Content changes of assimilative pigments in leaves after fertilizer Mg-Titanit application

© Slovak University of Agriculture in Nitra Faculty of Agriobiology and Food Resources, 2014 http://www.fapz.uniag.sk/ DOI: 10.15414/afz.2014.17.02.58–64


Introduction
The photosynthesis plays the direct role in the production process of plants (Ducsay, 2011;Olšovská et al., 2013).It depends on the size of photosynthesizing surfaces and the efficiency of the photosynthetic apparatus which is determined by the quality of the pigmental system.The most important pigments are chlorophyll a and chlorophyll b which are worn out in the process of photosynthesis therefore they are renewed permanently (Baker, 1996).The mineral nutrition determines significantly the photosynthetic activity of plants.The leaves grow old with the insufficient nutrition and lose the ability of the photosynthetic activity.The effect of particular nutrients on the process of photosynthesis is very different (Larcher, 1988).The positive relation is usually recorded between the nutrients supply in a plant and the size of the leaf surface and the photosynthesis speed (Nátr and Lawlor, 2005;Namvar et al., 2013), or between the content of nitrogen, magnesium, sulphur and the content of the total chlorophyll in a plant (Filová and Krivosudská, 2013;Marschner, 2005;Tůma and Tůmová, 2006).The other authors as Ram et al. (1983) but also Carvajal and Alcaraz (1995), Kužel et al. (2003), Kováčik et al. (2012) recorded the increase of content of the total chlorophyll in the cultivated plants after the application of the fertilizer containing titanium.Similarly, Traetta-Mosca (1913), Pais (1983), Simon et al. (1988), Matuškovič (1995), Carvajal and Alcaraz, (1998) observed that the well-balanced titanium application increases not only the chlorophyll content but also the yield of the cultivated crops, in particular from 5 to 50 %.On the other hand, the excessive application of titanium decreases yield (Kužel et al. 2007).The negative effects of titanium are evident on the plants more frequently if the plants are cultivated on the soils with the insufficient magnesium content, with the content of soil organic carbon less than 0.7% and the pH less than 6.
The first evidence of the excessive plant nutrition with titanium is the decreased content of the total chlorophyll (Carvajal and Alcaraz, 1998;Hrubý et al., 2002).The numerical value of the reasonable and unreasonable dose of titanium is different depending on the particular crops in the different cultivating conditions.Therefore, the objective of these experiments was to determine the impact of two doses of the fertilizer containing titanium, which were applied on two different crops (cereals and oil plants) during the growing season (in the first treatmentdouble application and in the second treatment -triple application) on total chlorophyll content in the leaves of winter wheat and winter rape.
The wheat and rape trials involved 5 variants.Var.1: 0Ti -control, MGT unfertilized variant; var.2: 2 × Ti 0.2 -spraying twice by MGT in the dose 0.2 l ha -1 ; var.3: 3 × Ti 0.2 -spraying thrice by MGT in the dose 0.2 l ha -1 ; var.4: 2 × Ti 0.4 -spraying twice by MGT in the dose 0.4 l ha -1 , var.5: 3 × Ti 0.4 -spraying thrice by MGT in the dose 0.4 l ha -1 .The fertilizer Mg-Titanit contained 3 % magnesium, 4 % sulphur, and 8.5 g of titanium in 1 litre, where titanium was in the form of titanium ascorbate and sulphur and magnesium in the form of magnesium sulfate (MgSO 4 ).The fertilizer Mg-Titanit, which has been categorized as a biostimulator since 2013, was applied at two or three growth stages: at wheat in the phenophases BBCH 29, BBCH 32, BBCH 55; at rape in the phases BBCH 50 -52, BBCH 59, BBCH 66 -67.14 to 21 days after the fertilizer application by spraying the samples of the youngest, fully developed sound leaves were analysed from both crops and the pigment contents were determined.The spectrophotometric method was used to determine the content of the assimilative pigments (chlorophyll a, chlorophyll b and carotenoids) in the acetonic extract.Extract was centrifuged 2 minutes at 2500 rpm.Absorbance (A) of the solution was measured by UV-VIS spectrophotometer (Jenway, UK), at 470 nm (carotenoids), 647 nm (chlorophyll b), and 663 nm (chlorophyll a), with correction for scattering at 750 nm; the measurements were done in three repetitions.The concentrations of chlorophyll a (Chl a), chlorophyll b (Chl b) and carotenoids (Car) in mg l -1 were determined by using the equations of Lichtenthaler ( 1987 The concentrations of the pigments were calculated in mg l -1 ; A n was the absorbance at given wavelengths (n) after correction for scattering at 750 nm; D was the optical thickness of cuvette; results were also recalculated in mg.m -2 using the volume of solution and the area of leaf segments: [mg m -2 ] = V/1000 × 1/A, when V is volume of 80% acetone and A is area of leaf segments.The sampling was carried

