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

Ozone may cause functional alterations on plants without, before and besides the onset of visible foliarsymptoms. While field assessment techniques for foliar symptoms have been developed and appliedeven at the large-scale, methods to detect functional alterations were mostly used under controlledexperiments with little (if any) formalized field test. During a five-month field survey, two populationsof Viburnum lantana L. plants growing at sites characterized by different ozone levels (+62.6% at thehigh-ozone with respect to the low-ozone, in terms of cumulated exposure) and visible foliar ozonesymptoms (from negligible to frequent) were examined in Trentino (North Italy). For each site, fivehaphazardly selected leaves from six randomly selected plants were monitored to evaluate the temporaltrend in the chlorophyll content (ChlSPAD) and chlorophyll a (Chl a) fluorescence transient (FT) in relationto 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 arelinked to the development of visible ozone injury on native vegetation; (ii) whether they can be an earlyindicator 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) ChlSPADand FMdecreased concurrently with the onset and time development of foliar injury (suggesting a degradation ofchlorophyll due to an excess of the total oxidative stress), with a much stronger reduction when the injurybecomes widespread (reduction of chlorophyll due to increased necrotic surface). (ii) At the high ozonesite visible foliar symptoms were frequent and appeared on plants displaying significant differences inmost of the Chl a FT variables even before the onset of symptoms. (iii) Given the reported variability ofthe ChlSPADand 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 samplingfour leaves for each V. lantana plant and five plants per site. Under these conditions, the concurrentmeasurements of Chl a FT variables together with ozone-specific visible foliar symptoms and ChlSPADcanprovide a set of valuable diagnostic indicators for the early identification and assessment of ozone effectson native vegetation and – potentially – for the phenotyping of ozone-sensitive individuals.