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© by PSP Volume 27 – No. 11/2018 pages 7409-7415 Fresenius Environmental Bulletin
7409
EFFECT OF EFFECTIVE MICRO-ORGANISMS ON THE
PROLINE AND MDA CONTENTS IN HERB PLANT
MATERIAL OF OCIMUM BASILICUM L. VAR. PICCOLINO
Alicja Auriga*, Jacek Wrobel
Department of Plant Physiology and Biochemistry, Faculty of Environmental Management and Agriculture, West Pomeranian University of
Technology in Szczecin, ul. Słowackiego 17, 70-310 Szczecin, Poland
ABSTRACT
Research on effective micro-organisms (EM)
mainly focuses on their effect on crop yield and
crop quality. On the other hand, knowledge on the
effect of EM on metabolic processes that take place
in plants at the cellular level is not sufficiently sys-
tematised. The subject of this study was to evaluate
the effect of an EM preparation on two oxidative
stress parameters, i.e. free proline and malondialde-
hyde (MDA) contents in the green parts of sweet
basil Ocimum basilicum L. var. Piccolino grown in
pots. The concentration of free proline was deter-
mined by the ninhydrin reaction and the malondial-
dehyde concentration based on the reaction with
thiobarbituric acid. Analysis of variance for select-
ed factors showed a significant effect of interaction
both EM and time on the decrease of proline and
MDA concentration. Effect of singular factor i.e.
EM has shown a favourable influence of the prepa-
ration on the oxidative stress parameters in sweet
basil by lowering the concentration of proline and
significant slowing down the process of lipid perox-
idation in the plant tissues. EM can be used in crop
growing as a preparation to facilitate the adaptation
of plants to changing climatic and habitat condi-
tions.
KEYWORDS:
biochemical parameters, cultivation, oxidative stress
reduction, sweet basil
INTRODUCTION
Due to the progressing environmental degrada-
tion as a result of crop chemization, the organic
farming, natural fertilisers and preparations are
becoming more popular. Effective microorganism
(EM) technology consisting biological preparations
composed of specially selected, naturally occurring
microorganisms, is one of the alternative for mod-
ern agriculture[1, 2].
Various environmental factors cause changes
in plants metabolism [3,4]. Prolonged or increased
exposure to stress factor results in an imbalance
between the generation of reactive oxygen species
(ROS) and its antioxidant abilities, what in conse-
quences can lead to plants death [5,6]. Proline and
malondialdehyde (MDA) are good indicators of
oxidative stress in plants [7-9].
Proline participates in the stabilisation of pro-
teins and cell membranes [10,11]. It also serves as
an osmoprotectant and is a reservoir of nutrients for
plants [9]. MDA induces changes in the structure of
the cell membrane leading to its disintegration and
uncoupling of phosphorylation in the mitochondria
[12]. Its concentration depends on the level of ROS
in tissues – the greater the production of free radi-
cals, the higher the concentration of malondialde-
hyde [13].
Scientific reports to date mainly focus on the
evaluation of the effect of EM on crop yield and
crop quality [e.g. 14-17] but do not explore the
issues that concern the effectiveness of their protec-
tive properties based on the metabolic mechanisms
taking place in plants at the cellular level. Most of
this type research is conducted on crop plants. On
the other hand, there are only few reports on herba-
ceous plants that enjoy the growing interest in Po-
land due to their high biological value. A very valu-
able herbaceous plant, of a wide range of applica-
tion, is sweet basil (Ocimum basilicum L.) and its
new varieties.
Therefore, the study determines the effect of
EM on the level of free proline and MDA, the bio-
chemical indicators adequate in the evaluation of
biochemical activity and general physiological
condition of sweet basil var. Picollino.
MATERIAL AND METHODS
Material. In 2014-2015, during the growing
season, a two-year pot experiment with sweet basil
(Ocimum basilicum L.) var. Piccolino was conduct-
ed. The plant material came from a private horticul-
tural farm in Szczecin. Biochemical analyses on the
acquired material were performed at the laboratory
of the Department of Plant Physiology and Bio-
chemistry, Faculty of Environmental Management
and Agriculture, West Pomeranian University of
Technology in Szczecin.
© by PSP Volume 27 – No. 11/2018 pages 7409-7415 Fresenius Environmental Bulletin
7410
The experimental material was sweet basil var.