Results and discussion
The pigment biosynthesis in plants is influenced by a whole range of external and internal factors.Apart from the genetic factors also the light, temperature, water and last but not least nutrition rank among the important factors.The Figure 1 shows the fact that in the first year of the trial the wheat leaves treated with the fertilizer Mg-Titanit proved a higher content of the total chlorophyll (chlorophyll a + chlorophyll b) in each sampling than the wheat leaves cultivated in the control unfertilized variant.The highest and statistically significant increase of the contents of total chlorophyll was achieved at the growing stage BBCH 55, i.e. after the second spraying applied at the growing stage BBCH 32.The growth rates varied in the interval from 33.5 % to 37.3 %.A higher increase was recorded in the variant where a lower dose of fertilizer (0.2 l ha -1 ) was applied.The first and third spraying (application at the growing stages BBCH 29 and BBCH 55) impacted on the content of the total chlorophyll insignificantly (Table 2).The first spraying tended to increase the contents of the total chlorophylls (crop samplings at BBCH 32) and the third spraying (var. 2 versus var. 3 and var. 4 versus var.5) had the tendency to decrease the contents of the total chlorophylls (crop samplings at BBCH 67).Despite the recorded slightly negative impact of the third spraying on the content of the total chlorophylls their contents were higher compared with the contents of the total chlorophyll in the unfertilized variant.
The Table 2 shows that the increase of the total chlorophyll contents after spraying by Mg-Titanit was carried out through both chlorophyll a and chlorophyll b.The fertilizer Mg-Titanit also tended to increase the carotenoids content, except for the sampling after the first spraying.
The content changes of the total chlorophyll in the wheat leaves proved the single-apical progress (Figure 1) in the first year of the trial.At the stage of the massive vegetative growth and ear formation, i.e. from April to May the chlorophyll content was increased.It was decreased with the progressive senescence of the leaf system and the plants themselves.The differences related to the contents of the total chlorophyll between the plants sprayed by the fertilizer Mg-Titanit and unfertilized plants were gradually decreased.
In the second year of the trial, at the growth stage BBCH 32, i.e. 14 days after the first wheat spraying by the fertilizer Mg-Titanit, which was applied at the growth stage BBCH 29, unlike in the first year generally supposed (Kužel et al., 2003;Dobromilska, 2007) positive impact of fertilizers containig titanium on the total chlorophyll content was not recorded (Figure 2).The following samplings of the variants treated by Mg-Titanit proved higher contents of the total chlorophyll compared with the unfertilized variant, however, the differences were not significant statistically (Figure 2 and Table 3).Higher contents of the total chlorophylls were detected in the variants where a higher dose of the tested fertilizer was applied.In the same year the application of Mg-Titanit had the insignificant impact on the content of the particular pigments (chlorophyll a, chlorophyll b, carotenoids).
The similar findings about the conclusive and also inconclusive impact of the fertilizer containing titanium on the chlorophyll content in plants were detected by Vician et al. (2012).
The Figure 3 and Table 4 demonstrate that 5.4 % (var. 1 versus var. 2 and 3) and 11.4 % (var. 1 versus var. 4 and 5) content increase of the total chlorophylls was recorded at the growth stage of winter rape BBCH 59, i.e. 14 days after the first spraying.The highest content of chlorophylls after the first spraying, unlike wheat (year 2010 -Table 2), was detected in the rape leaves sampled from the variants where the highest dose of fertilizer was applied (0.4 l ha -1 ).The differences in the content of total chlorophyll between the variants 4 and 5 and the other variants were statistically significant (Table 4).The second spraying had also the positive impact on the content of the total chlorophylls but only with the application dose 0.2 l ha -1 of the fertilizer Mg-Titanit.The dose 0.4 l.ha -1 did not influence their quantity (Table 4).The third spraying by ) decreased the total content of chlorophylls where their number was lower than the chlorophyll content on the unfertilized variant.The detected negative impact, still statistically insignificant, of the third spraying on the content of the total chlorophyll corresponds with the findings related to wheat which were recorded in the first year of the trial.The indicated findings prove that in the given year the foliar application of Mg-Titanit, regardless the model crop, did not lead to the increase of the chlorophyll contents at the later growth stages BBCH 55 (wheat) and BBCH 67 (rape).
In the second year of the winter rape trial the different development was recorded in the contents of the total chlorophyll in the rape leaves in comparison with the first year of the trial.However, the positive impact of the fertilizer Mg-Titanit on the formation of the total chlorophyll was proved (Figure 4).The given figure shows statistically significant differences in the content of the total chlorophyll immediately after the first spraying by the fertilizer (Table 5).The differences were decreased after the second spraying but the succession was retained.The lowest content was detected in the unfertilized variant and the highest one in the variants fertilized by the lower application dose (Figure 4).This trend was retained, apart from two exceptions, till the last plant sampling carried out after the third spraying by the fertilizer.The exceptions were the variants 4 and 5 where the highest dose of Mg-Titanit was used.In the variant 4 without the third spraying the highest content of the total chlorophyll was recorded.In the variant 5 with the third spraying the content of the total chlorophyll was nearly identical with the content of the variant 1.The comparison of the contents of the total chlorophylls between the variants 2 and 3 and the variants 4 and 5 proved again that the third application dose of the fertilizer Mg-Titanit tends to decrease the content of the total chlorophyll in the leaves of the cultivated crops.Taking into consideration the support of the chlorophyll formation in the leaves of winter rape and winter wheat these facts show that the spraying by Mg-Titanit should not be applied at the given growth stages or it should be carried out earlier.
The chlorophyll a and chlorophyll b did not participate by the same rate in the increase of the contents of the total chlorophylls.After the first spraying the contents increase was carried out predominantly by the chlorophyll a.After the second spraying it mainly by the increase of the chlorophyll b content.
In general, the results of the quantitative changes of chlorophyll a, b, and a + b in the leaves of both model plants are identical with the findings of the author (Wang, 2009) who claimed that the leaves had a higher chlorophyll a content than b (Table 2).The average value of the chlorophylls a : b ratio was 2.73 : 1 in the wheat leaves and 2.45 : 1 in the rape leaves during the observed periods.

