Bridging experimental and monitoring research for visible foliar injury as bio-indicator of ozone impacts on forests

ABSTRACT

Tropospheric ozone (O₃) is a phytotoxic air pollutant and the O₃-induced visible foliar injury (O₃ VFI) is a biomarker. A recently developed Free-air O₃ eXposure (FO₃X) is a promising facility to verify field-observed “O₃-like” VFIs and to establish a flux-based threshold for the O₃ VFI onset. The present study compared O₃-like VFI registered in the southern European forest sites with actual O₃ VFI observed in a FO₃X experiment. The O₃-like VFIs were evaluated by eye in forests and thus it was subjective. According to the imaging analysis, we firstly demonstrated that major parts of the colors were similar in the field and the FO₃X. The color pallets for O₃ VFI was species-specific and considered a advanced tool for the O₃ VFI diagnosis. In addition, we calculated a flux-based threshold for the O₃ VFI onset at the FO₃X based on a Phytotoxic Ozone Dose (POD₁), which ranged from 4.9 to 18.1 mmol m⁻² POD₁. This FO₃X-derived threshold partly explained but did not necessarily match with the observation for several tree species in actual forests. The multivariate analysis showed that O₃ VFI was decreased by the presence of various species and suggested the importance of continuous monitoring activities in the field for the further analysis.

KEYWORDS:

• Ozone
• visible foliar injury
• FO3X
• MOTTLES
• forest monitoring

Introduction

Tropospheric ozone (O₃) is a significant greenhouse gas and phytotoxic air pollutant, which has a deleterious effect on forest health (Watanabe et al. 2021). Therefore, monitoring forest health and functionality under climate change and air pollution is crucial for estimating forest vulnerability to the ongoing changes and for establishing mitigation measures (Paoletti et al. 2022). Furthermore, the recently revised National Emission Ceilings Directive of the European Union (2016/2284/EU, hereafter NEC Directive 2016 targets a 30% reduction in air pollutant emissions by 2030, compared with 2005 levels, and it is necessary to ensure the monitoring of air pollution impacts on ecosystems in EU member states as specified in Art. 9 of NEC Directive. Various O₃ metrics such as AOT40 (Accumulated exposure Over a Threshold of 40 ppb; abbreviations are listed in Table S1) have been proposed to examine forest responses to O₃ (Lefohn et al. 2018). However, scientific evidence has pointed out that O₃ damage is closely related to O₃ uptake through stomata (CLRTAP 2017). Therefore, Phytotoxic Ozone Dose above a stomatal-flux threshold Y (POD_Y), which is derived from a cumulative O₃ uptake over the growing season, is now included in the NEC Directive for the assessment of quantitative O₃ impacts on forest trees (de Marco et al. 2019).

When plants are exposed to O₃, they often show specific O₃-induced visible foliar injury (O₃ VFI), which has been used as a bio-marker to assess potential negative impacts of O₃ (Feng et al. 2014; Hoshika et al. 2018). Ozone-like VFIs have been found in various tree species across forest sites in Europe, indicating a possible phytotoxicity of the ambient level of O₃ (annual O₃ mean concentrations: 35 to 50 ppb) in forests (Paoletti et al. 2019; Vollenweider et al. 2019; Sicard et al. 2020).

Traditionally, the O₃-like VFIs have been evaluated by eye and, thus, the approach was rather subjective, although much efforts have been made to harmonize the VFI monitoring through the inter-calibration courses by international organizations such as ICP-Forests (Schaub et al. 2016). A manipulative O₃ exposure experiment is therefore considered as one of the key approaches for the validation of O₃-like VFI to diagnose and identify foliar symptoms caused by O₃ (Moura et al. 2018; Vollenweider et al. 2019). Previously, O₃-like VFIs have been verified in a wide variety of settings, including greenhouses and chambers of various sizes and designs (e.g., Bussotti et al. 2003). Although those experiments provided an insight into potential mechanisms, a difference in meteorological factors such as light, air temperature, and wind speed between chambers and natural conditions may also affect a quantitative analysis of O₃ VFI relative to actual field conditions (Nussbaum and Fuhrer 2000). Free-air O₃ eXposure (FO₃X) has been established in Italy since 2015, which is a unique facility in Mediterranean Europe to assess the effects of O₃ on vegetation within the framework of the European AnaEE (Analysis and Experimentation on Ecosystems) Research Infrastructure. As is the case with free-air CO₂ enrichment, experiments by free-air O₃ enrichment are considered as the best approach to provide realistic estimates of tree responses under real-world conditions (Paoletti et al. 2017). Symptoms of O₃ VFIs were reported in several tree species at the FO₃X (Hoshika et al. 2018, 2020). However, there is a need for validation studies to confirm whether the O₃-like VFIs observed in actual forests are similar enough to real O₃ VFIs produced in the FO₃X. In addition, little is known whether the FO₃X-derived thresholds for O₃ VFI can be applied to actual forest conditions, although such manipulative experiments have been used to develop standards for forest protection against O₃ (Büker et al. 2015). A flux-based threshold for the O₃ VFI onset is considered essential for the evaluation of regional O₃ pollution (Sicard et al. 2020). According to the forest monitoring data, Sicard et al. (2020) proposed flux-based thresholds for O₃-like VFI of 5 and 12 mmol m⁻² POD₁ in dominant conifers and broadleaved trees, respectively.

The present study compared O₃ VFI for forest tree species at the FO₃X facility with the symptoms observed at actual forest sites with the main aim of validating the field observations. For this purpose, a dataset obtained at southern European forest sites (France, Italy, and Romania) was utilized thanks to the European project MOTTLES (MOnitoring ozone injury for seTTing new critical LevelS) (Paoletti et al. 2019). A novel color pallet approach was developed to analyze the color composition of symptoms for categorizing and standardizing the O₃ VFI. The onset of O₃ VFI was also analyzed on a stomatal flux basis using POD₁. This study aimed to answer the following specific questions: 1) Are O₃-like foliar symptoms observed in the field comparable to actual O₃ VFIs produced at the FO₃X? and 2) Does the FO₃X-derived flux-based threshold also explain the incidence of O₃ VFI at the forest sites?

