Use of effective micro-organisms to enhance the productivity and quality of dry biomass of the basil cultivar “Sitnolisni aromatični“

Abstract

The trials conducted during 2014 and 2015 on plots and at the seed laboratory at the Institute of Medicinal Plant Research ˝Dr Josif Pančić˝ from Belgrade, located in Pančevo, (Vojvodina, Serbia) investigated the impact of pre-planting application and foliar application (pre-planting soil treatment and foliar treatment) of the product called EM-AKTIV on morphological, productive and qualitative properties of the basil (Ocimum basilicum L.) cultivar ˝Sitnolisni aromatični˝, which is grown and propagated at the Institute. The plots on which the product was not applied were used as a control variant. The trail was set up in a complete randomised block design with four replications and the size of basic plots was 3.25 m2 (5.0 m x 0.65 m). The research investigated the impact of the EM-AKTIV on plant height, root length, plant width, the number of inflorescences, above-ground fresh biomass yield, dry biomass yield, essential oil content and essential oil yield per hectare. The highest yield of dry biomass was obtained in the first year in the soil treatment variant (3,134 kg ha-1). The same variant in the second year of research gave the highest content of essential oil (25.9 kg ha-1). These years were quite different in terms of climatic conditions, and precipitation sums, distribution and average daily temperatures in the first year favoured the formation of higher values for most of the investigated parameters.

Full text

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10.7251/AGRENG1607163
USE OF EFFECTIVE MICRO-ORGANISMS TO ENHANCE THE PRODUCTIVITY
AND QUALITY OF DRY BIOMASS OF THE BASIL CULTIVAR “SITNOLISNI
AROMATIČNI“
Vladimir FILIPOVIĆ1, Gorica CVIJANOVIĆ2, Vladan UGRENOVIĆ3, Milica AĆIMOVIĆ4,
Vera POPOVIĆ5, Dragoja RADANOVIĆ1, Slađan STANKOVIĆ6*
1Institute for Medicinal Plant Research ˝Dr Josif Pančić˝, Tadeuša Košćuška 1, 11000 Belgrade, Serbia
2Megatrend University in Belgrade, Faculty of Bio-farming, Maršala Tita 39, 24300 Bačka Topola, Serbia
3Agricultural Advisory Service - ˝Tamiš˝ Institute, Novoseljanski put 33, 26000 Pančevo, Serbia
4Institute of Food Technologies, Bulevar cara Lazara 1, 21000 Novi Sad, Serbia
5Institute of Crop and Vegetable Production, Maksima Gorkog 30, 21000 Novi Sad, Serbia
6Institute of Science Application in Agriculture, Despota Stefana 68b, 11000 Belgrade, Serbia
*Corresponding author: sstankovic@ipn.bg.ac.rs
Abstract
The trials conducted during 2014 and 2015 on plots and at the seed laboratory at the Institute of
Medicinal Plant Research ˝Dr Josif Pančić˝ from Belgrade, located in Pančevo, (Vojvodina,
Serbia) investigated the impact of pre-planting application and foliar application (pre-planting
soil treatment and foliar treatment) of the product called EM-AKTIV on morphological,
productive and qualitative properties of the basil (Ocimum basilicum L.) cultivar ˝Sitnolisni
aromatični˝, which is grown and propagated at the Institute. The plots on which the product was
not applied were used as a control variant. The trail was set up in a complete randomised block
design with four replications and the size of basic plots was 3.25 m2 (5.0 m x 0.65 m). The
research investigated the impact of the EM-AKTIV on plant height, root length, plant width, the
number of inflorescences, above-ground fresh biomass yield, dry biomass yield, essential oil
content and essential oil yield per hectare. The highest yield of dry biomass was obtained in the
first year in the soil treatment variant (3,134 kg ha-1). The same variant in the second year of
research gave the highest content of essential oil (25.9 kg ha-1). These years were quite different
in terms of climatic conditions, and precipitation sums, distribution and average daily
temperatures in the first year favoured the formation of higher values for most of the investigated
parameters.
Key words: basil, above-ground biomass, plant height, yield, essential oil content, EM-AKTIV
product
Introduction
Due to its aroma, basil (Ocimum basilicum L.) has long been used in this region as a medicinal,
sacred herb and a spice. Basil is grown for its above-ground parts (Basilici herba), from which
essential oil is extracted (Basilici aetheroleum). Dry matter of basil biomass contains 0.5-1.5% of
essential oil, characteristic for its pleasant and pungent smell (Stepanović and Radanović, 2011).
In our country, the most commonly grown basil cultivar is “Sitnolisni aromatični”, good as a
culinary herb and as an ornamental plant. According to its general characteristics, “Sitnolisni
aromatični” yields about 3,000 kg ha-1 of dry biomass (Anonymous, 2016). Moreover, it contains
significant amounts of good-quality essential oil suitable for use in the pharmaceutical industry.
Biomass productivity/yields depend on a series of factors. In order to achieve desired results for a
certain plant cultivar, it is necessary to meet all agro-ecological, agro-technical, edaphic,

