Acta agriculturae Slovenica, 117/2, 1–15, Ljubljana 2021
doi:10.14720/aas.2021.117.2.1819 Original research article / izvirni znanstveni članek
Response of hemp (Cannabis sativa L.) to integrated application of chemi-
cal and manure fertilizers
Samaneh LALEH, Majid JAMI AL-AHMADI
*
, Soheil PARSA
 
Received August 09, 2020; accepted April 21, 2021.
Delo je prispelo 9. avgusta 2020, sprejeto 21. aprila 2021.
Response of hemp (Cannabis sativa L.) to integrated applica-
tion of chemical and manure fertilizers
Abstract: The investigation of various nutrition systems 
in hemp plays an influential role in improving its production. 
An experiment was conducted in University of Birjand, Iran, 
during 2013-2014, in which manure (0, 10, 20, and 30 t.ha
-1
 of 
cow manure) was considered as the main plot and the combina-
tion of nitrogen (0, 50, and 100 kg N ha
-1
 as urea) with phos-
phorus (0 and 80 kg P ha
-1
 as triple superphosphate) fertilizers 
was considered as factorial in subplots. The type of soil fertil-
ity management had no significant effect on the percentage of 
female plants. Applying 20 t.ha
-1
 of manure plus 100 kg N ha
-1
 
produced the highest biological yield, seed, and leaf extract. The 
highest oil content was obtained by applying a maximum of 50 
kg N ha
-1
 without the use of phosphorus. The 30 t ha
-1
 manure 
plus 100 kg N ha
-1
 increased the leaf harvest index and decreased 
seed harvest index. Nitrogen consumption also increased the 
seed oil content and yield. Phosphorus increased the biomass 
and extracts of seed and leaves, also biological, seeds and oil 
yield. It seems hemp responds well to the combined application 
of nitrogen fertilizer and animal manure, while its response to 
P fertilization was limited. 
Key words: cow manure; hemp; nitrogen; oil yield; phos-
phorus; seed yield 
Department of Agronomy and Plant Breeding, Faculty of Agriculture, University of Birjand, Birjand, Iran
mjamialahmadi@birjand.ac.ir
Odziv konoplje (Cannabis sativa L.) na hkratno uporabo mi-
neralnih in organskih gnojil 
Izvleček: Preučevanje različnih gnojilnih režimov igra pri 
konoplji pomembno vlogo za izboljšanje pridelave. Na univerzi 
v Birjandu (University of Birjand, Iran) je bil v letih 2013-2014 
izveden gnojilni poskus, v katerem je bilo gnojenje s kravjim 
gnojem glavno obravnavanje (0, 10, 20, in 30 t ha
-1
) in kombi-
nirano gnojenje z dušikovimi (0, 50, in 100 kg N ha
-1
 kot urea) 
in fosforjevimi gnojili (0 in 80 kg P ha
-1
 kot trojni superfosfat) 
kot faktorski poskus na podploskvah. Način gnojenja ni imel 
značilnega vpliva na odstotek ženskih rastlin. Uporaba 20 t 
ha
-1
 kravjega gnoja z dodatkom 100 kg N ha
-1
 je dala največji 
biološki pridelek, pridelek semena in izvlečkov listov. Največja 
vsebnost olja je bila dosežena pri uporabi največ 50 kg N ha
-1
 
brez dodatka fosforjevega gnojila. Uporaba 30 t ha
-1
 gnoja z 
dodatkom 100 kg N ha
-1
 je povečala žetveni indeks listov in 
zmanjšala žetveni indeks semena. Uporaba dušikovih gnojil 
je tudi povečala vsebnost olja v pridelku. Dodatek fosforjevih 
gnojil je povečal biomaso, vsebnost snovi v izvlečkih iz listov 
in semen, kot tudi biološki pridelek, pridelek semena in olja. 
Izgleda, da se konoplja odziva dobro na kombinirano gnojenje 
z mineralnimi dušikovimi gnojili in živinskimi gnojili, med 
tem, ko je njen odziv na gnojenje s fosforjevimi mineralnimi 
gnojili omejen.
Ključne besede: kravji gnoj; konoplja; dušikova gnojila; 
pridelek olja; fosforjeva gnojila; pridelek semena

Acta agriculturae Slovenica, 117/2 – 2021 2
S. LALEH et al.
1 INTRODUCTION
Cannabis sativa L. is one of the oldest domesticated 
plants in human history and has been used since 10,000 
years ago or more (Faux et al., 2013; Da Porto et al., 
2014). Today, its cultivation is exposed to limitations due 
to the use of cannabis as a narcotic. Recently, the use of 
cannabis as a medicinal plant has been taken into consid-
eration and the motive of this approach is to identify the 
effectiveness of whole plant cannabis in relieving some 
chronic diseases, although there is still debate about the 
medical benefits of cannabis worldwide (Madras, 2015). 
The seeds of this plant have a high nutritional value with 
a content of 22 to 35 % of oil (Peiretti, 2009), and has 
attracted the attention of nutritionists due to its high nu-
tritional quality (Leizer et al., 2000; García-Tejero et al., 
2014). There are high concentration of bioactive compo-
nents, such as cannabinoids, in Cannabis sativa L., with 
nutritional value associated with health benefits, which 
can be isolated from different parts of the plant. While 
large amounts of Δ
9
-THC accumulate in plant leaves, 
non-psychoactive cannabinoids are predominantly pres-
ent in seeds (Fathordoobady et al., 2019). Meanwhile, ex-
traction with ethanol is the preferred extraction method 
used for medicinal formulations (Thomas and Pollard, 
2016).
The evaluation of different plant nutrition systems is 
one of the important needs in agronomic planning in or-
der to achieve higher yields of plants such as hemp, as an 
oil or food crop. Nitrogen is one of the essential elements 
affecting the plants growth (Shams et al., 2013), which is 
required for proteins, enzymes, coenzymes, nucleic ac-
ids, cytochromes, and metabolic processes contributing 
to the synthesis and transfer of energy (Hoffman & Cl-
eemput, 2004). In hemp, the application of 240 kg N ha
-1
 
