Correlation between airborne pollen data and the risk of tick-borne encephalitis in northern Italy.

Tick-borne encephalitis (TBE) is caused by a flavivirus that infects animals including humans. In Europe, the TBE virus circulates enzootically in natural foci among ticks and rodent hosts. The abundance of ticks depends on the abundance of rodent hosts, which in turn depends on the availability of food resources, such as tree seeds. Trees can exhibit large inter-annual fluctuations in seed production (masting), which influences the abundance of rodents the following year, and the abundance of nymphal ticks two years later. Thus, the biology of this system predicts a 2-year time lag between masting and the incidence of tick-borne diseases such as TBE. As airborne pollen abundance is related to masting, we investigated whether inter-annual variation in pollen load could be directly correlated with inter-annual variation in the incidence of TBE in human populations with a 2-year time lag. We focused our study on the province of Trento (northern Italy), where 206 TBE cases were notified between 1992 and 2020. We tested the relationship between TBE incidence and pollen load collected from 1989 to 2020 for 7 different tree species common in our study area. Through univariate analysis we found that the pollen quantities recorded two years prior for two tree species, hop-hornbeam (Ostrya carpinifolia) and downy oak (Quercus pubescens), were positively correlated with TBE emergence (R2 = 0.2) while a multivariate model with both tree species better explained the variation in annual TBE incidence (R2 = 0.34). To the best of our knowledge, this is the first attempt at quantifying the correlation between pollen quantities and the incidence of TBE in human populations. As pollen loads are collected by widespread aerobiological networks using standardized procedures, our study could be easily replicated to test their potential as early warning system for TBE and other tick-borne diseases.

A simple linear model for estimating ozone AOT40 at forest sites from raw passive sampling data.

A rapid, empirical method is described for estimating weekly AOT40 from ozone concentrations measured with passive samplers at forest sites. The method is based on linear regression and was developed after three years of measurements in Trentino (northern Italy). It was tested against an independent set of data from passive sampler sites across Italy. It provides good weekly estimates compared with those measured by conventional monitors (0.85 ≤R(2)≤ 0.970; 97 ≤ RMSE ≤ 302). Estimates obtained using passive sampling at forest sites are comparable to those obtained by another estimation method based on modelling hourly concentrations (R(2) = 0.94; 131 ≤ RMSE ≤ 351). Regression coefficients of passive sampling are similar to those obtained with conventional monitors at forest sites. Testing against an independent dataset generated by passive sampling provided similar results (0.86 ≤R(2)≤ 0.99; 65 ≤ RMSE ≤ 478). Errors tend to accumulate when weekly AOT40 estimates are summed to obtain the total AOT40 over the May-July period, and the median deviation between the two estimation methods based on passive sampling is 11%. The method proposed does not require any assumptions, complex calculation or modelling technique, and can be useful when other estimation methods are not feasible, either in principle or in practice. However, the method is not useful when estimates of hourly concentrations are of interest.

Leaf trait plasticity and site-specific environmental variability modulate the severity of visible foliar ozone symptoms in Viburnum lantana.

The assessment of Visible Foliar Symptoms (VFS) is commonly adopted by forest monitoring programs to evaluate ozone impact on vegetation. The occurrence of ozone VFS may differ among individuals of the same species at the same site, and within leaves of the same individual. The aim of this study was to identify site and plant characteristics as well as functional leaf traits associated with the occurrence and severity of VFS in Viburnum lantana (an ozone-sensitive species) and at the scale of an individual site. V. lantana plants growing at one site of the ViburNeT monitoring network (Trentino, North Italy) experiencing high ozone levels were surveyed in relation to 1) sun exposure, 2) shading effect from neighbor vegetation, 3) plant height and 4) presence and severity of VFS. Leaves from three different sections of each plant were subjected to a phenotypic characterization of leaf area, dry weight, specific leaf area (SLA), chlorophyll content (ChlSPAD), percentage of VFS, and adaxial and abaxial trichome density (Tr). We showed that plants at high irradiation levels had significantly lower SLA (p<0.05), higher Tr (p<0.01) and greater ChlSPAD (p<0.01) when compared to shaded and/or west- and north-exposed plants, thus indicating a strong influence of site-specific characteristics on leaf trait plasticity. Similar differences were observed for taller vs. shorter plants and apical vs. basal branches (p<0.05). Ozone-induced VFS at leaf level were associated with lower SLA (p<0.001) and higher Tr in the abaxial leaf surface (p<0.05). Both leaf traits showed significant differences also within the south and east exposed plant category, thus suggesting the increase in leaf thickness and Tr as a potential adaptive strategy under multiple stress conditions. Our results provide evidence of a strong relationship between VFS, leaf traits and site-specific variables, offering new insights for interpreting data on the impact of ozone on vegetation.