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genotypic, seeding and other factors that affect chances of obtaining expected quantities of raw
material of satisfactory quality (Glamočlija et al., 2015).
By using effective micro-organisms in basil fields, one can achieve intensive proliferation of
micro-organisms, especially in early stages of plant growth, which in some cases leads to an
increased intake of mineral nitrogen (N) and inhibits growth of plants (Frąszczak et al., 2012).
Over the last twenty years the use of effective micro-organisms (EM) has been intensified. Higa
(2003) claims that EMs are capable of producing anti-oxidants and preventing the emergence and
growth of free radicals with oxygen. Free radicals contribute to the development of certain
diseases, whereas anti-oxidants produced by EM eliminate those diseases, combat them or
enhance the effects of oxygen activity. Microbiological products with active PGM micro-
organisms diminish negative effects of water deficit; especially in combined inoculation where
many useful micro-organisms can affect the direction and dynamics of micro-organisms and
hence maintain and improve fertility in the soil (Heidari and Golpayegani, 2011).
Aside from getting vigorous plants with high quality biomass, the role of basil reflects in the fact
that soil remains clear after harvest, with no weed but with good physical properties, which
makes basil a great preceding crop for most crop species, especially if it is included in crop
rotation on fields with organic production (Glamočlija et al., 2015).
The use of EM-based products is one more way to produce organic herbs. Hence the goal of this
paper was to determine the possible impact of the product EM-AKTIV on morphological,
productive and qualitative properties of basil biomass.
Material and methods
This research investigated the basil (Ocimum basilicum L.) cultivar ˝Sitnolisni aromatični˝ grown
and propagated at the Institute for Medicinal Plant Research ˝Dr Josif Pančić˝ in Belgrade. The
trials were carried out during 2014 and 2015 on plots and in the seed laboratory at the Institute in
Pančevo (44°52'20"N; 20°42'06"E; 74 m altitude). The trial was set up in a complete randomised
block design with four repetitions, and the size of basic plots was 3.25 m2 (5.0 m x 0.65 m). The
researchers investigated the impact of the EM-AKTIV (Effective Micro-organisms) on
morphological parameters, productivity and the quality of basil biomass and essential oil content.
The EM-AKTIV is a product containing a mix of lacto-acid bacteria (Lactobacillus casei, L.
plantarum and Streptococcus lactis), photosynthetic bacteria (Rhodopseudomonas palastrus and
Rhodobacter spaeroides), yeasts (Saccharomyces carevisiae and Candida utilis), actinomycetes
(Streptomyces albus and S. griseus) and moulds (Aspergillus aryzae and Mucor hiemalis). In this
research it was used twofold. In the first variant, the soil was treated with the EM-AKTIV three
days before harvest, when the product was raked directly into the soil. In four repetitions, 0.4 dl
of the EM-AKTIV with 7 l water was applied into the soil. In the second variant, the EM-AKTIV
was used foliarly – three weeks after transplanting, when 0.13 dl of the EM-AKTIV with 7 l
water was applied. A plot without the EM-AKTIV was used as a control variant.
Meteorological data for the vegetation period of basil were retrieved from the Meteorological
station of the ˝Tamiš˝ Institute, Pančevo (Table 1).