nitrogen was effective in increasing the plant biomass, 
stem dry mass, and inflorescence mass, compared to the 
control (without any fertilization) (Papastylianou et al., 
2018). Poisa & Adamovics (2010) reported an increase 
in the applied nitrogen fertilizer increased the yield of 
hemp seed, while the content of seed oil decreased in 
such a way that the content of hemp seed oil decreased 
from 42 % in control plants to 40.5 % in in those plants 
that received 100 kg N ha
-1
.
After nitrogen, phosphorus is the second most im-
portant nutrient for plant growth, which contributes to 
activating coenzymes producing amino acids (Hendawy 
& Khalid, 2011). Phosphorus is effective on the number 
of young cells in roots and stems and is highly needed 
in those places where cellular metabolism is greater and 
cells are dividing. Further, phosphorus plays a significant 
role in starting flowering, developing seeds and fruits, 
reducing diseases, increasing the quality of some crops, 
and root growth, especially the lateral roots, and devel-
oping fibrous root system (Hendawy et al., 2014; Kareem, 
2013).
Although the use of chemical fertilizers for supply-
ing nitrogen and phosphorus required by the plant has 
many benefits, there is an increasing demand for or-
ganic fertilizers, due to the soil and water pollution, and 
community health threats caused by synthetic chemical 
materials (Akande et al., 2010). The organic fertilizers 
(in particular, animal manures) have large amounts of 
organic matter, which could be used as a source of nu-
trients elements, especially nitrogen, phosphorus, and 
potassium (Cvetkov et al., 2010). Despite the benefits of 
using organic fertilizers, the chemical fertilizers cannot 
be removed from agricultural ecosystems and replaced 
by the organic fertilizers simultaneously, as the sustain-
ability in agriculture is ensured by adequate income and 
food security. In this regard, the use of renewable and 
natural materials with organic sources, along with the 
optimal use of chemical fertilizers, can be of great im-
portance in preserving fertility, biological building and 
activity, cation exchange capacity , water retention and 
ultimately, improving the physical and chemical struc-
ture of the soil (Ghosh et al., 2004). A large number of 
studies have been conducted on the integrated applica-
tion of organic- with chemical fertilizers on different 
plants, including bean (Phaseolus vulgaris L.), soybean 
(Glycine max L.) (Rurangwa et al., 2018), corn (Zea mays 
L.) (Arif et al., 2016), sunflower (Helianthus annus L.) 
(Akbari et al., 2011), and coriander (Coriandrum sativum 
L.) (Mallanagouda et al., 1995). Generally, nutritional 
studies on medicinal plants indicate the positive effect of 
organic fertilizers on the both quantitative and qualita-
tive yields of medicinal plants. In saffron (Crocus sativus 
L.), for example, the highest stigma yield was reported 
0.45 g m
-2
 in combined treatment of manure and chemi-
cal fertilizers (23 kg N ha
-1
 + 20 kg P
2
O
5
 ha
-1
 + 20 t ha
-1
 
animal manure) (Amiri, 2009). The study of different fer -
tilizer treatments on the German chamomile (Matricaria 
chamomilla L.) indicated that the combined application 
of manure and chemical fertilizers (23 kg N ha
-1 
+ 23 kg 
P
2
O
5
 ha
-1
 + 7.5 t ha
-1
 cow manure) increased the biologi-
cal yield (188.6 %), harvest index (18.69 %), and essential 
oil content (84.27 %), compared to the control (Shams et 
al., 2012).
It seems that the integrated application of appropri-
ate amounts of organic and chemical fertilizers positively 
affect the oil yield of plants. For example, 50 % cow ma-
nure plus 50 % nitrogen in sunflower, produces the high-
est oil yield (1275.9 kg ha
-1
), and 100 % application of 
cow manure produces the highest oil percentage (49.4 %) 
(Akbari et al., 2011). In another study, the use of 50 % 

Acta agriculturae Slovenica, 117/2 – 2021 3
Response of hemp (Cannabis sativa L.) to integrated application of chemical and manure fertilizers
cow manure plus 50 % of chemical fertilizer (N, P and K 
at 220, 150 and 100 kg ha
-1
, respectively), produces the 
highest yield (1482.4 kg ha
-1
) and oil content (39.7 %) in 
sunflower (Esmaeilian et al., 2011). The combined appli-
cation of organic fertilizers (15 t ha
-1
 vermicompost) with 
100 kg N ha
-1
 was effective in increasing the seed yield and 
oil content of rapeseed (Brassica napus L.) (Zahedifard et 
al., 2014). In a study with chemical fertilizers (nitrogen 
and phosphorus), and bio-fertilizer, the combination of 
bio-fertilizer with 50 % of total nitrogen (200 kg ha
-1
 am-
monium nitrate 33 % ) and 50 % of total phosphorus (100 
kg ha
-1
 of calcium superphosphate 15.5 %), produced the 
highest oil yield in the fennel (Foeniculum vulgare Mill.) 
(Dadkhah, 2012). It was reported that nitrogen fertilizer 
reduces the oil content of the juniper (Juniperus commu-
nis L.) (Hendawy et al., 2014), although it increases the 
oil yield in the thyme medicinal plant (Thymus vulgaris 
L.) (Baranauskienne et al., 2003).
To the best of our knowledge, the effect of the com-
bined application of chemical and organic fertilizers on 
the yields of seed oil and extracts of different parts of 
the hemp has not yet been studied. Regarding the global 
trend towards the production and propagation of low-
demand crops in sustainable agricultural systems and the 
importance of reducing chemical inputs in agriculture 
on community health, more studies are necessary on the 
impact of chemical and organic fertilizers on neglected 
and new crops such as hemp, to be introduced in sustain-
able farming systems especially in marginal lands. This 
valuable plant has not yet found its true position among 
cash crops due to legal constraints despite the thousands 
of years of planting hemp (Da Porto et al., 2014). The 
present study aimed to investigate the effect of chemical 
(nitrogen and phosphorus) and organic (cow manure) 
fertilizers on biological, seed, leaf extract, and oil yields 
of hemp in two cropping years.
2 MATERIAL AND METHODS
The integrated application of chemical (nitrogen, 
phosphorus) and organic (rotted cow manure) fertiliz-
ers on hemp (Khusf landrace) was investigated during an 
experiment in two consecutive cropping years (2013 and 
2014) in the Research Farm of Faculty of Agriculture (32° 
52ʹ N, 59° 12ʹ E, 1491 meters above the sea level), Uni-
versity of Birjand, Birjand, Iran. The seeds were obtained 
from smallholder farmers who grow this crop in their 
subsistence farming systems in the Khusf (a small town 
near Birjand, eastern Iran). Plant height and the length 
of main inflorescence of this local variety are 150 and 22 
cm, respectively, with 25-20 nodes in the main stem, on 
average. The stem diameter is 2-3 cm and the height of 
the first flowering node is 80 cm. Its growth period lasts 
160 to 180 days (Riahi et al., 2016)
Tables 1 and 2 represent the trends of temperature 
and rainfall during experiment and the characteristics 
of soil of experimental site and applied manure of each 
year, respectively. In order to determine the physical and 
chemical characteristics of the soil in each year, the five 
samples were taken prior to planting from five points 
through experimental site within a depth of 0-20 cm. The 
samples were mixed and a sub-sample was taken. The 
electrical conductivity of the saturated extract and pH 
of soil and manure were measured. The nitrogen content 
of the soil and manure was calculated by using the Kjel-
dahl method (Jackson, 1958). Further, the carbon and 
the phosphorous of the soil and manure were measured 
by using Walkley-Black (Walkley and Black, 1934) and 
spectrophotometry (Bouyoucos, 1951), respectively. In 
addition, the soil texture was determined based on Bicas’ s 
hydrometer method.
The experiment was conducted in a factorial split-
plot arrangement with three replications. The manure (0, 
10, 20, and 30 t ha-1 of rotted cow manure) was consid-
ered as the main plot and the combination of nitrogen 
(0, 50, and 100 kg N ha
-1
 as urea) with phosphorus (0 
and 80 kg P
2
O
5
 ha
-1
 as triple superphosphate) fertilizers 
was considered as factorial in subplots. Generally, 100 kg 
ha
-1
 of potassium sulfate (50 kg K
2
O ha
-1
) was used for all 
plots before sowing. All phosphorus fertilizers and half of 
the nitrogen fertilizers were applied before sowing below 
the seed planting depth. After thinning, the other half of 
the nitrogen fertilizer treatments (except control) were 
applied.
In both years, sowing was on plots that were under 
fallow the previous year. The manure and potassium fer-
tilizer were spread on the soil surface and then mixed 
into the soil during soil preparation for sowing. The plot 
size consisted of 5 rows with row length of 4 m. In each 
plot the hemp was sown with a density of six pl m
-2
 (with 
60 and 30 cm space between rows and on the rows) in 
five rows with a depth of 3-4 cm. The seeds were hand 
sown with high density beside row bed on June 7, 2013, 
and May 5, 2014, and then emerged plants were thinned 
in two stages (when plants had 2-4 leaves and two weeks 
later) to achieve the final density. The irrigation interval 
was adjusted to 10 days. During the experiment, no her-
bicides, fungicides, and fertilizers (except for fertilizer 
treatments) were used, and all weeds were controlled by 
twice hand weeding early in plant growth. At final har-
vest, the side rows and one meter at all row ends were 
discarded to avoid border effects. The whole plants were 
harvested on November 11, 2013, and October 7, 2014, 
when 50 % of the grains were hard. 

