Effect of mixed infections of Potato virus Y and Pepino mosaic virus on amout of viruses and symptoms in different host plants

Mešane okužbe rastlin z virusi so pogoste tako v naravi kot v gojenih rastlinah. Zanimal nas je odnos med pri nas razširjenim virusom Y krompirja (PVY) in virusom mozaika pepina (PepMV), ki se v zadnjem času hitro širi po Evropi, v Sloveniji pa še ni prisoten. Virusa imata podoben nabor gostiteljev in teoretično lahko okužita isto rastlino. Ker se vplivi mešanih okužb razlikujejo med gostitelji, smo izbrali 3 vrste testnih rastlin: krompir (Solanum tuberosum), paradižnik (Solanum lycopersicum) in vrsto tobaka Nicotiana benthamiana. Na različnih sortah krompirja smo rastline inokulirali na 3 različne načine (samo s PVY, samo s PepMV in z obema), da bi ugotovili, če izbrane sorte sploh lahko okužimo s PepMV. Pri paradižniku in tobaku smo vzpostavili 8 različnih načinov inokulacije, saj nas je zanimalo ali na uspešnost okužbe in bolezenska znamenja vpliva tudi časovni potek inokulacije. 4 tedne po mehanski inokulaciji smo popisali bolezenska znamenja, ki so se med vrstami razlikovala, in izolirali celokupno RNA iz izbranih vzorcev. Količino virusa v teh vzorcih smo določili s pomočjo kvantiativne verižne reakcije s polimerazo, pri čemer smo v raziskavo vključili tudi primerjavo normalizacij s pomočjo referenčnega gena COX in količino celokupne RNA ter ugotovili, da sta zelo primerljivi. Nobene od 6 sort krompirja nismo uspeli okužiti s PepMV. Paradižnik in tobak sta se okužila ne glede na to ali je bila inokulacija sočasna ali zaporedna. V nekaterih primerih smo pri mešanih okužbah zaznali mnogo močnejša bolezenska znamenja. Med nekaterimi načini inokulacije smo opazili razlike v količini virusov.Mixed infections of plant viruses are common in nature as well as in agriculturally important plants. We were interested in relationship between Potato virus Y (PVY) which is wide-spread in Slovenia and Pepino mosaic virus (PepMV) which is spreading fast across Europe. These two viruses have partially overlapping host ranges and can theoretically appear in the same plant. Effects of mixed infections differ in different hosts, thus we used 3 test hosts: potato (Solanum tuberosum), tomato (Solanum lycopersicum) and tobacco species Nicotiana benthamiana. On different varieties of potato, we established 3 treatments (only PVY, only PepMV and mixed infection)on tomato and tobacco we esatblished 8 inoculation treatments, to investigate if different time course of infection has an effect on relationship bewtween viruses in mixed infection and on the developed disease symptoms. 4 weeks after mechanical inoculation we checked disease symptoms, which differed between inoculation treatments, and isolated total RNA from plant samples. We determined virus titer in samples using quantitative polymerase chain reaction, and normalised the amount of viruses in two different ways: using reference gene COX and the amount of total RNA measured with fluorometry. We confirmed that the normalization methods are comparable. None of the potato varieties was infected with PepMV but in tomato and tobacco we observed mixed infection in case of coinfection and superinfection. In mixed infected plants we observed the strongest disease symptoms. There were differences in virus accumulation between some of the different inoculation treatments

Development and validation of a new TaqMan real-time PCR for detection of Candidatus phytoplasma pruni

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 PCR detection system was developed for ‘Ca. P. pruni’ using TaqMan chemistry. This test was designed to detect ‘Ca. P. pruni’, by amplifying the species-specific secY gene. In addition, a test to amplify the group-specific 16S rRNA gene region was also developed. The performances of both tests were evaluated. The test that amplifies the secY gene provided reliable and quick detection of ‘Ca. P. pruni’. Using the newly developed and validated test, ‘Ca. P. pruni’ was not found in any of the 434 field samples collected from different plants species grown in different regions of Slovenia