Piccolino and EM. Sweet basil is an annual plant of
the family Lamiaceae; the variety being tested is
characterised by small leaves and a high content of
essential oils. EM are natural preparations to sup-
port plant growth, being widely used in organic
farming. They are composed of: lactic acid bacteria
(Lactobacillus casei), photosynthetic bacteria (Rho-
dopseudomonas palustrus), yeasts (Saccharomyces
albus), actinomycetes (Streptomyces albus) and
mould (filamentous) fungi (Aspergillus oryzae).
A two-factor pot experiment was set up fol-
lowing the randomised complete block design in
three replications. The first factor was 2 levels of
EM application (level 1 involved the use of EM in
cultivation, while level 2 is a control, without EM).
The second factor was times of measurement (3
levels).
Sweet basil seeds, in the amount of 10 seeds
per pot, were sown into a ready-made peat-based
substrate with pH 5.5-6.5, salinity of 1.9 g
NaCl∙dm-3 and with a starter dose of NPK com-
pound fertiliser 14-16-18 in the amount of 0.6 kg∙m-
3. From the moment the seeds were sown, the
objects intended for the application of effective
micro-organisms were watered with an aqueous EM
solution at a 1:100 dilution, every 7 days, in ac-
cordance to the manufacturer’s recommendations.
On other days, the plants were watered without
addition of the EM preparation. On the other hand,
the objects not intended for EM application were
watered with plain water only.
The plant material for analyses was collected
three times at monthly intervals, i.e. at the begin-
ning of June, July and August. On all the dates, free
proline and MDA contents in fresh herb parts of
sweet basil were determined.
Proline (Pro) determination. The concentra-
tion of free proline in fresh green parts of sweet
basil was determined by the ninhydrin reaction
according to the method developed by Bates et al.
[18]. Approximately 0.5 g of fresh plant tissue was
homogenised in the presence of 3% aqueous solu-
tion of salicylic acid (10 ml), and the resultant ho-
mogenate was filtered through a filter paper. To the
upper aqueous phase, 2 ml of acidic ninhydrin and 2
ml of glacial acetic acid were added. Next, the re-
sultant solution was mixed thoroughly and, after
pouring it into the closed tubes, placed in an incuba-
tor set at 90-100°C. After 1 hour, the tubes were
transferred into an ice bath for 15 minutes to cool
them. Then, 4 ml of toluene was added to each tube
and they were shaken for 30 minutes. The samples
prepared this way were left to allow the phases to
separate. The upper phase (toluene) was sampled to
determine the absorbance of chromatophore, against
the blank, at the wavelength λ = 520 nm.
Malondialdehyde (MDA) determination.
The concentration of malondialdehyde was deter-
mined by a slightly modified method according to
Sudhakar et al. [19] that is based on the reaction of
MDA with thiobarbituric acid.
The acquired plant material (1g) was homoge-
nised with 0.1% TCA, then the resultant homoge-
nate was filtered. To 1 cm3 of the supernatant, 4
cm3 of 0.5% TBA (in 20% TCA) was added. The
closed tubes were placed in a water bath at 90-
100°C and shaken for 30 minutes. Next, the tubes
were placed in an ice bath for 15 minutes to cool
them. The samples prepared this way were filtered
once again, and then the absorbance against the
reagent blank was determined in them at the wave-
lengths λ = 532 nm and λ = 600 nm. After removing
the nonspecific turbidity being measured at λ = 600
nm, the MDA concentration was calculated using
the mili-molar absorbance coefficient 155 mM–
1∙cm–1.
Both determinations were made using a Shi-
madzu 1800 UV-Vis spectrophotometer (Shimadzu
Scientific Instruments Inc., Columbia, Md., USA).
Statistics. The findings with regard to the ef-
fect of effective microorganisms on proline and
malondialdehyde concentrations in the plant mate-
rial were subjected to a two-way analysis of vari-
ance (ANOVA). Homogeneous groups were deter-
mined by the Tukey’s test at the significance level α
= 0.05.
RESULTS
A significant effect of EM application on the
concentration of free proline in the analysed plant
material was shown (Tab. 1). The analysis of vari-
ance showed that the most significant statistical
factor affecting the proline level in sweet basil herb
was the time of taking measurements – 92.8 %
(Tab. 1). The interaction of both factors, i.e. EM
and time, had a significant effect on the analysed
parameter – 4.7%.
Figures 1 and 2 present the effect of individual
factors on the proline content. Under control condi-
tions (without EM), the proline content was slightly
higher than after EM application, the difference
being however not significant. On the other hand,
significant differences were found in the proline
content depending on the time of measurement. The
lowest proline concentration, ranging from 0 to 0.02
µmol∙g-1 f.w., was determined on the first and the
second date of taking measurements for the two
experimental variants. Its highest concentration was
observed on the third date of taking measurements,
regardless of the EM level, i.e. 1.48 µmol∙g-1 f.w.
