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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 DanaIONITA2), Marina Cirjaliu-MURGEA2),
Viorica CHITU1), Laurentiu FILIPESCU2)
1) RIFG Pitesti, Maracineni, Arges, Romania;[email protected]) Politehnica" University of Bucharest, Faculty of Applied Chemistry and Material Science,
Bucharest, Romania; [email protected]
Abstract.Water and nutrients are the most yield limiting factors in fruit crops. Many
studies have shown the chlorophyll (Chl) fluorescence parameters provide good indicators of
nutrient deficiency and stress. The effect of four foliar nutritive fluids considered as completeformulations 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 withweather data taken from May 2007 and June 2009 period. As nutrientstress 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 fluorescence 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 chemicalstress.
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 fluorescencetechnique, 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
fluorescence level(T1/2), and fast dark adapted tests using OJIP protocol (F0, FJ, FI, FV, FV, VJ,
mailto:[email protected]:[email protected]:[email protected]:[email protected]:[email protected] -
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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/FMimpart diurnal and seasonal variations in photosynthesis rate under natural or induced
conditions of plant growth. Reversible inactivation of photosystem II reaction centers underhigh 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 fluorescencetechnique 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 comingout 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 asthe rest of foliar treated plots; V2 foliar treated plot with the nutritive fluid Nutrinaft A; V3
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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) chlorophyllfluorometer, 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 checksfor 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 R
2
=0.381***), like drought and overheat (solar radiation (rpartial=-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 (Schreiberet 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 ifFV/FM correlates negatively with F0 (r= -0.877**) and FM (r= -0.249**), the experimental data
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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 excedentaryenergy 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 potentialquantum 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 ondifferent position against light. Above observations seem to sustain non quenching energy
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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
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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 emulsifiednutritive fluids (partial correlations)
OJIP
parametersNitrogen Phosphorus Potassium
Naphthenic
acids
Multiple correlation
coefficient(R)F0 0.118* -0.090 0.088 -0.050 0.147
FM 0.082 -0.080 0.092* -0.065 0.128
Fix Area 0.086 -0.084 0.095* -0.070 0.128
SS -0.074 0.087 -0.096* 0.062 0.125
ET0/RC 0.066 -0.090 0.109* -0.063 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 influencedby nitrogen content of the applied emulsified nutritivefluids (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 ET0/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 inthe 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).
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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 evidencingthe active components carried by the emulsified nutritive fluids have been adsorbed, and
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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.
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