Comparing Soil Substrates of Low Cost for the Production of Calabrian
Pine (Pinus brutia Ten) Seedlings Resilient to Unfavorable Conditions
having in Mind the Climatic Change Phenomenon
ANTONIOS TAMPAKIS1, PAPAIOANNOU EVGENIA1, THEOCHARIS CHATZISTATHIS2,
PARASKEVI KARANIKOLA3
1Faculty of Agriculture, Forestry and Natural Environment, School of Forestry and Natural
Environment, Aristotle University of Thessaloniki, 54124 Thessaloniki, GREECE
2Hellenic Agricultural Organization "DEMETER" Soil Institute, Thermi 57001 Thessaloniki, GREECE
3Department of Forestry and Management of the Environment and Natural Resources and in
Democritus University of Thrace, 193 Pantazidou Street, 68200 Orestiada, GREECE
Abstract: For the production of Pinus brutia seedlings resilient to dry climatic conditions of Mediterranean
ecosystems and better adapted to climatic change, the laboratory of Forest Soil seedlings of P. brutia on the
first year of their growth, replanted in bigger sized plastic pots. As fulfilled material used forest soil from 90%
gneiss rock and 10% from different low-cost materials like cow manure, goat manure, forest floor of
broadleaved forests and Calabrian pine. The research was conducted to the greenhouse of the Laboratory of
Forest Soils. To evaluate the results the development of the seedlings and conciseness of different nutrients
were measured. The measurements were analyzed with One Way Anova test and the results indicate the soil
substrate most suitable for the production of second year Calabrian pine seedlings with greater probability of
survival in dry climate conditions.
Key-Words: - nutrients, soil substrates, Calabrian pine seedlings, One Way Anova Test
Received: May 27, 2021. Revised: April 15, 2022. Accepted: May 12, 2022. Published: June 2, 2022.
1 Introduction
Soil fertility constitutes the capacity of the soil to
supply nutrients to growing plants [1]. This
capability of forest soils is not stable, and it relies on
the fact that nutrients are accessible through the
release of decomposing organic matter,
precipitation, activity of microorganisms and
dissolution of minerals [2]. Every nutrient is
paramount for the plant metabolism and each
nutrient is physiologically irreplaceable. Proper
growth of the vegetation is feasible only if each
nutrient can be accessed in a satisfactory amount
and in a chemical form that can be absorbed by the
plants [3]. This is more apparent when a major
element N, P, Ca, Mg, S and less obvious when a
trace element is in shortage [4]. The uneven
distribution in the supply of nutrients contributes in
the abnormal development of vegetation and make
plants susceptible to pathogens [5].
Nitrogen together with Potassium and
Phosphorus are the three most basic elements in the
soil. Deficiencies of nitrogen result in stunted
growth [6]. Plants can absorb nitrogen mainly in the
form of nitrate ions and secondarily in the form of
ammonium ions from the soil. In situations that
don’t allow the supply of the needed amount of
nitrogen, the leaves are smaller and take a yellow
color while also having smaller amounts of
chlorophyll [7]. Branches are thinner with reduced
photosynthesis occurring in their leaves and
production of organic matter is decreased while in
comparison, the root system is developed further
and the development cycle being reduced [3].
Phosphorus can be found in the soil in both
organic and inorganic forms, while both forms of
Phosphorus originate exclusively from magmatic
and metamorphic mineral [8].
P most commonly creates bonds with Al, Fe, Ca
and Mg. Forest soil is loaded with great amounts of
P (2,5-12 kg/ha) due to the decomposition of
organic matter [9] with only a small portion of
Phosphorus being soluble in water while also being
absorbable with the rest of the P being inaccessible
to the vegetation [10]. In the case of phosphorus
deficiency in pine trees, the tree’s oldest needles
have a red- chestnut color [11].
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K that is contained in the soil originates from the
dissolution of minerals containing it, with large
amounts of K being released after the
decomposition of organic matter [12]. K has a great
role in the metabolic process, particularly in
enzymes that metabolize nitric components and
hydrocarbons [3].
