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Research Article
doi: 10.1590/01047760202228013018
vol(28), 2022
AYAN, S.; ÇALIŞKAN, E.; ÖZEL, H. B.; YER ÇELİK, E. N.; YILMAZ, E.; GÜLSEVEN, O., AKIN, S. S. The inuence of effective microorganisms
on physiological characteristics of containerized taurus cedar (Cedrus libani A. Rich.) seedlings. CERNE, v. 28, e-103018, doi:
10.1590/01047760202228013018
✤Corresponding author e-mail: esranurtenyer@gmail.com Received: 24/10/2021 Accepted: 28/01/2022
FOREST MANAGEMENT
SILVICULTURE
1 Kastamonu University, Faculty of Forestry, Kastamonu, Turkey.
2 Kastamonu University, Institute of Science, Kastamonu, Turkey.
3 Bartın University, Faculty of Forestry, Bartın, Turkey.
The influence of effective microorganisms on
physiological characteristics of containerized
taurus cedar (
Cedrus libani
A. Rich.) seedlings
Sezgin Ayan1iD, Ebru Çalışkan2iD, Halil Barış ÖZEL3iD Esra Nurten Yer Çelik1*iD,
Ergin Yılmaz2iD, Orhan Gülseven2iD , Şeyma Selin Akın2iD
ABSTRACT
Background: In this study, the impact of Effective Microorganism (EM) applications on some
physiological characteristics of 2+0 aged, containerized Taurus cedar (
Cedrus libani
A.Rich.)
seedlings were investigated. EM-1, EM-A, EM-5, and EM-Gold were used as effective microorganism
varieties. EM application was repeated twice at two different times. The first application was carried
out in April-May 2017 at the beginning of the vegetation period, and the second application was
carried out in June-July, when the growth of seedlings was the most active period, according to a
randomized plot design with three replications.
Results: Physiological measurements on seedlings were carried out at the end of the second vegetation
period. According to the results of the study, it was determined that the EM type, dose and application
time factors had a significant effect on physiological characteristics such as Chlorophyll a, Chlorophyll b,
Total Chlorophyll, Photosynthesis Rate, Transpiration and Relative Humidity. EM-A had a positive effect
on photosynthesis rate and EM-5 had a positive effect on all other measured physiological parameters.
High dose of EMs had the most positive effect on relative humidity, while medium dose of EMs had the
most positive effect on chlorophyll values, transpiration rate and photosynthesis rate. In addition, it was
determined that EM application at the beginning of the vegetation period had an increasing effect on
all measured physiological parameter values.
Conclusion: According to the results of the study, it was concluded that EMs have a positive effect on
the physiological quality characteristics of Taurus cedar seedlings and can contribute to the production
of seedlings with higher adaptability in afforestation.
Keywords: Effective microorganism,
Cedrus libani
, Taurus Cedar,seedling quality, physiology.
HIGHLIGHTS
EM application positively affects photosynthetic pigments and increases the rate of photosynthesis,
coinciding with the period when the plant awakens, and physiological processes accelerate.
EMs effected on chlorophyll a, chlorophyll b, total chlorophyll, relative humidity (%) and transpiration
rate, significantly.
EMs play a critical role in improving physiological characteristics that significantly affect seedling quality.
So, it should be used for the seedling propogation, effectively.
EM treatments may also be used in forestry to cultivate high quality seedlings and achieve high quality
plantation as well as serve as a tool to increase the performance of forestation.
Research Article
doi: 10.1590/01047760202228013018
vol(28), 2022
Ayan et al.
2CERNE (2022) 28: e-103018
INTRODUCTION
Cedrus libani
A. Rich., Taurus cedar, a forest tree that
establishes pure and mixed stands in the Taurus orogenic
belt in Turkey, makes its wide, vertical amplitude natural
expansion happen from the sea level up to 1,400-2,200
m altitude in the mountains of Syria and Lebanon outside
Turkey (Quézel and Médaıl, 2003; Ayan et al., 2018). Taurus
cedar is a very tolerant variety to harsh winter conditions
and high temperatures in summer. Its high tolerance has
made the cedar tree a frequently preferred variety in
afforestation (Ata, 1995). Apart from the natural distribution
areas of the Taurus cedar, the existence of successful
plantations within the borders of 25 different provinces in
five different geographical regions of Turkey confirms that it
is a variety with high plasticity (Ayan et al., 2017).