Integration of reference data from different Rapid-E devices supports automatic pollen detection in more locations.

Pollen is the most common cause of seasonal allergies, affecting over 33 % of the European population, even when considering only grasses. Informing the population and clinicians in real-time about the actual presence of pollen in the atmosphere is essential to reduce its harmful health and economic impact. Thus, there is a growing network of automatic particle analysers, and the reproducibility and transferability of implemented models are recommended since a reference dataset for local pollen of interest needs to be collected for each device to classify pollen, which is complex and time-consuming. Therefore, it would be beneficial to incorporate the reference dataset collected from other devices in different locations. However, it must be considered that laser-induced data are prone to device-specific noise due to laser and detector sensibility. This study collected data from two Rapid-E bioaerosol identifiers in Serbia and Italy and implemented a multi-modal convolutional neural network for pollen classification. We showed that models lost their performance when trained on data from one and tested on another device, not only in terms of the recognition ability but also in comparison with the manual measurements from Hirst-type traps. To enable pollen classification with just one model in both study locations, we first included the missing pollen classes in the dataset from the other study location, but it showed poor results, implying that data of one pollen class from different devices are more different than data of different pollen classes from one device. Combining all available reference data in a single model enabled the classification of a higher number of pollen classes in both study locations. Finally, we implemented a domain adaptation method, which improved the recognition ability and the correlations of transferred models only for several pollen classes.

A pilot study of eDNA metabarcoding to estimate plant biodiversity by an alpine glacier core (Adamello glacier, North Italy).

Current biodiversity loss is a major concern and thus biodiversity assessment of modern ecosystems is compelling and needs to be contextualized on a longer timescale. High Throughput Sequencing (HTS) is progressively becoming a major source of data on biodiversity time series. In this multi proxy study, we tested, for the first time, the potential of HTS to estimate plant biodiversity archived in the surface layers of a temperate alpine glacier, amplifying the trnL barcode for vascular plants from eDNA of firn samples. A 573 cm long core was drilled by the Adamello glacier and cut into sections; produced samples were analyzed for physical properties, stable isotope ratio, and plant biodiversity by eDNA metabarcoding and conventional light microscopy analysis. Results highlighted the presence of pollen and plant remains within the distinct layers of snow, firn and ice. While stable isotope ratio showed a scarcely informative pattern, DNA metabarcoding described distinct plant species composition among the different samples, with a broad taxonomic representation of the biodiversity of the catchment area and a high-ranking resolution. New knowledge on climate and plant biodiversity changes of large catchment areas can be obtained by this novel approach, relevant for future estimates of climate change effects.

Plant biodiversity assessment through pollen DNA metabarcoding in Natura 2000 habitats (Italian Alps).

Monitoring biodiversity is of increasing importance in natural ecosystems. Metabarcoding can be used as a powerful molecular tool to complement traditional biodiversity monitoring, as total environmental DNA can be analyzed from complex samples containing DNA of different origin. The aim of this research was to demonstrate the potential of pollen DNA metabarcoding using the chloroplast trnL partial gene sequencing to characterize plant biodiversity. Collecting airborne biological particles with gravimetric Tauber traps in four Natura 2000 habitats within the Natural Park of Paneveggio Pale di San Martino (Italian Alps), at three-time intervals in 1 year, metabarcoding identified 68 taxa belonging to 32 local plant families. Metabarcoding could identify with finer taxonomic resolution almost all non-rare families found by conventional light microscopy concurrently applied. However, compared to microscopy quantitative results, Poaceae, Betulaceae, and Oleaceae were found to contribute to a lesser extent to the plant biodiversity and Pinaceae were more represented. Temporal changes detected by metabarcoding matched the features of each pollen season, as defined by aerobiological studies running in parallel, and spatial heterogeneity was revealed between sites. Our results showcase that pollen metabarcoding is a promising approach in detecting plant species composition which could provide support to continuous monitoring required in Natura 2000 habitats for biodiversity conservation.