(Fig. 1).
© by PSP Volume 27 – No. 11/2018 pages 7409-7415 Fresenius Environmental Bulletin
7411
TABLE 1
Analysis of variance for selected factors and interaction between the factors affecting the proline content.
Factor / interaction
SS
Df
MS
F
p
X
EM
0.11
1
107
11.45
0.005
1.22
Time
8.15
2
4074
436.13
0.000
92.80
EM x Time
0.41
2
207
22.11
0.000
4.70
Error
0.11
12
9
1.28
SS – sum of squared deviations from the mean, Df – degrees of freedom, MS – mean square (MS=SS/Df), F – F-test value, p
– probability of error, X – percent effect of factors on the analysed property.
FIGURE 1
Average Pro concentrations in sweet basil herb for individual experimental factors.
FIGURE 2
Pro content [µmol×g-1 f.w.] in the green parts of EM-treated sweet basil and under control conditions
(without EM) depending on the time of measurement.
TABLE 2
Analysis of variance for selected factors and interaction between the factors affecting the MDA content.
Factor / interaction
SS
Df
MS
F
p
X
EM
130.07
1
130.073
361.45
0.000
22.89
Time
240.38
2
120.189
333.98
0.000
42.30
EM x Time
193.47
2
96.735
268.81
0.000
34.05
Error
4.32
12
0.360
0.76
SS – sum of squared deviations from the mean, Df – degrees of freedom, MS – mean square (MS=SS/Df), F – F-test
value, p – probability of error, X – percent effect of factors.
© by PSP Volume 27 – No. 11/2018 pages 7409-7415 Fresenius Environmental Bulletin
7412
Also on the third date of taking measurements
– at the end of the growing season, a significant
difference was found between the control plants
(1.74 µmol∙g-1 f.w.) and those with EM added (1.18
µmol∙g-1 f.w.) (Fig. 2.). The EM addition decreased
the proline content.
The EM preparation applied in the experiment
also significantly decreased the malondialdehyde
concentrations in the analysed plant material (Tab.
2.). The percent effect of this factor amounted to
around 23%, with the time of taking measurements
having once again the most significant effect on the
MDA content in the plant tissue – 42.3%. The in-
teraction of the two factors also showed a signifi-
cant effect on the analysed parameter – around
34%.
Figures 3 and 4 present the effect of individual
factors on the MDA content in the analysed materi-
al. The application of EM resulted in a 2-fold de-
crease in the MDA content in sweet basil herb
compared to the control (Fig. 3). The time of taking
measurements also significantly affected the aver-
age proline content in the analysed material. On the
1st date, its content was the lowest, whereas on the
3rd one the highest.
A significant interaction between the analysed
experimental factors was observed (Fig. 4). The
lowest MDA concentration was found in the plants
collected on the 1st date and after EM application
(3.17 nmol∙g-1 f.w.), whereas by far the highest
concentration was observed in the control plants
collected on the 3rd date (20.22 nmol∙g-1 f.w.).
FIGURE 3
Average MDA concentration in sweet basil herb for individual experimental factors.
0
5
10
15
20
25
III III
MDA [nmol∙g -1 f.w.]
Term
C
EM
b b
a
a
a
c
FIGURE 4
MDA content [nmol∙g-1 f.w.] in the green parts of EM-treated sweet basil and under control condi-
tions (without EM) depending in the time.
© by PSP Volume 27 – No. 11/2018 pages 7409-7415 Fresenius Environmental Bulletin
7413
When comparing the MDA content in control
plant herb and after EM application on respective
dates of taking measurements, it was observed that
the lowest concentration of this chemical compound
was characteristic of the sweet basil plants being
collected on the 1st date. However, the plants with-
out EM contained significantly more MDA (5.01
nmol∙g-1 f.w.), while those with EM less (3.17
nmol∙g-1 f.w.). On the 2nd date, the MDA concen-
tration increased and, regardless of the variant with
EM, was at a similar level, i.e. 9.57 - 9.84 nmol∙g-1
f.w. On the other hand, the MDA concentration in
the control plants on the 3rd date was the highest
(20.22 nmol∙g-1 f.w. ) and was significantly higher,
4-fold, than in the plants being treated with EM
preparation (5.65 nmol∙g-1 f.w.) – Fig. 4.