The use of containerized seedlings (Ayan, 2007),
different growing medium substrates (Ayan, 1999, 2001,
2002a, 2002b; Ayan and Tüfekçioğlu, 2006; Ayan and Tilki,
2007), slow working fertilizers (Ayan, 1998), different growing
processes – “greenhouse-open area-shaded area” (Ayan et
al., 2000), many cultural processes based on the seedling
development stages determined according to the ecological
conditions of the nursery (Yer and Ayan, 2011) have been
reported in various studies to have an effect on the quality
of seedlings to be used in afforestation. In particular, soil
fertility is effective on the quality of seedlings to be used
in afforestation areas. However, one of the most important
problems of nurseries in Turkey is the low fertility of nursery
soils (Atik, 2013). Long-term mismanagement on nurseries,
lack of organic matter in nursery lands and malpractices
in the use of artificial fertilizers reduce the quality of the
seedlings and cause a decrease in the productivity (Yılmaz,
1988). Furthermore, the use of chemical fertilizers in forest
nurseries, as in agricultural areas, directly or indirectly
affects the natural structure of the soil. Therefore, the
need for biological and natural materials has gained more
importance in recent years to increase the current yield of
nurseries and to produce quality seedlings.
Some outcomes have been expressed those
chemical fertilizers used in forest nurseries and agricultural
production give positive results in meeting plant
nutrients (Özdemir, 1989; Kulaç, 2016). However, Effective
Microorganism (EM), i.e., effective, active, or beneficial
microorganism technology has started to develop in recent
years due to the harmful effects of chemical fertilizers or
pesticides on human health and nature -it was established
by the Japanese philosopher and naturalist Mokichi Okada
in the 1930s, considering the long-term harm of chemicals.
EM consists of mixed cultures of natural and
beneficial microorganisms that are widely used in soil and
is applied to plants as inoculants to increase soil quality,
plant growth and yield (Higa and Parr, 1994; Iwaishi,
1994; Iwahori and Nakagawara, 1996). EMs, which are
mostly used in agriculture, have become an important
part of natural agriculture (Calıskan, 2018). Although EM
consists of numerous microbial varieties, the predominant
populations are lactic acid bacteria, yeasts, actinomics,
and photosynthetic bacteria.
It is known that EM preparations have positive effects
on the improvement of soil production properties (Valarini
et al., 2003) and that EMs accelerate the mineralization of
organic matter in the soil (Daly and Stewart, 1999). EMs
are explained to be natural products that have positive
effects on increasing beneficial soil microflora, improving
vegetative growth of plants, and increasing resistance
to insect diseases and pests (Daly and Stewart, 1999).
Although the positive effects of EM on plant growth, yield
and quality have been suggested by many studies, there
are still many questions as to which EM cultures or which
combinations of these cultures are most effective for
alleviating certain chemical, physical and microbiological
problems in existing soils (Calıskan, 2018).
Many studies have been conducted on the positive
effects of EM on crop yield in agricultural areas (Chaudhry
et al., 2005; Piskier, 2006; Javaid, 2006). On the extraordinary
effects of these microorganisms, some studies have been
conducted on chamomile (
Chamomilla spp
. or
Chamomilla
sp.
) on grass in the Netherlands and Austria (Daly and
Stewart, 1999; Fujita, 2000) and on apples in Japan (Fujita,
2000). Although EM is widely used especially in agriculture,
the information about the effectiveness of EMs in forestry is
quite limited. Atik (2013) has studied the effect of EM on seedling
height and root collar diameter in
Pinus nigra
J.F.Arnold.
In this research, it was aimed to evaluate the effect of
EM application on the physiological quality characteristics of
Taurus cedar seedlings, which is one of the most preferred
species in afforestation investigations in Turkey and has high
plasticity, showing high adaptability in different environments
both within and outside its natural range.
MATERIAL AND METHODS
Material
The study, which was conducted on 2+0 aged,
polyethylene-containerized (Dimensions: 11 x 25 cm) Taurus
cedar seedlings originating from Eğirdir, Isparta, was carried
out in Daday State Forest Nursery affiliated to Kastamonu
Regional Directorate of Forestry. Brief information about
the nursery where the study was carried out is given in Table 1.
Table 1 General information of Daday State Forest Nursery
FEATURES VALUES
Latitude 41° 22' 16"
Longitude 33° 46' 38"
Exposure South
Elevation from Sea Level 800 mt
Annual Average Temperature 9,9 oC
Annual Maximum Temperature 19,9 oC
Annual Minimum Temperature --0,3 oC
Annual Precipitation 611 mm
Annual Average Relative Humidity % 50
Vegetation Period May-October
A mortar mixture consisting of 60% mineral soil,
5% fine sand, 20% humus and 15% burnt barn manure was
used for seedlings grown in polyethylene containers.
