus Phytoplasma prunorum'). ESFY phytoplasma is transmitted to the host plants of Prunus spp. by the vector Cacopsylla pruni. The disease is graft-transmissible as well. The occurence of ESFY phytoplasma was monitored from 2004 to 20...