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

Figures & data

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

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

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].

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.

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

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).

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

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).