Begomovirusi uvrščeni v točko 13.F. Priloge II Uredbe 2019/2072/EU

Virusi iz rodu begomovirusov (družina Geminiviridae) lahko okužijo številne vrste rastlin. Opisanih je več kot 400 različnih begomovirusov, od tega jih več kot 200 lahko okuži bučevke in/ali paradižnik. Vsi begomovirusi z izjemo Abutilon mosaic virus, Sweet potato leaf curl virus, Tomato leaf curl New Delhi virus, Tomato yellow leaf curl virus, Tomato yellow leaf curl Sardinia virus, Tomato yellow leaf curl Malaga virus in Tomato yellow leaf curl Axarquia virus, so uvrščeni v točki 13. F priloge II/A Uredbe 2019/2072/EU

Testing of begomoviruses capable of infecting tomatoes and plants of the family Cucurbitaceae by PCR

Detection of begomoviruses capable of infecting tomatoes and plants of the family Cucurbitaceae

Development of efficient methods and identification of barcodes for discriminating Grapevine flavescence dorée sensu-stricto from other related phytoplasmas and investigation of potential correlation between taxonomic identity and grapevine, alders and hazelnut plant hosts

Development of efficient methods and identification of barcodes for discriminating Grapevine flavescence dorée sensu-stricto from other related phytoplasmas and investigation of potential correlation between taxonomic identity and grapevine, alders and hazelnut plant host

Evaluation of methods and processes for robust monitoring of SARS-CoV-2 in wastewater

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 wastewater coupled with a lack of standardized methods led us to evaluate each step of the analysis individually and see which approach gave the most robust results for SARS-CoV-2 monitoring in wastewater. In this article, we present a step-by-step, retrospective view on the method development and implementation for the case of a pilot monitoring performed in Slovenia. We specifically address points regarding the thermal stability of the samples during storage, screening for the appropriate sample concentration and RNA extraction procedures and real-time PCR assay selection. Here, we show that the temperature and duration of the storage of the wastewater sample can have a varying impact on the detection depending on the structural form in which the SARS-CoV-2 target is present. We found that concentration and RNA extraction using Centricon filtration units coupled with Qiagen RNA extraction kit or direct RNA capture and extraction using semi-automated kit from Promega give the most optimal results out of the seven methods tested. Lastly, we confirm the use of N1 and N2 assays developed by the CDC (USA) as the best performing assays among four tested in combination with Fast Virus 1-mastermix. Data show a realistic overall process for method implementation as well as provide valuable information in regards to how different approaches in the analysis compare to one another under the specific conditions present in Slovenia during a pilot monitoring running from the beginning of the pandemic

Looking beyond virus detection in RNA sequencing data

High-throughput sequencing (HTS), more specifically RNA sequencing of plant tissues, has become an indispensable tool for plant virologists to detect and identify plant viruses. During the data analysis step, plant virologists typically compare the obtained sequences to reference virus databases. In this way, they are neglecting sequences without homologies to viruses, which usually represent the majority of sequencing reads. We hypothesized that traces of other pathogens might be detected in this unused sequence data. In the present study, our goal was to investigate whether total RNA-seq data, as generated for plant virus detection, is also suitable for the detection of other plant pathogens and pests. As proof of concept, we first analyzed RNA-seq datasets of plant materials with confirmed infections by cellular pathogens in order to check whether these non-viral pathogens could be easily detected in the data. Next, we set up a community effort to re-analyze existing Illumina RNA-seq datasets used for virus detection to check for the potential presence of non-viral pathogens or pests. In total, 101 datasets from 15 participants derived from 51 different plant species were re-analyzed, of which 37 were selected for subsequent in-depth analyses. In 29 of the 37 selected samples (78%), we found convincing traces of non-viral plant pathogens or pests. The organisms most frequently detected in this way were fungi (15/37 datasets), followed by insects (13/37) and mites (9/37). The presence of some of the detected pathogens was confirmed by independent (q)PCRs analyses. After communicating the results, 6 out of the 15 participants indicated that they were unaware of the possible presence of these pathogens in their sample(s). All participants indicated that they would broaden the scope of their bioinformatic analyses in future studies and thus check for the presence of non-viral pathogens. In conclusion, we show that it is possible to detect non-viral pathogens or pests from total RNA-seq datasets, in this case primarily fungi, insects, and mites. With this study, we hope to raise awareness among plant virologists that their data might be useful for fellow plant pathologists in other disciplines (mycology, entomology, bacteriology) as well

Epidemiology of flavescence dorée and hazelnut decline in Slovenia: geographical distribution and genetic diversity of the associated 16SrV phytoplasmas

