Acta Sci. Pol.

, Agricultura 8(2) 2009, 43-50

EFFECT OF NITROGEN FERTILIZATION

AND METHODS OF MAGNESIUM APPLICATION
ON CHLOROPHYLL CONTENT, ACCUMULATION
OF MINERAL COMPONENTS, AND MORPHOLOGY OF
TWO MAIZE HYBRID TYPES
IN THE INITIAL GROWTH PERIOD
PART I. CONTENT OF CHLOROPHYLL AND MINERAL
COMPONENTS

Piotr Szulc
Poznan University of Life Sciences1

Abstract. Field experiment was carried out in the Didactic and Experimental Department
in Swadzim (52o26’ N; 16o45’ E) near Pozna in years 2004-2007. The experiment was
established in a „split-plot” design with 3 factors and 4 field replications. Reactions of
two maize hybrid types to differentiated nitrogen and magnesium fertilization and to the
method of magnesium application were studied. The influence of these factors on
chlorophyll content and mineral components in the dry matter of maize plants in the phase
of 5-6 leaves (BBCH 15-16) was estimated. On the basis of chloroplast pigments content,
it was found that the hybrid LG 2244 stay-green type was better nourished with nitrogen,
in comparison with the traditional hybrid Anjou 258. The highest nitrogen content in dry
plant matter was found after the application of a 90 kg N·ha-1 nitrogen dose, while for
magnesium of a 150 kg N·ha-1 dose. A better method of magnesium application has been
shown to be the sowing in row method, in comparison with the broadcasting method. As
a result of magnesium sown in rows, the Mg content in plant dry matter was higher
compared with the broadcasting method and with a zero dose of magnesium.
Key words: BBCH 15-16, fertilizer application method, magnesium, maize hybrid types,
nitrogen, stay-green

Corresponding author – Adres do korespondencji: dr Piotr Szulc, Department of Soil and Plant
Cultivation of Pozna University of Life Sciences, Mazowiecka 45/46, 60-623 Pozna, Poland
e-mail: [email protected]
44 P. Szulc

INTRODUCTION

Maize (Zea mays L.) uptakes great amounts of nutritive components from the soil.
They exert an influence not only on the size of the obtained yields, but also on their
fodder and consumption values [Costa et al. 2002, Scharf et al. 2002]. In order to
achieve full utilization of the yield-creating possibilities of maize, the farmer must apply
nutritive components so that the plant can uptake them according to the rhythm of plant
growth. In maize development, a series of phases occurs where each of them plays
a definite role in maize life cycle. In the phase of 6-12 leaves, maize builds its potential
yield structure because at that time the ear setting takes place and the number of grain
rows in the ears is determined [Grzebisz 2008]. This feature is genetically determined.
Nevertheless, as a result of the action of environmental and agronomic stresses, there
comes a decrease in the number of rows in the ears. According to Subedi and Ma
[2005], nitrogen malnutrition of plants, before the phase of the 8th leaf, leads to
irreversible reduction (even up to 30%) in the number of cobs and in the potential grains
in the cob. Binder et al. [2000] showed that the delay in nitrogen application until the
phase of the 6th leaf reduced the grain yield by 12%. The above results were obtained
on the basis of field studies with the application of conventional hybrids only.
The hypothesis of the present experiment assumed that the cultivars of stay-green
type, because of more abundant leafage and big biomass [Andrews et al. 2000], may
have other requirements for nutritive components, in comparison with the traditional
hybrid. Furthermore, in case of maize in the phase of 5-10 leaves, on the basis of the
content of the particular mineral components in dry matter, one can make a final
correction of the faulty fertilization method chosen earlier.
In this connection, studies were undertaken whose objective was to define the
nutritional status of two maize hybrids in the phase of 5-6 leaves, depending on
different nitrogen fertilization levels and on the magnesium dose application method.

