Continental-scale Evaluation of Downy Birch Pollen Production: Estimating the Impacts of Global Change.

The high prevalence of hay fever in Europe has raised concerns about the implications of climate change-induced higher temperatures on pollen production. Our study focuses on downy birch pollen production across Europe by analyzing 456 catkins during 2019-2021 in 37 International Phenological Gardens (IPG) spanning a large geographic gradient. As IPGs rely on genetically identical plants, we were able to reduce the effects of genetic variability. We studied the potential association with masting behavior and three model specifications based on mean and quantile regression to assess the impact of meteorology (e.g., temperature and precipitation) and atmospheric gases (e.g., ozone (O3) and carbon-dioxide (CO2)) on pollen and catkin production, while controlling for tree age approximated by stem circumference. The results revealed a substantial geographic variability in mean pollen production, ranging from 1.9 to 2.5 million pollen grains per catkin. Regression analyses indicated that elevated average temperatures of the previous summer corresponded to increased pollen production, while higher O3 levels led to a reduction. Additionally, catkins number was positively influenced by preceding summer's temperature and precipitation but negatively by O3 levels. The investigation of quantile effects revealed that the impacts of mean temperature and O3 levels from the previous summer varied throughout the conditional response distribution. We found that temperature predominantly affected trees characterized by a high pollen production. We therefore suggest that birches modulate their physiological processes to optimize pollen production under varying temperature regimes. In turn, O3 levels negatively affected trees with pollen production levels exceeding the conditional median. We conclude that future temperature increase might exacerbate pollen production while other factors may modify (decrease in the case of O3 and amplify for precipitation) this effect. Our comprehensive study sheds light on potential impacts of climate change on downy birch pollen production, which is crucial for birch reproduction and human public health.

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.

Between Habitats: Transfer of Phytopathogenic Fungi along Transition Zones from Kettle Hole Edges to Wheat Ears.

Kettle holes are able to increase the soil and air humidity around them. Therefore, they create a perfect habitat for phytopathogenic fungi of the genera Fusarium and Alternaria to develop, sporulate, and immigrate into neighboring agricultural fields. In our study, we establish transects from the edges of different kettle holes and field edges up to 50 m into the fields to analyze the abundance and diversity of pathogenic fungi in these transition zones by culture-dependent and culture-independent methods. However, in 2019 and 2020, low precipitation and higher temperatures compared to the long-time average were measured, which led to limited infections of weeds in the transition zones with Fusarium and Alternaria. Therefore, the hypothesized significantly higher infection of wheat plants next to the kettle holes by a strong spread of fungal spores was not detected. Infestation patterns of Fusarium and Alternaria fungi on weeds and wheat ears were spatially different. In total, 9 different Fusarium species were found in the transition zone. The species diversity at kettle holes differed from 0 to 6 species. The trend toward increased dryness in the northeast German agricultural landscape and its impact on the changing severity of fungal infections is discussed.

Molecular Population Genetics of Aspen Mosaic-Associated Virus in Finland and Sweden.

Aspen mosaic-associated virus (AsMaV) is a newly identified Emaravirus, in the family Fimoviridae, Bunyavirales, associated with mosaic symptoms in aspen trees (Populus tremula). Aspen trees are widely distributed in Europe and understanding the population structure of AsMaV may aid in the development of better management strategies. The virus genome consists of five negative-sense single-stranded RNA (-ssRNA) molecules. To investigate the genetic diversity and population parameters of AsMaV, different regions of the genome were amplified and analyzed and full-length sequence of the divergent isolates were cloned and sequenced. The results show that RNA3 or nucleoprotein is a good representative for studying genetic diversity in AsMaV. Developed RT-PCR-RFLP was able to identify areas with a higher number of haplotypes and could be applied for screening the large number of samples. In general, AsMaV has a conserved genome and based on the phylogenetic studies, geographical structuring was observed in AsMaV isolates from Sweden and Finland, which could be attributed to founder effects. The genome of AsMaV is under purifying selection but not distributed uniformly on genomic RNAs. Distant AsMaV isolates displayed amino acid sequence variations compared to other isolates, and bioinformatic analysis predicted potential post-translational modification sites in some viral proteins.

Annual (2023) taxonomic update of RNA-directed RNA polymerase-encoding negative-sense RNA viruses (realm Riboviria: kingdom Orthornavirae: phylum Negarnaviricota).

In April 2023, 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 one new family, 14 new genera, and 140 new species. Two genera and 538 species were renamed. One species was moved, and four were abolished. This article presents the updated taxonomy of Negarnaviricota as now accepted by the ICTV.

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.

Pollen production of downy birch (Betula pubescens Ehrh.) along an altitudinal gradient in the European Alps.