Ayan et al.
3CERNE (2022) 28: e-103018
Method
Preparation of Effective Microorganisms
Routine nursery cultural processes were applied to
the seedlings, which were considered as research objects
in the nursery, and irrigation was carried out once a week
based on observation with a sprinkler system. The research
was established according to the “Random Plots Trial
Design” in open area conditions.
Four different EM products, EM-1, EM-5, EM-A, and
EM-Gold, were used in the study. Solutions of 30%, 60%,
90% from products EM-1, EM-A, and 10%, 20% and 30%
from EM-5, EM-Gold products (adjusted as low, medium
and high dose) were prepared in 1.5 litre containers. They
were applied to the seedlings as a total of 12 different
treatments of three different concentrations of four
different products (Table 2).
EM application was carried out by directly pulverizing
to the mortar on the surface of the container and the above-
ground parts of the 2+0 aged, containerized seedlings,
which continue their development in the containers; in April
before they enter the vegetation period, and in the first week
of June, when the growth of the seedlings is most active
(rapid development stage). The trial was set up with three
replications of each treatment, with each plot represented
by 10 seedlings. Four different types of EM were applied
in three different doses at two different application times
(before and during the vegetation period).
For the first trial, EM solutions were treated by
spraying on each plot in the first week of April 2017 before
entering the vegetation period. The second application was
repeated one month after this procedure, that is, in the first
week of May. For the second trial, each plot was treated with
EM solution by spraying in the first week of June 2017 during
the vegetation period. The second application was repeated
one month after this procedure in the first week of July.
Seedling Physiological Features
The needles were taken and chlorophyll a,
chlorophyll b, total chlorophyll, photosynthesis rate and
relative humidity (NNI%) were determined after EM
applications, at the end of January, when the seedlings
were in true dormancy at the end of the vegetation period.
Measurements of the amount of cumulative transpiration
(S) were carried out after the pluck.
Determination of chlorophyll analysis: For the
measurement of chlorophyll amounts, needle leaves were
taken from the terminal shoot of the seedling, crushed in
a pestle and treated with ethanol (1 g pure chlorophyll, 25
ml ethanol) to form a solution. Using the FEK-M method,
the amount of chlorophyll in the solution was determined
with the help of photoelectrocolorimeter (Dmitriyeva and
Kefeli, 1991). By means of this method, the amount of
chlorophyll in the solution was determined by how much of
the applied radiation was absorbed by the solution, that
is, its optical frequency (density) (Dutton et al., 1943; Atik,
2008; Aydınoğlu, 2014).
The determination of Photosynthesis rate:Photosynthesis
rate was analysed with the LICOR-6200 portable
photosynthesis measuring device. Photosynthesis rate
was measured in the exposed grown needle leaves of 5
sample seedlings taken from each plot. Measurements
were recorded between 9:00-12:00. The results obtained are
expressed as µmol m–2 s–1.
EM Type Explanation of EM Contents Dose (%)
Low Medium High
EM-1
It is used effectively in
agriculture and includes all
the beneficial properties of
EM (EM Agrıton Turkey, 2018).
It contains 96% water, 3%
Molas, 1% lactic acid bacteria
Lactobacillus casei
(Orla-Jensen
1916) Hansen & Lessel 1971.
30 60 90
EM-A
It includes enzymes,
antioxidant substances,
organic acids, bioactive
substances, minerals,
natural hormones and other
beneficial substances that
increase the resistance of the
plant against diseases and
pests, low temperatures and
frosts (EM Agrıton Turkey,
2018). It contains lactic
acid bacteria (
Lactobacillus
fermentum
Beijerinck 1901),
L. plantarum (Orla-Jensen
1919),
L. rhamnosus
(Hansen
1968),
L. casei
(Orla-Jensen
1916) Hansen & Lessel 1971,
L. delbrueckii Weiss et al.,
1984), yeasts (
Saccharomyces
cerevisiae
Meyen ex EC
Hansen),
photosynthetic
bacteria
(
Rhodopseudomonas
palustris
(Molisch 1907) van
Niel 1944), heterotrophic
bacteria Bacillus subtilis
Ehrenberg, 1835 and
beneficial organisms that can
survive under pH 3.5 in EM-A
(EM-A, 2021).
