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* Corresponding author: swamprat@rambler.ru
Exogenous regulation of biological productivity of fenugreek
Olga M. Savchenko* and Firdaus M. Khazieva
All-Russian Scientific Research Institute of Medicinal and Aromatic Plants, 117216 Moscow, Russia
Abstract. In the article the results of the effect on Trigonella foenum-graecum L. productivity by organic
fertilizers EcoFus and Siliplant and presowing inoculation of seeds with the Rizotorphyn are presented. We
investigated effect of organic-mineral fertilizers “EcoFus” and “Siliplant” and presowing inoculation by
“Rhizotorphyn”. ”EcoFus” and “Siliplant” increase herb yield by 24.8 and 18.8 % compare to control.
“Rhizotorphyn” increasing yield of seeds production by 32.1 % compared to control. For the first time we
present the data of the study devoted to the effect of foliar treatments with micronutrients and presowing
seed inoculation on the quantitative content of flavonoids in fenugreek seeds. The highest flavonoid content
was found in seeds subjected to foliar treatments with a binary mixture of “EcoFus + Siliplant” (1.59 %).
Work is performed under the topic of the research project No. 0576-2019-0007.
1 Introduction
Fenugreek (Trigonella foenum-graecum L.) is an annual
herbaceous plant, well known in culture since ancient
times as valuable forage and medicinal plants.
The fenugreek (Trigonella) genus of the legume
family (Fabaceae) consists of 129 species in the world
flora, of which 44 are perennial and 85 are annual. On
the territory of the Russian Federation grow about 50
species of fenugreek (perennial and annual) [1].
Fenugreek grows naturally in Western Asia, Turkey,
Iraq and Iran, spreading to the Himalayas.
It is used for green fodder, hay and green fertilizer.
Seeds are used for concentrates, as well as in folk
medicine, veterinary medicine and technology. In its
pure form fenugreek is not recommended for use. Young
shoots are used as a salad, seeds - for cereals, flour. In
the southern regions of Russia it is cultivated in busy
pairs (for early feed, for green fertilizer) and as stubble
[1].
It positively responds to moisture and fertilizers. To
soils it is not demanding, unsuitable only to heavy,
floating, acidic, waterlogged soil. Many of its ecotypes
and geographical forms are not affected by diseases and
pests [1].
Fenugreek is a drought-resistant plant, characterized
as plastic to growing conditions. Therefore, some of its
forms and biotypes can be cultivated up to 60° North
latitude. Shoots appear in 7–8 days after germination of
seeds, with a lack of moisture in the soil – in 15–18 days.
The plant has a tap root. Stem erect, slightly branched,
round, hairy, green, length 25–60 cm. Leaves are
trifoliate and short. Leaflets are back-ovate or broadly
lanceolate, toothed, glabrous, 2-4 cm long. The corolla is
almost twice as long as the calyx. The fruit is a bean,
somewhat curved, glabrous, 6–16 cm long, brown after
ripening. Seeds are greenish and yellowish, 4–6 mm
long. Weight of 1000 seeds is 14–16 g. The whole plant
has an intense odor. [1, 2].
Mature fenugreek seeds contain up to 1.34 % of the
amount of steroid saponins (diosgenin, tigonin,
yamogenin) and they are used in official medicine in 8
countries [1–3].
Trigonella foenum-graecum L. is well known due to
pharmacology advantages: antidiabetic, antioxidant,
hypocholesterolemic, anti-tumor, anti-inflammatory,
anti-carcinogenic, antipyretic, immunomodulating and
anti-tumor. Fenugreek has a tonic effect [4–6].
Polyphenolic flavonoids, which are characterized by
hypoglycemic, hypocholesterolemic, hypotriglyceridemic
and antiperoxidative properties [7], steroidal saponins
exhibiting anti-inflammatory action on the uterus and
lactation-stimulating properties [8, 9], such as
anisanabarides, were identified and isolated in
Trigonella foenum-graecum L seeds [8, 9] have
antidiabetic effects [10], the amino acid 4-
hydroxyisoleucine has insulin-mimetic properties [11].
