Buletin UASVM, nr.

66 (1-2)/2009 Horticulture, Print ISSN 1843-5254; Electronic ISSN 1843-5394

Evaluation of Foliar Nutritive Fluids Effect on Apple Photosystem II Efficiency using Chlorophyll Fluorescence Emil CHITU1), Aura Dana IONITA2), Marina Cirjaliu-MURGEA2), Viorica CHITU1), Laurentiu FILIPESCU2) 1) RIFG Pitesti, Maracineni, Arges, Romania; emilchitu@gmail.com 2) Politehnica" University of Bucharest, Faculty of Applied Chemistry and Material Science, Bucharest, Romania; laurentiu_filipescu@yahoo.co.uk Abstract. Water and nutrients are the most yield limiting factors in fruit crops. Many studies have shown the chlorophyll (Chl) uorescence parameters provide good indicators of nutrient deficiency and stress. The effect of four foliar nutritive fluids considered as complete formulations of different classes of these products carrying nutrient, growth enhancing, and fungicide functions on the Jonathan cv. photosystem II (PSII) efficiency has been tested. A database of chlorophyll fluorescence (OS 30 and FP 100 fluorometers), determinations was used, together with weather data taken from May 2007 and June 2009 period. As nutrient stress tests, there were used the potential quantum yield of PS II (F V/FM), fast light adapted test (Yield), half rise time from minimal to maximal uorescence level (T1/2), and fast dark adapted tests using OJIP protocol (F0, FJ, FI, FV, FV, VJ, VI, FM/F0, FV/FM, M0, Area, Fix Area, SM, SS, N, P0, 0, E0, D0, Pav, PIAbs, ABS/RC, TR0/RC, ET0/RC, DI0/RC). The twenty six above parameters of the Chl fluorescence were measured under variable apple tree foliar feeding conditions related to the vegetative stage, measurements timing and meteorological factors dynamics. All data were used to assess and quantify the foliar nutritive fluids chemical stress. Keywords: apple, nutritive fluids, nutrient stress, OJIP protocol, photosynthesis INTRODUCTION Photosynthesis rate and its shifting around the normal values are important indicators of both plant overall fitness and extent of environmental plant stress. Most of the plant responses to unfriendly growth conditions are particularly resulting in quite proportional decreases in the photosynthesis rate (Reddy et al., 2004). Quenching analysis of modulated fluorescence by means of the saturation pulse method, known as the chlorophyll fluorescence technique, provides measured parameters reflecting the functioning of photosystem II (PSII) and in effect correlations with photosystem I (PSI) and the dark reactions (Schreiber et al., 1986). Actually, the chlorophyll fluorescence technique may generate the data concerning the impacts of light intensity and quality, water and mineral nutrition, disease control system responses, pollution and other environmental factors on the rate of CO2 assimilation, as well as, the plant health status. Nutrient deficiency, the water stress, long-term stress events and limiting growth factors could be monitored by chlorophyll fluorescence measurements coupled with analysis of the chlorophyll (Chl) and carotenoid content in leaves (Freedman et al. 2002). There are several surveying parameters from chlorophyll fluorescence measurements, but in this study only the following were analyzed: potential quantum yield of PS II (FV/FM), fast light adapted test (Yield), half rise time from minimal to maximal uorescence level (T1/2), and fast dark adapted tests using OJIP protocol (F0, FJ, FI, FV, FV, VJ,