MATERIAL AND METHODS

Field studies were carried out at the Didactic and Experimental Farm in Swadzim
(52o26’ N; 16o45’ E), near Pozna in years 2004-2007. The results of the studies carried
out in 2006 were disqualified because of prevailing drought in the period of maize
growth and development. The experiment was carried out in a „split-plot” design with
3 factors in 4 field replications. Two hybrids were studied: Anjou 258 and LG 2244
stay-green type. Six nitrogen doses were used: 0, 30, 60, 90, 120, and 150 kg N·ha-1, and
the doses of magnesium of: 0 kg Mg·ha-1, 15 kg Mg·ha-1 (in rows), and 15 kg Mg·ha-1
(broadcasting), in the form of kieserite were applied. A detailed method of the
experiment and the description of thermal and moisture conditions are both contained in
an earlier work by the authors [Szulc et al. 2008b].
Measurement of the content of chloroplast pigments and of mineral components was
carried out in the phase of 5-6 leaves (BBCH 15-16). Samples for the chemical
determinations consisted of 8 randomly taken plants from each plot. The content of
chlorophyll was determined by two methods: the direct and the indirect one. In the case
of the direct method, the leaf weighed portion was cut into 2-3 mm sections, which were
poured with 5 ml DMSO (dimethyl sulfoxide). The samples were kept in the dark at
room temperature and then incubated at 65°C (water bath) for 30 minutes. In the

Acta Sci. Pol.

Effect of nitrogen... Part I. Content of chlorophyll... 45

obtained extract, after cooling down, the a and b chlorophyll contents were
spectrophotometrically determined.
The content of chlorophyll pigments was spectrophotometrically determined using
Spekol spectrophotometer at an adequate wave length. For chlorophyll a, the
measurement of extract absorbance was carried out at the wave length of 663 nm, while
for chlorophyll b, the same was done at the wave length of 656 nm. For carotenoids,
470 nm wave length was used. The amount of a and b chlorophyll and the amount of the
sum of a + b chlorophyll, as well as the number of carotenoids were calculated with the
use of the formulae contained in the paper of Arnon [1949]:

Chlorophyll a = (12.7 · A663 – 2.7 · A645) · V · (1000 W)-1

Chlorophyll b = (22.9 · A645 – 4.7 · A663) · V · (1000 W)-1
Chlorophyll a + b sum = (20.2 · A645 + 8.02 · A663) · V · (1000 W)-1
Carotenoids = (1000 · A470 – 1.9 · chlorophyll a – 63.14 · chlorophyll b)/214

where:
A – absorbance at a given wave length,
V – total volume of the extract, cm3,
W – sample weight, g.

The number of the particular pigments is quoted in g·g-1 of fresh matter, while the
weight of carotenoids is given in mg·g-1 of fresh weight.
In case of the indirect method, maize nutritional status with nitrogen was defined
using an optical apparatus known in Europe as Hydro N-Tester, while in the USA as
SPAD-502 apparatus. This apparatus operates by measuring light absorption by a leaf at
the wave lengths of 650 and 940 nm. The quotient of these differences indicates the
chlorophyll content, and it is defined in SPAD units [Soil and Plant Analysis
Development]. A high determination coefficient (R2) was shown, depending on the
species, between the readings of the apparatus and the extracted amount of chlorophyll
[Piekielek et al. 1995, Scharf et al. 2006].
The analysis of the content of mineral components (N, P, K, Ca, Mg, Na) in dry
matter in the phase of 5-6 leaves (BBCH 15-16) was carried out in the laboratory of the
Department of Soil and Plant Cultivation, University of Life Sciences in Pozna,
according to the methods described by Gawcki [1994]. Furthermore, potassium and
calcium were determined in flame spectrophotometer Flapho 40, while phosphorus and
magnesium were identified in Spekol 11 colorimeter. Nitrogen and sodium were
determined after previous mineralization of plant material and the determination of
mineralizates concentration.