Acta agriculturae Slovenica, 117/2 – 2021 4
S. LALEH et al.
Table 1: Average precipitation and temperature during the growth season of hemp in 2013 and 2014
Table 2: Physicochemical properties of the soil (0-20 cm depth) and animal manure used in 2013 and 2014
May June July August September October November
2013
Average temperature (°C) 21.1 26.7 27.2 27.2 23.6 20.9 10.4
 Average precipitation (mm) 24.8 0.2 0 0 0 1.1 4.4
2014
 Average temperature (°C) 22.3 25.7 28.5 25.3 22.4 19.6 13.4
 Average precipitation (mm) 3.8 0.4 0 0 0 6.2 11.2
Ye ar pH EC
Organic 
carbon
N
C/N 
ratio
P Texture Silt Clay Sand
(dS m
-1
) ------- (%) ------- (ppm) ---------- (%) ----------
2013
Soil 7.98 9.75 0.52 0.06 8.66 10.3 Loamy sandy clay 26 20 54
Manure 8.5 6.9 12.6 0.66 19.09 1240 - - - -
2014
Soil 7.79 7.48 0.31 0.04 7.75 9.8 Loamy sandy clay 26 22 52
Manure 8.01 6.03 12.4 0.58 21.15 1180 - - - -
2.1 MEASURED CHARACTERISTICS
In this experiment, biological and seed yield, thou-
sand seeds mass, leaf and seed harvest indices, the yield 
of leaf and seed extract, percentage and yield of seed oil, 
and specific leaf area (SLA) were measured. Considering 
the same planting density in all plots, the number of all 
female plants per plot was counted.
At final harvest, whole plant were harvested from 
three m
2
, and after completely drying in shading condi-
tions and free air, were weighed on a scale (± 0.01 g) to 
measure biological yields of female plants. After that, 
seeds were separated and weighted to calculate seed 
yields. Five replicates of 100 seeds then were weighted 
with a 0.001 g scale to determine the mass of 1000 seeds. 
Also the seed and leaf harvest indices were calculated for 
female plants based on the leaf and seed dry yields to the 
biological yield ratio, respectively. 
Another random sample was taken from one square 
meter of each plot to measure the leaf area and dry mass 
of female plants, and then SLA was calculated. The seed 
and leaves of these plants also used to measure their ex-
tracts. In this regard, we used ethanol to prepare an alco-
holic extract of leaves and seeds (Khan et al., 2014). To 
do this, the leaves and seeds were separately grinded and 
50 g of powdered material, after adding ethanol 70 %, 
were placed for 48 hours in a shaker incubator at 25 °C 
and the speed of 100 rpm. At the end of this period, the 
solution was filtered by Watten’s No. 1 filter paper. In or-
der to remove the solvent from the extract, the samples 
were placed in a 40 °C oven for 48 hours. The obtained 
extract had no alcohol and contained a sticky and green 
substance. Then, the extracts were weighed with a 0.001 
gram scale.
The extraction of seed oil was performed with the 
soxhlet method according to the AOAC (1990). A 
sample of 10 g of seeds flour was extracted using 100 ml 
hexane as solvent, for 2 h. The solvent was removed with 
a rotary evaporator and the residue was placed in oven 
at 100 ºC for 4 h and then transferred to a desiccator 
and weighed up to constant value. The seed oil percent-
age was obtained by equation [1]:
Where m
1
 represents the initial mass of the sample 
(g), m
2
 indicates the initial mass of the container (g), and 
m
3
 is the secondary mass of the container (container + 
oil). The oil yield was simply obtained by multiplying 
seed yield in the oil percentage.
The specific leaf area (cm
2
 g
-1
) was calculated from 
the equation [2] (Amanullah et al., 2007):
Where LA represents the leaf area (cm
2
 pl
-1
) and LM 
indicates the leaf mass per plant (g pl
-1
). For this purpose, 

Acta agriculturae Slovenica, 117/2 – 2021 5
Response of hemp (Cannabis sativa L.) to integrated application of chemical and manure fertilizers
in the seed filling stage, female plants were harvested 
from one m
2
 of each plot, and their leaves were separated. 
Leaf area was determined using a leaf area meter (Delta-
T Devices, UK).
2.2 STATISTICAL ANALYSIS
In the present study, check the normality of data, 
plotting, and analysis of regressions, as well as the cor-
relations of traits were done by IBM SPSS Statistics 22 
software. Before the combined analysis of the data, Bar-
tlett’s test was used to ensure the uniformity of the test 
error variance. Data analysis was performed using SAS 
9.2 software considering the effect of year as random and 
the effect of experimental treatments for desired traits as 
constant. Comparison of meanings was done by FLSD 
test at 5% probability level.
3 RESULTS AND DISCUSSION
3.1 RESULTS
Table 3 shows the results of analysis of variance of 
traits. As shown in Table 4, the studied traits were not 
significantly different between two years. The fertilizer 
treatments had no significant effect on the percentage of 
female plants in the hemp. However, an increase in the 
manure level and also applying 50 kg N ha
-1
 increased 
the percentage of female plants. Bigger increase in the 
nitrogen fertilizer level reduced the percentage of female 
plants, which was not significant (Table 4).
By comparing biological yield among different ma-
nure levels, the most beneficial effect of manure on in-
creasing biological yield was achieved from 10 to 20 t ha
-1
 
applied manure, that was on average 148.94 kg ha
-1
 bio-
logical yield per each one t ha
-1
 of more manure; although 
a further increase in the manure amount reduced its ef-
fectiveness in increasing the biological yield (T able 4). An 
increase in the nitrogen amounts also reduced its effect 
on enhancing biological yield, such that the increase in 
biological yield per each kg more nitrogen between 0-50 
kg N ha
-1
 (44.4 kg ha
-1
) was more than 50-100 kg N ha
-1
 
