Quantifying Plant Viruses: Evolution from Bioassay to Infectivity Dilution Curves along the Model of Tobamoviruses.

This review describes the development of the bioassay as a means of quantifying plant viruses, with particular attention to tobamovirus. It delves into various models used to establish a correlation between virus particle concentration and the number of induced local lesions (the infectivity dilution curve), including the Poisson, Furumoto and Mickey, Kleczkowski, Growth curve, and modified Poisson models. The parameters of each model are described, and their application or performance in the context of the tobacco mosaic virus is explored. This overview highlights the enduring value of the infectivity dilution curve in tobamovirus quantification, providing valuable insights for researchers or practitioners of bioassays and theoreticians of modeling.

Combining micro- and portable-XRF as a tool for fast identification of virus infections in plants: The case study of ASa-Virus in Fraxinus ornus L.

Plant viruses can affect micro- and macro-nutrients homeostasis in woody plants, with fluctuation in the concentration of some elements at the leaf level due to the pathogen activity and/or the plant physiological response to the infection.Leaves of Fraxinus ornus L. (flowering ash) were sampled for three consecutive years in the city of Hamburg (Germany), from both trees showing the typical symptoms of the ash shoestring associated virus infection (ASaV+) and healthy trees (ASaV-). Such leaves were analyzed by µ-XRF, using both laboratory and synchrotron X-ray sources, and large differences between symptomatic and not symptomatic leaves were observed: ASaV+ samples showed uneven element distribution and regions of the lamina with severe depletions of P, S, and Ca. Differently, K appeared more concentrated. Thus, 139 leaflets sampled from various healthy and infected ash trees over the three-year period were analyzed for K and Ca concentration with a portable XRF instrument. We found that the K:Ca concentration ratio was always significantly higher in ASaV+ samples, and this trend was verified for all the samplings over the tree years. We conclude that the K:Ca ratio parameter has potential in the frame of trendsetting diagnostics and could be used, together with visual symptoms, for a rapid, non-destructive, on-site and cheap indirect ASaV detection.

Applicability of Different Methods for Quantifying Virucidal Efficacy Using MENNO Florades and Tomato Brown Rugose Fruit Virus as an Example.

After entry of a quarantine/regulated pathogen, infected plants shall be destroyed, and the cultivated area (e.g., greenhouse) shall be disinfected. Therefore, the selection of an effective disinfectant plays an important role. With the availability of different methods for virus quantification, we investigated the application of quantitative ELISA (qELISA), RT-qPCR (reverse transcription-quantitative polymerase chain reaction), and bioassays for the quantification of disinfectant efficacy. Therefore, we estimated the titer reduction in tomato brown rugose fruit virus (ToBRFV), a regulated pathogen, in plant sap and on germ carriers after treatment with MENNO Florades 4% for 16 h. The virus load before and after the treatment was measured with the mentioned methods. The RT-qPCR and qELISA methods showed very low efficacy in the presence of the disinfectant. Although bioassays are time-consuming, need purified particles for establishing the quantification models, and are less sensitive than RT-qPCR, they were able to quantify the differences in virus titer in the presence/absence of disinfectant. Interestingly, the bioassays reached at least the lower limit sensitivity of a qELISA. By being less sensitive to the presence of the disinfectant, bioassays proved to be the only technique for the determination of the disinfectant efficacy against ToBRFV on different germ carriers as well as on virus-infected plant sap.

Comparison of Models for Quantification of Tomato Brown Rugose Fruit Virus Based on a Bioassay Using a Local Lesion Host.

Considering the availability of serological and molecular biological methods, the bioassay has been paled into insignificance, although it is the only experimental method that can be used to demonstrate the infectivity of a virus. We compared goodness-of-fit and predictability power of five models for the quantification of tomato brown rugose fruit virus (ToBRFV) based on local lesion assays: the Kleczkowski model, Furumoto and Mickey models I and II, the Gokhale and Bald model (growth curve model), and the modified Poisson model. For this purpose, mechanical inoculations onto Nicotiana tabacum L. cv. Xanthi nc and N. glutionosa L. with defined virus concentrations were first performed with half-leaf randomization in a Latin square design. Subsequently, models were implemented using Python software and fitted to the number of local lesions. All models could fit to the data for quantifying ToBRFV based on local lesions, among which the modified Poisson model had the best prediction of virus concentration in spike samples based on local lesions, although data of individual indicator plants showed variations. More accurate modeling was obtained from the test plant N. glutinosa than from N. tabacum cv. Xanthi nc. The position of the half-leaves on the test plants had no significant effect on the number of local lesions.