K takes place in the process of lignification of
the seedlings and increases resistance to both
drought and cold [13]. Ca while having an important
influence in the traits of the soil it also nullifies the
acidity of acidic organic compounds that are
produced by the decomposing organic matter. With
abundance of Ca resulting in smaller absorption of
K [14].
Organic matter has a positive in maintaining
fertility while also being a material composing the
soil, source of energy for the microorganisms
inhabiting it and as reservoir of water. The organic
material is composed of vegetation, manure of
animals e.tc. [15].
Organic material in favorable conditions while in
the presence of a variety of microorganisms breaks
down and becomes humus [16].
The forest floor constitutes one of the major
variables in the improvement of the physical,
chemical and biological traits of the soil [17]. In
rural environments the use of peat is particularly
popular for the improvement of parks. The
effectiveness of peat diminishes in forest areas of
urban and peri-urban soils [18] especially in dry
conditions when peat droughts totally during the
development cycle of the seedlings [3].
Manure is fairly affordable and is used by
farmers to fertilize their crops to provide nutrients
and organic matter. It is a cheap and affordable
fertilizer that can be obtained by farmers with great
ease [19]. The fertilizer greatly improves the quality
of the soil in the long-term [20]. The chemical
composition of manure depends on the diet and
species of animal and the straw covering the living
spaces of the animals. Manure contains 50% of both
organic matter and nitrogen while also 60% of P
and K originating from the animal feed [14]. In
general, Phosphorus is contained in small amounts,
with continuous use of manure in particular
situations creating symptoms of Phosphorus
malnutrition in vegetation [21].
Use of peat intensifies the activity of
microorganisms of soil with high corrosion while
achieving an increase in fertility [22].
An alternative method for the production of
seedlings with resilience to the harsh conditions
with high survivability is the use of inoculated with
mycorrhizae seedlings [23-26].
An early start in the absorption of nutrients,
water increase the chances of survival of the
seedlings [27].
The use of inoculated with mycorrhizae
seedlings is going to increase the success of the
efforts made by the country to reinstall tree
vegetation in areas with harsh climatic conditions
[1].
The process can be considered to be an imitation
of the natural process of regeneration [27] with
research proving the positive impact of symbiotic
fungi at the first stages of acclimatization of forest
species [10].
The current conditions have led to the creation of
advanced methods of seedling production, which
was achieved through experimentation and the study
of alternative methods of seedling production. The
study described in this paper is one of them.
The aim of the study was the use of organic
material (forest floor, manure) to improve the
properties of inorganic soil and to inoculate the root
system of the seedlings with mycorrhizae originated
by the use of forest floor.
2 Materials and Methods
2.1 Study Area
The study was conducted in the research facilities of
the laboratory of forest soils in the School of
Forestry and Natural Environment, Aristotle
University of Thessaloniki, in which the data were
collected (photo 1).
2.2 Experimental Design
Water is very valuable to Mediterranean ecosystems
[28-29]. For multitude of reasons like climatic
change and eroded soils, in Greece the half million
of one year seedlings planted after wild fires are not
succeeded in high degree [30]. The approach of this
research was developed by the need to develop
plants tolerant to the conditions of the
Mediterranean forest environment:
1. In order to replace peat that has some
particular positive qualities (low weight, can
hold a large amount of water, does not contain
weeds, has large alternative capacity) and a
major setback (in dry environment shrinks and
separates with the soil resulting in air entering in
the mass of the soil, not allowing any humidity
increase.
2. The production of plants with greater
resilience, capable of overcoming the problems
that appear when peat is used.
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3. In order to decrease the cost by using solid
mixed with organic materials instead of peat, and
increase the totally volume of the substrate the
plant will have during reforestation.
Photo 1. Seedlings of Calabrian pine in the
laboratory of forest soils, in Aristotle University of
Thessaloniki
The seedlings of Calabrian pine (Pinus brutia)
were used for the experiment of the study originated
from the nursery of Halkidona, 37.2 km near the
city of Thessaloniki, with the plants being one year
old. The soil was used, originated from gneiss and
was collected from the area of Agia Anastasia
(Basilika village, 31 km near Thessaloniki). The
cow and goat manure originated from the area of
Gomati located in Chalkidhiki. The hummus of
deciduous evergreen trees and hummus of Pinus
brutia were collected by the area of Gomati and
Thasos respectively.