(13.61 kg N ha
-1
) (Table 4).
The highest biological yield was obtained in the 
combined treatment of 20 t ha
-1
 manure plus 100 kg N 
ha
-1
, which did not show any significant difference with 
the combined treatment of 30 t ha
-1
 manure plus 50 kg N 
ha
-1
 (Table 5). Without the applying manure, for increas-
ing each kg N ha
-1
 from the 0 to 50 and 50 to 100 kg N ha
-
1
, the biological yield increased, on average by 29.4 and 
21.4 kg ha
-1
, respectively. When 10 t ha
-1
 cow manure was 
used, the increase in the biological yield was 35.22 and 
17.9 kg ha
-1
 per each kg N in 0-50 and 50-100 kg ha
-1
 ap-
plied N, respectively. These increases in biological yield 
for 20 t ha
-1
 of manure were 54.28 and 20 kg ha
-1
, respec-
tively. At the level of 30 t ha
-1
 manure, the biological yield 
increased by an average of 57.25 kg ha
-1
 per each kg N 
ha
-1
 in the range from 0 to 50 kg N ha
-1
 while it decreased 
by 18.38 % per each kg of nitrogen in the range 50-100 kg 
N ha
-1
. This indicates the use of manure has been effec-
tive in increasing the effectiveness of nitrogen fertilizer. 
In each level of manure, the efficiency of adding first 50 
kg N ha
-1
 (0-50 kg N ha
-1
) to improve biological yield was 
more than that of the second 50 kg (50-100 kg N ha
-1
), 
confirming that the response of biological yield to the 
amount of consumed nitrogen follows law of diminish-
ing returns. The results also indicated that an increase in 
the nitrogen levels to 100 kg ha
-1
 could have a negative 
effect on the biological yield of hemp for levels of more 
than 20 t ha
-1
 of manure (Table 5). 
Based on the results, the beneficial effect of nitro-
gen on the seed yield increased under the influence of 
manure, and accordingly, no significant difference was 
observed between 0 and 50 kg N ha
-1
 when manure was 
not used. However, manure was applied at rates of 10, 20, 
and 30 t ha
-1
, the use of 50 kg N ha
-1
 could significantly 
increase the seed yield by 29.33 %, 33.9 %, and 33.75 %, 
respectively, compared to non-application of nitrogen 
(Table 5). The highest seed yield was achieved when 20 
t ha
-1
 manure was used along with 100 kg N ha
-1
 , which 
increased by 40.95 % compared to when the manure was 
not used. Further, there was no significant difference be-
tween the combined applications of 20 t ha
-1
 of manure 
plus 100 kg N ha
-1
 with 20 t ha
-1
 manure with 50 kg N 
ha
-1
, and 30 t ha
-1
 manure plus 50 kg N ha
-1
 (Table 5). It 
is worth noting that an increase in the nitrogen fertilizer 
level increased the seed yield at 0, 10, and 20 t ha
-1
 of ma-
nure, while the use of 100 kg N ha
-1
 could not positively 
affect the seed yield when combined with 30 t ha-1 of 
manure. This combined treatment reduced the seed yield 
by 17.11 %, compared to a combined use of 30 t ha
-1
 ma-
nure plus 50 kg N ha
-1
 (Table 5).
The response of seed yield to manure was followed 
a second-order function under the influence of phospho-
rus consumption, upon which, when phosphorus was 
applied, the highest seed yield was obtained with the ap-
plication of 20 t ha
-1
 manure; however when phosphorus 
was not consumed, the response of the seed yield to ma-
nure was linear (Figure 1). Therefore, in the case of phos-
phorus application, the use of more than 20 t ha
-1
 manure 
reduced the effectiveness of manure to increase the seed 
yield, while, in the case of non-application of phospho-
rus, as the amount of manure increased, seed yield also 

Acta agriculturae Slovenica, 117/2 – 2021 6
S. LALEH et al.
Table 3: The Results of combined analysis of variance of measured traits (mean squares) in cannabis during 2013 and 2014 as affected by manure, nitrogen and phosphorus ferti-
lizers
ns: no significant difference, * and ** significant difference at the level of one and five percent, respectively
Mean squares
Seed oil 
yield
Seed 
oil
Seed 
extract 
yield
Leaf 
extract 
yield
Seed harvest 
index
Leaf harvest 
index
Seed mass Seed yield Biological yield
female 
plant
df Source of Variation
2.87
 ns
4.34
 ns
11.30
 ns
78.54
ns
1.25
 ns
38.10
 ns
0.18
 ns
16197.14
 ns
478005.7
 ns
107.22
ns
1 Y ear (Y)
11.02 2.67 1.70 2.59 18.50 77.79 2.01 11751.22 694989.4 33.29 4 Replication (Y)
1816.24
**
48.58
**
3.19
*
23.29
*
64.39
**
203.46
**
35.38
**
1735544.27
**
47084929.5
**
17.00 
ns
3 Animal manure (M)
69.87
 ns
3.82
 ns
0.04
 ns
0.20
 ns
26.37
 ns
73.29
 ns
2.09
 ns
115785.15
 ns
289722.6
 ns
1.34
ns
3 Y × M
57.61
 ns
4.75
 ns
0.62
 ns
3.90
 ns
18.12 51.50 10.79
 ns
88101.52
 ns
680624.2
 ns
27.84 12 Error a
2585.62
**
29.21
*
8.35
**
78.42
**
141.72
**
260.07
**
79.64
**
3896713.79
**
109253454
**
30.97
 ns
2 Nitrogen (N)
805.65
**
4.34
 ns
0.77
 *
6.37
 *
5.70
 ns
37.40
 ns
150.06
**
1575995.26
**
29378275.4
**
35.16
 ns
1 Phosphorus (P)
157.32
 ns
11.42
ns
0.27
 ns
1.68
 **
11.00
 ns
30.02
 ns
8.36
 ns
179094.96
*
3472068.2
**
17.05
 ns
6 M×N
203.11
 ns
3.37
 ns
2.72
 ns
5.99
 ns
46.48
 ns
88.15
 ns
1.31
 ns
232450
*
492938.4
ns
32.07
 ns
3 M×P
218.10
 ns
27.17
*
0.67
 ns
0.49
 ns
5.04
 ns
12.93
 ns
0.36
 ns
73550.68
 ns
679811.2
 ns
8.14
 ns
2 N×P
70.21
 ns
2.90
 ns
1.79
 ns
5.39
 ns
27.13
 ns
45.24
 ns
3.19
 ns
79057.64
 ns
871692.2
 ns
47.04
 ns
6 M×N×P
98.09
 ns
7.96
 ns
1.25
 ns
5.53
 ns
8.23
 ns
58.72
 ns
7.06
 ns
145898.51
 ns
1419729.1
 ns
8.41
 ns
2 Y×N
27.68
 ns
10.56
ns
1.09
 ns
0.69
 ns
1.84
 ns
38.61
 ns
0.17
 ns
3344.92
 ns
9586.7
 ns
3.62
 ns
1 Y×P
135.78
 ns
1.30
 ns
0.46
 ns
0.97
 ns
27.62
 ns
9.37
 ns
8.68
 ns
162175.15
 ns
613936.5
 ns
63.81
 ns
6 Y×M×N
147.53
 ns
3.67
 ns
1.14
 ns
7.23
 ns
9.87
 ns
9.98
 ns
2.71
 ns
1322860.66
ns
1351238.3
 ns
81.13
 ns
3 Y×M×P
119.29
 ns
21.06
ns
0.39
 ns
2.65
 ns
35.51
 ns
29.55
 ns
0.04
 ns
125542.09
 ns
810736.0
 ns
3.09
 ns
2 Y×N×P
113.10
 ns
12.25
ns
1.61
 ns
4.91
 ns
12.21
 ns
7.50
 ns
5.11
 ns
42360.76
 ns
605625.4
 ns
73.76
 ns
6 Y×M×N×P
79.63 7.68 0.97 8.24 20.57 45.04 6.93 78291.42 757591.5 45.02 80 Error b
18.20 10.21 12.60 14.82 17.64 12.13 14.13 15.49 12.11 12.12
Coefficient of variation 
(%)