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Table 1. Meteorological data for the vegetation period of basil, 2014 and 2015
Parameters
V
VI
VI
I
VIII
IX
X
Sum
Ave
r. T
Precipitation 2014
220.2
52.1
87.1
113.7
140.6
39.9
653.6
-
Precipitation 2015
88.2
20.1
4.8
69.1
86.4
68.3
336.9
-
Temperatures 2014
16.4
21.5
22.7
22.4
18.1
13.6
-
19.1
Temperatures 2015
18.5
23.3
27.5
25.5
20.9
10.7
-
21.1
Precipitation 1994-
2013
61.4
81.7
61.8
49.6
65.2
49.7
369.4
-
Temperatures 1994-2013
18.6
21.9
23.8
23.5
18.0
12.6
-
19.7
Basil seedlings were produced in a plastic greenhouse. The production was standard, like
production of vegetable seedlings. Seeds for future seedlings were sawn in late February. Three
months later, in mid-May, the seedlings were transplanted. At that time the seedlings had 3-4
pairs of leaves and were 10-12 cm high, being at the stage when seedlings have the best chances
to grow after transplantation. The marigold (Calendula officinalis L.), a medicinal plant species,
was a preceding crop on the trial plots. Planting was done manually, on May 8 in the first year,
and on May 18 in the second year. Row spacing was 50 cm, and crop spacing 30 cm. The first
watering was carried out the day before planting, and the second watering right after planting,
whereas other watering was done when needed. A watering norm in each of four watering was 25
mm/m2.
Standard crop tending measures were applied during the period of vegetation. Weeds were
eliminated mechanically, without using pesticides. During vegetation, no disease or pest
infestations were recorded, and therefore there was no need for protective measures.
The first harvest of green biomass was conducted at full bloom, in late July. In that stage, basil
contains most of essential oil and green biomass yields are highest. The second harvest was
conducted in early October. Prior to harvest, a sample of five plants from each plot was taken to
measure plant height, root length, plant width and the number of inflorescences. Harvest was
carried out manually, with a sickle, at the cut height of 8 cm, in nice and sunny weather. After
harvest, weight of fresh biomass was measured, and yield per hectare was calculated and
expressed in kilograms. The green biomass was air-dried in a draught place, after which dry
biomass yield was measured and samples were taken for determining the content of essential oil.
Essential oil was extracted in the process of hydro distillation of dry material by using a
Clevenger apparatus, following the procedure described in Ph. Jug. IV (Ph. Jug. IV, 1984). 20 g
of the fragmented plant material was hydro distilled in a balloon of 1000 ml with 400 ml of
water. The mass was then heated to the point of boiling and such distilled for two and a half
hours. After distillation, the volume (ml) of the essential oil was detected and the content of the
essential oil (in %) in 100g of dry biomass (v/w) was calculated according to the following
formula:
C = (b : a) x 100
Where: a – measured biomass quantity (in g); b – essential oil content (in ml); C – content
(in %) of essential oil in 100 g of biomass. Results of determining the content of essential oil in
basil biomass are the mean value of three continuous determinations. The yield of essential oil
per hectare was determined by calculating the percentage of oil in biomass weight per hectare and
expressing it in kilograms.

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Testing the significance of differences between the mean values of the investigated factor
(use of the EM-AKTIV) was conducted by using the model of the analysis of variance, in the
following mathematical form:

ijk
ij
jkiijk
y


(i=1,2,3;...,r; j=1,2,...s; k=1,2,3,4)
All the assessments of significance were based on the F-test and LSD-test at the level of 5%
and 1%. The analysis of variance (ANOVA, in Randomized Blocks) was done with Statistica for
Windows 10 (STATISTICA, 2010). The same software was used to calculate the coefficient of
variation (Cv).
Results and discussion
High variation in morphological and productive parameters of the basil cultivar “Sitnolisni
aromatični“ is the result of a long dry period recorded in the second year (Table 1, 2 and 3).
Table 2. Morphological parameters of the basil cultivar ˝Sitnolisni aromatični˝
Years
Treatment
Plant height
(cm)
Root length
(cm)
Plant width
(cm)
Number of
inflorescences
2014
Control (no product used)
47.3
36.7
26.1
92.3
Soil treatment
54.2
40.2
33.2
106.7
Foliar treatment
52.9
41.3
31.0
102.0
2015
Control (no product used)
37.7
28.9
23.7
76.7
Soil treatment
44.4
31.5
24.6
85.6
Foliar treatment
43.8
32.4
23.9
79.7
2014
Average
51.5
39.4
30.1
100.3
2015
Average
42.0
30.9
24.1
80.7
Total average
46.7
35.2
27.1
90.5
The coefficient of variation
13.2
14.3
14.9
13.6
LSD
0.05
22.2
11.7
15.4
30.5
0.01
29.7
16.4
20.6
40.2
Plant height is an important property both from the agro-technical and the production aspect.
Knowing this parameter allows us to define and assess more precisely the habitus of basil. In this
research, the highest values for plant height, in both years of research, were obtained in the
variant treated with the EM-AKTIV, 54.2 cm on average (in 2014), which was statistically much
higher than values obtained in the control variant, i.e. the variant with lowest height (37.7 cm, in
2015). In the research of Labra et al. (2004), the height of tiny-leaf forms of basil ranged from 30
cm to 35 cm, and the height of large-leaf forms ranged from 40 cm to 45 cm. Analysing the
results obtained for root length, one can see that the use of the EM-AKTIV (in both variants)
increases root length, whereby the recorded differences were not statistically significant. Plant
width and plant height together define plant habitus. In other words, by comparing plant height
and plant width one can determine plant habitus. Plant width in this research ranged from 23.7
cm (control in 2014) 33.2 cm (soil treatment variant in 2015). Eckelmann (2002) claims that basil
height and width mostly depends on environmental conditions, whereas its shape, leaf area and
tips are genetically determined and represent identifying properties of cultivars. This opinion is
backed by Polish researchers (Nurzyńska-Wierdak and Borowski, 2011), who treated basil with
foliar fertilisers. Basil develops dichasial inflorescences of different length. The number of
inflorescences in this research ranged from 106.7 (recorded in the soil treatment variant in 2014),