Fenugreek was conducted at the all-Union research
Institute of medicinal and aromatic plants (VILAR,
Moscow region), in the Crimean, North Caucasus
(preserved branch VILAR) and Ukrainian, the
Przheval'skaya zonal experimental stations of VILAR.
All these data confirm the need to continue the study
of fenugreek as a source of raw materials for the
production of diosgenin. In VILAR has been initiated to
restore fenugreek as promising medicinal crops.
In 1980-s medicine “Pasenin” being analog
“Polisponin” and “Tribusponin” was developed in
Russian State Medicinal and Aromatic Plant research
Institute. The “Pasenin” was developed from Trigonella
foenum-graecum L. seeds. [12].
Fenugreek hay can be used as a forage plant (due to
the presence of protein green mass and hay have high
© The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0
(http://creativecommons.org/licenses/by/4.0/).
BIO Web of Conferences 17, 00193 (2020) https://doi.org/10.1051/bioconf/20201700193
FIES 2019
nutritional properties), and is a good precursor for all
vegetable plants.
Research purpose – the studies of non-root
treatments effect of micro-fertilizers and presowing
inoculation of seeds on yield and flavonoid content in
Trigonella foenum-graecum L. seeds.
2 Materials and methods
2.1 Characteristics of the place of the
experiment
The studies were conducted in 2017–2019 on the
experimental field of the Department of Agrobiology
and Breeding according to the methods adopted for
experiments with medicinal plants [13].
The soil of the site is heavy loamy: humus (by
Tyurin) - 2.23 %, the mass fraction of nitrogen nitrates -
<2.80 million-1, the mass fraction of phosphorus
compounds (by Kirsanov) P2O5-386.28 million-1, the
amount of absorbed bases (by Cappen) - 6.3 mmol /
100g, salt pH-5.15.
Allocation of plots was randomized. Repeat 4-fold,
the square of the each experimental plot was 24 m².
2.2 Characteristics of weather conditions during
the experiment
Unstable weather conditions have a negative impact on
yields and contribute to the deterioration of the quality of
the products. Under these conditions, there is a decrease
in plant resistance to stress factors and the inability to
obtain the potential productivity of cultivated varieties
and populations.
The temperature in the growing seasons of
2017–2019 varied within 3–5 ºC. In 2017, the
temperature during the growing season was on average
below the norm by 1.3–3.4 C.
In 2018, during the flowering phase and the
beginning of fruiting, the air temperature was relatively
low and amounted to 16.2–17.3 °C. Precipitation fell in
the amount of 60 mm. in the phase of seed ripening
(July), the temperature increased (to 20. 5°C),
precipitation fell 93 mm. Weather conditions in 2019 at
the beginning of seed ripening deviated from the norm
by –2.4° [13].
2.3 Micronutrients and inoculants
In order to increase productivity, we used a binary
mixture of organic fertilizer “EcoFus” (5 ml/L) and
“Siliplant” microfertilizers (1 ml/L), which have proven
themselves in medicinal plants [15, 16].
“Siliplant” is a microfertilizer with a high content of
silicium (6–7 %) in an accessible form for plants.
Silicium, which is a part of “Siliplant”, activates
synthesis of auxins necessary for root system growth.
The solution was applied at a concentration of 1 ml/L.
[15, 16].
“EcoFus” – organic-mineral fertilizer based on fucus
algae, contains physiologically active substances
possessing immunostimulating, antiviral, antibacterial
and fungicidal action. The solution was applied at a
concentration of 5 ml/L. Consumption 300–400 l/hectare.
The inoculation of seeds was carried out with the
biological product “Rhizotorphyn” (200 g/hectare).