VI, FM/F0, FV/FM, M0, Area, Fix Area, SM, SS, N, P0, 0, E0, D0, Pav, PIAbs, ABS/RC, TR0/RC, ET0/RC, DI0/RC). Significant changes in chlorophyll fluorescence parameter, FV/FM impart diurnal and seasonal variations in photosynthesis rate under natural or induced conditions of plant growth. Reversible inactivation of photosystem II reaction centers under high light conditions conjoined with antenna heat dissipation and photorespiration were found by chlorophyll fluorescence measurements responsible for delayed CO2 assimilation as recoil for the photodamage alleviation. According to recent researches, the parameter FV/FM is not intensely affected by nitrogen and phosphorus stress (Nowak and Stroka, 2001), mainly when these nutrients are applied at moderate rates and concentrations. Also, some other parameters like K Step parameter, PI parameter and the qP parameter show unquestionable cases of nitrogen deficiency. Comprehensible parameters related to PS II efficiency like quantum yield in darkness (FV/FM) or under the light exposure (F/FM or PSII) are relevant in assessing transient and steady state nutrient plant stress, even if their changes could be associated either to the photo protective energy dissipation or to photochemistry rate changes (Baker and Rosenquist, 2004). Besides understanding the plant functions, chlorophyll fluorescence technique is a powerful research method which enables in vivo run tests for the new technologies designed to improve crop yield and quality or in agricultural ecology researches and certifications of the new products for agricultural uses. This paper regards the experiments conducted at Research Institute for Fruit Growing Pitesti, Maracineni, Romania during 2007 and 2009 seasons over the sensitivity of chlorophyll fluorescence parameters to the nutritional and climatic stress factors, and the possibility of employing these parameters as a trustful evidence of some new foliar nutritive fluids effects on photosystem II efficiency and yield. MATERIALS AND METHODS Experimental field. Experimental plots were located 6 km north of Pitesti town, on third ledge of the terraced Arges river meadow at 200-280 meters altitude, 2455 east longitude and 4455 north latitude. Soil of these ledges was typically sloppy cambic with sandy-loam or sandy-clayey texture, poor in ion exchange capacity (11.6 meq/100g soil) and humus reserve (70.1 t ha-1). Basic foliar diagnosis has shown impaired macronutrients ratios with satisfactory levels only in phosphorus and potassium. Biological material studied was represented by apple (Malus domestica Borkh.) Jonathan cv. with trees aligned in simple rows (3.3 x 1.0 m) on a 30 years old high density orchard (3,367 tree/ha). The experiments cover 2007 and 2009 years. Foliar nutritive fluids. Run tests were carried out with four different products coming out from the same baseline of emulsified foliar nutritive fluids transporting to the foliage surface variable NPK macronutrient formulas overcharged with mezzo nutrients (Nutrinaft A), growth enhancers (Nutrinaft B), micronutrients (Amokem) and fungicides (Frucol). These fluids are hydrolyzing through dilution and carbonation, leaving on the leaf surface a freshly precipitated layer of new biological active moieties. Their composition and hydrolyzed components identified on the foliage surface after the application was given elsewhere (Cirjaliu et al., 2007, 2008; Chitu et al., 2004, 2009; Gaidau et al., 2009). Foliar application. Before application the foliar nutritive sample were diluted with hard water up to 1.0% mass concentration and applied in the first part of the day using a low pressure sprayer. Experimental plots were set on 5 variants for each apple cultivar: V1 untreated blank plot, just spread with water at the same rate and at the same time intervals as the rest of foliar treated plots; V2 foliar treated plot with the nutritive fluid Nutrinaft A; V3