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 range has recently been extended to hazelnuts. In Slovenia, hazelnut orchards are declining due to 16SrV phytoplasma infections, where large populations of the non-autochthonous leafhopper Orientus ishidae have been observed. To better characterise the phytoplasma-induced decline of hazelnut and possible transmission fluxes between these orchards and grapevine, genetic diversity of 16SrV phytoplasmas in grapevine, hazelnut and leafhoppers was monitored from 2017 to 2022. The nucleotide sequence analysis was based on the map gene. The most prevalent map genotype in grapevine in all wine-growing regions of Slovenia was M54, which accounted for 84 % of the 176 grapevines tested. Besides M54, other epidemic genotypes with lower frequency were M38 (6 %), M51 (3 %), M50 (2 %) and M122 (1 %). M38, M50 and M122 were also detected in infected cultivated hazelnuts and in specimens of O. ishidae leafhopper caught in declining hazelnut orchards. It suggests that this polyphagous vector could be responsible for phytoplasma infection in hazelnut orchards and possibly for some phytoplasma exchanges between hazelnuts and grapevine. We hereby describe new genotypes: M158 in grapevine as well as four never reported genotypes M159 to M162 in hazelnut. Of these four genotypes in hazelnut, one (M160) was also detected in O. ishidae. Analysis of additional genes of the new genotypes allowed us to assign them to the VmpA-III cluster, which corresponds to the 16SrV-C strains previously shown to be compatible with S. titanus transmission

Highly specific qPCR and amplicon sequencing method for detection of quarantine citrus pathogen Phyllosticta citricarpa applicable for air samples

The fungus Phyllosticta citricarpa 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 market-ability, while higher disease pressure can cause premature fruit drop, significantly increasing crop losses. The wind-dispersed spores of P. citricarpa are especially prob-lematic for rapid pathogen dispersal, but also provide an opportunity for early detec-tion of the disease spreading into a new area. In this study we have developed and validated a quantitative PCR (qPCR) assay based on the TEF1-α sequence. Specificity testing demonstrated that it is currently the only qPCR assay that does not cross- react with closely related Phyllosticta species. The assay is sensitive and can detect a single copy of the TEF1 gene in a reaction, it is highly repeatable and reproducible and can be used for testing of the sticky tapes from spore traps as well as citrus fruit sam-ples. High-throughput sequencing (HTS) of the DNA barcodes ITS1 and TEF1 was also explored for the detection and discrimination of P. citricarpa. The limit of detection of the HTS was 1000 spores on a daily spore trap tape. This study makes an important improvement to the diagnostics of the CBS and the methods developed can also be applied to improve the surveillance and early detection of the pathogen when linked to spore samplers in the field

Epidemiology of flavescence dorée and hazelnut decline in Slovenia: geographical distribution and genetic diversity of the associated 16SrV phytoplasmas

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 range has recently been extended to hazelnuts. In Slovenia, hazelnut orchards are declining due to 16SrV phytoplasma infections, where large populations of the non-autochthonous leafhopper Orientus ishidae have been observed. To better characterise the phytoplasma-induced decline of hazelnut and possible transmission fluxes between these orchards and grapevine, genetic diversity of 16SrV phytoplasmas in grapevine, hazelnut and leafhoppers was monitored from 2017 to 2022. The nucleotide sequence analysis was based on the map gene. The most prevalent map genotype in grapevine in all wine-growing regions of Slovenia was M54, which accounted for 84% of the 176 grapevines tested. Besides M54, other epidemic genotypes with lower frequency were M38 (6%), M51 (3%), M50 (2%) and M122 (1%). M38, M50 and M122 were also detected in infected cultivated hazelnuts and in specimens of O. ishidae leafhopper caught in declining hazelnut orchards. It suggests that this polyphagous vector could be responsible for phytoplasma infection in hazelnut orchards and possibly for some phytoplasma exchanges between hazelnuts and grapevine. We hereby describe new genotypes: M158 in grapevine as well as four never reported genotypes M159 to M162 in hazelnut. Of these four genotypes in hazelnut, one (M160) was also detected in O. ishidae . Analysis of additional genes of the new genotypes allowed us to assign them to the VmpA-III cluster, which corresponds to the 16SrV-C strains previously shown to be compatible with S. titanus transmission