Materials and methods

Ozone fumigation experiments at the FO₃X facility

Ozone fumigation studies were carried out at the FO₃X facility in Italy (43° 49“N, 11° 12” E, 55 m a.s.l.). We set three levels of O₃ treatments (ambient O₃ concentration [AA], 1.2 times ambient concentration [1.2×AA], and 1.4 times ambient concentration [1.4×AA], in 2015; AA, 1.5 times ambient concentration [1.5×AA], and twice ambient concentration [2.0×AA] in 2017, 2018, 2019, 2020). The experimental design was a split-plot with three replicated blocks (L × W × H: 5 × 5 × 2 m) for each O₃ treatment. The specific detail of the fumigation system can be found in Paoletti et al. (2017). Targeted species were three oaks (Quercus ilex L., Quercus pubescens Willd., Quercus robur L.) in 2015, strawberry tree (Arbutus unedo L.) in 2017, black alder (Alnus glutinosa (L.) Gaertn.), phillyrea (Phillyrea angustifolia L.), mountain ash (Sorbus aucuparia L.) and bilberry (Vaccinium myrtillus L.) in 2018, three pines (Pinus halepensis Mill., P. pinaster Ait., P. pinea L.) in 2019 and elmleaf blackberry (Rubus ulmifolius Schott) in 2020. Those species were common in the MOTTLES monitoring sites. All plants were transplanted into plastic pots (oaks: 10 L, phillyrea; bilberry, pines, and elmleaf blackberry: 25 L; strawberry tree: 30 L; black alder and mountain ash: 50 L) with a mixture of sand: peat: soil (1:1:1 as volume). Before the O₃ fumigation, the pots were irrigated to keep the well-watered conditions, i.e., volumetric soil water content was kept to field capacity (0.295 m³ m⁻³, Paoletti et al. 2017). Target species, exposure period, hourly mean O₃ concentrations, and mean meteorological parameters during the experiments are shown in Table 1.

Table 1. Exposure period, target species, and environmental conditions during the experiment at the free-air O₃ eXposure (FO₃X). n.A. denotes not available. Ambient O₃ concentration (AA), 1.2 times ambient concentration (1.2×AA), 1.4 times ambient concentration (1.4×AA), 1.5 times ambient concentration (1.5×AA) and twice ambient concentration (2.0×AA).

	2015	2017	2018	2019	2020
Exposure period	1 Jun − 15 Oct	12 Jun − 15 Oct	1 May − 31 Oct	15 May −20 Oct	15 May − 31 Oct
Target species	Quercus ilex	Arbutus unedo	Alnus glutinosa	Pinus halepensis	Rubus ulmifolius
	Q. pubescens		Phillyrea angustifolia	P. pinaster
	Q. robur		Sorbus aucuparia	P. pinea
			V. myrtillus
Hourly mean O3 concentration (ppb)
AA	35.2	40.3	35.2	38.8	37.5
1.5×AA	n.a.	57.1	53.1	56.2	52.3
2.0×AA	n.a.	71.8	65.2	68.6	73.3
For the year 2015
1.2×AA	42.9	n.a.	n.a.	n.a.	n.a.
1.4×AA	48.9	n.a.	n.a.	n.a.	n.a.
Meteorological parameters
Daily mean air temperature (°C)	24.4	24.5	22.8	23.5	22.1
Daily mean solar radiation (W m^−2)	250	252	230	236	192
Daily mean relative humidity (%)	52.9	47.6	55.6	55.1	61.8
Total precipitation (mm)	592	195	136	161	313

The symptoms were carefully checked using a × 10 hand lens for the closer examination of injuries, and all leaves were targeted for each plant. Two-well experienced observers periodically assessed O₃ VFI to determine the date for first-symptom onset. The observers were involved in validation activities, attended field courses and performed annual inter-comparison exercises, organized by ICP-Forests. Foliar injury was compared with the reference picture atlas provided by the validation center for central Europe (www.wsl.ch) and identified according to the previous O₃ VFI studies in tree species (Calatayud et al. 2010; Hoshika et al. 2013; Schaub and Calatayud 2013).

MOTTLES forest sites and field observations

The study is based on data recorded by the MOTTLES O₃ monitoring network from 2017 to 2021 for Italian sites, and 2017–2019 for other countries. The network includes various biogeographical regions in southern European forest sites in three countries (France, Italy, and Romania). The target forest sites cover representative species in these regions, such as Fagus sylvatica in mountainous Alpine areas and Quercus ilex in Mediterranean forest areas. As a result, the MOTTLES network considered 7 broadleaved and 4 coniferous dominant tree species (Table 2).

Table 2. Site information of the 17 monitoring ozone injury for seTttting new critical LevelS (MOTTLES) sites in France, Italy and Romania. Daily mean air temperature (T), Daily mean solar radiation (RAD), Daily mean relative humidity (RH), Annual total precipitation (Prep.) and Hourly mean ozone concentration (O₃) are calculated from 2017 to 2021 for Italian sites and from 2017 to 2019 for France and Romania (± standard deviation).