Responsiveness of Viburnum lantana L. to tropospheric ozone: field evidence under contrasting site conditions in Trentino, northern Italy.

Specific visible foliar injuries were demonstrated to occur on plants of Viburnum lantana L. (wayfaring tree) when exposed to ozone in open-top chamber experiments. However, although evidence of visible injury was reported even for native plants, no comprehensive testing has been carried out under real field conditions. Thus, the extent to which V. lantana may match the requirements to be used as an in situ bioindicator is not fully known. To investigate the actual responsiveness of native V. lantana plants to ozone under field condition, two 1 × 1 km quadrates (named "Margone" and "Lasino"), for which the occurrence of different ozone levels was known, were considered. There, a fully randomized design was adopted to ensure within-quadrate replications and to select V. lantana plants. Measurements confirmed different exposure levels (Margone, Accumulated ozone concentrations Over a Threshold of 40 ppb h (AOT40): 31 952 ppb h; Lasino, AOT40: 23 259 ppb h). Ozone visible foliar symptoms (i) matched the known symptomatology, (ii) were easy to be identified, (iii) confirmed by microscopical validation, and (iv) observed at both quadrates. However, higher frequency of symptoms, earlier date of onset and faster development occurred at the quadrate with the highest ozone exposure (Margone), although not always proportionally with the difference in ozone exposure. This may be partly due to inherent covariation of environmental variables (higher ozone exposure occurred at the sites with higher relative humidity and cooler air temperature, a set of conditions that may promote ozone uptake), and partly due to a set of (unmeasured) other factors that may cause additional oxidative stress to plants. Implications for biomonitoring are discussed.

Sampling bias and sampling errors in pollen counting in aerobiological monitoring in Italy.

Information about airborne pollen concentration is of concern for health authorities across Europe. The reliability of data estimates depends on the accuracy and precision of pollen counts. In Italy, pollen counts are carried out on slides for microscopic evaluation and are regulated by the national Standard UNI 11108:2004. Our results showed that counts performed according to the Italian standard may result in a significant bias in the number of pollen grains counted and this will have an impact on final estimates of pollen concentration. For the same sample size, confidence intervals vary in relation to pollen abundance, either in terms of number of grains or of number of species. The sample size suggested by the standard (20% of the target surface) may result in errors in pollen counts ranging from 7-55% of the mean value, and in missing 22-54% of the taxa present on the slide.

Ambient levels of nitrogen dioxide (NO2) may reduce pollen viability in Austrian pine (Pinus nigra Arnold) trees--correlative evidence from a field study.

A fully randomized sampling design was adopted to test whether pollen viability of Austrian pine (Pinus nigra Arnold) was impacted by NO(2) pollution. Spatial strata (500500 m each) with high (41.9-44.6 microg m(-3)) and low (15.4-21.0 microg m(-3)) NO(2) were selected from a defined population in a small area (236.5 km(2), <200 m range in elevation) in Northern Italy. Pollen viability was measured by means of the Tetrazolium (TTC) test. Analysis of variance by means of a generalised linear model showed that NO(2) was a significant factor (P=0.0425) affecting pollen viability. Within the treatment, no significant differences were detected among replicates. Within each replicate, sampling unit data were significantly different (P=0.000) and this suggested some improvement in the applied sampling design was needed. Pollen viability was significantly related to pollen germination (P<0.01) and tube length (P<0.01). This suggested a possible impact of NO(2) on the regeneration of Austrian pine in polluted environments.

In search for evidence: combining ad hoc survey, monitoring, and modeling to estimate the potential and actual impact of ground level ozone on forests in Trentino (Northern Italy).