DISCUSSION
Due to the ongoing climate and habitat chang-
es, the plants are exposed to the effects of abiotic
factors that induce stress in them. To adapt to ad-
verse conditions, the plants have developed some
defence mechanisms that allow them to survive.
Oxidative stress which is a response of the plant
organism to the effects of stress-inducing stimulus
is the phenomenon being most studied by scientists
and best illustrates the condition of the test object.
Bearing in mind the results presented in this paper,
the significant increase of free proline content in
sweet basil herb for the two variants of the material
collected being collected on the third date should be
taken into account in respect to other dates of mak-
ing measurements. According to Koralewski [3],
the proline level depends on both the internal envi-
ronmental factors, such as plant age and its devel-
opment stage, and the external ones, i.e. tempera-
ture, insolation, humidity, etc. Syversten and Smith
[20] have demonstrated that the Pro content in the
young plants is at the highest level than in the older
ones – unlike in the present experiment. This may
indicate the effects of a stress factor that activated
the defence mechanism against free radicals. Since
the function of proline includes, among others,
osmoregulation, stabilisation of cell membranes and
protection of plan tissues against degradation, the
relevance of EM application in the analysed crop
becomes essential. The obtained results show a
significant reduction in the proline level in the
plants being treated with EM preparation in relation
to the control variant for the third date. Lower lev-
els of this enzyme in the EM-treated plants may be
justified by the presence of photosynthetic bacteria
in the preparation which in co-operation with other
micro-organisms provide plants with essential nu-
trients: amino acids, nucleic acids, bioactive sub-
stances and sugars [2]. The constant access to nutri-
ents during stress allows protein degradation pro-
cesses to slow down. In addition, EM are rich in
micro-organisms that produce antioxidants, as well
as in enzymes and hormones that support active cell
division [1]. Talaat [2] in his experiment, has prov-
en a mitigating effect of EM on the salt stress in-
duced in common bean by increasing the protein
synthesis and changing the composition of polyam-
ines. The effect of biologically active substances,
i.e. bio-stimulators, on the proline content, other
than that being observed in the present study, has
been shown by Borowski and Blamowski [21].
They have observed a significant increase in the
proline concentration in the leaves of Ocimum ba-
silicium L. in the plants being treated with a bio-
preparation compared to the control plants.
The MDA content in the analysed control var-
iant is characterised by an upward trend over three
months, which is consistent with the mechanism of
organism aging. While on the first and the third date
the MDA content in the EM-treated plants is signif-
icantly lower than in the control, the level of the
analysed indicator on the second date seems to be
striking. A similar level of this parameter for the
two variants may indicate achieving optimum grow-
ing conditions and an adequate phenological phase
by the plant in which the application of EM prepa-
ration does not bring significant changes. A signifi-
cant reduction in the MDA level in the EM-treated
plants in relation to the control on the third date
points to the effects of antioxidants which, as re-
ported by Higa [1], are the major product of EM.
A reduction in the values of oxidative stress
parameters under optimum conditions as a result of
the application of effective micro-organisms may be
a confirmation of the protective properties of this
preparation. According to Janas and Grzesik [22,23]
biological conditioning of the seeds of some species
of medicinal plants and vegetable crops enhances
the health of seeds and improves their sowing val-
ue. On the other hand, Xu [24] and Chaudhry [25]
have demonstrated the positive effects of the appli-
cation of effective micro-organisms in maize grow-
ing. The bio-preparation has stimulated the growth
of plants and induced their resistance and the pro-
cess of photosynthesis [26].
CONCLUSION
The results obtained in this experiment con-
firm numerous scientific reports about the positive
effect of EM on the growth and development of
plants, not only under stress conditions. The study
has shown a favourable effect of EM on the oxida-
tive stress parameters in sweet basil by lowering the
concentration of free proline and significant slow-
ing down the process of lipid peroxidation in the
plant tissues. Effective micro-organisms can be
used in crop growing as a preparation to facilitate
the adaptation of plants to changing climatic and
habitat conditions.
© by PSP Volume 27 – No. 11/2018 pages 7409-7415 Fresenius Environmental Bulletin
7414
ACKNOWLEDGEMENTS
We thank the Faculty of Environment Man-
agement and Agriculture of West Pomeranian Uni-
versity of Technology in Szczecin for providing
laboratory facilities.
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CORRESPONDING AUTHOR
Alicja Auriga
West Pomeranian University of Technology in
Szczecin, ul. Słowackiego 17, 70-310 Szczecin,
Poland
e-mail: alicja.auriga@zut.edu.pl