30 60 90
EM-5
It is an important group of
effective microorganisms
that increase the resistance
against diseases and pests,
reduce the need for chemical
pesticides when applied
continuously, and are used
after germination, before the
emergence of diseases and
pests (EM Agrıton Turkey,
2018). It contains lactic
acid bacteria (Lactobacillus
delbrueckii Weiss et al., 1984,
L. plantarum (Orla-Jensen
1919), L. rhamnosus (Hansen
1968), L. casei (Orla-Jensen
1916) Hansen & Lessel 1971),
yeasts (Saccharomyces
cerevisiae Meyen ex EC
Hansen), phototrophic bacteria
(Rhodopseudomonas palustris
(Molisch 1907) van Niel 1944)
(PTTAVM.com, 2020).
10 20 30
EM-Gold
It contains antioxidant
substances and various
vitamins produced for humans
(EM Agrıton Turkey, 2018).
It is a special product based
on sugar cane molasses and
yeast extract fermented by
microorganisms in a mixture
of water, coral lime and nigari
(PRODUKTE.DE, 2021).
10 20 30
Table 2 Application levels of EM types, contents and doses.
Ayan et al.
4CERNE (2022) 28: e-103018
Relative humidity percentage (RH%):
Needle leaves
were cut in certain sizes (to cover an area of 1 cm2). They
were weighed with the help of precision scales immediately
after being cut and their wet weights (WW) were measured.
Then, these needle leaves were kept in pure water until they
became turgescent (approximately 4-5 hours). Turgorized
needle leaves were weighed again with a precision scale
and their turgor weights (TA) were recorded. Needle leaves
in turgorized state were taken into an oven at a temperature
of 102 ± 3 °C, and left for 24 hours to become oven-dry,
and then the oven-dry weights were recorded (KA). The
relative humidity of the needles taken was determined with
the help of the following formula (Dhanda and Sethi, 1998).
N NI ( % ) = [(YA - K A ) / ( TA - K A ) ] x 1 00 [ 1 ]
Cumulative transpiration (S):
5 seedlings belonging to
each application were removed from the containers, then
washed and cleaned, and brought to the laboratory. A
sensitive cleaning process was carried out again without
damaging the stem and roots of the seedlings. After this
process, the seedlings were cut from the NWF level and the
stems of the seedlings were placed in distilled water at +4 °C
until they were fully saturated (approximately 24 hours). The
stems of the seedlings that became fully saturated (FS) were
purified from the water with the help of a paper towel. The
fully saturated seedlings, whose weights were measured,
were immediately taken into the air-conditioning cabinet at
25 °C ambient temperature, 60-65% relative humidity and
4000-4500 lux light intensity. Since the moisture content
of the seedlings would decrease rapidly after being placed
in the cabinet, weight measurements were continued at
15-minute intervals, and the measurement intervals were
extended to 30, 45, and 60 minutes since moisture loss
would slow down further in the future.
Finally, the seedlings, which were measured at 420
minutes intervals, were taken from the air-conditioning cabinet
and their weight (DDA) was measured, and the water pressure
values were measured with the help of the Scholander device.
The seedlings were placed in an oven that was kept ready at
104 °C, and their oven-dried weight (KA) was measured after
the seedlings which were dried for 24 hours were taken out
of the oven. Cumulative Transpiration values were calculated
with the help of the following formula (Dirik, 1994).
S=(TDH-DDA)/KA x 100 gr (H2O/100 gr dry weight) [2]
Statistical Evaluations
Multiple variance analysis was applied to reveal the
individual and interactive effects of EM type, EM dose, and
application time factors on all measured and calculated
physiological characteristics, and Duncan’s multiple test was
applied with the help of SPSS package program for process
comparison according to variables. The data determined
as a percentage and counted before the variance analysis
were subjected to variance analysis after the necessary
transformations were applied.
RESULTS AND DISCUSSION
The EM type, dose and application time factors
applied to the seedlings created a significant difference
on the individually measured variables chlorophyll a,
chlorophyll b, total chlorophyll, photosynthesis rate, relative
humidity, and cumulative transpiration while the effects
of the double and triple interactions of the factors on the
physiological characteristics of the seedlings were found to
be statistically insignificant (Table 3).
It was determined that EM 5 variety caused the
highest values on chlorophyll a, chlorophyll b, total
chlorophyll, cumulative transpiration and relative humidity,
while EM-A had the highest effect on photosynthesis rate.