High-altitude environments are highly susceptible to the effects of climate change. Thus, it is crucial to examine and understand the behaviour of specific plant traits along altitudinal gradients, which offer a real-life laboratory for analysing future impacts of climate change. The available information on how pollen production varies at different altitudes in mountainous areas is limited. In this study, we investigated pollen production of 17 birch (Betula pubescens Ehrh.) individuals along an altitudinal gradient in the European Alps. We sampled catkins at nine locations in the years 2020-2021 and monitored air temperatures. We investigated how birch pollen, flowers and inflorescences are produced in relation to thermal factors at various elevations. We found that mean pollen production of Betula pubescens Ehrh. varied between 0.4 and 8.3 million pollen grains per catkin. We did not observe any significant relationships between the studied reproductive metrics and altitude. However, minimum temperature of the previous summer was found to be significantly correlated to pollen (rs = 0.504, p = 0.039), flower (rs = 0.613, p = 0.009) and catkin (rs = 0.642, p = 0.005) production per volume unit of crown. Therefore, we suggest that temperature variability even at such small scales is very important for studying the response related to pollen production.

Betula pendula trees infected by birch idaeovirus and cherry leaf roll virus: Impacts of urbanisation and NO2 levels.

Viruses are frequently a microbial biocontaminant of healthy plants. The occurrence of the infection can be also due to environmental stress, like urbanisation, air pollution and increased air temperature, especially under the ongoing climate change. The aim of the present study was to investigate the hypothesis that worsened air quality and fewer green areas may favour the higher frequency of common viral infections, particularly in a common tree in temperate and continental climates, Betula pendula ROTH. We examined 18 trees, during the years 2015-2017, the same always for each year, in the region of Augsburg, Germany. By specific PCR, the frequency of two viruses, Cherry leaf roll virus (CLRV, genus Nepovirus, family Secoviridae), which is frequent in birch trees, and a novel virus tentatively named birch idaeovirus (BIV), which has been only recently described, were determined in pollen samples. The occurrence of the viruses was examined against the variables of urban index, air pollution (O3 and NO2), air temperature, and tree morphometrics (trunk perimeter, tree height, crown height and diameter). Generalized Non-linear models (binomial logit with backward stepwise removal of independent variables) were employed. During the study period, both CLRV and BIV were distributed widely throughout the investigated birch individuals. CLRV seemed to be rather cosmopolitan and was present independent of any abiotic factor. BIV's occurrence was mostly determined by higher values of the urban index and of NO2. Urban birch trees, located next to high-traffic roads with higher NO2 levels, are more likely to be infected by BIV. Increased environmental stress may lead to more plant viral infections. Here we suggest that this is particularly true for urban spaces, near high-traffic roads, where plants may be more stressed, and we recommend taking mitigation measures for controlling negative human interventions.

Identification of the Coding-Complete Genome Sequence of a Novel Cytorhabdovirus in Tilia cordata Showing Extensive Leaf Chloroses.

Here, we report the coding-complete sequence (14,152 nucleotides [nt]) of a novel cytorhabdovirus detected in Tilia cordata and tentatively named cytorhabdovirus tiliae. The assumed genome organization is 3'-N-P-P3-M-G-p6-p6'-L-5'. The N gene encodes the putative nucleoprotein (59.1 kDa), P encodes the phosphoprotein (34.7 kDa), P3 encodes the movement protein (23.1 kDa), M encodes the matrix protein (23.1 kDa), G encodes a glycoprotein (64.4 kDa), and L encodes the viral RNA polymerase (247 kDa). P6 and P6' are overlapping open reading frames (ORFs), which may encode gene products of 7.9 and 9.5 kDa, respectively, of unknown functions.

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.

Environmental Filtering Drives Fungal Phyllosphere Community in Regional Agricultural Landscapes.

To adapt to climate change, several agricultural strategies are currently being explored, including a shift in land use areas. Regional differences in microbiome composition and associated phytopathogens need to be considered. However, most empirical studies on differences in the crop microbiome focused on soil communities, with insufficient attention to the phyllosphere. In this study, we focused on wheat ears in three regions in northeastern Germany (Magdeburger Börde (MBB), Müncheberger Sander (MSA), Uckermärkisches Hügelland (UKH)) with different yield potentials, soil, and climatic conditions. To gain insight into the fungal community at different sites, we used a metabarcoding approach (ITS-NGS). Further, we examined the diversity and abundance of Fusarium and Alternaria using culture-dependent and culture-independent techniques. For each region, the prevalence of different orders rich in phytopathogenic fungi was determined: Sporidiobolales in MBB, Capnodiales and Pleosporales in MSA, and Hypocreales in UKH were identified as taxonomic biomarkers. Additionally, F. graminearum was found predominantly in UKH, whereas F. poae was more abundant in the other two regions. Environmental filters seem to be strong drivers of these differences, but we also discuss the possible effects of dispersal and interaction filters. Our results can guide shifting cultivation regions to be selected in the future concerning their phytopathogenic infection potential.

Infected grasses as inoculum for Fusarium infestation and mycotoxin accumulation in wheat with and without irrigation.

