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The impact of mixed infection of grapevine leafroll-associated virus 1 and 3 (GLRaV-1&-3) on physiological performance of the Portuguese grapevine variety 'Touriga Nacional' was evaluated during 3 years with the main purpose of understanding the drastic reduction in yield. Overall, gas exchange was negatively affected in leaves with these leafroll virus infections. Particularly at ripeness stage, the reduction in stomatal conductance (g _s) was higher than in net CO ₂ assimilation rate (A), leading to higher intrinsic water use efficiency (A/g _s) in infected leaves. However, the decrease in g _s and A were not a consequence of the decrease in bulk water potential, as the water index/normalised difference vegetation index ratio suggested similar magnitude for both treatments. The maximum quantum efficiency of photosystem II was unaffected by GLRaV-1&-3, whereas quantum effective efficiency of PSII, apparent electron transport rate and photochemical quenching significantly decreased in infected leaves and these was paralleled by a significant increase of non-photochemical quenching. Relative to carbon metabolism, the analyses of the net CO ₂ assimilation rate/photosynthetic photon flux density (A/PPFD) and net CO ₂ assimilation rate/internal CO ₂ concentration (A/C _i) curves revealed that virus infection had a negative effect on light saturated rate of CO ₂ fixation at high irradiances and carboxylation efficiency but, in contrast, apparent quantum yield of CO ₂ fixation was significantly higher. Meanwhile, the presence of GLRaV-1&-3 resulted in a marked decrease in photosynthetic pigments, soluble sugars and soluble proteins contents, while starch and anthocyanins were significantly improved. N, P, Ca, S and Fe leaf concentrations significantly decreased, while K, Mg, B, Cu, Zn and Mn were unaffected by these two leafroll virus species. Infected plants showed a significant decrease in yield, mainly due to a lower cluster weight. These results emphasised the important role of GLRaV-1&-3 as a biotic stress for the grapevine physiology and consequently to yield attributes. © 2012 Association of Applied Biologists.