1089
to 76.7 (in the control variant in 2015). Basil is morphologically diverse in terms of size, shape,
colour, leaves and inflorescence (Szabó et al., 1996). Statistically significant discrepancies in
certain properties of basil were recorded in the research of Rahimi et al. (Rahimi et al., 2013),
when different types of bio-stimulators and bio-fertilisers were used.
In the second year of the research more dry weight was recorded, which can be seen from the
fresh-dry matter ratio that averaged 4.1 : 1. Unfortunately, low humidity in 2015 resulted in
31.5% lower dry biomass yield (Table 1 and 3).
Table 3. Yield, content and yield of essential oil from dry biomass of the basil cultivar ˝Sitnolisni
aromatični˝
Years
Treatment
Yield of fresh
biomass
(kg ha-1)
Yield of dry
weight biomass
(kg ha-1)
Essential oil
content
(%)
Essential oil
yield
(kg ha-1)
2014
Control (no product used)
13,728.0
2,965
0.74
21.8
Soil treatment
12,880.7
3,134
0.75
23.6
Foliar treatment
12,831.0
2,820
0.66
18.5
2015
Control (no product used)
6,896.1
1,810
1.05
19.0
Soil treatment
9,635.3
2,333
1.11
25.9
Foliar treatment
8,560.8
1,968
1.13
22.2
2014
Average
13,146.6
2,973
0.71
21.3
2015
Average
8,364.1
2,037
1.10
22.4
Total average
10,755.3
2,505
0.91
21.8
The coefficient of variation
25.8
21.9
23.6
12.8
LSD
0.05
4,638.3
1,374.5
0.089
4.21
0.01
6,388.8
1,687.2
0.128
5.72
The highest yield of fresh biomass (13,728.0 kg ha-1) was recorded for the control variant (the
variant without the EM-AKTIV) achieved in the first year of research. It was twice as higher
value than the minimum value recorded for the same variant but a year later (6,896.1 kg ha-1).
Previous experience has shown that the maximum weight of biomass is achieved in the period
from the end of bloom to the beginning of seed maturity. Depending of agro-ecological
conditions, that period can last from 150 to 180 days after planting (Putievsky and Galambosi,
1999). After drying, the highest biomass yield was recorded in the soil treatment variant (3,134
kg ha-1) in the first year of research, and the lowest yield in the control variant in the second year
of research (1,810 kg ha-1). The average yield of dry biomass in this research was 34.0% lower
than in the research of Adamović (Adamović, 2012), where it was 3,800 kg ha-1 in early harvest,
and 2,500 kg ha-1 in late harvest – what was in line with our research. The content of essential oil
in dry biomass averaged 0.91%, whereby the coefficient of variation was 23.6%. The highest
content of essential oil was achieved in the second year in the variant with the foliar EM-AKTIV
treatment (1.13%). The same variant in the first year of research gave the lowest content of
essential oil (0.66%). In general, the content of essential oil in dry biomass was significantly
higher than in some of the latest national research (Bulut, 2012; Filip, 2014). Essential oil yield
had the most uneven values of all investigated parameters. For that situation a reason can be
found in climatic factors, which are in correlation between yield and essential oil content. The
highest content of essential oil was obtained in the soil treatment variant in the second year (25.9
kg ha-1), and the lowest in the foliar treatment variant in the first year of research (18.5 kg ha-1).
Plant development stages affect the yield of oil to a great extent. A study conducted in the late

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20th century showed that the maximum concentration of oil was achieved at full bloom
(Putievsky and Galambosi, 1999), which was also conducted in this research.
Conclusions
The goal of this research was to determine whether there is an impact of the EM-AKTIV product
on morphological and yield parameters, and on the content of essential oil of the basil cultivar
“Sitnolisni aromatični”. When compared the years and all morphological parameters, yield of
fresh biomass and yield of dry biomass, one can see a statistically significant differences in 2014
and 2015. Precipitation sums and average daily temperatures during the first year were
favourable for having higher values for the investigated parameters. Agro-ecological indicators in
the second year were more favourable for the content of essential oil and its higher yields. When
observed both years of the research, the soil treatment variant with the EM-AKTIV obtained the
highest values for plant height, root length, plant width, the number of inflorescences, yield of
green biomass and yield of dry biomass. However, the highest values for root length (in 2014 and
2015) and the content of essential oil in 2015 were recorded in the foliar variant.
Acknowledgement
This paper presents part of the results obtained within Project III 46006, funded by the
Ministry of Education, Science and Technological Development of the Republic of Serbia.
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