Chlorophyll-related indicators are linked to visible ozone symptoms: Evidence from a field study on native Viburnum lantana L. plants in northern Italy

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 (ChlSPAD) and chlorophyll a (Chl a) fluorescence transient (FT) in relation to the onset and development of foliar injury. The FT variables considered were: basal (F0) and maximal (FM) fluorescence in the dark-adapted state; maximum quantum yield efficiency (FV/FM); J phase; I-P phase; performance index total (PITOT). 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) ChlSPAD and FM 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 ChlSPAD 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 ChlSPAD 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.