The hummus was used for a multitude of reasons
a) In order to improve the natural properties of
soil, especially to the absorption of water.
b) For providing nutrients
c) In order to colonize the soil with mycorrhiza,
that increases the uptake of nutrients
The experiment was performed at the 10th of
February 2017, with the measurements of height
starting at the 15th of March 2017.
1. Analysis of the sub layer at the start of the
study
2. Both the height of every plant and after a
while the diameter above the root system were
measured every 15 days.
3. When the study ended every plant was
separated in its over ground and underground
part. The two parts were weighted being both
fresh and dry. The plant tissue was analyzed
macronutrients (N, P, Ca, Mg, K, Na) and
micronutrients (Fe, Cu, Zn, Mn)
The plants were separated to above ground and
underground parts on the root node.
The seedling of Pinus brutia where grown in 3lt
bags. With 5 experimental treatments used as sub
layer for the seedlings
1. 90% gneiss +10 cow manure
2. 90% gneiss +10% goat manure
3. 90% gneiss + 10% humus of evergreen
broadleaf
4. 100% gneiss (control group)
5. 90% gneiss + 10% Pinus brutia humus
The experiment was performed at 10-2-17, with
the measurements of height starting at 15-2-17.
These measurements were about:
1. Analysis of the sub layer at the start of the
experiment.
2. Both the height of every plant and after a
while the diameter above the root system were
measured every 15 days.
3. When the study ended every plant was
separated in its over ground and underground
part. The two parts were weighted being both
fresh and dry. The plant tissue was analyzed
macronutrients (N, P, Ca, Mg, K, Na) and
micronutrients (Fe, Cu, Zn, Mn).
The plants were separated to aboveground and
underground parts on the radical node. The analyses
of the aboveground part of the seedlings, needles
and stem were included, while the underground part
constituted by the root system
2.3 Laboratory Analyses
The plants tissue was made in a powdery and
homogeneous material that was created after the
grinding of the dry sample. To the samples the total
N was measured using the Kjeldahl method [31].
The chemical elements Ca, K, Na, Fe, Mn, Zn and
Cu were measured with the use of an Atomic
Absorption Spectrophotometer in a diluted solution
that was produced after the dissolution by H2SO4,
HNO3 και HClO4 of powdered sample. In the same
solution P was determined by the method
Molybdenum Blue.
The alkalinity (pH) of the soil suspended in
water (1:1 ration) was determined using a
potentiometer [31]. For the measurement of Carbon
the method of liquid oxidation was used [32].
Organic N was measured with the Kjeldahl method
[29]. For the measurement the extractable P Olsen
method was used. The alternative cations Ca, Mg,
K, Na, were measured with the use of a solutions of
CH3COONH4 1N, pH 7 [35]. With the trace
elements Fe, Mn, Zn and Cu being measured with
the use of DTPA, pH 7.3. And the extractable ions
Ca, Mg, K, Na, Fe, Mn, Zn and Cu were measured
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with the use of Atomic Absorption
Spectrophotometer [34].
2.4 Statistical Analyses of the Data
The data of the study was collected, categorized and
statistically modified with SPSS. The results of the
study were depicted in diagrams with the use of
excel. The data was analyzed using both one way
anova and two step cluster analysis.
In particular one way anova was used to examine
if there is significant deference in a dependent
variable between individuals that differentiate from
one another in one independent variable [36]. The
method uses the F distribution to compare the
estimation of the dispersion between samples [37].
Particularly in the comparison of more than one
sample means.
By comparing the five samples means (five
layers) μ1, μ2, μ3, μ4 and μ5 we formulate the Ho
and H1 in the following way:
Ho There is no significant difference between the
five means
H1 There is significant difference between at
least to a couple of means.
The assumptions of one way anova according to
[38] are: a) Randomness and independence, b)
normality and variance equality.
The Shapiro-Wilk test was used to check
normality while in order to test assess the equality
of variances for a variable Levene test was used
[38].