Conclusions
In the cultivation of winter wheat the content of the total chlorophyll was increased with both application doses of Mg-Titanit (0.2 and 0.4 l ha -1 ) in each year if the spraying was applied at the growth stage BBCH 32 (the second spraying).
Winter rape reacted positively to the spraying by Mg-Titanit applied at the growth stage BBCH 50 -52 (the first spraying) through the chlorophyll formation in both years of the trial.
The third spraying by both application doses Mg-Titanit tended mostly to decrease the content of the total chlorophyll in both crops.
The content of the total chlorophyll was predominantly higher in the plants fertilized by the dose 0.2 l.ha -1 than in those fertilized by the dose 0.4 l ha -1 .
The positive effect of Mg-Titanit on the content of the total chlorophyll was carried out by the increase of the amount of both assimilative pigments (chlorophyll a and chlorophyll b).In the most cases the application of the fertilizer Mg-Titanit determined more the content of chlorophyll b than chlorophyll a.The average value of chlorophyll ratio a : b in the wheat leaves was 2.73 : 1 and ): Chl a = 12.25 × (A 663 -A 750 ) -2.79 × (A 647 -A 750 ) × D Chl b = 21.50 × (A 647 -A 750 ) -5.10 × (A 663 -A 750 ) × D Chl a + b = 7.15 × ( A663 -A 750 ) + 18.71 × (A 647 -A 750 ) × D Car = [(1,000 × (A 470 -A 750 ) -1.82 × (Chl a) -85.02 × × (Chl b)/198] × D

Table 2
Impact of trial variants on dynamics of changes of pigment contents in leaves of winter wheat in 2010 Chlor. a -chlorophyll a, chlor.b -chlorophyll b, no.-number, des.-designation, LSD 0.05 -Least Significant Difference at the level α = 0.05

Table 3
Impact of trial variants on dynamics of changes of pigment contents in leaves of winter wheat in 2011 Chlor. a -chlorophyll a, chlor.b -chlorophyll b, no.-number, des.-designation, LSD 0.05 -Least Significant Difference at the level α = 0.05

Table 4
Impact of trial variants on dynamics of changes of pigment contents in leaves of winter rape in 2010

Table 5
Impact of trial variants on dynamics of changes of pigment contents in leaves of winter rape in 2011 no.-number, des.-designation, LSD 0.05 -Least Significant Difference at the level α = 0.05 Chlor. a -chlorophyll a, chlor.b -chlorophyll b,