A 5-year project was carried out over the period 2007-2011 to estimate the potential and actual ozone effect on forests in Trentino, Northern Italy (6207 km2) (Ozone EFFORT). The objective was to provide explicit answers to three main questions: (i) is there a potential risk placed by ozone to vegetation? (ii) are there specific ozone symptoms on vegetation, and are they related to ozone levels? (iii) are there ozone-related effects on forest health and growth? Different methods and techniques were adopted as follows: monitoring ozone levels, ad hoc field survey for symptoms on vegetation and chlorophyll-related measurements, modeling to upscale ozone measurements, ozone flux estimation, statistical analysis, and modeling to detect whether a significant effect attributable to ozone exists. Ozone effects were assessed on an ad hoc-introduced bioindicator, on spontaneous woody species, and on forest trees. As for question (i), the different ozone-risk critical levels for both exposure and stomatal flux were largely exceeded in Trentino, evidencing a potentially critical situation for vegetation. As for question (ii), specific ozone foliar symptoms related to ozone exposure levels were observed on the introduced supersensitive Nicotiana tabacum L. cv Bel-W3 and on the spontaneous, ozone-sensitive Viburnum lantana L., but not on other 33 species surveyed in the field studies. Regarding question (iii), statistical analyses on forest health (in terms of defoliation) and growth (in terms of basal area increment) measured at 15 forest monitoring plots and tree rings (at one site) revealed no significant relationship with ozone exposure and flux. Instead, a set of factors related to biotic and abiotic causes, foliar nutrients, age, and site were identified as the main drivers of forest health and growth. In conclusion, while ozone levels and fluxes in the investigated region were much higher than current critical levels, evidence of impact on vegetation-and on forest trees in particular-was limited.

Scarce evidence of ozone effect on recent health and productivity of alpine forests-a case study in Trentino, N. Italy.

We investigated the significance of tropospheric ozone as a factor explaining recent tree health (in terms of defoliation) and productivity (in terms of basal area increment, BAI) in 15 ICP Forests level I and one level II plots in alpine forests in Trentino (N. Italy). Mean daily ozone summer concentrations varied between 30 and 72 parts per billion (ppb) leading to large exceedance of concentration-based critical levels set to protect forest trees. Phytoxic ozone dose (POD0) estimated at the level II plot over the period 1996-2009 was 31-61 mmol m-2 projected leaf area (PLA). The role of ozone was investigated taking into account other site and environmental factors. Simple linear regression, multiple linear regression (MLR, to study mean periodical defoliation and mean periodical BAI), and linear mixed models (LMM, to study annual defoliation data) were used. Our findings suggest that-regardless of the metric adopted-tropospheric ozone is not a significant factor in explaining recent status and trends of defoliation and BAI in the alpine region examined. Both defoliation and BAI are in turn driven by biotic/abiotic damage, nutritional status, DBH (assumed as a proxy for age), and site characteristics. These results contrast with available ozone-growth dose response relationships (DRRs) and other observational studies. This may be due to a variety of concurrent reasons: (i) DRRs developed for individual saplings under controlled condition are not necessarily valid for population of mature trees into real forest ecosystems; (ii) some observational studies may have suffered from biased design; and (iii) since alpine forests have been exposed to high ozone levels (and other oxidative stress) over decades, possible acclimation mechanisms cannot be excluded.

Suppression Substractive Hybridization and NGS Reveal Differential Transcriptome Expression Profiles in Wayfaring Tree (Viburnum lantana L.) Treated with Ozone.