RESULTS AND DISCUSSION

In the phase of 5-6 leaves (BBCH 15-16), the content of chloroplast pigments,
chlorophyll a, chlorophyll a + b, chlorophyll expressed in SPAD units, and carotenoids
depended exclusively on the type of hybrid (Table 1). Significantly higher amounts of
chlorophyll a, chlorophyll a + b, and SPAD units were found for the hybrid LG 2244
stay-green type, in comparison with the traditional Anjou 258 hybrid. The respective
differences were: 0.1 g·g-1, 0.11 g·g-1, and 15. In the case of carotenoids, the LG 2244

Agricultura 8(2) 2009

46 P. Szulc

hybrid showed a significantly smaller amount of these pigments in leaf blades (smaller
by 0.29 mg·g-1 of dry matter), in comparison with Anjou 258 hybrid. The obtained
results were confirmed by the author in his earlier studies [Szulc et al. 2008a]. He found
that the hybrid of stay-green type, on the basis of chlorophyll content in the phase of 5-6
leaves, was better nourished with nitrogen in comparison with the traditional hybrid.
Thomas and Smart [1993] explained the feature of the permanent green colour (stay-
green) i.e. the phenotypes showing a delayed senescence – by the fact that they possess
a higher content of water and chlorophyll in leaf blades in comparison with traditional
maize hybrids. The identified smaller amount of carotenoids in LG 2244 hybrid testifies
to a higher photooxidation of chlorophyll (a smaller protection barrier) in comparison
with the traditional hybrid Anjou 258.

Table 1. Content of chloroplast pigments and chlorophyll in SPAD units in the phase of 5-6
leaves (BBCH 15-16)
Tabela 1. Zawarto barwników chloroplastowych i chlorofilu w jednostkach SPAD w fazie 5-6
lici (BBCH 15-16)

Chlorophyll – Chlorofil Carotenoids

Specification
a b a+b in SPAD units Karetonoidy
Wyszczególnienie
w jednostkach
g·g-1 SPAD mg·g-1 f.m
Anjou 258 1.58 0.29 1.88 283 7.11
Hybrid
LG 2244 1.68 0.31 1.99 298 6.82
Odmiana
LSD0.05 – NIR0.05 0.070 ns 0.090 9.9 0.117
0 1.58 0.31 1.89 283 7.63
30 1.52 0.28 1.81 287 6.67
Dose of N 60 1.56 0.28 1.85 294 6.83
Dawka N
-1 90 1.62 0.30 1.92 297 7.06
kg·ha
120 1.65 0.29 1.95 296 6.86
150 1.63 0.32 1.96 285 6.72
LSD0.05 – NIR0.05 ns ns ns ns ns
0 1.61 0.30 1.92 290 6.91
Dose of Mg
15 in rows – rzdowo 1.61 0.29 1.91 293 7.16
Dawka Mg
15 broadcasting – rzutowo 1.60 0.30 1.91 289 6.83
kg·ha-1
LSD0.05 – NIR0.05 ns ns ns ns ns
ns – non-significant differences – rónice nieistotne

In the performed experiment, no effect of any of the studied experimental factors

was exerted on the contents of phosphorus and potassium in the dry matter of plants in
the phase of 5-6 leaves (BBCH 15-16). Hybrid type modified merely the contents of
calcium and sodium in plant dry matter (Table 2). A significantly higher amount of
these mineral components was found in LG 2244 hybrid compared with Anjou 258.
These differences showed: 0.93 g·kg-1 of dry matter for calcium and 0.43 g·kg-1 of dry
matter for sodium. Nitrogen dose exerted a significant influence on the contents of
nitrogen and magnesium in the dry matter of the aboveground parts of maize in the
phase of 5-6 leaves (BBCH 15-16). Under the influence of increased fertilization, the N
content increased from 36.0 g·kg-1 dry matter for the dose of 0 kg N·ha-1 to 37.8 g·kg-1
d.m. for the dose of 90 kg N·ha-1. In the discussed developmental phase, the application
of a higher fertilization level with this microelement caused a drop in the nitrogen
content in the dry matter of plants.

