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SPÁČILOVÁ, V., ŠAFRÁNKOVÁ, I.: Potential of chlorophyll fl uorescence and VIS/NIR spectroscopy measurement use for the detection of nitrogen content and disease infection of apple leaves. Acta univ. agric. et silvic. Mendel. Brun., 2011, LIX, No. 6, pp. 317–328 A possibility of using spectral methods for determining a nutritional status and detecting pathogens in apple-tree cvs. ’Jonagold’ and ’Idared’ was verifi ed in an orchard and pot experiments in 2007– 2010. Treatments diff ered in the fertilizer or fungicide dose. Leaf samples were collected from the experimental variants to determine nitrogen content and to measure spectral refl ectance (spectrophotometer Avantes USB 2000) and chlorophyll fl uorescence imaging (FluorCam). Results of the measurements were correlated to leaf analyses for nitrogen content in dry matter. At the same time, a health status (the occurrence of fungal pathogens Venturia inaequalis and Podosphaera leucotricha) was assessed and changes of photochemical effi ciency of PSII of infected leaves were evaluated. The parameters providing the best description of diff erences in the photosynthetic activity of leaves depending on treatments (parameter Fv/Fm and parameter GENTY, known as PSII – eff ective quantum yield of PSII) were selected. The values of correlation coeffi cients of Fv/Fm and PSII depending on fertilization treatments were as follows: Fv/Fm: r = −0.4735, p<0.000089,  = 0.05; PSII: r = 0.755; p < 0.00038,  = 0.05. Data obtained from measuring with a spectrophotometer was used for the calculation to normalized diff erence vegetation indices NDVI; a signifi cant relationship was found for the index GNDVI (r = 0.4691, p < 0,0002,  = 0.05). The signifi cant diff erence between healthy leaves and leaves infected by the pathogens V. inaequalis and P. leucotricha was confi rmed using the spectrophotometer, and the largest diff erences in refl ectances were found in wavelengths around 400 nm. The values of indices GNDVI, RNDVI and NDVI 450 obtained from measuring refl ectance of leaves with symptoms of V. inaequalis and P. leucotricha infections were signifi cantly lower compared to the indices of healthy leaves. The values of indices NDVI were as follows: GNDVI 0.930; RNDVI 0.912; NDVI 450 0.917 for healthy leaves and GNDVI 0.519/0.623; RNDVI 0.428/0.540; NDVI 450 0.432/0.499 for leaves infected by pathogens V. inaequalis/P. leucotricha, respectively. There was found signifi cant diff erence between infected and healthy leaves for all indices ( = 0.05). Also, the PSII exhibited signifi cant responses to the presence of V. inaequalis and P. leucotricha (PSII: healthy leaves 0.182; V. inaequalis/P. leucotricha presence 0.232/0.222;  = 0.05). apple tree, spectral refl ectance, chlorophyll fl uorescence, Venturia inaequalis, Podosphaera leucotricha, nitrogen content Light energy absorbed by chlorophyll molecules in chloroplasts of green plant leaves is used in the three ways: for synthesis of simple sugars in photosynthesis, for thermal energy, and it is released to the environment or emitted in the form of red fl uorescence radiation. These 318 V. Spáčilová, I. Šafránková processes are competitive among each other and stress conditions (high temperatures, drought, frost, defi cient or abundant nutrients, infection by pathogens, herbicide eff ects) induce changes in the activity of the photosynthetic process. Use of photochemical energy decreases resulting in increased release of thermal energy, and especially fl uorescence radiation. There is an inversely proportional relationship between the effi ciency of photosynthesis and chlorophyll fl uorescence. Upon UV-A excitation, the fl uorescence spectrum of green leaves presents not only the red and farred emission bands respectively centred near to 690 and 740 nm due to the emission of the proteinbound chlorophyll a in the chloroplasts of the subepidermal mesophyll cells, but also a maximum (430–450 nm) in the blue region and visible near to 520 nm (CHAPELLE et al., 1984). A complex description of the photosynthetic apparatus activity, including distribution of loss processes of photosynthesis into photochemical and thermal is provided by quenching analysis, the most widespread technique of measuring chlorophyll fl uorescence. The quenching analysis is based on highly-intensive fl ashes of a xenon lamp, so-called saturation light pulses. The measurement is usually carried out on leaves that were adapted to dark (for at least 15 min). Analysis of quenching kinetics for small regions can be used to diagnose distribution of the photosynthetic activity in leaves. During the growing season, apple trees are exposed to diff erent stress factors. In plant physiology, two basic groups of stress are identifi ed: abiotic stress (chemical factors or mechanical damage) and biotic stress (it is caused by living organisms). Chlorophyll fl uorescence imaging can determine changes in leaf photosynthesis induced by both types of stresses abiotic and biotic. Biotic factors infl uencing the plant metabolism and decreasing yield of