Code	Country	Lat. ^1 N	Long. ^1 E	Elevation a.s.l.	Dominant species	Slope aspect	Soil type	T (°C)	RAD (W m^−2)	RH (%)	Prep. (mm)	O3 (ppb)
LCAS	France	44.99703	6.48802	1755	Larix decidua	30° E	Orthic Luvisols	7.95 ± 6.8	402.06 ± 176.7	63.43 ± 7.3	415.52 ± 248.0	48.18 ± 6.6
MNTFR	France	45.80500	2.06200	810	Pinus sylvestris	Plain	Dystric Cambisols	10.57 ± 6.3	299.31 ± 173.7	77.17 ± 1.8	745.64 ± 492.2	38.46 ± 5.6
MORV	France	47.27491	4.09921	620	Alnus glutinosa	6.5° SE	Chromic Cambisols	9.51 ± 6.1	327.16 ± 191.5	82.48 ± 7.6	186.64 ± 116.2	35.02 ± 5.3
REV	France	49.90778	4.62972	390	Picea abies	2°	Calcaric Leptosol	10.88 ± 6.1	284.52 ± 176.6	80.72 ± 10.6	635.10 ± 387.9	32.30 ± 7.2
ABR1	Italy	41.86064	13.57482	1500	Fagus sylvatica	5–10° S	Humic Acrisols(	8.02 ± 7.1	369.71 ± 186.0	78.33 ± 7.4	663.83 ± 522.4	54.87 ± 9.9
CPZ1	Italy	41.70423	12.35719	0	Quercus ilex	Plain	Fluvisol	16.71 ± 6.2	424.28 ± 224.9	78.95 ± 3.5	455.40 ± 342.6	33.05 ± 6.3
CPZ2	Italy	41.70429	12.35732	0	Phillyrea latifolia	Plain	Fluvisol	16.71 ± 6.2	424.28 ± 224.9	78.95 ± 3.5	455.40 ± 342.6	33.05 ± 6.3
CPZ3	Italy	41.68068	12.39084	0	Pinus pinea	Plain	Fluvisol	16.71 ± 6.2	424.28 ± 22.9	78.95 ± 3.5	455.40 ± 342.6	33.05 ± 6.3
EMI1	Italy	44.71998	10.20345	200	Quercus petraea	Plain	Haplic Luvisols	11.60 ± 7.6	321.76 ± 209.4	70.62 ± 12.3	484.47 ± 311.9	39.68 ± 14.7
LAZ1	Italy	42.82746	11.89817	690	Quercus cerris	5° WNW	Dystric Cambisols	13.75 ± 7.3	388.65 ± 181.8	73.16 ± 11.2	1027.06 ± 1060.3	46.99 ± 7.3
PIE1	Italy	45.68374	8.06994	1150	Fagus sylvatica	30° WNW	Haplic Podzols	7.42 ± 6.2	309.64 ± 140.4	71.77 ± 8.7	1138.12 ± 1151.0	47.72 ± 8.7
TRE1	Italy	46.35825	11.49405	1800	Picea abies	10° NNW	Haplic Podzols	5.76 ± 7.2	337.89 ± 173.3	71.54 ± 8.2	674.76 ± 403.7	48.24 ± 8.7
VEN1	Italy	46.06335	12.38810	1100	Fagus sylvatica	5° E	Haplic Luvisol	8.25 ± 7.2	329.93 ± 179.8	86.54 ± 5.2	1137.28 ± 705.3	35.46 ± 8.0
FAG	Romania	45.43305	25.26972	1300	Fagus sylvatica	20° W	Rendzin	7.21 ± 7.1	370.26 ± 155.0	78.25 ± 7.8	584.36 ± 363.0	41.28 ± 6.6
GORUN	Romania	45.02874	24.99576	500	Quercus petraea	10° W	Luvosol	11.02 ± 7.7	444.63 ± 201.3	78.84 ± 6.4	539.94 ± 243.7	20.17 ± 6.6
MOLID	Romania	45.50694	25.58916	1185	Picea abies	20° E	Eutricambosol	7.44 ± 7.2	271.76 ± 148.4	80.46 ± 6.1	438.97 ± 274.0	26.23 ± 5.9
STEJAR	Romania	44.50567	26.17300	86	Quercus robur	Plain	Preluvosol	12.47 ± 7.9	434.89 ± 191.3	75.98 ± 8.2	541.67 ± 429.0	21.22 ± 7.2

Each site consisted of i) an open area (open field – OFD) to record remotely and continuously meteorological and O₃ values with active sensors, ii) a nearby Light Exposed Sampling Site – LESS (Schaub et al. 2016), located in the light-exposed forest edge closest to the OFD station (maximum radius of 500 m) LESS area, and iii) an “in the plot” (ITP) area, where soil moisture and forest-health indicators are recorded into the forest. Specific details on MOTTLES active O₃ monitoring sites are available in Paoletti et al. (2019). The field protocols for assessing O₃ VFI by MOTTLES surveyors followed the ICP-Forests manual (Schaub et al. 2016). Surveys were carried out by the same two people in each country from August to early-mid September, i.e., when O₃ VFI are more likely to be observed in the MOTTLES sites (Dalstein et al. 2005; Paoletti et al. 2010). Two campaigns of trans-country inter-calibration were carried out within MOTTLES, and the surveyors had taken part in the international cross-calibration courses organized by ICP Forests.

At each site, the assessment of O₃ VFI was conducted every year within the ITP and the LESS. In ITP, the evaluation of O₃ VFI were performed at each plot on five trees randomly selected. For each tree, five light-exposed branches with ≥30 needles/leaves per branch or needle age class were removed from the upper crown. For deciduous species, current year (C) leaves/needles were assessed. For evergreen species, C, one – year–old (C + 1) and two – year–old (C + 2) leaves/needles were assessed. On each leaf/needle, the extent of O₃ VFI (as a percentage of the total leaf area) was visually scored by using the actual percentage, and results were then averaged for the five branches, resulting in one value per tree; finally, the tree values were averaged per plot. In the MOTTLES network, the LESS is 50 m long and divided into 25 × 2 m² non-overlapping quadrats. In 20 of the 25 quadrants, which were randomly chosen, all plant species were listed, and the presence or absence of O₃ VFI was recorded on the same day in which the ITP survey was carried out.

For the species with validated O₃ VFI, the available literature (Innes, Skelly, and Schaub 2001) and atlas provided by the validation center for central Europe (www.wsl.ch) were used to support the observations. Here, O₃ VFI datasets at MOTTLES sites during 2017–2021 were used for the analysis.

Calculation of ozone indices

Both exposure- and flux-based O₃ metrics were already calculated at MOTTLES sites over the study period from measured parameters (Sicard et al. 2020, 2021). In addition, the metrics, i.e., AOT40 and species-specific POD₁, were calculated by using meteorological and O₃ concentration data measured at the FO₃X facility. Accumulated exposure over a threshold of 40 ppb (AOT40) was calculated using the hourly O₃ data during daytime (solar radiation >50 W m⁻², CLRTAP, 2017) measured at each site:

(1) $AOT40=\sum max\left(\left[O_3\right]-40,\hspace{0.17em}0\hspace{0.17em}\right)\cdot dt$(1)

where [O₃] is the hourly concentration of O₃ (ppb), and dt is the time step (1 h).

