Ecological Indicators 39 (2014) 65–74 Contents lists available at ScienceDirect Ecological Indicators j o ur na l ho me page: www.elsevier.com/locate/ecolind Chlorophyll-related indicators are linked to visible ozone symptoms: Evidence from a field study on native Viburnum lantana L. plants in northern Italy Elena Gottardini a,∗ , Antonella Cristofori a , Fabiana Cristofolini a , Cristina Nali b , Elisa Pellegrini b , Filippo Bussotti c , Marco Ferretti d a Department of Sustainable Agro-Ecosystems and Bioresources, Research and Innovation Centre, Fondazione Edmund Mach (FEM), Via E. Mach 1, 38010 San Michele all’Adige, Italy b University of Pisa, Department of Agriculture, Food and Environment, Via del Borghetto 80, 56124 Pisa, Italy c University of Florence, Department of Agri-Food Production and Environmental Sciences, Piazzale delle Cascine 28, 50144 Firenze, Italy d TerraData Environmetrics, via L. Bardelloni 19, 58025 Monterotondo M.mo, Grosseto, Italy a r t i c l e i n f o Article history: Received 29 July 2013 Received in revised form 11 October 2013 Accepted 19 November 2013 Keywords: Viburnum lantana L. Ozone Foliar injury Chlorophyll content Fluorescence transient JIP-test a b s t r a c t Ozone may cause functional alterations on plants without, before and besides the onset of visible foliar symptoms. While field assessment techniques for foliar symptoms have been developed and applied even at the large-scale, methods to detect functional alterations were mostly used under controlled experiments with little (if any) formalized field test. During a five-month field survey, two populations of Viburnum lantana L. plants growing at sites characterized by different ozone levels (+62.6% at the high-ozone with respect to the low-ozone, in terms of cumulated exposure) and visible foliar ozone symptoms (from negligible to frequent) were examined in Trentino (North Italy). For each site, five haphazardly selected leaves from six randomly selected plants were monitored to evaluate the temporal trend in the chlorophyll content (Chl SPAD ) and chlorophyll a (Chl a) fluorescence transient (FT) in relation to the onset and development of foliar injury. The FT variables considered were: basal (F 0 ) and maximal (F M ) fluorescence in the dark-adapted state; maximum quantum yield efficiency (F V /F M ); J phase; I- P phase; performance index total (PI TOT ). We investigated (i) whether the physiological indicators are linked to the development of visible ozone injury on native vegetation; (ii) whether they can be an early indicator of ozone effect on a sensitive species before the onset of visible foliar symptoms; and (iii) what is the appropriate sample size for their reliable measurements. Results show that (i) Chl SPAD and F M decreased concurrently with the onset and time development of foliar injury (suggesting a degradation of chlorophyll due to an excess of the total oxidative stress), with a much stronger reduction when the injury becomes widespread (reduction of chlorophyll due to increased necrotic surface). (ii) At the high ozone site visible foliar symptoms were frequent and appeared on plants displaying significant differences in most of the Chl a FT variables even before the onset of symptoms. (iii) Given the reported variability of the Chl SPAD and Chl a FT data, and assuming a randomized sampling design, an acceptable precision level (defined as SE = 10% of the mean estimated value at P = 95%) can be achieved in most cases by sampling four leaves for each V. lantana plant and five plants per site. Under these conditions, the concurrent measurements of Chl a FT variables together with ozone-specific visible foliar symptoms and Chl SPAD can provide a set of valuable diagnostic indicators for the early identification and assessment of ozone effects on native vegetation and – potentially – for the phenotyping of ozone-sensitive individuals. © 2013 Elsevier Ltd. All rights reserved. Abbreviations: Chl a, chlorophyll a; Chl SPAD , chlorophyll content measured with the chlorophyll meter SPAD; DOY, day of year; F0, minimal fluorescence from dark adapted leaves; FM, maximal fluorescence from dark adapted leaves; FT, fluorescence transient; FV/FM, maximum quantum yield efficiency of primary photochemistry; I-P phase, 1 - VI amplitude of the I-P phase, i.e. the efficiency of electron transport around the PSI; J phase, 1 - VJ efficiency with which a trapped exciton can move an electron into the electron transport chain from QA- to the intersystem electron acceptors; LII, Leaf Injury Index; PITOT, performance index total, potential performance index for energy conservation from photons absorbed by PSII to the reduction of PSI end acceptors; PSI, photosystem I; PSII, photosystem II. ∗ Corresponding author. Tel.: +39 0461 615362; fax: +39 0461 650956. E-mail addresses: elena.gottardini@fmach.it (E. Gottardini), antonella.cristofori@fmach.it (A. Cristofori), fabiana.cristofolini@fmach.it (F. Cristofolini), cristina.nali@unipi.it (C. Nali), elisa.pellegrini@for.unipi.it (E. Pellegrini), filippo.bussotti@unifi.it (F. Bussotti), ferretti@terradata.it (M. Ferretti). 1470-160X/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ecolind.2013.11.021