3 Results
We used a one-way anova to assess differences in
variables (length shoot, and diameter e.tc.) among
the different substrate treatments.
The results of the analysis are presented on table 1.
The null hypothesis was tested in order to
determine if it was true of false, and the statistical
difference between substrates was tested with
Tukey’s range test and testing of assumptions.
Variables with green color are statistically
significant, with a significance level of 0.05.
Kitikidou et al. [39], didn’t find any significant
interaction between seedlings and substrates while
researching a similar topic in four different forest
tree species seedlings
In order to check if there is any significant
statistical difference between the different substrates
the Tukey test is used. The results of the test are
demonstrated on table 2. Means that are underlined
indicate statistical deference. Means that are
accompanied by bold letters are statistically
different (a=0.05) according to the Tukey test. The
letter b and less transparent coloring indicates the
group with statistically greater values, while the
letter and the transparent coloring indicate the
group with statistically smaller values. When a
substrate is placed in both groups it is represented
with ab and the according coloring. The substrates
that produce optimal results should be considered to
be the ones with less transparent coloring and the
letter b for the factors concerning plant growth (the
fresh and dry weight).
In the first line of table 2 there is no statistical
difference between the substrates with all of them
being placed in the same group.
The variable dry weight gr, underground presents
significant variance between substrate 5 (90%
gneiss + 10% Pinus brutia humus), substrate 2 (90%
gneiss +10% goat manure), substrate 3 (90% gneiss
+ 10% humus of evergreen broadleaf) and substrate
1 (90% gneiss +10 cow manure). All in substrate 5
is statistically superior to substrates 1, 2 and 3 for
the variable fresh weight. While substrate 4 (control
group) is placed in both groups cause.
There is a significant difference between
substrates 5 and 2 for the variables dry weight for
both over and underground plant tissue. Substrates
3, 1 and 4 are grouped and placed in both groups
and they don’t have any statistical difference.
N % concentration for the above ground tissue
of the seedlings is significantly differentiated
between the group of substrates 5, 4, 2, 1 and
substrate 3. With all substrates being statistically
superior to layer 3 for the variable above ground
with lower concentration of N %.
N % concentration for the above ground tissue of
the seedlings is significantly differentiated between
the group of substrates 5, 4, 2, 1 and substrate 3.
With all substrates being statistically superior to
layer 3 for the variable above ground N %
concentration.
N % concentration for the underground tissue if
the seedlings is significantly differentiate between
the group of substrate 5 and substrate 3. With
substrate 5 being statistically superior to substrates 3
while substrates 1,2 and 4 are placed in both groups.
The variable concentration of P mg/gr of the
above ground tissue of the seedlings creates 2
groups of substrates with one being substrate 1 and
the other group being substrate 4 and 5. With
substrate 1 being statistically superior in the
concentration of P mg/gr to substrates 4 and 5 while
substrates 2 and 3 are placed in both groups
Respectively the variable concentration of P
mg/gr of the underground tissue of the seedlings
creates 2 groups of substrates with one being
substrate 1 and with the other group being substrate
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5 concentration of P mg/gr rate 5. With substrate 1
and 4 being statistically superior to substrate 5,
while substrates 2 and 3 are placed in both groups.
The variable concentration of Mg mg/gr of the
above ground tissue of the seedlings creates 2
groups of substrates with one being substrate 1, 2
and the other group being substrate 3 and 4. With
substrates 1 and 2 are statistically superior to
substrates 3 and 4 while substrate 5 is placed in both
groups.
To the variable concentration of Mg mg/gr of the
underground tissue there is sufficiently separation
between substrates 1 and 3. This means that
statistically substrate 1 contains more quantity of
Mg from the substrate 3 Substrates 3 and 4 and 5
are placed in both groups.
The variable concentration of K mg/gr of the
above ground tissue of the seedlings creates 2
groups of substrates with one being substrate 1, and
the other group being substrate 3. With substrate 1
being statistically superior to substrate 3 while
substrates 2, 4 and 5 are placed in both groups.
The variable concentration of Ca mg/gr of the
above ground tissue of the seedlings creates 2
groups of substrates with one being substrates 5, 1
and 2 and the other group being substrates 4 and 3.