Acta Sci. Pol.

Effect of nitrogen... Part I. Content of chlorophyll... 47

Table 2. Content of mineral components in the aboveground maize parts in the phase of 5-6
leaves (BBCH 15-16)
Tabela 2. Zawarto skadników mineralnych w czciach nadziemnych kukurydzy w fazie 5-6
lici (BBCH 15-16)

Content of nutrients, g·kg-1 d.m.

Specification Zawarto skadników, g·kg-1 s.m.
Wyszczególnienie
N P K Mg Ca Na
Anjou 258 37.0 3.21 42.29 2.60 4.90 1.07
Hybrid
LG 2244 36.4 3.08 41.31 2.61 5.83 1.50
Odmiana
LSD0.05 – NIR0.05 ns ns ns ns 0.932 0.236
0 36.0 3.27 42.48 2.51 5.41 1.32
30 36.4 3.10 41.04 2.52 5.46 1.28
Dose of N 60 36.9 3.04 41.33 2.51 5.28 1.23
Dawka N 90 37.8 3.09 41.51 2.59 5.17 1.26
kg·ha-1 120 36.9 3.17 41.65 2.67 5.40 1.33
150 36.2 3.19 42.80 2.80 5.48 1.29
LSD0.05 – NIR0.05 1.70 ns ns 0.220 ns ns
0 36.5 3.13 41.91 2.56 5.42 1.30
Dose of Mg
15 in rows – rzdowo 37.0 3.14 42.18 2.65 5.21 1.30
Dawka Mg
15 broadcasting – rzutowo 36.7 3.16 41.31 2.58 5.46 1.26
kg·ha-1
LSD0.05 – NIR0.05 ns ns ns 0.061 ns ns
ns –non-significant differences – rónice nieistotne

Nitrogen content in dry matter of maize depended also on the interaction of the
nitrogen dose size and the hybrid type. The plotted curves have been described by
equations of the 2nd degree, whereby for Anjou 258 cultivar, the curve course was on
a higher level in relation to that of the LG 2244 stay-green type. The maximal nitrogen
content (38.1 g·kg-1 d.m.) for Anjou 258 hybrid was obtained with the dose of 71 kg
N·ha-1, while for LG 2244 hybrid, the maximal content was smaller by 1.5 g·kg-1 d.m.,
with the nitrogen dose higher by 4 kg N·ha-1 (Fig. 1).

Fig. 1. Content of nitrogen in maize dry matter in the phase of 5-6 leaves (BBCH 15-16)
Rys. 1. Zawarto azotu w suchej masie kukurydzy w fazie 5-6 lici (BBCH 15-16)

Magnesium content in dry matter increased in a linear way together with the
nitrogen dose increasing from 2.51 g·kg-1 d.m. (0 kg N·ha-1) to 2.80 g·kg-1 d.m. (150 kg
N·ha-1). Furthermore, the magnesium content in the dry matter of maize depended also

Agricultura 8(2) 2009

48 P. Szulc

on the size of magnesium dose and on the method of its application. The greatest
amount of Mg was accumulated by maize fertilized with 15 kg Mg·ha-1 applied in rows
(2.65 g·kg-1 d.m.), in comparison with 0 kg and 15 kg Mg·ha-1 (by broadcasting),
between which no significant difference was found (Table 2).
From soil solution, plants uptake Mg2+ cations, which reach the root surface in two
ways, i.e. by way of direct contact (12%) and by way of water transpiration current
(88%). In a soil abundant in assimilable magnesium, the number of ions reaching the
root surface exceeds almost three-fold the metabolic requirements of plants in relation
to this element [Grzebisz and Härdter 2006]. This fact explicitly stresses the importance
of magnesium concentration in soil solution, which is a necessary condition for Mg
accumulation in the plant. This fact explains the higher content of magnesium in the dry
matter of the plant, which occurs as a result of the Mg fertilization in rows. This
happens because in the case of fertilization in rows, the fertilizer is placed below the
seed into a moist, not loosened soil layer. Subsequently, because of water ascension,
magnesium contained in the fertilizer is available to the plants from the very beginning
of plant vegetation. In turn, the broadcasting method causes the fertilizer to be spread on
the surface of the loosened soil layer which, in case of rain shortage, becomes quickly
dry and the uptake of magnesium is limited.
The content of magnesium in maize plant dry matter depends also on the interaction
of the nitrogen dose with the hybrid type. The plotted curves have been described by
equations of the 1st degree, whereby, for Anjou 258 hybrid, the increment of
magnesium content under the influence of the increased nitrogen dose was higher in
relation to the hybrid LG 2244 stay-green type – Fig. 2.