 foliar treated plot with the nutritive fluid Nutrinaft B; V4 foliar treated plot with the nutritive fluid Amochem dual B; V5 foliar treated plot with the nutritive fluid Frucol. Chlorophyll fluorescence measurements. During experiments carried out in 2007, the chlorophyll fluorescence data were collected with an OS 30 (Opti-Sciences) chlorophyll fluorometer, with the test length of 2 seconds and the excitation source intensity of 2,000 uE, by placing the leaves successively in dark (15 minutes with clips) and then in light. Over the 2009 year, the chlorophyll fluorescence data acquisition was made with portable FluorPen FP 100 chlorophyll fluorometer (Photos Systems Instruments). According to the instrument manual, 25 parameters and their variation for each of the four basic emulsified nutritive fluids were computed on the grounds of 354 measurements taken from 3 different experiments made in 3 successive days. Supplementary information over air temperature (averaged mean, 2 m over the soil level), air relative humidity, solar radiation (1-1,250 W m-2), and water potential in soil (0.3 m depth) were recorded with Spectrum WatchDog 900ET meteorological station and used to correlate with the chlorophyll fluorescence data. Treatment effects on chlorophyll fluorescence were determined by one-way analyses of variance (ANOVA) following checks for normality and equal variance distributions. Differences between treatment variants were rated by the Duncans multiple range test, at the 95% confidence level (P > 0.05) using the SPSS 14.0 software for Windows (SPSS, Inc.). Multiple regression and partial statistical correlation coefficients (describing the relationship between two variables while adjusting for the effects of one or more additional variables) between chlorophyll fluorescence parameters, emulsified nutritive fluids composition and meteorological factor were computed according Morrison method (Morrison, 1976). RESULTS AND DISCUSSIONS During the first experiments in 2007, the apple crop was crossing most critical energetic and nutritional period of the entire vegetative cycle, when flowering reached its end (29 April) and the fruitlets abscission was beginning (15 May). Due to the consumption of fall accumulated reserves, mineral charge of the foliar fluids prove to be as important as foliage extension and light contribution. Also, over the April of 2007 there were recorded significant water shortages (82.9 mm rainfall deficit), higher temperature (average, 23.4C, in the experimental period), which results in low relative humidity (20.7 48.9%) and high light intensity from 129 to 882 watt m-2 (average, 645 watt m-2). The dark adapted leaves fluorescence indicators shows that only the half-rise time of FM (T1/2), which is responsible for the size of the plastoquinone pool, was sensitive to the environmental factors (multiple R2 =0.381***), like drought and overheat (solar radiation (r partial=-0.564***) and relative humidity (r partial=-0.219**), fig. 1), and its significant decay is a proof the system could not keep in line a constant electron flow over the entire day. Value of the T1/2 parameter ranged from 375 ms in low radiation conditions to near zero (20 ms) at midday (figure 1), sustaining the previous findings concerning the minimal stress under mild drought conditions (Osorio et al., 2006). Others indicators, excepting photochemical quenching, maintain normal variations around the customary values under weak environmental stress (figure 1). In dark adapted test, the measured FV/FM ratio, or the maximum potential quantum efficiency of Photosystem II (PSII), finely correlates with carbon fixation under any of circumstantial conditions (Schreiber et al., 1986; Baker & Rosenkuist, 2004). Mean value of this indicator was found to be 0.755, very close to optimal accepted figure (0.830). Even if FV/FM correlates negatively with F0 (r= -0.877**) and FM (r= -0.249**), the experimental data

point out to a low level of stress, induced by all the emulsified nutritive fluids. Nevertheless, figure 2 clearly unveil certain significant jumps in FV/FM indicator values immediately in hours after application on the foliage for all the products. This behavior might be explained by fast penetration of nutritive and growth enhancing fluids through cuticula, and excedentary energy comsumption during this short period of overfeeding. After some 24-48 hours, this unharmful transitory overfeeding stress settles down to common potential quantum efficiency of Photosystem II (0.750-0.800), reflecting the safety health state of foliage. Also, it is not excluded the assumption of the leaf cooling contribution (during fluids spraying) to the temporary changes in potential quantum efficiency FV/FM.

Fig. 1. Correlation matrix of the chlorophyll fluorescence indicators with meteorological factors

The light adapted leaves, the fluorescence indicator yield F/F M (Y(II),PSII, where F = FM FS)/ FM) is measuring the magnitude of effective quantum photosynthesis yield under steady-state photosynthetic lighting conditions. Closure of reaction centers and heat dissipation are non-quenching energy consumers and share with yield Y(II) the total potential quantum efficiency FV/FM. Yield Y(II) low values (average mean 0.310) recorded during run tests for emulsified nutritive fluids under experimental conditions could not be easily explained (figure 3). Chiefly, the use of nutritive fluids raise just temporarily the level of potential quantum efficiency FV/FM. Afterwards, the changes in FV/FM are keeping on with normal values as 0.830. No one from the chlorophyll fluorescence indicators displayed any correlation with meteorological factors. The only positive correlation was found between the yield Y(II) and T1/2 indicator, but this last indicator did not correlate with meteorological factors. The same values for yield Y(II) and the same dependencies with Frucol product performing better have been assigned for measurements on trees bourse, shots and leaves on different position against light. Above observations seem to sustain non quenching energy

consumption for nutritive moieties transport under leaf overfeeding in first hours after emulsified nutritive fluids application.

Fig. 2. Variation of the FV/FM indicator versus the time elapsed since foliar application of the new emulsified nutritive fluids.

Fig. 3. Variation of yield Y(II) versus the time elapsed since foliar application of the new emulsified nutritive fluids.