2022 taxonomic update of phylum Negarnaviricota (Riboviria: Orthornavirae), including the large orders Bunyavirales and Mononegavirales.

In March 2022, following the annual International Committee on Taxonomy of Viruses (ICTV) ratification vote on newly proposed taxa, the phylum Negarnaviricota was amended and emended. The phylum was expanded by two new families (bunyaviral Discoviridae and Tulasviridae), 41 new genera, and 98 new species. Three hundred forty-nine species were renamed and/or moved. The accidentally misspelled names of seven species were corrected. This article presents the updated taxonomy of Negarnaviricota as now accepted by the ICTV.

A novel emaravirus comprising five RNA segments is associated with ringspot disease in oak.

We report the complete nucleotide sequence of the genome of a novel virus in ringspot-diseased common oak (Quercus robur L.). The newly identified pathogen is associated with leaf symptoms such as mottle, chlorotic spots and ringspots on diseased trees. High-throughput sequencing (HTS, Illumina RNASeq) was used to explore the virome of a ringspot-diseased oak that had chlorotic ringspots of suspected viral origin on leaves for several years. Bioinformatic analysis of the HTS dataset followed by RT-PCR enabled us to determine complete sequences of four RNA genome segments of a novel virus. These sequences showed high similarity to members of the genus Emaravirus, which includes segmented negative-stranded RNA viruses of economic importance. To verify the ends of each RNA, we conducted rapid amplification of cDNA ends (RACE). We identified an additional genome segment (RNA 5) by RT-PCR using a genus-specific primer (PDAP213) to the conserved 3´ and 5´termini in order to amplify full-length genome segments. RNA 5 encodes a 21-kDa protein that is homologous to the silencing suppressor P8 of High Plains wheat mosaic virus. The five viral RNAs were consistently detected by RT-PCR in ringspot-diseased oaks in Germany, Sweden, and Norway. We conclude that the virus represents a new member of the genus Emaravirus affecting oaks in Germany and in Scandinavia, and we propose the name "common oak ringspot-associated emaravirus" (CORaV).

Kinetics of inactivation and dilution effects on the mass balance of fungal phytopathogens in anaerobic digesters.

Knowledge of fate and behavior of plant pathogens in the biogas production chain is limited and hampers the estimation and evaluation of the potential phytosanitary risk if digestate is spread on arable land as a fertilizer. Therefore, simulation is an appropriate tool to demonstrate the effects which influence the steady state of pathogen infected plant material in both digesters and digestate. Simple approaches of kinetics of inactivation and mass balances of infected material were carried out considering single-step as well as two-step digestion. The simulation revealed a very fast to fast reduction of infected material after a singular feeding, reaching a cutback to less than 1% of input within 4 days even for D90-values of 68 h. Steady state mass balances below input rate could be calculated with D90-values of less than 2 h at a continuous hourly feeding. At higher D90-values steady state mass balances exceed the input rate but are still clearly below the sum of input mass. Dilution further decreases mass balances to values 10(-5) to 10(-6) Mg m(-3) for first-step digestion and 10(-8) to 10(-9) for second-step.

Impact of Piriformospora indica on tomato growth and on interaction with fungal and viral pathogens.

Piriformospora indica is a root endophytic fungus with plant-promoting properties in numerous plant species and induces resistance against root and shoot pathogens in barley, wheat, and Arabidopsis. A study over several years showed that the endophyte P. indica colonised the roots of the most consumed vegetable crop tomato. P. indica improved the growth of tomato resulting in increased biomass of leaves by up to 20%. Limitation of disease severity caused by Verticillium dahliae by more than 30% was observed on tomato plants colonised by the endophyte. Further experiments were carried out in hydroponic cultures which are commonly used for the indoor production of tomatoes in central Europe. After adaptation of inoculation techniques (inoculum density, plant stage), it was shown that P. indica influences the concentration of Pepino mosaic virus in tomato shoots. The outcome of the interaction seems to be affected by light intensity. Most importantly, the endophyte increases tomato fruit biomass in hydroponic culture concerning fresh weight (up to 100%) and dry matter content (up to 20%). Hence, P. indica represents a suitable growth promoting endophyte for tomato which can be applied in production systems of this important vegetable plant not only in soil, but also in hydroponic cultures.