Fig. 2. Content of magnesium in maize dry matter in the phase of 5-6 leaves (BBCH 15-16)
Rys. 2. Zawarto magnezu w suchejmasie kukurydzy w fazie 5-6 lici (BBCH 15-16)

CONCLUSIONS

1. LG 2244 stay-green type hybrid, in the phase of 5-6 leaves (BBCH 15-16),
contained a greater amount of chloroplast pigments a, chloroplast pigments a + b, and
SPAD units but a smaller amount of carotenoids in comparison with the traditional
hybrid Anjou 258.
2. LG 2244 stay-green type in the phase of 5-6 leaves (BBCH 15-16) in dry matter
contained a higher content of calcium and sodium in comparison with the traditional
hybrid Anjou 258.

Acta Sci. Pol.

Effect of nitrogen... Part I. Content of chlorophyll... 49

3. The lowest amount of nitrogen in maize dry matter, in the phase of 5-6 leaves,
was found for the dose of 0 kg N·ha-1, while the highest content was found for the dose
of 90 kg N·ha-1.
4. Magnesium content in dry matter increased together with the increase of nitrogen
dose from 0 to 150 kg N·ha-1.
5. The greatest content of magnesium was found for the dose of 15 kg Mg·ha-1 (sown
in rows), in comparison with the doses of 0 and 15 kg Mg·ha-1 (by broadcasting).
6. Anjou 258 hybrid accumulated a greater content of nitrogen in dry matter in
comparison with the hybrid LG 2244 stay-green type.
7. Magnesium content in both studied hybrids increased in a linear way together
with the increase of the nitrogen fertilization size, whereby the increase of Mg in Anjou
258 hybrid was higher than in LG 2244 stay-green type.

