1. The International Symposium “PROSPECTS FOR THE 3 rd MILLENNIUM AGRICULTURE” Emil CHITU 1), Aura Dana IONITA 2), Marina Cirjaliu-MURGEA 2), Viorica CHITU 1), Laurentiu FILIPESCU 2) 1) RIFG Pitesti, Maracineni, Arges, Romania 2) „Politehnica" University of Bucharest, Faculty of Applied Chemistry and Material Science, Bucharest, Romania

2. 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. There are several surveying parameters from chlorophyll fluorescence measurements, but in this study only the following were analyzed: potential quantum yield of PS II (F V /F M ), fast light adapted test (Yield), half rise time from minimal to maximal fluorescence level (T 1/2 ), and fast dark adapted tests using OJIP protocol (F 0, F J, F I, F V, F M, V J, V I, F M /F 0, F V /F M, M 0, Area, Fix Area, S M, S S, N, ΦP 0, Ψ 0, ΦE 0, ΦD 0, Φ Pav, PI Abs, ABS/RC, TR 0 /RC, ET 0 /RC, DI 0 /RC). 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. The International Symposium “PROSPECTS FOR THE 3 rd MILLENNIUM AGRICULTURE”

3. Experimental plots were located 6 km north of Pitesti town, on third ledge of the terraced Arges river meadow at 200-280 meters altitude. 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. 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. The International Symposium “PROSPECTS FOR THE 3 rd MILLENNIUM AGRICULTURE” MATERIALS AND METHODS

4. 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). 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 Duncan’s 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. The International Symposium “PROSPECTS FOR THE 3 rd MILLENNIUM AGRICULTURE”

5. The International Symposium “PROSPECTS FOR THE 3 rd MILLENNIUM AGRICULTURE” RESULTS AND DISCUSSIONS The dark adapted leaves fluorescence indicators shows that only the half-rise time of F M (T 1/2 ), which is responsible for the size of the plastoquinone pool, was sensitive to the environmental factors (multiple R 2 =0.381***), like drought and overheat (solar radiation (r partial= - 0.564***) and relative humidity (r partial= -0.219**), and its significant decay is a proof the system could not keep in line a constant electron flow over the entire day. Figure 1. Correlation matrix of the chlorophyll fluorescence indicators with meteorological factors

6. The International Symposium “PROSPECTS FOR THE 3 rd MILLENNIUM AGRICULTURE” Figure 2. Variation of the F V /F M indicator versus the time elapsed since foliar application of the new emulsified nutritive fluids. - Figure clearly unveil certain significant jumps in F V /F M 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 consumption 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.

7. Figure 3. Variation of yield Y(II) versus the time elapsed since foliar application of the new emulsified nutritive fluids The light adapted leaves, the fluorescence indicator yield ΔF/F M ’ (Y(II),Ф PSII, where ΔF = F M ’– F S )/ F M ’) is measuring the magnitude of effective quantum photosynthesis yield under steady-state photosynthetic lighting conditions. 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. The International Symposium “PROSPECTS FOR THE 3 rd MILLENNIUM AGRICULTURE”

8. Table 1. Intensity of the correlations between the OJIP test parameters and the composition of emulsified nutritive fluids (partial correlations) - 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; - 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 F40µs 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. The International Symposium “PROSPECTS FOR THE 3 rd MILLENNIUM AGRICULTURE”

9. The International Symposium “PROSPECTS FOR THE 3 rd MILLENNIUM AGRICULTURE” Figure 4. Variation of the ET 0 /RC parameter over three day treatment with emulsified nutritive fluids - 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 ET 0 /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.

10. The International Symposium “PROSPECTS FOR THE 3 rd MILLENNIUM AGRICULTURE” CONCLUSIONS - In dark adapted test (unfriendly environment conditions), the measured F V /F M 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 F V /F M indicator values in hours after application were noticed. But this transitory overfeeding stress was fading out after 24-48 hours, when the F V /F M indicator was turning back to normal rates, reflecting the safety health state of foliage. - In the light adapted test, the ET 0 /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.

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13. The International Symposium “PROSPECTS FOR THE 3 rd MILLENNIUM AGRICULTURE” ACKNOWLEDGMENT The work was carried out with the financial support of the CNCSIS Program, ‘Ideas’, within the Research Project 1035/2007.

14. The International Symposium “PROSPECTS FOR THE 3 rd MILLENNIUM AGRICULTURE” Acknowledgment The work was carried out with the financial support of CNCIS, Program Idei, project 1035/2007.