Acta agriculturae Slovenica, 117/2 – 2021 7
Response of hemp (Cannabis sativa L.) to integrated application of chemical and manure fertilizers
Table 4: Simple effects of animal manure, nitrogen and phosphorus on measured traits in hemp. For fertilization 
treatments, numbers are means of three replication ±SEM
Table 5: Mean comparisons for interaction effects of animal manure and nitrogen levels on measured traits in hemp. 
For fertilization treatments, numbers are means of three replication ±SEM
In each column, the values that share at least one letter have no significant differences according to LSD test at 5 percent 
of probability.
Seed oil 
yield (kg 
ha
-1
)
Seed oil 
(%)
Seed 
extract 
yield (kg 
ha
-1
)
Leaf 
extract 
yield (kg 
ha
-1
)
Seed 
harvest 
index 
(%)
Leaf 
harvest 
index 
(%)
Seed 
mass 
(g 1000 
seed)
Seed 
yield (kg 
ha
-1
)
Biological 
yield (kg 
ha
-1
)
female 
plant 
(%)
Level Treatments
488.77
a
27.30
a
88.83
a
787.40
a
25.61
a
54.79
a
18.58
a
1795.29
a
7242.34
a
54.48
a
2013
Ye ar
491.59
a
26.95
a
78.14
a
807.32
a
25.74
a
55.82
a
18.66
a
1816.50
a
7127.11
a
56.20
a
2014
394.02
c
25.52
b
66.52
b
599.50
b
27.03
a
52.76
c
17.47
b
1544.87
d
5858.80
c
54.62
a
0
Animal 
manure       (t 
ha
-1
)
483.94
b
28.22
a
74.95
b
685.19
b
26.31
a
53.88
bc
18.13
ab
1710.41
c
6602.00
b
55.00
a
10
558.77
a
27.66
a
98.67
a
939.89
a
25.56
ab
56.72
ab
19.47
a
2027.85
a
8091.40
a
55.54
a
20
524.01
ab
27.11
a
93.81
a
964.88
a
23.91
b
57.84
a
19.44
a
1940.47
b
8186.70
a
56.21
a
30
406.08
b
27.33
ab
61.88
b
538.02
c
27.45
a
52.79
b
17.17
b
1475.00
b
5488.20
b
54.72
a
0
Nitrogen 
fertilizer  (kg 
ha
-1
)
541.27
a
27.79
a
86.75
ab
872.65
b
25.63
ab
55.73
a
19.08
ab
1946.55
a
7690.30
a
56.25
a
50
523.20
a
26.27
b
100.57
a
981.43
a
24.02
b
57.38
a
19.63
a
1993.15
a
8375.70
a
55.06
a
100
466.53
b
27.30
a
76.76
b
734.54
b
25.90
a
54.79
a
17.61
b
1701.28
b
6733.05
b
55.84
a
0
Phosphorus 
fertilizer  (kg 
ha
-1
)
513.83
a
26.95
a
90.21
a
860.19
a
25.50
a
55.81
a
19.65
a
1910.51
a
7636.41
a
54.85
a
80
increased, although there was not a significant difference 
between of 20 and 30 t ha
-1
 manure (Figure 1).
With the use of 20 and 30 t ha
-1
 manure, the grain 
mass increased by 11.44 and 11.26 %, respectively, com-
pared to the control. The most beneficial effect of manure 
was achieved in the range of 10 to 20 t ha
-1
 (0.11 g per 
ton of manure, on average), although the more increase 
in manure reduced the effectiveness of manure on seed 
Leaf extract yield   
kg ha
-1
)
Seed yield(kg ha
-1
)
Biological yield           
(kg ha
-1
)
Nitrogen fertilizer 
(kg ha
-1
)
Animal manure (t ha
-1
)
41.05 ± 17.23
f
1159.98 ± 20.67
e
4522.29 ± 85.10
i
0
0 63.20 ± 13.14
def
1455.86 ± 25.51
cde
5992.23 ± 68.64
fgh
50
75.59 ± 11.80
cd
1718.76 ± 19.05
bc
7061.83 ± 56.72
de
100
48.68 ± 13.25
ef
1273.54 ± 11.81
de
5129.13 ± 78.41
hi
0
10 68.93 ± 13.81
cde
1647.19 ± 30.48
c
6890.58 ± 92.57
def
50
87.93 ± 14.09
bc
1710.48 ± 29.07
c
7786.29 ± 86.14
cd
100
57.30 ± 10.26
def
1519.01 ± 23.02
cd
5848.32 ± 65.88
gh
0
20 101.98 ± 16.79
ab
2034.00 ± 31.75
ab
8562.79 ± 86.22
bc
50
122.67 ± 19.96
a
2130.54 ± 29.25
a
9563.08 ± 120.53
a
100
63.06 ± 11.45
def
1559.47 ± 32.94
cd
6453.02 ± 90.05
efg
0
30 115.57 ± 12.98
a
2085.84 ± 19.64
a
9315.63 ± 52.46
ab
50
106.36 ± 29.36
ab
1728.82 ± 30.54
bc
8396.21 ± 121.13
bc
100