Grasses growing next to agricultural fields influence the Fusarium abundance, the species composition, and the mycotoxin accumulation of wheat plants, especially the field parts directly adjacent to grasses, are highly affected. Grasses are a more attractive and suitable habitat for Fusarium fungi compared to other arable weeds and occur at mostly every semi-natural landscape element (e.g., kettle holes, hedgerows, field-to-field-borders). In our study, we analyzed the ability of a highly Fusarium infected grass stripe (F. graminearum, F. culmorum, F. sporotrichioides) to infect an adjacent wheat field with these species. Results show that the primary inoculated Fusarium species were as well the dominant species isolated from the wheat field. Regarding transects originating from the grass stripe going into the field, the results demonstrate that wheat ears next to the infected grass stripe have a higher Fusarium abundance and furthermore show higher mycotoxin accumulation in the wheat kernels. This effect was highly promoted by irrigation. Therefore, grass stripes next to arable fields must be considered as reservoirs for fungal infections and as a source for a contamination with mycotoxins.

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.

Is Pollen Production of Birch Controlled by Genetics and Local Conditions?

Intraspecific genetic variation might limit the relevance of environmental factors on plant traits. For example, the interaction between genetics and (a-)biotic factors regulating pollen production are still poorly understood. In this study, we investigated pollen production of 28 birch (Betula pendula Roth) individuals in the years 2019−2021. We sampled catkins of eleven groups of genetically identical trees, which were partially topped, but of the same age and located in a seed plantation in southern Germany characterized by similar microclimatic conditions. Furthermore, we monitored environmental factors such as air temperature, characterized air quality (NO2, NOx and O3), and assessed potential solar radiation. We especially checked for differences between years as well as between and within clones and assessed the synchronicity of years with high/low pollen production. We present a robust mean for the pollen production of Betula pendula (1.66 million pollen grains per catkin). Our findings show temporal (H(2) = 46.29, p < 0.001) and clonal variations (H(4) = 21.44, p < 0.001) in pollen production. We conclude that synchronized high or low pollen production is not utterly site-specific and, in addition, not strictly dependent on genotypes. We suggest that appropriate clone selection based on application (seed plantation, urban planting) might be advantageous and encourage a long-term monitoring.

Determination of spider mite abundance in soil of field-grown cucumbers and in plants under predatory mite pressure in invasive infestations using HRM real-time PCR assay.

The two spotted spider mite, Tetranychus urticae Koch L. (Acari: Tetranychidae), is a plant pest that can lead to severe economic losses in open field cucumber cultivation. Between 2017 and 2019 we studied the abundance of spider mites in the soil to estimate the potential infestation pressure of soil colonizing spider mites. The spider mites were heterogeneously distributed in small concentrations in the soil. Soil colonizing spider mites did not affect spider mite abundance on plants and reversed. We observed that spider mite migration occurred primarily from the edge of the field adjacent to the weed strip. In 2020 and 2021, we investigated the efficacy of the predatory mite Neoseiulus californicus (McGregor) for suppressing spider mite hotspots in the cropland. We compared untreated spider mite hotspots with N. californicus treated hotspots and showed that a single release of predatory mites could result in a high level of control when spider mite infestation density was initially high. With this study, soil can be ruled out as a habitat for spider mites, and attention to spider mite pest control can be directed to plant infestations. The highly sensitive HRM real-time PCR assay was used for the quantification of the spider mites.

2021 Taxonomic update of phylum Negarnaviricota (Riboviria: Orthornavirae), including the large orders Bunyavirales and Mononegavirales.

In March 2021, 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 four families (Aliusviridae, Crepuscuviridae, Myriaviridae, and Natareviridae), three subfamilies (Alpharhabdovirinae, Betarhabdovirinae, and Gammarhabdovirinae), 42 genera, and 200 species. Thirty-nine species were renamed and/or moved and seven species were abolished. This article presents the updated taxonomy of Negarnaviricota as now accepted by the ICTV.

Towards the Forest Virome: High-Throughput Sequencing Drastically Expands Our Understanding on Virosphere in Temperate Forest Ecosystems.

Thanks to the development of HTS technologies, a vast amount of genetic information on the virosphere of temperate forests has been gained in the last seven years. To estimate the qualitative/quantitative impact of HTS on forest virology, we have summarized viruses affecting major tree/shrub species and their fungal associates, including fungal plant pathogens, mutualists and saprotrophs. The contribution of HTS methods is extremely significant for forest virology. Reviewed data on viral presence in holobionts allowed us a first attempt to address the role of virome in holobionts. Forest health is dependent on the variability of microorganisms interacting with the host tree/holobiont; symbiotic microbiota and pathogens engage in a permanent interplay, which influences the host. Through virus-virus interplays synergistic or antagonistic relations may evolve, which may drastically affect the health of the holobiont. Novel insights of these interplays may allow practical applications for forest plant protection based on endophytes and mycovirus biocontrol agents. The current analysis is conceived in light of the prospect that novel viruses may initiate an emergent infectious disease and that measures for the avoidance of future outbreaks in forests should be considered.

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).