Tropospheric ozone (O3) is a global air pollutant that causes high economic damages by decreasing plant productivity. It enters the leaves through the stomata, generates reactive oxygen species, which subsequent decrease in photosynthesis, plant growth, and biomass accumulation. In order to identify genes that are important for conferring O3 tolerance or sensitivity to plants, a suppression subtractive hybridization analysis was performed on the very sensitive woody shrub, Viburnum lantana, exposed to chronic O3 treatment (60 ppb, 5 h d(-1) for 45 consecutive days). Transcript profiling and relative expression assessment were carried out in asymptomatic leaves, after 15 days of O3 exposure. At the end of the experiment symptoms were observed on all treated leaves and plants, with an injured leaf area per plant accounting for 16.7% of the total surface. Cloned genes were sequenced by 454-pyrosequencing and transcript profiling and relative expression assessment were carried out on sequenced reads. A total of 38,800 and 12,495 high quality reads obtained in control and O3-treated libraries, respectively (average length of 319 ± 156.7 and 255 ± 107.4 bp). The Ensembl transcriptome yielded a total of 1241 unigenes with a total sequence length of 389,126 bp and an average length size of 389 bp (guanine-cytosine content = 49.9%). mRNA abundance was measured by reads per kilobase per million and 41 and 37 ensembl unigenes showed up- and down-regulation respectively. Unigenes functionally associated to photosynthesis and carbon utilization were repressed, demonstrating the deleterious effect of O3 exposure. Unigenes functionally associated to heat-shock proteins and glutathione were concurrently induced, suggesting the role of thylakoid-localized proteins and antioxidant-detoxification pathways as an effective strategy for responding to O3. Gene Ontology analysis documented a differential expression of co-regulated transcripts for several functional categories, including specific transcription factors (MYB and WRKY). This study demonstrates that a complex sequence of events takes place in the cells at intracellular and membrane level following O3 exposure and elucidates the effects of this oxidative stress on the transcriptional machinery of the non-model plant species V. lantana, with the final aim to provide the molecular supportive knowledge for the use of this plant as O3-bioindicator.

Ozone risk and foliar injury on Viburnum lantana L.: a meso-scale epidemiological study.

A stratified random sampling design was adopted to contrast sites with different ozone exposure levels (≤ 18,000 and >18,000 µg m(-3) h) in order to define whether and to what extent a relationship exists between potential risk (estimated by exposure to ozone) and the response of Viburnum lantana L. in terms of foliar symptoms. The study was designed over a meso-scale (6200 km(2)), carried out in 2010 and repeated in 2012 on a subset of sites. No difference was found between the occurrences of symptoms in relation to soil moisture or plant size. Although no direct significant exposure-response function could be identified, when data were aggregated according to ozone exposure levels the symptoms (in terms of number of symptomatic plants and symptomatic leaves per plant) were found to be significantly more frequent at sites with higher exposure (AOT40>18,000 µg m(-3) h), especially at high elevations (>700 ma.s.l.). The 2012 results confirmed the 2010 findings. Although ozone levels in the region were almost similar between 2010 and 2012, symptoms were significantly less frequent in 2012. This was likely due to drier conditions in 2012 (+1.1 °C; -23% precipitation), a situation that may have prevented in part ozone uptake and therefore the expression of symptoms. These results are useful in several respects: (i) for identifying areas where ozone is likely to impact vegetation; (ii) for testing the appropriateness of EU standards to protect vegetation from ozone; and (iii) for designing biomonitoring surveys. We suggest that V. lantana is a suitable indicator for assessing qualitatively (but not quantitatively) the potential risk of ozone damage to vegetation over remote, large areas.

Biomolecular identification of allergenic pollen: a new perspective for aerobiological monitoring?

BACKGROUND: Accurate and updated information on airborne pollen in specific areas can help allergic patients. Current monitoring systems are based on a morphologic identification approach, a time-consuming method that may represent a limiting factor for sampling network enhancement. OBJECTIVE: To verify the feasibility of developing a real-time polymerase chain reaction (PCR) approach, an alternative to optical analysis, as a rapid, accurate, and automated tool for the detection and quantification of airborne allergenic pollen taxa. METHODS: The traditional cetyl trimethyl ammonium bromide-based method was modified for DNA isolation from pollen. Taxon-specific DNA sequences were identified via bioinformatics or literature searches and were PCR amplified from the matching allergenic taxa; based on the sequences of PCR products, complementary or degenerate TaqMan probes were developed. The accuracy of the quantitative real-time PCR assay was tested on 3 plant species. RESULTS: The setup of a modified DNA extraction protocol allowed us to achieve good-quality pollen DNA. Taxon-specific nuclear gene fragments were identified and sequenced. Designed primer pairs and probes identified selected pollen taxa, mostly at the required classification level. Pollen was properly identified even when collected on routine aerobiological tape. Preliminary quantification assays on pollen grains were successfully performed on test species and in mixes. CONCLUSIONS: The real-time PCR approach revealed promising results in pollen identification and quantification, even when analyzing pollen mixes. Future perspectives could concern the development of multiplex real-time PCR for the simultaneous detection of different taxa in the same reaction tube and the application of high-throughput molecular methods.