Acta agriculturae Slovenica, 117/2 – 2021 8
S. LALEH et al.
mass (Table 4). The application of 100 kg N ha
-1
 increased 
the seed mass by 14.27 % compared with the control. De-
spite the increase of the seed mass under the influence of 
the nitrogen, the results indicated that the average effec-
tiveness of nitrogen on the seed mass in the range of 0 to 
50 kg N ha
-1
 (0.04 g per kg N ha
-1
), was more than 50 to 
100 kg N ha
-1
 (0.01 g per kg N ha
-1
) (Table 4).
Based on a linear relationship, an increase in the leaf 
harvest index in hemp was consistent with decreasing the 
seed harvest index (Figure 2). The application of manure 
was effective in increasing the leaf harvest index and the 
levels of 20 and 30 t ha
-1
 manure, compared to the con-
trol, increased the leaf harvest index by 7.5 and 9.62 %, 
respectively. However, the use of 30 t ha
-1
 of manure 
reduced the seed harvest index by 8.87 %, as compared 
with the control (Table 4). Thus, it seems applying 30 t 
ha-1 of manure stimulate assimilate partitioning to plant 
leaves and thereby reduced allocation ratio to seeds.
It is worth noting that the use of nitrogen fertilizer 
in hemp also increased the leaf harvest index, as 50 and 
100 kg N ha
-1
 increased this index by 5.56 and 8.7 %, re-
spectively, compared with the control treatment, while 
using 100 kg N ha
-1
 resulted in a 12.49 % reduction in the 
seed harvest index, compared to its non-application (Ta-
ble 4). So, like the highest level of manure, the level of 100 
kg N ha
-1
 reduced allocation of assimilates to the seeds by 
more biomass partitioning to the plant leaves.
The highest and the lowest leaf extract yields were 
obtained with integrated application of 20 t ha-1 manure 
with 100 kg N ha
-1
 and in no-fertilizer treatment, respec-
tively (Table 5). The use of nitrogen alone (without ma-
nure) reduced the efficiency of nitrogen in increasing the 
yield of leaf extract, as in the ranges of 0-50 and 50-100 
kg N ha
-1
, the average yield of hemp leaf extract increased 
by 0.44 and 0.27 kg per kg N applied, respectively. These 
values were 0.40 and 0.38 kg leaf extract per kg N applied 
Figure 1: Response of hemp seed yield to animal manure at 0 () and 80 () kg P ha
-1
. The points that share at least one letter 
have no significant differences according to FLSD test at 5 percent of probability. 
for the level of 10 t ha
-1
 manure and 0.89 and 0.41 kg leaf 
extract per kg N applied for the level of 20 t ha
-1
 manure, 
respectively. With 30 t ha
-1
 manure, the leaf extract yield 
increased by 1.05 kg per kg N applied in the range of 0-50 
kg N ha
-1
, while it decreased by 0.18 kg per kg N applied 
from 50 to 100 kg N ha
-1
 (Table 5). The results suggested 
that at all levels of manure, the effectiveness of nitrogen 
on the leaves extract yield was more in the first 50 kg than 
that of the second one. The combination of the highest 
levels of manure and nitrogen had a negative effect on the 
yield of hemp leaf extract.
There was a relation between yield of hemp leaf ex-
tract with specific leaf area (SLA), as an increase in the 
manure level reduced the SLA and increased the yield of 
leaf extract. Further, the SLA of most treatments was in 
the range of 20 to 40 cm
2
 g
-1
. (Figure 3).
The 20 and 30 t ha
-1
 manure increased seed extract 
yield by 48.33 and 41.02 %, respectively, in comparison 
with the non-application of manure (Table 4). The level 
of 20 t ha
-1
 of manure was the most effective in increasing 
the yield of seed extract, since the yield of seed extract 
increased by 16.55 and 31.74 kg t
-1
 manure applied in the 
range of 0-10 and 10-20 t ha
-1
 manure, respectively. Nev-
ertheless, the yield of seed extract was reduced by 8.73 kg 
t
-1
 manure applied from 20 to 30 t ha
-1
 manure (Table 4).
Seed extract yield increased in response to higher 
nitrogen fertilizer levels, so applying 50 and 100 kg N ha
-1
 
increased the seed extract yield by 40.19 and 62.52 %, 
compared to the control (non-application of nitrogen), 
respectively (Table 4). The positive effect of nitrogen 
utility on seed extract yield decreased by increasing the 
nitrogen levels, so the amount of increase in the seed ex-
tract per nitrogen consumed in the range of 0-50 kg N 
ha
-1
 (0.5 kg kg
-1
 nitrogen) was more than that of 50-100 
kg N ha
-1
 (0.28 0.5 kg kg
-1
 nitrogen) (Table 4).
The use of manure led to higher seed oil percentage, 

Acta agriculturae Slovenica, 117/2 – 2021 9
Response of hemp (Cannabis sativa L.) to integrated application of chemical and manure fertilizers
Figure 2: Changes of seed harvest index (%) to leaf harvest index (%) in hemp.
Figure 3: Changes of Leaf extract yield (kg ha
-1
) to SLA (cm
2
 g
-1
) for 0 (), 10 ( ), 20 () and 30 () ton animal manure ha
-1
so that applying 10, 20, and 30 t ha
-1
 of manure increased 
seed oil by 10.58 %, 8.38 % and 6.23 %, respectively, com-
pared to the control. An increase in manure level by more 
than 10 t ha
-1
 decreased the profitability of manure to in-
crease the percentage of seed oil (Table 4).
The highest percentage of seed oil (28.85 %) was 
achieved by applying 50 kg N ha
-1
 and not using phos-
phorus (Figure 4). When no phosphorus was used, an in-
crease in the nitrogen levels up to 50 kg N ha
-1
 improved 
its efficiency to enhance the seed oil percentage, although 
efficiency was reduced with more nitrogen consumption, 
so that, in the absence of phosphorus application, the lev-
el of 100 kg N ha
-1
 decreased the percentage of seed oil by 
4.77 %, compared to the 50 kg N ha
-1
. When phosphorus 
was used, the nitrogen effectiveness on the seed oil per-
centage decreased by increasing its level, however there 
was not any significant differences between 50 and 100 
kg N ha
-1
 with in terms of seed oil percentage (Figure 4).
The slopes of regression lines fitted between the oil 
yields versus the seed yields were significant (p ≤ 0.01), 
indicating any increase in the seed yield is accompanied 
with the oil yield, the use of manure, especially at levels 
of 10 and 20 t ha
-1
 (compared with control), was more ef-
fective in increasing the yield of oil than grain yield (Fig-
ure 5).All levels of manure were effective in increasing 
the yield of seed oil as 10, 20 and 30 t ha
-1
 of manure in-
creased the seed oil yield by 22.82 %, 41.81 %, and 33 %, 
respectively, compared to the control. Compared to the 
application of 20 t ha
-1
 of manure, the 30 t ha
-1
 manure 
caused a slight and non-significant decline in seed oil 
yield (Table 4).
The 50 and 100 kg N ha
-1
 levels improved the seed 
oil yield by 33.30 and 28.84 %, respectively, compared to 