Phytotoxic Ozone Dose above a detoxification threshold of 1 nmol m⁻² s⁻¹ (POD₁) was calculated according to the standard methodology suggested by CLRTAP (2017).

(2) $PO{D}_1=\sum max\left(F_{st}-1,\hspace{0.17em}0\hspace{0.17em}\right)\cdot dt$(2)

where F_st is the hourly stomatal uptake of O₃ (nmol m⁻² s⁻¹), which is derived from leaf surface resistance (r_c) and boundary layer resistance (r_b) (CLRTAP, 2017). F_st is thus given by:

(3) $F_{st}={\left[O_3\right]}_i\cdot g_s\cdot \frac{r_c}{r_b+r_c}$(3)

where g_s is the stomatal conductance (mmol O₃ m⁻² projected leaf area [PLA] s⁻¹). r_b is calculated by wind speed and leaf dimension, and r_c is defined as 1/(g_s+g_ext) (s m⁻¹). g_ext is the cuticular conductance (0.0004 s m⁻¹). For the details of the calculation of r_b and r_c, see CLRTAP (2017).

A simplified formula of the multiplicative stomatal conductance model was applied because all plants were grown under well-watered conditions at the FO₃X (Hoshika et al. 2020). It is given by:

(4) $g_s=g_{max}\ast f_{light}\ast max\left\{f_{min},\hspace{0.17em}\left(f_{temp}\ast f_{VPD}\right)\right\}$(4)

where g_max is the maximum stomatal conductance to O₃ expressed on a total leaf surface area (mmol O₃ m⁻² PLA s⁻¹), f_min is the minimum conductance, f_light, f_temp, and f_VPD indicate the stomatal response functions to photosynthetic photon flux density (PPFD), air temperature (T) and vapor pressure deficit (VPD), respectively. For several species (A. glutinosa, Q. ilex, Q. pubescens, Q. robus, S. aucuparia, V. myrtillus), species-specific stomatal conductance parameters were already reported in our previous studies (Hoshika et al. 2018, 2020). Stomatal conductance for the other species was parameterized according to the measurements with various meteorological conditions using a portable leaf gas exchange measurement system (CIRAS-2 PP Systems, Herts, UK). Measurements were carried out at least once a month (7 to 19 days in total for each species) throughout the experimental period (Table 1). Pooled data (A. unedo: 128 data, P. angustifolia: 374 data, P. halepensis: 174 data, P. pinaster: 303 data, P. pinea: 164 data, R. ulmifolius: 249 data) were used for the species-specific parameterization according to the boundary line technique (Elvira et al. 2004; Büker et al. 2015; Hoshika, Paoletti, and Omasa 2012). To draw the boundary line (i.e., upper limits of point g_s data in a scatter diagram) for each environmental variable, the data were divided into the following stepwise classes: PPFD: 200 μmol m⁻² s⁻¹ steps (when PPFD <200 μmol m⁻² s⁻¹, 50 μmol m⁻² s⁻¹ steps were applied), T: 2°C steps, VPD: 0.2 kPa steps. Each model function was then fitted according to 95^th percentile values per each stepwise class of environmental factors. g_max and f_min were estimated as the 95^th and 5^th percentile, respectively (Bičárová et al. 2019; Hoshika et al. 2020). For the detailed formula of f_light, f_temp, and f_VPD functions, see CLRTAP (2017).

Data analysis

For three species, Rubus ulmifolius, Vaccinium myrtillus, and Sorbus aucuparia, an O₃ VFI image analysis was developed to compare the symptoms observed in the field with those developed at the FO₃X. Three pictures with minimum 180 dpi (RGB – red, green, blue color mode) of leaves presenting O₃ VFI of each species were selected from the field and FO₃X conditions. First, the mid-part of each leaf was cut off from the original picture with the same size for each species. Next, each cut of the sample picture was transformed in Indexed Color mode, and a Local (Perceptual) and an 8-color pallet was generated. Next, the color pallets were merged, forming a general 8-color pallet for the O₃ VFI pictures obtained from MOTTLES or FO₃X conditions. The final 8-color pallets, which characterize the color composition of O₃ VFIs, were then compared between field and FO₃X conditions, considering the percentage of the same colors appearing in both conditions. We surveyed the significant O₃ VFI colors of the final color pallet (50% of the total colors range) as the ones better describing the O₃ VFI (O₃ VFI/Color), while the other colors were related to leaf typical chlorophyll pigments (LTCP). All analyses were conducted using Adobe Photoshop CC 2017, and the color names were verified in the Hex dictionary (https://www.hexdictionary.com/). All pictures analyzed with its, respectively, 8-color pallet are available as supplementary material (Fig. S1).

In order to detect main predictors for symptoms in the natural environment, we correlated symptom variables with site variables. We applied two linear models by using the linear model (lm) function of R software (Team R Development Core, 2018): in the first model we considered the percentage of symptomatic species within the LESS as response variable; in the second model we considered the O₃ VFI (merging ITP and LESS data) as response variable. The following site variables were included in the model as predictors: year, country, slope aspect, dominant species, and biogeographical region (as categorical), elevation (as continuous), and the total number of species within the LESS (as count variable); all interactions among site variables were also considered in the model. Selection of the optimal model, among those generated by all possible combinations, was based on the Akaike Information Criterion (AIC) values to assess the quality of the model (Barton et al. 2012). For the best model selected, we also calculated R² and parameter-specific p-values for each predictor level (e.g., value).

Results

Ozone visible injury at MOTTLES forest sites

The O₃ VFI occurring in the MOTTLES sites were characterized by the following categories: Homogeneously distributed interveinal reddish (Rd), Reddish interveinal stippling (RdSt), Homogeneously distributed interveinal brownish (Brw), Dark brownish interveinal stippling (BrwSt), Bronzing (Br), and chlorosis (Cl) (Table 3). The 23 symptomatic species presented, in most of the cases, a combination of these symptoms. The most common O₃ VFIs were Cl and BrwSt, occurring in 43% of the species, followed by RdSt, detected in 35% of the species. Rd, Br, and Brw occurred in 26%, 13%, and 9% of the species, respectively. Some pictures were selected to show each of the categorical O₃ VFI occurring in symptomatic species at MOTTLES sites (Figure 1.).