All the 354 measurements of OJIP parameters taken from the dark adapted leaves, during the three experiments made in three successive days in June, 2009, were correlated with the content of nitrogen, phosphorus, potassium and naphthenic acids in emulsified nutritive fluids. Values of partial correlation coefficients (separated from the influence of other independent factors) given in the table 1 particularly illustrate the only few from the measured 25 OJIP fluorescence parameters provide information about the contribution of mineral and growth enhancers charge in nutritive fluid to the photosynthetic processes. Thus, the table 1 parameters are suggesting the efficiency of the PSII photochemistry was not

significantly affected by phosphorus applications, which is in good agreement with other authors data (Nowak and Stroka, 2001).
Tab. 1 Intensity of the correlations between the OJIP test parameters and the composition of emulsified nutritive fluids (partial correlations)

OJIP parameters F0 FM Fix Area SS ET0/RC

Nitrogen Phosphorus Potassium 0.118* 0.082 0.086 -0.074 0.066 -0.090 -0.080 -0.084 0.087 -0.090 0.088 0.092* 0.095* -0.096* 0.109*

Naphthenic acids -0.050 -0.065 -0.070 0.062 -0.063

Multiple correlation coefficient (R) 0.147 0.128 0.128 0.125 0.169*

* Partial correlation is significant at the 0.05 level

Parameter F0, encompassing a range from 235 and 521 (average mean 380) have been significantly and positively influenced by nitrogen content of the applied emulsified nutritive fluids (r partial=0.118*). Thus, if the FM parameter takes a constant value over the variation range of nitrogen content, the above correlation is equivalent with a cutback in PSII efficiency. Other OJIP parameters were mainly dependent only of the potassium content in the applied emulsified nutritive fluids. The FM parameter (r partial=0.092*), as well as the total area above the OJIP fluorescence transient between F40s and F1s (r partial=0.095*) and ET0/RC, electron transport flux per reaction centre at t=0 (r partial=0.109*) were very sensitive to potassium variation in the applied fluids. Only SS parameter, standing for the smallest SM single turn-over (r partial=-0.096*), has been diminished by potassium ion in nutritive fluids. By far, the parameter ET0/RC is certainly expressing with fine accuracy the potassium contribution to increase in the achieved efficiency of photosystem PS II. An explanation of the above findings about sensitivity of chlorophyll fluorescence to potassium foliar feeding may stand, also, in the well known high potassium requirements during late June intensive fruit growth. Foliar treatment effects of the emulsified nutritive fluids on ET 0/RC chlorophyll parameter, which quantifies the flux of electrons beyond QA during the initial fluorescence rise after actinic light impulse, are imparted by the figure 4. The raise in electron transport per reaction centre (expressed by ET0/RC level) is significant for all the treatments, and for all the products this raise is continual till the second day after application. Slight decrease in the third day is factually evidencing the active components have been adsorbed and consummated in the metabolic process. Moreover, the consumption was not occurring on the spot. Even if active components are there spread on the leaf or on their way to penetrate cuticle and tissular membranes, they are metabolized at a rate controlled by the specific plant nutrition mechanism. Thus, the overfeeding and leaf burning under excessive foliar nutritive products could not come about with the frequency met to the common NPK foliar fertilizers. It seems the products Amokem and Frucol perform better than the other, but it is rather difficult to name the component which is able to produce the raise in ET0/RC level. All the above data back up the formulation principles of emulsified nutritive fluids and sustain their capacity to promote growth stimulation and alleviate mineral stress at the foliage surface (Cirjaliu et al., 2007, 2008).

Fig. 4. Variation of the ET0/RC parameter over three day treatment with emulsified nutritive fluids