While all applied EM doses caused higher values in all
physiological variables compared to the Control treatment,
medium level EM application between three doses was more
effective than low and high dose applications. The highest
value was obtained with high-dose EM application only on
the relative humidity variable. In terms of the application
time of EMs, it was determined that the application during
the April-May period, which is the rapid development
phase of the seedlings and before the vegetation starts, has
an effect on increasing the physiological characteristics of
all seedlings measured compared to the application in the
June-July period (Table 4).
Various studies have been carried out on the
seedling quality and afforestation performance of Taurus
cedar, which is the most commonly used coniferous
variety in afforestation together with Anatolian black pine
(
Pinus nigra
J.F.Arnold subsp. pallasiana (Lamb.) Holmboe)
in Turkey. Semerci (2005), in his study on Taurus cedar
seedlings, emphasized that the physiological characteristics
have a strong effect on the survival potential of the
seedlings. Sarı and Deligoz (2019), in their study on the
effect of the physiological state of Taurus cedar seedlings
on the time of pluck, stated that appropriate seedling pluck
should be done not only on the basis of morphological
characteristics, but also on physiological parameters.
Mercan (2010) indicated that in the studies conducted
in Mediterranean countries such as Spain, the success
of both seedling holding and seedling growth is higher
in arid afforestation areas of seedlings that are well fed
in the nursery environment. Ritchie and Shula (1984)
stated that morphological parameters and physiological
parameters show parallelism.
Pigments such as chlorophyll, anthocyanins and
carotenoids in plants perform very important functions.
Especially chlorophyll has an important function in
photosynthesis, which is necessary for the growth and
development of the plant (Schaefer and Wilkinson, 2004;
Gould and Lee, 2002; Gouldet al., 2002). Photosystem
I (PSI-P700) and Photosystem II (PSII-P680) are
photoreceptors that contain chlorophyll
a
and chlorophyll
b
and have an important function in photosynthesis
(Poethig, 2013). In his study of the effects of drought on
photosynthesis and Photosystem II heat tolerance in
Cedrus
libani
and
C. atlantica
(Endl.) Manetti ex Carrière seedlings,
Epron (1997) stated that
C. libani
exhibited a significantly
Ayan et al.
5CERNE (2022) 28: e-103018
Measurement Application Time EM dose EM Type App. Time x
EM Dose App. Time x EM
Type
EM Dose x EM
Type App. Time x EM
Dose x EM Type
FPFPFPFPFPFPF
P
Chlorophyll
a
1099.6 0.00** 261.98 0.00** 673.76 0.00** 0.875 0.42 4.032 0.09 3.154 0.07 0.166 0.988
Chlorophyll
b
419.2 0.00** 89.98 0.00** 202.49 0.00** 0.502 0.607 1.43 0.236 2.476 0.28 0.021 0.96
Total Chlorophyll 196.6 0.00** 476.9 0.00** 476.9 0.00** 0.855 0.423 3.05 0.32 2.94 0.11 0.03 1.00
Photosynthesis rate 353.8 0.00** 206.6 0.00** 2526.5 0.00** 0.139 0.870 0.77 0.513 7.20 0.06 0.14 0.95
Relative humidity 165.2 0.00** 110.7 0.00** 47.19 0.00** 0.19 0.828 0.083 0.97 1.08 0.38 0.22 0.94
Cumulative trn. 1049.8 0.00** 664.9 0.00** 554.9 0.00** 66.16 0.06 120.5 0.07 2.08 0.177 4.58 0.67
Table 3 The results of variance analysis of the physiological characteristics of the seedlings.
Table 4 Duncan test results for physiological measurements according to EM type, dose and application time.