“Rhizotorphyn” (strains of nodule bacteria of the genera:
Bradyrhizobium, Rhizobium, Sinorhizobium,
Mesorhizobium), highly concentrated inoculants with
increased biological efficiency based on gamma sterile
peat. 1 g contains at least 2.5 billion Rhizobia. Inoculants
have increased nodulation ability of the root system of
legumes. As a result of the using of the inoculant,
nodulation form on the roots which fix molecular
nitrogen (N2) from the air and convert it into NH4 + form
which accessible for plants. This process allows reducing
or abandoning mineral fertilizers introduced into the soil
without reducing yield, increases the yield of plants and
their resistance to adverse environmental factors [17–19].
The biological product was developed by the Russian
State Research Institute of Agricultural Microbiology.
2.4 Quantitative analysis of flavonoids
Quantitative analysis of flavonoids in the seeds of
fenugreek we have developed methodology based on
differential spectrophotometry on a LAMBDA-950
spectrophotometer using a technique for determining
flavonoids in combination with Aluminum chloride in
the range of 385–460 nm. For quantitation of flavonoids
in raw we have developed methodology based on
bacteria’s ability to form colored complex of C2H5OH
and AlCl3 what causes a bathochromic shift of the long-
wavelength absorption band and at the same time gives
the main absorption maximum at a wavelength of 393 ±
2 nm [20–22].
The extract was left to soak for 24 hours and then the
extraction was carried out in a boiling water bath for 30
minutes twice. For develop methods for the quantitative
determination of the sum of flavonoids, a reaction with
an alcoholic solution of Aluminum chloride was used.
In order to develop the manual we determinate
optimal conditions for extractions: ethanol 70 %, as
extracting. Ratio raw / extracting 1:50. Sum of
flavonoids measured in conversion with Apigenin using
standard solution of Apigenin CAS№520-36-5 in 95 %
ethanol.
The degree of flavonoid content (%) was calculated
by formula:
(1)
Х – flavonoid content conversioned to Apigenin
А – optic density of test solution
А0 – optic density of standard solution ;
m – raw mass, gramm
m0 – mass standard solution ruthin, gramm;
– mass loss on drying of raw materials
Concentrations of Apigenin and Aluminium chloride
from 0,0002 to 0,0020 % of are match to meanings of
optic density from 0,1 to 1,0 %.
This dependence is described by linear equation:
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BIO Web of Conferences 17, 00193 (2020) https://doi.org/10.1051/bioconf/20201700193
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(2)
Mean of coefficient of absorption of Apigenin and
Aluminium chloride complex is 491 with relative error 3 %.
3 Results and discussions
In 2017–2019 years sowing of Trigonella foenum-
graecum L was carried out in the first ten days of May in
a wide-row method (row spacing 60 cm). The sowing
rate is 6 kg/ha, the seed placement depth is 3 cm. The
predecessors are black steam. After 7–10 days, seedlings
appeared. The field germination of seeds in all variants
was 94 % after 20–30 days from the plants began to
branch. In the last decade of June, mass budding of
plants was observed. In the phase of the onset of seed
ripening, the height of the plants had been 39-43 cm was
not increased further.
The length of the growing season of fenugreek plants
after treatment by micronutrients was lengthened. From
germination to full maturation of seeds in the control
variant was 74 days. The time from germination to
harvesting of fenugreek plants after treatment by
micronutrients was 77–80 days. The time from
germination to harvesting in control variant was 85–95
days. The main phenological phases took of the
fenugreek plants, after the influence of non-root
treatments with a binary mixture of “EcoFus + Siliplant”
take the place on 2–4 days earlier than the plants from
the control site.
Foliar treatments and seed inoculation also had a
noticeable effect on the intensity of the color of
fenugreek leaves and stems. Plants in the variant
“EcoFus + Siliplant” were characterized by intensive
growth, dark green color of leaves (Fig. 1).