With substrate 5, 1 and 2 being statistically
superior (contain more quantity of Ca) comparing to
substrates 3 and 4 while substrates 3 and 4 are
placed in group 3 of substrates.
Concentration of Cu ppm of the above ground
tissue of the seedlings there is differentiation
between substrate 4 and substrate 2. With substrate
4 being statistically superior to substrate 2, while
substrates 1, 3 and 5 are placed in both groups.
Adding up the results from Table 2 and 3, we can
extrapolate a relationship between the growth
characteristics of and the nutritional situation of the
plants of P. brutia with the soil analyses of
substrates.
Table 1. Concentrated results of dispersion analysis with one factor
1-2
1-3
1-4
1-5
2-3
2-4
2-5
3-4
3-5
4-5
Randomness -
Independence
sign. 1 = 0.038 < 0.05
sign. 2 = 0.025 < 0.05
for significance level a = 0.05
Aboveground
Underground
Characterists
Observing level of
statistical significance
Statistically significant difference of
substrates (Tukey method)
Check of assumptions
Length shoot 26 of June
Diameter (mm) 26 of June
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Table 2. Table of checking the totally homogeneity of the groups
Table 3. Substrate analysis
In particular, for substrate 5 in table 2 the
seedlings seem to have greater value of N% for
both above and underground tissue while also
having high values of N% in analysis of soil (table
3). Also in substrate 5, Ca cmol/Kg presents
statistically greater values in the over ground tissue
while Ca cmol/Kg in the underground tissue
doesn’t differ with from any of the other substrates
(table 3).
4 Conclusion
Taking into account the results of table 2 and 3 in
come to the following conclusions.
The study used one way anova and found major
differences between the manifested characteristics
of the seedlings. More specifically the fresh weight
of the shoot and diameter of the seedlings of the
substrates presented small differences with no
substrate that can be considered superior.
Substrate 5 provides better results than
substrates 1 2 and 3 in increase of fresh weight of
shoot and leaves of the seedlings.
Substrate 4 (control group) provided average
results.
With substrate 5 proving more effective than
sub substrate 2 in the increase of dry weight of both
length shoot, diameter and roots with neutral results
in substrates 4,3 and 1
A reliable solution that can be used in Pinus
brutia seedlings is substrates 5 using 90% gneiss
and 10% humus of Pinus brutia with sub layer 3
(90% gneiss and 10% goat manure) being an
acceptable alternative.
90% gneiss + 10%
cow manure
90% gneiss + 10%
goat manure
90% gneiss +10%
humus of evergreen
broadleaf
100% gneiss (control
group) from St.
Anastasia
90% gneiss from St.
Anastasia +10% Pinus
brutia humus
Fresh weight (gr) - Aboveground 27.9448 19.2300 20.8500 32.2675 42.6040
Fresh weight (gr) - Underground
23.0700 a16.0700 a 17.3920 a25.7650 ab 45.9066 b
Dry weight (gr) - Aboveground
10.1940 ab 7.5580 a8.2740 ab 12.8125 ab 16.6700 b
Dry weight (gr) - Underground
4.5840 ab 3.3180 a3.9140 ab 6.2025 ab 8.4260 b
N% - Aboveground
1.2978 b1.3262 b0.9917 a1.3713 b 1.4805 b
N% - Underground
1.2506 ab 1.1473 ab 0.8433 a1.0627 ab 1.3551 b
P mg/gr - Aboveground
1.5367 b1.2647 ab 1.2294 ab 1.1808 a1.07275 a
P mg/gr - Underground
1.2505 b1.0885 ab 1.1029 ab 1.2546 b0.8505 a
Mg mg/gr - Aboveground
1.7165 b1.7080 b1.1132 a0.9752 a1.3924 ab
Mg mg/gr - Underground
2.4835 b1.9818 ab 1.4365 a1.8710 ab 1.996 ab
K mg/gr - Aboveground
6.3123 b5.7747 ab 4.3072 a 5.0271 ab 5.1812 ab
Ca mg/gr - Aboveground
4.6169 c3.9484 bc 3.3046 ab 2.5932 a4.8173 c
Cu ppm ή μg/ml - Underground
13.3735 ab 7.6990 a 9.6931 ab 16.0150 b10.6932 ab
for significance level a = 0.05
Substrate
90% gneiss + 10%
cow manure
90% gneiss + 10%
goat manure
90% gneiss +10%
humus of evergreen
broadleaf
100% gneiss (control
group) from St.