Figure 1. Examples of ozone visible foliar injury (O3 VFI) from Monitoring Ozone injury for seTTtting new critical LevelS (MOTTLES) sites and the Free-air O3 eXposure (FO3X), A and G) Homogeneously distributed interveinal reddish (Rd); B, C, F, H and I) Reddish interveinal stippling (RdSt); D, K and L) Dark brownish interveinal stippling (BrwSt); E and J) Chlorosis (Cl). K and L) Homogeneously distributed interveinal brownish (Brw); C, F) Bronzing (Br).

Table 3. Species showing ozone-induced visible foliar injury (O₃ VFI) at the MOTTLES (Monitoring Ozone injury for seTttting new critical LevelS) sites (ITP – in the plot and LESS - Light Exposed Sampling Site) in France (FR), Italy (IT) and Romania (RO) or from the Free-air O₃ eXposure (FO₃X). O₃-induced visible foliar injury (O₃ VFI) classifies as homogeneously distributed interveinal reddish (Rd), Reddish interveinal stippling (RdSt), Homogeneously distributed interveinal brownish (Brw), Dark brownish interveinal stippling (BrwSt), chlorosis (Cl) and Bronzing (Br). Leaf habit (evergreen [Ev]/deciduous [De], conifer [C]/broadleaf [Br].

In MOTTLES forest sites, merging ITP and LESS data, O₃ VFI was observed on 23 species, consisting of 14 trees, 8 shrubs and 1 liana (Clematis vitalba) (Table 3). Fagus sylvatica was the species found to be most frequently symptomatic (21 times across all sites and years), followed by the shrubs Rubus ulmifolius and Corylus avellana, recorded 12 and 11 times, respectively. Most of the symptomatic species were deciduous angiosperms, while only two conifers, i.e., Pinus cembra and Picea abies, were found to be symptomatic 2 times and once, respectively. Regarding the forest LESS, linear models showed that the percentage of symptomatic species, despite a significant influence of the country (Table 4; Figure 2.), decreased with the increment in the total number of tree and shrub species within the LESS (Figure 1.). The same trend was observed for O₃ VFI (Figure 3.); however, in this case, species richness in the LESS was the unique driver among the selected site characteristics.

Figure 2. Visual results of the selected linear model for the percentage of symptomatic species within the Light Exposed Sampling Site – LESS (symptomatic_sps) presented in table 3. Significant effects on symptomatic_sps determined by a) by the total number of species in the LESS (SR_LESS) plotted as linear regression with confidence interval and b) by the Country (FR, France; IT, Italy; RO, Romania) plotted as violin box plot.

Figure 3. Visual results of the selected linear model for the percentage of ozone visible foliar injury (O₃_VFI) per plot, presented in table 3. Significant effect on O₃_VFI determined by the total number of species in the Light Exposed Sampling Site – LESS (SR_LESS) plotted as linear regression with confidence interval.

Table 4. Optimal linear model structures relating the percentage of symptomatic species (symptomatic_sp) over the LESS (Light Exposed Sampling Site) and the percentage of ozone visible foliar injury (O₃ VFI) in the plot (ITP). Response variables to the total number of species in the LESS (SR_LESS), the country, and the year [R syntax of the starting model: y ~ SR_LESS * year * country * elevation * aspect * dominant species * biogeographical region]. Values for the predictor variables: SR_LESS, country (level Italy and Romania compared with France), are parameter estimates. R² refers to the fraction of the variation explained by the model structure, and R² adjusted takes into account the number of independent variables used for predicting the target variable.

	Symptomatic_sps	O3 VFI
SR_LESS	−0.015***	−3.256**
	(0.006)	(1.362)
Italy	−0.381***
	(0.110)
Romania	−0.363***
	(0.116)
SR_LESS: Italy	0.014
	(0.013)
SR_LESS:Romania	0.006
	(0.008)
Constant	0.584***	51.619***
	(0.072)	(10.321)
Observations	61	23
R^2	0.426	0.231
Adjusted R^2	0.374	0.191
Residual Std. Error	0.158 (df = 55)	14.878 (df = 19)
F Statistic	8.165*** (df = 5; 55)	5.710** (df = 1; 19)

Significance level: *p < 0.05, **<p < 0.01, ***p < 0.001.

Ozone visible injury at the FO₃X

Pictures of the O₃ VFI for the symptomatic species at FO₃X are shown in Figure 1.. Ozone exposure caused O₃ VFI in all target deciduous species, while only two of six evergreen species were symptomatic, i.e., a Mediterranean shrub, A. unedo, and a Mediterranean pine, P. halepensis (Table 5). Among the symptomatic species, A. glutinosa, S. aucuparia, and V. myrtillus showed first symptoms even under real-world O₃ conditions (18 May to 21 June), while O₃ VFI was found only later for A. unedo and Q. pubescens (3 to 25 September).

Table 5. Onset of ozone visible foliar injury (O₃ VFI) at the Free-air O₃ eXposure (FO₃X). Ambient O₃ concentration (AA), 1.2 times ambient concentration (1.2×AA), 1.4 times ambient concentration (1.4×AA), 1.5 times ambient concentration (1.5×AA) and twice ambient concentration (2.0×AA).