crops are various infections (fungal, viral or bacterial). Pathogen infections induce changes in the primary metabolism of the aff ected plant tissue (CSÉFALVAY et al., 2010). A lot of plant pathogens damage photosynthetic tissues (necrotic lesions, chloroses, yield reduction) (BEDBROOK & MATTHEWS, 1973). Tobacco mosaic virus probably acts on chloroplasts as a parasite damaging the photosynthetic apparatus indirectly through depleting nitrogen supplies which are used for the synthesis of ribulose-1,5bisphospate carboxylase oxygenase (Rubisco). A similar principle of infl uence can be found in various virus species (REINERO & BEACHY, 1989). CHAERLE et al. (2007) tested the use of chlorophyll fl uorescence imaging for screening resistance to Cercospora beticola in sugar beet. The method is useful for automatic quantifi cation of the stress induced by damage during diagnosis using fl uorescence, i.e. before visual symptoms of infection, and for detection of pre-visual expressions of the pathogen presence in leaves of infected plants of sugar beet. Abiotic factors causing stress can be added heat, drought or nutrient defi ciency. They negatively infl uence the plant metabolism but as well as leaf photosynthesis. SCHAPENDONK et al. (1992) verifi ed a possibility of using chlorophyll fl uorescence for fast assessment of a photosynthetic status in plants. His experiments with maize plants exposed to water and cold stress confi rmed a possibility to employ this method and demonstrated that it is a non-destructive technique detecting damage of the photosynthetic apparatus of plants. Cold stress was determined by a decrease in maximum quantum yield of PSII – Fv/Fm, water stress was determined by increased qn. ZULINI et al. (2007) conducted experiments aimed at detecting drought infl uence on chlorophyll fl uorescence and photosynthetic pigments in grapevine (cv. White Riesling). He compared leaves of grapevine grown under optimum conditions and those exposed to water stress. Leaf pigment concentrations (chlorophyll a, chlorophyll b and carotenoids) were not infl uenced by water stress, but in stressed plants, the correlation between Fv/Fm and chlorophyll concentrations was signifi cant. They concluded that photosynthetic effi ciency was only aff ected at a high intensity of drought. Fv/Fm could therefore be a good indicator to defi ne threshold between moderate and excessive drought stress in the fi eld. HEISEL et al. (1999) investigated detecting of other stress factors which were caused especially by a lack (N, K, Mg) or excess (Ca) of nutrients in wheat, grapevine and apple trees. The eff ect of nitrogen supply on chlorophyll emission intensity in apple cv. ’Jonagold’ was studied. Under 355 nm excitation, the intensities at 690 and 740 nm of the chlorophyll emission, as well as the blue/red and blue/far-red ratios, were modifi ed by the nitrogen fertilization in an opposite way for rosette leaves (much lower intensities and slightly higher ratios when nitrogen was supplied) than for the 1-year short leaves (weak intensities increase and important decrease of the ratios). For 532 nm excitation, the fertilization decreased the red emission and increased the farred intensities for both leaves, markedly for rosette leaves. BREDEMIER & SCHMIDHALTER (2001) assumed a close relationship between nitrogen content in plants and chlorophyll synthesis, and a possibility to detect its content basing on changes in the fl uorescence spectrum of plants. An important component of tetrapyrole chlorophyll core is nitrogen and the chlorophyll component in leaves is correlated with nitrogen concentration. The author not only confi rmed a relationship between fl uorescence intensity and chlorophyll content in leaves, especially at 680 nm, but also a negative relationship between values of the fl uorescence ratio 680/740 nm and chlorophyll values in winter wheat leaves. DALEY (1994) exposed tobacco plants to slight stress of nitrogen defi ciency. Due to this stress, fl uorescence quenching considerably changed, particularly during stomata movement. Potential of chlorophyll fl uorescence and VIS/NIR spectroscopy measurement use for the detection of nitrogen 319 LIMA et al. (1999) tested the eff ect of nitrogen and phosphorus defi ciency on chlorophyll fl uorescence in beans. He demonstrated that the phosphorus and nitrogen defi ciency aff ected the growth and parameters of photosynthesis, and decreased maximum fl uorescence yield by up to 25 %. Detection of physiological defects of apples using chlorophyll fl uorescence during storage was studied by CISCATO et al. (2001). In apple cvs. ’Golden Delicious Smoothe’ and ’Jonagold’ he proved a possibility to use chlorophyll fl uorescence for assessing physiological damage of fruits depending on storage potential prior to emerging symptoms. He found a relationship between total chlorophyll content and chl

acta universitatis agriculturae et silviculturae mendelianae brunensis

Potential of chlorophyll fluorescence and VIS/NIR spectroscopy measurement use for the detection of nitrogen content and disease infection of apple leaves