Anastasia
90% gneiss from St.
Anastasia +10% Pinus
brutia humus
pH 5.86 6.25 6.27 6.14 6.29
C% 1.189 1.494 1.565 0.908 1.621
Organic matter 2.050 2.575 2.652 1.265 2.794
N% 0.124 0.086 0.110 0.109 0.165
C/N 9.562 17.417 14.014 8.359 9.824
Pmg/100gr of soil 1.480 1.250 0.360 0.410 2.060
Ca cmol/kg 9.931 10.403 11.695 9.417 10.883
Mg cmol/kg 4.593 4.813 4.079 4.426 5.210
K cmol/kg 0.476 0.214 0.146 0.183 0.850
Na cmol/kg 0.609 0.332 0.251 0.229 0.300
Cu ppm or μg/ml 0.376 0.242 0.250 1.858 1.934
Fe ppm 37.42 20.06 44.74 62.38 78.63
Zn ppm 0.906 1.048 0.950 0.568 0.596
Mn ppm 10.98 5.84 11.16 21.80 8.46
Substrate
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Use of Pinus brutia humus is beneficial cause of
the existence of fungi that can create mycorrhizae.
The main benefit is the easy absorption of nutrients
needed by the seedlings. Substrates with results of
average impact from best to worst are substrate 4
(100 % gneiss control group), substrate 1 (90 %
gneiss + 10% cow manure) and substrate 3 (90 %
gneiss + 10% humus of evergreen broadleaf).
The highest interest presents substrate 5 with
plants cultivated in it having the largest amounts of
N mg/gr for length shoot and diameter, above and
underground and large amounts of Ca mg/gr for
shoot and crown. With substrate presenting the
lowest value for P mg/gr for root, shoot and crown
even through the seedlings that grown on the
particular layer had greater development (in
weight) than their counterparts.
From the soil analysis of substrate 5 has high
values of N cmol/Kg while values of Ca cmol/Kg
are average in comparison to the other 5. Also
substrate 5 has the greatest values for P mg/100gr,
Mg cmol/Kg, K cmol/Kg and Cu ppm.
We should accept that the amount of nutrients
has no impact in the fertility of the soil with the
availability of those nutrients being of major
importance.
These findings reveal that the approach used
here is suitable for preliminary screening of the
impact of a forestry species on soil, to aid in
species selection and improve soil health for
afforestation and reforestation projects [40].
The conclusions we take from the results
constitutes preliminary results where we can based
and organize a future research using only the
substrate 5, with 90% gneiss and 10% Pinus brutia
hummus .and checking the kind of mycorrhiza
there are in the particular hummus in the area and
its contribution to the development of the seedlings.
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Contribution of Individual Authors to the
Creation of a Scientific Article (Ghostwriting
Policy)
-Tampakis Antonios designed the research and
coordinated all the phases for its effective
implementation.
-Papaionou Evgenia, designed the methods and
materials used for the research.
-Hatzistathis Theoharis designed and coordinated
the data collection.
-Paraskevi Karanikola was responsible for the
implementation of fieldwork.
All authors cooperated for the manuscript writing
and review and finally approved the final version
submitted for publication
Sources of Funding for Research Presented in a
Scientific Article or Scientific Article Itself
This research received no sources of funding.
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(Attribution 4.0 International, CC BY 4.0)
This article is published under the terms of the
Creative Commons Attribution License 4.0
https://creativecommons.org/licenses/by/4.0/deed.e
n_US
WSEAS TRANSACTIONS on ENVIRONMENT and DEVELOPMENT
DOI: 10.37394/232015.2022.18.62
Antonios Tampakis, Papaioannou Evgenia,
Theocharis Chatzistathis, Paraskevi Karanikola
E-ISSN: 2224-3496
661
Volume 18, 2022