Acta agriculturae Slovenica, 117/2 – 2021 10
S. LALEH et al.
Figure 4: Response of hemp seed oil percentage to applied nitrogen at 0 (white columns) and 80 (dark columns) kg P ha
-1
. The 
columns that share at least one letter have no significant differences according to FLSD test at 5 percent of probability. Vertical bars 
on columns indicate SEM.
Figure 5: Changes of seed oil yield (kg ha
-1
) to seed yield (kg ha
-1
) for 0 (), 10 (), 20 () and 30 () ton animal manure ha
-1
the control and with the application of 100 kg N ha
-1
 the 
yield of the hemp seed oil showed a slight and non-signif-
icant decrease (Table 4).
The consumption of 80 kg P ha-1 increased the bio-
logical yield (13.41 %), seed mass (11.6 %) and the seed 
oil yield (10.13 %), compared to the control (no phos-
phorus application) (Table 4). The results also indicated 
that the phosphorus improved the yield of leaf and seed 
extracts by 17.9 and 17.52 %, respectively. The phospho-
rus was more effective in increasing the leaf extract yield 
than seed extract, so that phosphorus increased the yield 
of leaf and seed extracts by 1.57 and 0.17 kg per kg P, 
respectively (Table 4).
3.2. DISCUSSION
In this experiment, the effect of year on the meas-
ured traits was not significant (Table 4). The average 
temperature for the cropping seasons was approximately 
the same for two years (the average temperature in 2013 
and 2014 was 22.66 and 23.96 °C, respectively) (Table 1). 
A little superiority which was observed in 2014 in some 
measured traits, such as the seed yield, seed mass, leaf 
and seed harvest indices, the yields of seed and plant oil 
extracts, is probably due to more suitable germination 
temperature in this year compared to 2013. The opti-
mum temperature for germination of hemp seeds is 20 
°C (Albu and Marti, 2008) and the lower temperature 
in May 2014 (22.3 °C), compared to the June 2013 (26.7 
°C, Table 1), which are the planting times in this experi-
ment, caused germination and early growth of the plant 
in 2014 to be occurred in a more appropriate condition. 
The more rainfall in June and October 2014, compared to 

Acta agriculturae Slovenica, 117/2 – 2021 11
Response of hemp (Cannabis sativa L.) to integrated application of chemical and manure fertilizers
2013, was also effective in improving the measured traits 
in this year (Table 1). Also the higher soil EC of the ex-
perimental site in 2013 could be one of the reasons for 
the overall decline in traits in this year compared to 2014 
(Table 2).
Chemical agents and environmental parameters 
have been identified are among the factors affecting the 
expression of sex genes in hemp (Milewicz and Sawicki, 
2012). Nitrogen is more or less effective on incidence of 
male sex in hemp (Truta et al., 2007); but to increase the 
female sex ratio in population, it is necessary to increase 
the nitrogen level (Hall et al., 2013). Generally, in this 
study, an increase in the levels of manure and nitrogen 
slightly increased the percentage of female plants, com-
pared to their control; which was not significant (Table 
4). This is probably due to the use of moderate levels of 
nitrogen fertilizer in integration with manure applica-
tion, and the effect of these moderate levels of fertilizers 
on increasing female sex in hemp. The use of phospho-
rus reduced the percentage of female plants in hemp, al-
though its effect was not significant in this study (Table 
4). In an experiment on sweet gale (Myrica gale L.), the 
application of phosphorus also reduced the abundance of 
female plants (Chang and Martin, 2014).
The increase of the biological, seed, and leaf ex-
tract yields under the combined effect of manure and 
the nitrogen fertilizer, compared to the application of 
manure alone, indicated the synergistic effect of manure 
and nitrogen (Table 5). The increase of plant yield in the 
integrated system of plant nutrition is due to the avail-
ability of the soil nitrogen when needed by plant. When 
chemical and organic fertilizers are used in combination 
with each other, the nutrients in the chemical fertilizers 
are quickly released and help the initial establishment of 
the plant. On the contrary the mineralization of organic 
fertilizer is gradually done during the growing season, 
which helps to achieve higher plant yields (Ayoola et al., 
2007). More than 70 nitrogenous compounds, including 
alkaloids, amides, lignanamide and proteins (edestin, 
zeatin and zeatin nucleoside), enzymes (edestinase, glu-
cosidase, polyphenoloxydase, peptidase, peroxidase and 
adenosine-5-phosphatase), and amino acids have been 
recognized in hemp (Bernneisen, 2007). The synthesis of 
these nitrogenous compounds existent in the hemp ex-
tract is influenced by the amount of nutrients absorbed 
by the plant, especially nitrogen.
The combined application of 30 t ha
-1
 of manure, 
with high levels of nitrogen had a negative effect on bio-
logical and seed yields (Table 5). In fact, adding manure 
was effective in increasing nitrogen absorption by plants. 
Despite the higher levels of phosphorus compared to the 
nitrogen in the most manures, plants absorb nitrogen 
about 2.4 to 4.5 times more than the phosphorus through 
animal manures (Mullins, 2009). The pools of nitrogen 
absorbed through the manure or chemical fertilizers by 
the plant, as protein or amino acids, requires carbon in-
puts to provide the structure of the carbon skeleton and 
energy supply (Cheng et al., 2004) and this can reduce 
the growth and yield of the plant. The nitrogen has a 
complex effect on the metabolism of plant carbohydrates. 
Sometimes it significantly increases the production of 
carbohydrates and in some cases reduces it significantly 
(Murata, 1969). Nitrogen requires some metabolites of 
Krebs cycle to stabilize amino acids. The continuation 
of this cycle involves the use of carbohydrates and their 
derivatives. The reduction of nitrite and nitrate also re-
quires a reducing power derived from photosynthesis 
and respiration. If this power is provided through res-
piration, it will reduce plant’s carbohydrates and, if it is 
provided through the photosynthesis, a less amount of 
CO
2
 is reduced and converted to carbohydrates (Minotti 
et al., 1969).
Based on the results, an increase in the levels of 
manure and nitrogen increased the leaf harvest index, 
while the seed harvest index decreased significantly at 30 
t ha
-1
 manure and also 100 kg N ha
-1
, compared to their 
controls (Table 4). Thus, it seems that the high levels of 
manure and nitrogen disturb the partitioning balance be-
tween leaves and seeds of hemp plant by further alloca-
tion to leaves and thus stimulated the vegetative growth 
of the plant. However, when lower levels of manure or 
nitrogen were applied, a more balanced trend was ob-
served in assimilates partitioning between the seed and 
the leaf. Maobe et al. (2010) also reported that the effect 
of nitrogen on increasing the photosynthesis rate in veg-
etative parts of the plant was effective in increasing dry 
matter accumulation and the ratio of vegetative parts to 
the plant seed and accordingly, reducing the seed harvest 
index. The existence of a negative and high correlation 
between the leaf harvest index and the seed yield index 
(r= -0.851, p < 0.01) suggests that an increase in assimi-
lates allocation to the leaf does not necessarily lead to an 
increase in biomass partitioning to the seed at the repro-
ductive stage in the hemp (Figure 2).
It seems that the addition of phosphorus fertilizer 
to the manure, up to 20 t ha
-1
, has a positive effect on 
supplying the nutrient requirements of plant, especially 
the phosphorus, and increases the seed yield. Applying 
more manure may diminish the positive effect of phos-
phorus fertilizer because the plant’s needs have already 
been achieved (Figure 1). The results of manure analy-
sis revealed high levels of phosphorus in manure in both 
years of the experiment (Table 2). Therefore, manure 
seems to be effective in supplying phosphorus needed 
by the plant. Generally, it is not necessary to use higher 
fertilizer (a combined application of 30 t ha
-1
 with 80 kg 