EM Type
Chlorophyll a (mcg/ml) Chlorophyll b
(mcg/ml)
Total Chlorophyll
(mcg/ml) Rate of
Photosynthesis
Relative Humidity
(RHI %) Cumulative Transpiration
(H2O/100 g)
Mean and Std. Error (X+Sx)
Control 1.94±0.03 e 1,80±0,04 e 3,74±0,07 e 670,50±3,45 e 38,06±0,8 e 83,68±0,9 e
EM-1 2.66±0.04 b 2,45±0,04 b 5,11±0,09 b 777,23±3,9 b 46,65±0,6 b 106,05±1,6 b
EM-5 2.90±0.04 a 2,67±0,04 a 5,57±0,09 a 722,07±3,84 c 47,96±0,6 a 115,34±1,8 a
EM-A 2.56±0.04 c 2,36±0,04 c 4,93±0,08 c 838,83±3,02 a 43,80±0,6 d 105,04±1 c
EM-GOLD 2.27±0.04 d 2,13±0,04 d 4,39±0,07 d 709,73±2,5 d 44,22±0,54 c 99,04±15 d
EM Dose Chlorophyll a
(mcg/ml)Chlorophyll b
(mcg/ml) Total Chlorophyll
(mcg/ml) Rate of
Photosynthesis
Relative Humidity
(RHI %) Cumulative Transpiration
(H2O/100 g)
Control 1.94±0.03 d 1.80±0.04 d 3.73±0.08 d 670.50±3.5 d 38.06±0.8 d 83.68±1.2 d
Low 2.60±0.04 b 2.41±0.04 b 5.01±0.09 b 756.80±8.29 b 43.11±0.5 c 105.52±1.6 b
Medium 2.74±0.04 a 2.52±0.04 a 5.26±0.09 a 778.47±9.1 a 45.49±0.5 b 113.13±1.2 a
High 2.46±0.05 c 2.26±0.04 c 4.72±0.09 c 750.62±7.9 c 48.37±0.44 a 100.44±0.9 c
Application
Time Chlorophyll a
(mcg/ml) Chlorophyll b
(mcg/ml) Total Chlorophyll
(mcg/ml) Rate of
Photosynthesis Relative Humidity
(RHI %) Cumulative Transpiration
(H2O/100 g)
April- May 2.73±0.04 a 2.52±0.04 a 5.26±0.07 a 766.58±7.19 a 46.98±0.46 a 109.69±1.47 a
June- July 2.36±0.03 b 2.18±0.03 b 4.55±0.06 b 743.27±6.9 b 43.16±0.44 b 99.55±1.06 b
higher heat tolerance than
C. atlantica
. As pointed out by
Havaux (1993), even a small difference in heat tolerance has
important consequences in the plant’s struggle for existence.
Epron (1997) stated in his study that the combination of
high light and high temperature stresses greatly changed
the PSII photochemistry in cedar needles. This is common
because high temperatures increase photoinhibition of
photosynthesis in many varieties (Ludlow, 1987; Al-Khatib
and Paulsen, 1989; Gamon and Pearcy, 1990). In his study on
the effects of drought preconditioning on the heat resistance
of Photosystem II and the sensitivity of photosynthesis to
heat stress in cedar seedlings (
Cedrus brevifolia
(Roxb.) G.
Don,
C. libani
,
C. atlantica
), Ladjal et al. (2000) indicated that
differences in heat tolerance of PSII reflect differences in
the sensitivity of photosynthesis to heat stress. Due to the
net CO2 assimilation rate is inhibited more by heat stress
in
C. atlantica
than in
C. libani
, well-watered seedlings are
more susceptible than drought-preconditioned seedlings.
PSII activity is one of the most heat sensitive functions
in plants and loss of PSII activity occurs in a very narrow
temperature range. This behavior is likely to contribute to
Mediterranean cedars’ ability to tolerate the adverse hot
and dry seasons that characterize their natural habitats
during the summer months. In addition, Ozel (2016) stated
that air pollution negatively affects the total chlorophyll
content of cedar seedlings in his study on the effects of
pollutant particles from Bartın Cement Factory on the
development of Taurus cedar seeds and leaves in the
planting area around the factory. This shows that not
only temperature and drought but also polluting factors
affect the physiological development of cedar seeds and
seedlings. In the findings obtained in the study, it was
determined that EM applications had a positive effect on
chlorophyll a and chlorophyll b, which have important
functions in photosynthesis. Especially, EM-5 variety gave
statistically significant results in the amount of chlorophyll a
and chlorophyll b compared to the Control treatment and
other EM varieties. It was determined that medium (20%)
dose was sufficient as an application dose. The positive
effect of EM application on chlorophyll a and chlorophyll
b will have a positive effect on PSII, which is important
in drought, and may increase the field performance of
seedlings for Taurus cedar.
Turkey is in a geography dominated by the
Mediterranean climate, where climate change is the most
risky, and as a result, summer drought emerges as an
Ayan et al.
6CERNE (2022) 28: e-103018
important abiotic factor affecting forest trees (Dirik, 1994).