Fig. 1. Plant of fenugreek variants of foliar treatment by:
1 – Presowing inoculation of fenugreek by “Rhizotorphyn”;
2 – Foliar treatment by a mixture “EcoFus + Siliplant”;
3 – Control
As it is shown in figure 1, seed inoculation
significantly stimulates the development of the symbiotic
apparatus. The plants of fenugreek have a more
branched root system, therefore, get more nutrition.
Foliar treatment by a mixture of organic fertilizer
“EcoFus” with microfertilizer “Siliplant” increased plant
growth. In 20 days the height of the plants exceeded the
control by 7.0 %, harvesting (third decade of August) by
4.3 % (Table 1). “Rhizotorphyn” increased plant height
by 7.8 % in budding phase compare to control and by
4.8 % in harvesting. If the purpose of production is the
cultivation of fenugreek for green food, the plants are
suitable for harvesting after treatment by “EcoFus +
Siliplant”.
Table 1. Effect of complex organic-mineral fertilizers and macrosimbionts of Trigonella foenum-graecum L. plants 2017–2019
Treatments
Height, cm (20 days
after treatment)
Height, cm
(Harvesting)
Yield productivity
(air dry mass) kg/hectare
Number of shoots per
1 plant, pcs
EcoFus + Siliplant
42.3±3.6 (107.0 %)
43.0±4.1 (104.3 %)
3120.2±278.3 (124.8%)
5.0±0.7 (131.5 %)
Rhizotorphyn
42.6±3.8 (107.8 %)
43.2±4.2 (104.8 %)
2970.4±185.6 (118.8 %)
4.5±0.5 (118.4 %)
Control
39.5±3.0
41.2±3.5
2500.6±127.9*
3.8±0.2*
*Values are significantly different at P ≤ 0.05.
Increasing of growing on the “EcoFus + Siliplant”
and “Rhizotorphyn” types of experiment contributed to
an increase in grass yield (air-dry mass) by 24.8 and
18.8 %, respectively, compared to the control.
Under the influence of organic-mineral fertilizers and
macrosimbionts, not only the height of fenugreek plants
increased, but also their bushiness by 18.4–31.5 %. This
increased the yield of the above-ground part of the
plants, and also led to increase the number of beans on
the plants (Table 2).
Presowing inoculation of “Rhizotorphyn” increased
yield productivity and quality of seeds by 32.2 %. Non-
root treatment with a binary mixture of “EcoFus +
Siliplant” (5 ml/l + 1 ml/l) increases the seed yield by
13.7 % compared to control (Table 2).
In the analysis of table 2 a significant increase by
18.4–21.7 % weight of 1000 seeds after application of
foliar treatments “EcoFus” + ”Siliplant” and inoculation
with “Rhizotorphyn” it is shown. After the presowing
inoculation of fenugreek seeds with “Rhizotorphyn”, there
is a significant (at P<0.05) increase (22.3 %) compared
with control the weight of the seeds from 1 bean.
The use of a binary mixture “EcoFus + Siliplant”
increased the number of beans on the plant, but the
weight and quality of seeds was influenced by pre-
sowing inoculation with “Rhizotorphyn”.
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Table 2. Influence of complex application of organo-mineral fertilizers and macro-symbionts of legumes on the productivity of
fenugreek, 2017–2019
Treatments
Seed yield
1000seed mass 1000,
gramm
The amount of beans
per plant, pcs
Amount seeds in bean,
pcs.
Seed mass, kg/hectare
Control
871.0±84.22
18.4±1.92
6±0.6
6.7±0.84
EcoFus + Siliplant
996.5±91.81 (113.7 %)
22.4±2.44 (121.7 %)
16±1.7
7.3±1.15 (108.9 %)
Rhizotorphyn
1150.3± 112.54 (132.2 %)
21.8±2.23 (118.4 %)
13±1.2
8.2±1.43 (122.3 %)*
*Values are significantly different at P ≤ 0.05.