Species	Date for the onset of O3 visible foliar injury
A. glutinosa	AA: 31 May, 1.5×AA: 31 May, 2.0×AA: 18 May
A. unedo	AA: no symptoms, 1.5×AA: 25 September, 2.0×AA: 18 September
P. angustifolia	No symptoms
P. halepensis	AA: no symptoms, 1.5×AA: no symptoms, 2.0×AA: 28 June
P. pinaster	No symptoms
P. pinea	No symptoms
Q. ilex	No symptoms
Q. pubescens	AA: no symptoms, 1.2×AA: 3 September, 1.4×AA: 22 September
Q. robur	AA: 19 August, 1.2×AA: 5 August, 1.4×AA: 28 July
R. ulmifolius	AA: no symptoms, 1.5×AA: 8 July, 2.0×AA: 28 June
S. aucuparia	AA: 21 June, 1.5×AA: 21 June, 2.0×AA: 21 May
V. myrtillus	AA: 21 June, 1.5×AA: 21 June, 2.0×AA: 26 May

Following the same classification used to identify O₃ VFI in the MOTTLES sites, a similar combination of symptoms was observed in the eight symptomatic species in the FO₃X facility (Table 4). The most common O₃ VFI observed was BrwSt occurring in 50% of the species, followed by BrwSt and RdSt, detected in 38% of the species. Rd occurred in 25%, while Cl was found in 13% of the species. So far, Br has not been observed in the FO₃X. The Cl mottling appeared on needles of P. halepensis, while the other evergreen species, A. unedo, presented RdSt occurring between the veins on the upper leaf surface. A similar symptom was found in V. myrtillus in which Rd was also observed. The deciduous broadleaf S. aucuparia presented RdSt, and also Brw and BrwSt. In addition, interveinal Rd appeared on the upper leaf surface in R. ulmifolius. Finally, Brw and BrwSt similarly occurred between the primary leaves veins in the two deciduous oak species.

Comparing ozone visible injury at the MOTTLES and FO₃X sites: A color analysis

For R. ulmifolius, 62.5% of colors selected in the final 8-color pallets were common colors presented in both MOTTLES and FO₃X samples (Figure 4A–D). Considering only the O₃ VFI/Color, 75% of the colors were common between the MOTTLES and FO₃X samples (Coral Reef − 512E10, Peat – C7BAA1, and Pesto − 6C6133). One specific O₃ VFI/Color was not identified at FO₃X samples and was recorded only at the MOTTLES site (Bracken − 512E10). Furthermore, 50% of the LTCP colors were common between the MOTTLES and FO₃X samples.

Figure 4. Examples of color composition in adaxial leaf blades with ozone visible foliar injury (O3 VFI) from Monitoring Ozone injury for seTTtting new critical LevelS (MOTTLES) sites and the Free-air O3 eXposure (FO3X), A and C) Homogeneously distributed interveinal reddish (Rd); B and C) Reddish interveinal stippling (RdSt) B) Dark brownish interveinal stippling (BrwSt) and Homogeneously distributed interveinal brownish (Brw). Colors better describing the O3 VFI (O3 VFI/Color) or Leaf typical chlorophyll pigments (LTCP).

For S. aucuparia, 50% of colors selected in the final 8-color pallets were present in both MOTTLES and FO₃X samples (Figure 4B–C). Considering only the O₃ VFI/Color, 25% of the colors were common between the MOTTLES and FO₃X sample (Yellow Metal − 6C6133). One O₃ VFI/Color was recorded only at the MOTTLES site (Saddle −472D22), whereas two O₃ VFI/Color was recorded at the FO₃X (Sepia Skin – B5A42, and Tan – D7B690). Furthermore, 75% of the LTCP colors were common between the MOTTLES and FO₃X samples.

For V. myrtillus, however, not all the considered O₃ VFI in the MOTTLES sites were similar to the FO₃X O₃ VFI pictures (Fig. S1), 75% of colors selected in the final 8-color pallets were present in both samples (Figure 4C,D). Considering only the O₃ VFI/Color, 75% of the colors were common between the MOTTLES and FO₃X samples (Leather − 8B674F, West Coast − 695328 and Mongoose – A3926A). One specific O₃ VFI/Color, was not identified at FO₃X samples and was recorded only at the MOTTLES site (Beaver – A97E6D). Furthermore, 75% of the LTCP colors were common between the MOTTLES and FO₃X samples.

Stomatal ozone uptake for visible injury onset at the FO₃X

New stomatal conductance model parameters for species (A. unedo, P. angustifolia, P. halepensis, P. pinaster, P. pinea, R. ulmifolius) are shown in Table S2 and Fig. S2, and the g_max values were dependent on species ranging from 95 (A. unedo) to 165 mmol O₃ m⁻² PLA s⁻¹ (P. halepensis, and R. ulmifolius). The stomatal light response function (f_light) followed a typical light-saturation curve with a saturation point above 1000 µmol m⁻² s⁻¹. The g_s response to temperature (f_temp) had a bell-shaped curve where g_s reached the maximum at the optimal temperature (22–26 °C). The f_VPD function indicated that stomatal closure was caused by VPD higher than 1.2 to 1.5 kPa regardless of the species. However, the VPD attaining minimum g_s (VPD_min) was relatively low in P. halepensis compared to the other species.

Two representative O₃ indices, i.e., POD₁ and AOT40, were calculated to find the values corresponding to the onset of the first O₃ VFI in the 12 species at the FO₃X experiment (Table 6). The AOT40 threshold for the onset of O₃ VFI was different among the symptomatic species (3.9 to 50.9 ppm h AOT40). The Mediterranean evergreen A. unedo required 50.9 ppm h AOT40 to show the first O₃ VFI, while the first O₃ VFI appeared on both deciduous A. glutinosa and S. aucuparia from low AOT40 values (3.9 to 6.5 ppm h AOT40). On the other hand, the onset of O₃ VFI was observed from 5 mmol m⁻² POD₁ for A. glutinosa and V. myrtillus, and from 18.1 mmol m⁻² POD₁ for Q. pubescens.

Table 6. Phytotoxic Ozone Dose (POD₁) and accumulated exposure over a threshold of 40 ppb (AOT40) values corresponding to the occurrence of the O₃ visible foliar injury (O₃ VFI) onset at the Free-air O₃ eXposure (FO₃X).