The rate of photosynthesis is determined by measuring the
amount of CO2 used or the amount of O2 released during
photosynthesis. The rate of photosynthesis decreases as
a result of water deficiency in plants. Linares et al. (2011)
stated that drought stress caused by heat in Morocco has a
limiting effect on the development of older individuals of
Cedrus atlantica
, and that the decrease in precipitation has
a great effect on the radial growth of young trees rather
than old trees. Photosynthesis, one of the most important
physiological processes, meets 90% of the plant dry matter.
In addition, the rate of photosynthesis and transpiration
reflects and quantifies the effects of environmental stress
(Ewers et al., 2008). Tree transpiration rate is largely
controlled by biological factors such as tree size and leaf
area index and by environmental factors such as radiation,
vapor pressure deficit (VPD), wind speed and soil moisture
availability (Ewers et al., 2008; Hernández-Santana et al.,
2008; Komatsu et al., 2006; Tang et al., 2006; Tognetti et al.,
2009). These biophysical factors affect transpiration on a
temporal and spatial scale. Plant water use and transpiration
rates may increase with precipitation (Mitchell et al.,
2009) and may decrease during periods of low soil water
availability (Gazal et al., 2006; Luis et al., 2005). Studies show
that stand transpiration increases in years with high rainfall
(Macfarlane et al., 2010; Zeppel et al., 2008). Low soil water
availability can increase the hydraulic resistance between
the soil and the root system, prevent the movement of water
from the soil to the plant leaves, and reduce transpiration
rate by triggering stomatal closure (Meinzer et al., 1993;
Sala and Tenhunen, 1996; Tognetti et al., 2009). Under these
conditions, plants adopt protective water use strategies
(Tognetti et al., 2009) and take advantage of water reserves
in deeper soil layers (David et al., 2004; Hernández-Santana
et al., 2008; Thomas et al., 2006). Deligöz et al. (2016) tried to
reveal the responses of Cedrus libani seedlings to recurrent
drought cycles by examining the morpho-physiological
parameters of the seedlings. In their study, they found
that drought stress caused a significant decrease in
growth parameters such as height, dry weight and root
collar diameter, but severe drought stress increased the
root/shoot ratio.
C. libani
seedlings showed less growth,
higher root/shoot ratio and proline accumulation as
an adaptation mechanism to repeated drought stress.
Under moderate water stress, carbohydrates obtained
by photosynthesis are accumulated by plants and can
be divided into differentiation processes (e.g., secondary
metabolism) rather than growth (Ayres, 1993). The positive
effects of EM applications on chlorophyll
a
, chlorophyll
b
and photosynthesis rate will have a positive effect on
primary and secondary metabolism and photosynthesis.
Thus plant secondary metabolites are involved in the
biological processes of plants, which generally do
not have primary functions in the maintenance of life
processes in plants but deal with environmental stress in
terms of adaptation and defence (Lavola and Julkunen-
Tiitto, 1994; David et al., 2004; Ladjal et al., 2007;
Ramakrishna and Ravishankar, 2011).
There is a strong and positive relationship between
the resistance of plants to biotic and abiotic stress factors
and the biochemical and physiological plant processes that
form the basis of plant metabolism such as photosynthesis,
respiration and transpiration (Atik and Aslan, 2015). Ladjal
et al. (2007) conducted a study on the effects of soil and
air drought on growth, plant water status and leaf gas
exchange in three Mediterranean cedar species,
Cedrus
atlantica, C. brevifolia
and
C. libani
, and they subjected the
three- and four-year-old seedlings to two different irrigation
regimes. Evaluating the data obtained, they found a close
functional link between height growth in cedars and water
availability during the growing season. Drought, which is an
important problem for the Mediterranean climate, affects
the plant-water balance and has negative consequences in
many physiological processes such as closure of stomata,
inhibition of gas exchange with the plant’s environment,
reduction of transpiration and inhibition of photosynthesis.
It also negatively affects photosynthesis, which is of vital
importance for plant growth and development. This leads to
premature aging and growth inhibition in plants (Siddique
et al., 2016). The rate of photosynthesis decreases, and
metabolic deterioration occurs severely with the closure
of stomata (Walawwe, 2014). Bayar and Deligöz (2019)
investigated the changes in water relations and biochemical
properties of the 22-year-old Taurus cedar and Anatolian
black pine in the afforestation area during the summer
drought period. In the data obtained, it was determined that
the osmotic potential at the wilting point and fully saturated
state of
Cedrus libani
decreased towards September. They
found that Taurus cedar has a lower osmotic potential at
the wilting point than Anatolian black pine in September.