A comparative study of productivity fenugreek hay
revealed the dependence of these vegetation processes
on the meteorological conditions of the growing season.
Thus, under cold stress (2017), fenugreek had a decrease
in plant growth, bushiness and yield compared to
optimal weather conditions of 2018–2019 years (Fig. 2).
Fig. 2. Influence of foliar treatments and presowing inoculation
on fenugreek crop elements depending on the conditions of the
year of observation
Processing binary mixture “EcoFus + Siliplant”
increased the number of beans per plant. In this case, the
growing season increased to 95–110 days, the seeds did
not have time for full mature. In contrast, the inoculation
by “Rhizotorphyn” has proven itself in cold and wet
weather conditions of 2017 and allowed obtaining high
yields of seeds (Fig. 2).
It should be noted, that under adverse weather
conditions in 2017, harvesting was carried out 15–20
days later than under optimal conditions for fenugreek
hay. “Rhizotorphyn” under hydrothermal stress (drought
conditions 2018 year) contributed to the improvement of
adaptive capabilities of fenugreek to adverse weather
conditions and reduced crop losses.
In the variant “EcoFus + Siliplant” was a secondary
flowering of plants on the background of the warm
period in August of 2018 (the average temperature was
19, 8° C). In this variant, uneven ripening of beans and
seeds was noted.
The highest content of flavonoids was found in seeds
witch had been treated by “EcoFus + Siliplant” (1.59 %).
“Rhizotorphyn” increased flavonoid content by 1.09 %.
Presowing inoculation by “Rhizotorphyn” has not effect
on flavonoid content. Non root treatment by “EcoFus +
Siliplant” increased flavonoid content by 45.8 %
(Table 3).
Presowing inoculation of Trigonella foenum-
graecum L. seeds by the Rhizotorphyn have not much
effect on the accumulation of flavonoids. Rizotorphyn
can be used to increase seed yield and aerial mass.
Table 3. Content of flavonoids in terms of Apigenin in fenugreek seeds
Treatments
рН
Optimal complexation time, min
λ max
Flavanoid content, %
Ffact
F
EcoFus + Siliplant
2.8
50–55
392.68
1.59* ± 0.03
6.025
5.14
Rhizotorphyn
3.05
55–60
393.61
1.09 ± 0.03
Control
4,03
55–60
393.77
1.06 ± 0.03
*Values are significantly different at P ≤ 0.05.
4 Conclusion
Non root treatment binary mixture “EcoFus + Siliplant”
increased grass yield (dry mass) by 24.8 % compare to
control. This treatment also increases the number of
beans on fenugreek plants.
Presowing inoculation increased yield of seeds by
32.2 %. After the presowing inoculation of fenugreek
seeds with “Rhizotorphyn”, there is a significant increase
on 22.3 % compared with control the weight of the seeds
from 1 bean.
Non root treatment by binary mixture “EcoFus +
Siliplant” have effect on flavonoid content in Trigonella
foenum-graecum L. seeds by 45.8 %.
The highest flavonoid content was found in seeds
subjected to foliar treatments with a binary mixture of
“EcoFus + Siliplant” (1.59 %). Seeds collected from the
samples treatment by “Rhizotorphyn” slightly exceed the
control variant in terms of flavonoid content (1.09 %).
Acknowledgments
The authors are grateful to Andrei Petrovich
Kozhemyakov, Ph.D., the head of the Ecology
Laboratory of Symbiotic and Associative Rhizobacteria
of the Russian State Scientific Research Institute of
Agricultural Microbiology for the provided Rizotorfin
biological product.
The authors express special gratitude to Irina
Evgenievna Stanishevskaya (Deputy Director for
Academic Affairs, RUDN University) for phytochemical
studies of Trigonella foenum-graecum L. seeds.
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BIO Web of Conferences 17, 00193 (2020) https://doi.org/10.1051/bioconf/20201700193
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The authors have no conflict of interest.
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