REFERENCES

Andrews C.J., Dwyer L.M., Stewart D.W., Dugas J.A., Bonn P., 2000. Distribution of
carbohydrate during grain-fill in leafy and normal maize hybrids. Can. J. Plant Sci. 80, 87-95.
Arnon D.I., 1949. Copper enzymem in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris.
Plant Physiology 24,1-15
Binder D.L., Sander D.H., Walters D.T., 2000. Corn response to time nitrogen application as
affected by level of nitrogen deficiency. Agron. J. 92, 1228-1236.
Costa C., Dwyer M.L., Stewart W.D., Smith L.D., 2002. Nitrogen effects on grain yield and yield
components of leafy and nonleafy maize genotypes. Crop Sci. 42, 1556-1563.
Gawcki K., 1994. wiczenia z ywienia zwierz t i paszoznawstwo [Exercise in animal feeding
and fodder studies]. Wyd. AR Pozna [in Polish].
Grzebisz W., 2008. Rola skadników mineralnych w realizacji potencjau plonotwórczego
kukurydzy [Role of mineral components in the realisation of the yield-forming potential of
maize]. Mat. Konf. Nauk. Problemy agrotechniki oraz wykorzystania kukurydzy i sorgo, UP
Pozna, 50-61 [in Polish].
Grzebisz W., Härdter R., 2006. Kizeryt – naturalny siarczan magnezu w produkcji rolinnej
[Kieserite – natural magnesium sulphate in plant production]. K&S GmbH, Kassel Germany
[in Polish].
Piekielek W.P., Fox R.H., Toth J.D., Macneal K.E., 1995. Use of chlorophyll meter at the early
dent stage of corn to evaluate nitrogen. Agron. J. 87, 403-408.
Scharf P.C., William J., Wiebold J., Lory J.A., 2002. Corn yield response to nitrogen fertilizer
timing and deficiency level. Agron. J. 94, 435-441.
Scharf P.C., Brouder M.S., Hoeft G.R., 2006. Chlorophyll meter readings can predict nitrogen
need and yield response of corn in the North-Central USA. Agron J. 98, 655-665.
Subedi K.D., Ma B.L., 2005. Nitrogen uptake and partitioning in stay-green and leafy maize
hybrids. Crop Sci. 45, 740-747.
Szulc P., Rybus-Zaj c M., Waligóra H., Skrzypczak H., 2008a. Ocena stanu odywienia dwóch
typów odmian kukurydzy na podstawie zawartoci chlorofilu w warunkach braku aplikacji
nawoenia azotem [Assessment of nutrition conditions of two maize hybrid types on the basis
of chlorophyll content with no nitrogen fertilisation application]. [W:] Wybrane zagadnienia
ekologiczne we wspóczesnym rolnictwie, t. 5, pod red. Z. Zbytka, Pozna, 260-267 [in
Polish].
Szulc P., Skrzypczak W., Waligóra H., 2008b. Improvement of the effectiveness of maize
fertilization with nitrogen by the application of magnesium. Part. I. Grain yield and its
structure. Acta Sci. Pol., Agricultura 7(4), 125-135.
Thomas H., Smart C.M., 1993. Crops that stay green. Ann. Appl. Biol. 123, 193-219.

Agricultura 8(2) 2009

50 P. Szulc

WPYW NAWOENIA AZOTOWEGO I METODY STOSOWANIA

MAGNEZU NA ZAWARTO CHLOROFILU, GROMADZENIE
SKADNIKÓW MINERALNYCH I NA MORFOLOGI DWÓCH TYPÓW
ODMIAN KUKURYDZY W POCZTKOWYM OKRESIE WZROSTU
CZ. I. ZAWARTO CHLOROFILU I SKADNIKÓW MINERALNYCH

Streszczenie. Dowiadczenie polowe przeprowadzono w Zakadzie Dydaktyczno-

-Dowiadczalnym w Swadzimiu koo Poznania w latach 2004-2007 (52o26’ N; 16o45’ E).
Dowiadczenie prowadzono w ukadzie „split-plot” z 3 czynnikami w 4 powtórzeniach
polowych. Badano reakcj dwóch typów odmian kukurydzy na zrónicowane nawoenie
azotem oraz magnezem (sposób aplikacji magnezu). Oceniano wpyw tych czynników na
zawarto chlorofilu oraz skadników mineralnych w suchej masie rolin kukurydzy
w fazie 5-6 lici (BBCH 15-16). Na podstawie zawartoci barwników chloroplastowych
stwierdzono, e odmiana LG 2244 typ stay-green bya lepiej odywiona azotem
w stosunku do tradycyjnego mieszaca Anjou 258. Najwiksz zawarto azotu w suchej
masie rolin stwierdzono po zastosowaniu dawki azotu 90 kg N·ha-1, natomiast magnezu
– 150 kg N·ha-1. Korzystniejszym sposobem aplikacji magnezu bya metoda rzdowa ni
rzutowa. W wyniku zastosowania takiej metody wysiewu magnezu w suchej masie rolin
stwierdzono istotnie wiksz zawarto tego skadnika mineralnego w porównaniu
z metod rzutow oraz obiektem z zerow dawk magnezu.
Sowa kluczowe: azot, BBCH 15-16, magnez, sposób aplikacji nawozu, stay-green, typy
odmian kukurydzy

Accepted for print – Zaakceptowano do druku: 27.03.2009

Acta Sci. Pol.