In the study of Semerci (2001) the osmotic potential values
in full turgor state of Taurus cedar were at the level of -1.80
MPa, -2.32 MPa and -2.39 MPa in June, July and August,
respectively; the water potential values at the wilting point
of the same periods were -2.90 MPa, -3.30 MPa and -3.50
MPa, respectively. Bayar and Deligöz (2019) determined the
proportional water content at the wilting point of Taurus
cedar as 85% and 87% in July and August, respectively. Dirik
(2000) states that, depending on the type of tree studied, the
osmotic potential at the wilting point decreases during dry
periods, and while the resistance to drought increases. The
results obtained from the study of Bayar and Deligöz (2019)
show that
C. libani
is a more drought tolerant variety than
Pinus nigra
due to its lower osmotic potential at the wilting
point. The lack of water in the plant due to drought stresses
the plant and also reduces the rate of photosynthesis. In
order to mitigate this phenomenon, plants synthesize
certain compounds from the soil and minimize water loss.
These structures, which consist of different groups such as
amino acids, organic acids and carbohydrates, provide the
continuity of photosynthesis by increasing the conductivity
of the stomata as they balance the leaf water pressure.
Thus, it also contributes to plant growth (Örs, 2015). EM-5
variety applications will also positively affect the growth of
seedlings by increasing transpiration and relative humidity,
positively affecting transpiration and water intake, and
positively affecting photosynthesis, that is, CO2 fixation.
Ayan et al.
7CERNE (2022) 28: e-103018
Among all EM varieties, especially EM-A caused an increase
in the rate of photosynthesis and positively affected the
growth of seedlings. The time of EM application, especially
before and at the beginning of the vegetation period,
increases the success of EM application. This is thought
to be related to the fact that EM application positively
affects photosynthetic pigments and increases the rate of
photosynthesis, coinciding with the period when the plant
awakens, and physiological processes accelerate.
It is thought that amino acids produced by
photosynthesis bacteria in the structure of EMs applied
to Taurus cedar seedlings ensure the continuity of
photosynthesis and promote plant growth by maintaining
the plant turgor balance. EM consists of mixed cultures
of beneficial, naturally occurring microorganisms such as
photosynthetic bacteria
(Rhodopseudomonas palustris,
Rhodobacter sphaeroides),
lactobacilli (
Lactobacillus
plantarum, L. casei
and
Streptococcus lactis
), yeasts
(
Saccharomyces sppces
) and actinomycetes. Condor
et al. (2007) defined these microorganisms as follows:
Phototrophic bacteria are self-supporting microorganisms.
They synthesize amino acids, nucleic acids, bioactive
substances and sugars using root secretions in the soil,
organic matter, sunlight and geothermal heat as energy
sources. Unlike plants, they use energy from the infrared
band of solar radiation (700-1,200 nm) to produce organic
matter, thereby increasing the efficiency of plant growth.
Metabolites produced in this way can be directly absorbed
by plants or serve as substrates for other bacteria, thereby
increasing the biodiversity of soil microflora. Photosynthetic
bacteria, which are the main components of EM, are known
to work synergistically with other microorganisms to
support the nutritional requirements of plants and reduce
the incidence of pathogenic microorganisms (Condor et al.,
2007). Subadiyasa (1997) states that EMs can interact with
the soil-plant ecosystem by suppressing plant pathogens
and other disease agents, dissolving minerals, conserving
energy, maintaining the microbial and ecological balance
of the soil, increasing photosynthetic productivity and
fixing biological nitrogen.
CONCLUSIONS
Effective microorganisms play a critical role in
improving physiological characteristics that significantly
affect seedling quality. Furthermore, they can support the
development of plants in conditions of increased stress due
to global climate change in recent years. In this study, it was
determined that EMs had a positive effect on physiological
variables such as chlorophyll
a
, chlorophyll
b
, total chlorophyll,
photosynthesis rate, relative humidity and transpiration rate.
In addition, this study sheds light on the use of different kinds
of EMs in different forest tree seedlings.
ACKNOWLEDGEMENTS
The authors thank Elif Erentürk for the language edit.
AUTHORSHIP CONTRIBUTION
Project Idea: SA, HBÖ
Funding: EÇ, ENYÇ, SA
Database: EÇ, ENYÇ, SA
Processing: EÇ, ENYÇ, SA
Analysis: EÇ, ENYÇ, OG, SA
Writing: SA, EY, ENYÇ, ŞSA, OG
Review: SA, HBÖ
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