Species	POD1 (mmol m^−2)	AOT40 (ppm·h)
A. glutinosa	4.9	3.9
A. unedo	5.3	50.9
S. aucuparia	8.6	6.5
V. myrtillus	5.6	8.4
P. angustifolia	no injury	no injury
P. halepensis	9.2	25.3
P. pinaster	no injury	no injury
P. domestica	no injury	no injury
R. ulmifolius	11.4	23.5
Q. ilex	no injury	no injury
Q. pubescens	18.1	28.6
Q. robur	12.0	16.4

We then applied these FO₃X-derived thresholds to the MOTTLES conditions. In theory, O₃ VFI should be present when POD₁ exceeds the threshold for the onset of O₃ VFI, whereas no O₃ VFI should be found when POD₁ is lower than this threshold. In fact, in the MOTTLES sites, the results were in line with this theory for A. glutinosa and Q. pubescens (Figure 5). However, plants were often asymptomatic for Q. robur, S. aucuparia and V. myrtillus even though POD₁ was higher than this threshold. On the contrary, R. ulmifolius was symptomatic even in most cases when POD₁ was lower than the threshold.

Figure 5. Phytotoxic Ozone Dose (POD1) and the presence or absence of ozone visible foliar injury (O3 VFI) for several forest species in Monitoring Ozone injury for seTTtting new critical LevelS (MOTTLES) sites. Both in the plot (ITP) and Light Exposed Sampling Site (LESS) data were considered. Red line shows the POD1 threshold for the onset of O3 VFI obtained from the Free-air O3 eXposure (FO3X) experiments (see Table 6 for a detail).

Discussion

Color analysis and comparisons of the foliar symptoms between FO₃X and actual forests

This first attempt to diagnose O₃ VFI by comparing actual field and manipulative experimental conditions based on the symptom color composition appeared promising, especially for two species where 75% of the O₃ VFI/Color were shared between MOTTLES and FO₃X samples. A leaf color chart is defined as a series of color swatches used to identify leaf physiological status and characteristics (Takebe and Yoneyama 0000). Color composition in terms of the color chart has been used in several studies, especially to estimate leaf and canopy chlorophyll content and their variation in field conditions (Nguy-Robertson et al. 2015) and for adjusting nitrogen (N) fertilization rate, thus improving N management in field conditions on the basis of leaf color.

Comparing the O₃ VFI between the field and experimental conditions using the color composition was here demonstrated to be a potential tool for the O₃ VFI diagnosis. For the three species compared in the presented study, 50% to 75% of the colors in the final 8-color pallets were commonly present in the field and under manipulative experimental conditions. This indicates that the O₃ VFI/Color are similar between field and FO₃X conditions, suggesting that it is possible to make successful validation of field-observed O₃ symptoms by using manipulative fumigation studies. However, it is important to follow standard procedures for the shooting of pictures used in the image analysis, as lighting conditions and picture resolution may affect the colors. The ICP-forest manual (Schaub et al. 2016) listed a series of indications for taking photographs of O₃ VFI, that must be followed for the quality assurance.

The O₃ VFI provides clear indications of O₃-induced oxidative stress and is reliable when verified by a combination of approaches, once the symptoms expression can be similar even when the stress factors are different (Vollenweider and Gunthardtgoerg 2006; Guerrero et al. 2013; Alves et al. 2016; Moura et al. 2018; Vollenweider et al. 2019). For instance, the adaxial St can appear following several other sources of oxidative stress (Vollenweider and Gunthardtgoerg 2006), and Cl is a common symptom that needs careful validation to be diagnosed as O₃ VFI (Vollenweider et al. 2019). Furthermore, usually observed O₃ VFI in field surveys are aspecific and difficult to interpret (Bussotti et al. 2006) because leaf colors may also be affected by the plant phenological nutritional and physiological status and other abiotic causes (Bussotti and Ferretti 2009). However, it should be noted that such a field-specific O₃ VFI/Color was found to be always lower than 25% of color composition.

The successful assessments of O₃ VFI are mainly dependent on the observer’s experience, and knowledge and color images such as photo guides of O₃ VFI are considered important tools, especially in field conditions where observation is required for several species and a large number of individuals. The validation of O₃ VFI is therefore crucial and must be conducted under experimental conditions and using microscopy observation (Vollenweider, Ottiger, and Günthardt-Goerg 2003; Bussotti et al. 2005, 2006; Moura et al. 2018) to ascertain an O₃ VFI diagnosis by providing a mechanistic understanding of O₃ effect (Günthardt-Goerg and Vollenweider 2007; Faoro and Iriti 2009). However, the new methodology proposed here based on color composition can informatize well-trained observer’s experience and digitalizes their visual information and knowledge of how to identify O₃ VFI. For example, the arithmetic manipulation of the RGB color channels has been used to detect a wide variety of plant disease symptoms (Barbedo 2017). Although further analyses for the specificity of O₃ VFI are still needed, a proper visual information for the O₃ VFI can be a simple informatic tool to establish an automated O₃ VFI identifier using a RGB image alternative to the traditional method of a time-consuming validation by microscopy, thus improving the assessment and identification of O₃ VFI.

Comparison of flux-based threshold for the ozone visible injury onset between the FO₃X and actual forests

In the 2000s, several field studies reported that O₃ VFI was shown in forest trees when AOT40 reached 10 ppm h (Vollenweider et al. 2019; Vanderheyden et al. 2001). However, as Vanderheyden et al. (2001) pointed out for forest tree species in Switzerland, the AOT40 threshold showing O₃ VFI varied from year to year. In fact, O₃ damage is closely related to stomatal O₃ uptake rather than O₃ exposure only (CLRTAP, 2017; Watanabe et al. 2021). Therefore, a consensus has increased in the scientific community to recommend the flux-based O₃ risk assessment on forest trees (Paoletti et al. 2022) suggesting that an equivalent stomatal O₃ dose results in a similar O₃ damage over various species with a different sensitivity to O₃ (Reich 1987; Feng et al. 2018). In fact, the AOT40-based threshold for the onset of O₃ VFI for the O₃ resistant evergreen species, A. unedo, was 10-fold higher than that for the O₃ sensitive deciduous species, A. glutinosa. Interestingly, the flux-based threshold corresponding to the first symptom onset for A. unedo was rather similar to that for A. glutinosa. In fact, A. unedo, which has a low g_max limiting stomatal O₃ uptake, showed O₃ VFI only in the autumn season, while A. glutinosa showed O₃ VFI in early summer even under ambient O₃ conditions because it shows a very high stomatal conductance and stomatal O₃ uptake easily exceeds the critical range of O₃ dose that can be detoxified.