CONCLUSIONS During the experimental periods, along the 2007-2009 seasons, the sensitivity of chlorophyll fluorescence parameters to the nutritional and climatic stress factors was studied in order to estimate the stress and possible disturbances in the electron transport of Photosystem II at Jonathan cv. apple trees under foliar treatments with four new emulsified nutritive fluids. Experiments were carried out mainly within two distinctive different environmental conditions: advanced drought and low air relative humidity, and mild disturbances in normal meteorological common data at the experiment location, respectively. In dark adapted test (unfriendly environment conditions), the measured FV/FM ratio, standing for the maximum potential quantum efficiency of Photosystem II (PSII), was found to be 0.755 (averaged mean), very close to optimal accepted figure (0.830). Experimental data point out to a low level of stress induced by all the emulsified nutritive fluids, even if certain significant jumps of FV/FM indicator values in hours after application were noticed. But this transitory overfeding stress was fading out after 24-48 hours, when the FV/FM indicator was turning back to normal rates, reflecting the safety health state of foliage. In the light adapted leaves (unfriendly environment conditions), the fluorescence indicator yield F/FM (Y(II), PSII, where F = FM FS)/FM), which is measuring the magnitude of effective quantum photosynthesis yield under steady-state photosynthetic lighting conditions, did take uncommon low values around the average mean of 0.310. Because the light reaction centers remained open and potential quantum efficiency Fv/Fm was normal (around 0.755 averaged mean), it is reasonable to consider the yield Y(II) was low due to the non quenching energy consumers. As far as heat dissipation do not seems to prevail as a plausible consumer due to missing of correlations with meteorological factors, the nutrient moieties transport to other tree organs for storage was supposed to be the main non quenching energy consumer. The ET0/RC chlorophyll fluorescence parameter (measured under mild environmental stress), which quantifies the flux of electrons beyond QA during the initial fluorescence rise after saturated light impulse, was found significantly increasing for all the emulsified nutritive fluids applied as diluted solutions. Its slight decrease in the third day is factually evidencing the active components carried by the emulsified nutritive fluids have been adsorbed, and

consummated in the metabolic process at a rate controlled by the specific plant nutrition mechanism. All the above data back up the formulation principles of emulsified nutritive fluids and acknowledge these products capacity to promote growth stimulation and alleviate mineral stress at the foliage surface. ACKNOWLEDGMENT The work was carried out with the financial support of the CNCSIS Program, Ideas, within the Research Project 1035/2007. REFERENCES Baker, N.R. and E. Rosenquist (2004). Applications of chlorophyll fluorescence can improve crop production strategies: an examination of future possibilities. Journal of Experimental Botany 55:1607-1621. Chitu, V., E. Chitu, A. Hororoi, M. Calogrea, M. Cirjaliu-Murgea and L. Filipescu (2004). Researches concerning Nutrinaft products effects on apple production and fruit quality, Annals of the University of Craiova, Vol. IX (XLV):123-128. Chitu, V., E. Chitu, S. Nicolae, L. Filipescu, A. D. Ionita and M. Cirjaliu-Murgea (2009). Relationships between shelf life, health and quality of apple fruit. Acta Hort. (ISHS) 825:539-546. Cirjaliu-Murgea, M., A. D. Ionita, E. Chitu and L. Filipescu (2007). Emulsified Nutritive Fluids. Overbasicity and Hydrolysis Control. Annals of the University of Craiova, Vol. XII (XLVIII):183. Cirjaliu-Murgea, M., A. D. Ionita, E. Chitu, V. Chitu and L. Filipescu (2008). Emulsified nutritive fluids and their proprieties control. 6th International ISHS Symposium on Mineral Nutrition, Faro, Portugal. Freedman, A., J. Cavender-Bares, P. L. Kebabian, R. Bhashkar, H. Scott and F. A. Bazzaz (2002). Remote sensing of solar-excited plant fluorescence as a measure of photosynthetic rate. Photosynthetica 40(1):127-132. Gaidau, C., E. Stepan, D. Taloi, M. Niculescu and L. Filipescu (2009). Additives and advanced biofertilizers obtained from leather industry by-products. The Chemistry Magazine (Bucharest), [in Romanian], 60(5):501-507. Nowak, J. and S. Stroka (2001). The effect of phosphorus nutrition on growth, flowering and chlorophyll fluorescence of New Guinea Impatiens Pago pago. Acta Hortic. 548:561566. Morrison, D.F. (1976). Multivariante statistical methods. New York, McGwaw-Hill. Osrio, M., B. E. Rodrigues, A. Osrio, J. Le Roux, X. Daudet, F. A. Ferreira, and M. M. Chaves (2006). Limitations to carbon assimilation by mild drought in nectarine trees growing under field conditions. Environmental and Experimental Botany 55(3):235-247. Reddy, A.R., K. V. Chaitanya and M. Vivekananda (2004). Drought-induced responses of photosynthesis and antioxidant metabolism in higher plants. J. Plant Physiology 161:1189-1202. Schreiber, U., Schliwa U., Bilger, W. (1986). Continuous chlorophyll fluorescence quenching with a new type of modulation fluorometer. Photosynth. Res. 10:51-62.