Acta agriculturae Slovenica, 117/2 – 2021 12
S. LALEH et al.
P ha
-1
) to increase grain yield and even cause loss of fer-
tilizer and increase the cost of fertilizer supply. Through 
the application of soluble forms of phosphorus (e.g. tri-
ple superphosphate), the P ions reacts with Ca, Fe or Al 
ions, and then may convert to an insoluble form, or ad-
sorb on clay particles in the soil. In this regard, the use of 
animal manure may be a better source to supply plant’s 
phosphorus needs. The manures can store phosphorus in 
their adsorption sites and provide plants with its soluble 
forms during growth season. Because of this, using ani-
mal manures is recommended in acidic and calcareous 
soils for optimal supply of phosphorus required by the 
plants (Abolfazli et al., 2010).
The seed mass showed a positive reaction to ap-
plication of manure, nitrogen, and phosphorus fertiliz-
ers (Table 4). Increasing seed mass is one of the effec-
tive factors in increasing the seed yield (Akongwubel et 
al., 2012). Further, a positive and significant correlation 
was observed between the seed mass and the seed yield 
(0.424, p < 0.01) in this study. In another study on isabgol 
(Plantago ovata Forsk), an increase in seed mass and seed 
yield were reported by adding organic matter (Yadav et 
al., 2002). In fact, the seed mass is determined during its 
filling period, and providing sufficient photosynthetic 
materials at this stage, reduces the competitive effect of 
seeds to get these materials, which is effective in increas-
ing the seeds mass (Reed et al., 1988).
Increasing the level of manure, especially at the lev-
els of 20 and 30 t ha
-1
, was effective in increasing the yield 
of leaf extract and hemp seed (Table 4). The highest leaf 
extract yield was obtained in the combined treatment of 
20 t ha
-1
 manure along with 100 kg ha
-1
 nitrogen (T able 5). 
The leaf and seed extract yield had the highest correlation 
with the biological (0.915, p < 0.01) and seed yield (0.771, 
p < 0.01), respectively. It seems that the leaf extract yield 
was more sensitive to adding nitrogen fertilizer than seed 
extract yield. While the application of 50 kg ha
-1
 nitrogen 
significantly increased the yield of hemp leaf extract, a 
significant increase in the yield of seed extract was ob-
tained by using 100 kg ha
-1
 nitrogen (Table 4).
By increasing the level of manure, SLA decreased 
and the yield of leaf extract increased (Figure 3). In oth-
er words, with decreasing SLA, leaf thickness increased 
(Dantas et al., 2017), and the synthesis of effective com-
pounds in the extract of hemp leaves increased by in-
creasing leaf dry mass (compared to leaf area).
In this experiment, with an increase in manure lev-
els in excess of 10 t ha
-1
, the rate of increase in seed oil 
decreased (Table 4). The highest seed and oil yield was 
obtained at 20 t ha
-1
. The use of 20 t ha
-1
 of manure (com-
pared to 10 t ha
-1
 of this fertilizer), increased grain yield 
more than reducing the percentage of seed oil, which in-
creased the yield of seed oil (Table 4).
It seems that low nitrogen levels are necessary to in-
crease the oil content and higher levels of this fertilizer, 
reduce the percentage of seed oil (Table 4). The reaction 
of the seed oil content to nitrogen was different in the 
case of application and non-application of phosphorus. 
With the application of phosphorus, the reaction of 
seed oil content decreased to nitrogen. In the absence of 
phosphorus application, consumption of 50 kg N ha
-1
 in-
creased and application of 100 kg N ha
-1
 of this fertilizer 
reduced the content of seed oil (Figure 4). The higher ni-
trogen absorption under phosphorus treatment increases 
the nitrogen content of plant (Graciano et al., 2006) and 
consequently reduces the percentage of seed oil, because 
the amount of carbohydrates needed to synthesize the 
protein is less than oil. Therefore, with the use of nitro-
gen, more carbohydrates are used to synthesize amino 
acids and proteins, and consequently the synthesis of fat-
ty acids and oils are reduced (Rathke et al., 2005). On the 
other hand, the results of this experiment indicated the 
main effect of phosphorus on reducing the oil content, al-
though no significant difference was observed (Table 4).
The high correlation coefficient of the oil yield with 
the seed yield (0.913, p < 0.01) revealed that the oil yield 
was more affected by seed yield, compared to the oil con-
tent, it had less effect. In other words, an increase in the 
seed yield, increased the oil yield. In the other studies 
also a positive and significant correlation was observed 
between the oil yield and the seed yield (Basalma, 2007; 
Marjanovic-Jeromela et al., 2008; Flajšman et al., 2019). 
It seems that with application of 100 kg ha
-1
 nitrogen, 
the seed oil content was decreased compared to control 
treatment, but a 35.12 % increase in seed yield, compen-
sated the reduction of oil percent and increased the oil 
yield (Table 4). In this experiment, the application of 
phosphorus and 100 kg ha
-1
 nitrogen was effective in in-
creasing the oil and biological yield of hemp. Based on 
the results of Rajesware Rao et al. (1989), the application 
of phosphorus and 100 kg ha
-1
 nitrogen was effective in 
increasing oil and biomass yield of the Artimisia pallens 
Wall. ex DC.. Despite the slight reduction in the percent-
age of seed oil due to the consumption of phosphorus, 
the oil yield increased because of increasing seed yield 
of the plant (Table 4). Phosphorus can increase the oil 
content of plants by increasing the photosynthesis and 
the enzyme activity (Mohammadi et al., 2013).
4 CONCLUSIONS
The results suggested that adjusting the amount of 
organic and chemical fertilizers are important, depend-
ing on the purpose of the application for planting hemp. 
The combined application of 20 t ha
-1
 of manure along 

Acta agriculturae Slovenica, 117/2 – 2021 13
Response of hemp (Cannabis sativa L.) to integrated application of chemical and manure fertilizers
with 100 kg
 
ha
-1
 of nitrogen, produced the highest bio-
logical, seed and leaf extract yield. In order to increase 
the yield of seed oil, 20 t ha
-1
 manure, as well as 100 kg 
ha
-1
 of nitrogen is suitable and the application of higher 
amounts of fertilizer will increase costs and waste of fer-
tilizer. In conclusion, given that the applied manure adds 
some nutrients to the soil that can be made available to 
the plant, it seems that hemp to respond well to nitrogen 
supplied by the combined use of chemical and animal 
fertilizers, in while its response to phosphorus fertiliza-
tion is limited. Therefore, the application of animal ma-
nure not only reduces the need for chemical fertilizers 
consumption by satisfying part of the plant demand for 
nitrogen, but also has positive effects on improving soil 
properties, and therefore combined application of ma-
nure and chemical fertilizers is strongly recommended 
for the sustainability of cannabis planting systems.
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