According to the field observations in MOTTLES sites, Sicard et al. (2020) proposed flux-based thresholds for O₃ VFI of 5 and 12 mmol m⁻² POD₁ in dominant conifers and broadleaved trees, respectively, while 11 mmol m⁻² POD₁ was required for the presence of O₃ VFI in various sensitive tree species present within the LESS (Sicard et al. 2021). Although categorizing plant types is useful for setting the critical standard for forest protection, plant responses to O₃ are rather species-specific, as confirmed by the high flux-based threshold reported for Pinus halepensis (e.g., 8.2 mmol m⁻² POD₁) in Southeastern France (Sicard et al. 2016). At the FO₃X, the flux-based threshold for the O₃ VFI onset was ranged from 4.9 to 18.1 mmol m⁻² POD₁. It seems that the FO₃X-derived threshold also explained well the presence of O₃ VFI for A. glutinosa and Q. pubescens in the MOTTLES conditions. However, the threshold observed for the injury onset at the FO₃X did not often match the presence of O₃ VFI in actual forests. The incidence of the VFI was lower for S. aucuparia and V. myrtillus in actual forests compared to the FO₃X condition. Indeed, S. aucuparia, and V. myrtillus showed O₃ VFI in early summer at FO₃X even under AA conditions, while they were not always found symptomatic in actual forests. Also, for the less sensitive species such as Q. robur, very limited O₃ VFIs were found under the MOTTLES conditions even though a relatively high POD₁ was observed (>20 mmol m⁻² POD₁). There is a possibility that g_s of field plants would be different with that of pot plants. In fact, Beikircher et al. (2021) suggested a lower g_s in field-grown mature Acer pseudoplatanus trees than in potted-seedlings. On contrary, Samuelson and Kelly (1997) indicate a greater g_s in mature Quercus rubra trees than seedlings. Actually, a site-specific g_s parameter may provide a more precise estimation of POD₁ in the MOTTLES sites. In addition, in a manipulative experiment, plants are usually potted, well-watered, isolated, and totally exposed to the atmospheric O₃ conditions; therefore, the higher incidence of symptoms can be related to the lack of stress compensation by an interaction of leaves within a canopy crown (Löw et al. 2006). Otherwise, a complex structure of forests with species mixtures may decrease herbivory, disease and other abiotic stresses, with increasing nutrient supply rates over the long term (Tilman, Isbell, and Cowles 2014). Interestingly, according to the multivariate analysis on MOTTLES datasets, O₃ VFI decreased with an increasing number of species in the LESS. LESS is a forest edge, and therefore it is generally characterized by a high number of species with different growth forms determining a multi-layered structure (Ranney, Bruner, and Levenson 0000; Łuczaj and Sadowska 1997). As a vertical O₃ gradient can be observed in a forest canopy (Ollinger, Aber, and Reich 1997), the presence of nonsensitive species in the upper layer can establish a protective mechanism for more sensitive species in the bottom layer. Such a hypothesis is in line with the assumption that mixed stands are more resilient to environmental stress than monospecific ones (Grossiord et al. 2014). Eichhorn et al. (2005) and Pollastrini et al. (2016) identified species diversity as a relevant factor that positively influences the crown conditions (i.e., reduced defoliation) in North European and Mediterranean forests.

Only R. ulmifolius showed the opposite behavior, i.e., it was symptomatic in the field even with a relatively low POD₁ below the level in which the VFI was observed at the FO₃X. Such differences can be explained by the strong competitive habit of the species for light (Gaudio, Balandier, and Marquier 2008). R. ulmifolius shrubs tend to overwhelm other species and, therefore, are more exposed to atmospheric conditions.

Conclusions

The results confirmed that a new generation free-air O₃ fumigation facility such as FO₃X is an appropriate tool for the validation activities of field-observed O₃ VFI. New imaging analysis of color composition for O₃ VFI indicates that a major part of the colors were similar between the field and the FO₃X. Such digitized information will provide an advancement in the approach for O₃ VFI assessment from the bio-informatic point of view such as a mobile application as diagnostic tool for field scientists. A further calibration for estimating O₃ VFI by color composition (i.e., differences between O₃ VFI and other visible injury, color distribution in the leaves) is recommended for a more extensive range of O₃-sensitive species because other abiotic stress factors may sometimes mask the O₃ VFI.

The flux-based threshold for the O₃ VFI onset at the FO₃X was ranged from 4.9 to 18.1 mmol m⁻² POD₁. Although this FO₃X-derived threshold also explained the presence of O₃ VFI for A. glutinosa and Q. pubescens under real-world conditions, it did not always match with the observation for the other species. Q. robur, S. aucuparia and V. myrtillus were relatively resistant in actual forests rather than the FO₃X experiment, given that they were not visibly affected in the MOTTLES sites even though POD₁ exceeded the FO₃X-derived threshold. On the other hand, R. ulmifolius exhibited the O₃ VFI even at a relatively low POD₁. Although the mechanisms are still unknown, the multivariate analysis indicated an interaction of the presence of various species on O₃ VFI and suggested the importance of biodiversity and continuous monitoring activities in the field.

European forests are considerable areas of the world terrestrial biodiversity. In MOTTLES, we investigated 23 tree species. The ozone FACE (Free Air Controlled Exposure) experiments need to be expanded to more species to realize a proper and representative assessment of forest health under O₃ pollution.

Acknowledgments

We are grateful for financial support to the Fondazione Cassa di Risparmio di Firenze (2013/7956), the LIFE projects MOTTLES (LIFE15 ENV/IT/000183) and MODERn (NEC) (LIFE20 GIE/IT/000091), and the CNR project OzonPlant - Relazioni tra piante e inquinamento da ozono (DTA.AD002.640) and 4ClimAir (SAC.AD002.173.019).

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

The work was supported by the Consiglio Nazionale delle Ricerche [4ClimAir (SAC.AD002.173.019),OzonPlant (DTA.AD002.640)]; European Commission [MODERn (NEC) (LIFE20 GIE/IT/000091),MOTTLES (LIFE15 ENV/IT/000183)]; Fondazione Cassa di Risparmio di Firenze [2013/7956]