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A
BSTRACT
In 2020-2023, a study was conducted to study the distribution and biological diversity of steinernematid
and heterorabtid entomopathogenic nematodes (EPN) in three regions of Uzbekistan.
Steinernematids are more common than heterorhabditids, and S. feltiae is the most common species.
Sampling sites were grouped into 4 habitats: barren land, gardens, fields, residential homesteads; the soil
composition of each plot was determined and the influence of the habitat and soil composition of each
species was evaluated.
K
EYWORDS
4 habitats: barren land, gardens, fields, residential homesteads; the soil composition.
I
NTRODUCTION
Pest control of agricultural crops is one of the
global problems of today's food production. In
this regard, it is important to develop
countermeasures by protecting the ecological
environment and soil health. The use of
entomopathogenic - beneficial nematodes as
biological control measures is used in world
experience and is highly effective [15].
Beneficial nematodes occur naturally in the soil
and are used to control insect pests and their
larvae. Beneficial nematodes enter and feed on
their hosts through the mouth, anus, or
Journal
Website:
http://sciencebring.co
m/index.php/ijasr
Copyright:
Original
content from this work
may be used under the
terms of the creative
commons
attributes
4.0 licence.
Research Article
FIRST REPORT ON LOCAL ENTOMOPATHOGENIC NEMATODE
STEINERNEMA FELTIAE IN UZBEKISTAN
Submission Date:
July 20, 2023,
Accepted Date:
July 25, 2023,
Published Date:
July 30, 2023
Crossref doi:
https://doi.org/10.37547/ijasr-03-07-38
Nafosat Kurbonova Sattor Kizi
Research Scientific Institute Of Plant Protection And Quarantine, Uzbekistan
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respiratory openings. This leads to the emergence
of specific bacteria from the intestinal tract of the
nematode [13]. These bacteria leave the div
cavity of nematodes, spread inside insects and
multiply very quickly. The bacteria convert the
insect tissue into food that is easily absorbed by
the nematodes, and the insects die within a few
days.
Beneficial nematodes can be used for biological
control of more than 200 insect pests belonging
to 100 genera that live in the soil and pass the
larval stage in the soil [3].
They are distributed globally, but the biodiversity
and distribution of their species in some
countries and regions are still unexplored.
In particular, until now, no specific scientific
information has been provided about scientific
research related to entomopathogenic nematodes
in Uzbekistan. This shows that the study of these
beneficial organisms and the development of
technologies for their use as objects of biological
control are relevant.
The purpose of this research is to identify local
entomopathogenic nematodes distributed in the
territory of Uzbekistan, to analyze them from a
molecular and genetic point of view, and to
publish preliminary information about them.
Entomopathogenic nematodes (EPN) range in
size from 0.3 to 10 mm. In Greek dictionaries, the
term entomopathogenic nematodes is derived
from the words "entomos", "pathe", "guenos" -
meaning a group of nematodes capable of causing
disease in insects.
They belong to two families, Steinernematidae,
i.e. Steinernema (100 valid species) and
Heterorhabditidae,
i.e.
Heterorhabditis,
comprising 16 well-characterized species
globally (Kaya & Stock, 1997).
MATERIALS AND METHODS
Soil samples were collected from the southern
part of Uzbekistan in 2020 and 2023. Sampling
was carried out in spring and autumn from
different fields. A total of 1200 soil samples were
taken. Soil was collected from the upper 10 - 30
cm layer and a total of 1 kg of soil was sampled
from five parts of each plot.
The samples were stored in polyethylene bags in
the refrigerator until extraction. Samples were
processed within two days of collection [2]. Each
sample was thoroughly mixed and small samples
were taken from each soil sample for soil type, pH
analyses.
Using wax moth larvae (Galleria mellonela) using
the "bait" method developed by Kaya and Stock
(1997), beneficial nematodes in the soil were
collected in special plastic containers with 5-10
larvae per soil sample and stored in the dark at
room temperature (20-22 ° C) was stored for 14
days [12].
At two-day intervals, dead insects from the
samples were collected and placed in a “modified
White trap” (Kaya & Stock, 1997) to collect
nematodes from the insect div. Collected
infective stage nematode larvae were stored in
special culture flasks in an isotonic solution in a
refrigerator at 4°C.
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All larvae and 50 adult nematodes obtained in the
experiment were fixed and transferred to
anhydrous glycerol. Nematodes were examined
under a special biological MBC 10 microscope,
morphological and morphometric analyzes were
performed.
Molecular genetic methods
Species were selected for molecular-genetic
research from the samples of nematodes
belonging to the genera Heterorhabditis Poinar,
1976 and Steinernema Travassos, 1927 collected
from the study area. Diatom DNA Prep kit was
used to extract genomic DNA from selected
samples [25].
DNA isolation using the Diatom DNA Prep kit
This method is one of the methods of extraction of
nucleotides using reagents or kits [1].
This kit allows for the isolation of DNA from
various natural materials, as well as the rapid
purification of DNA from clinical samples. This
method differs from the FX method by its speed
(30 min. - 1.5 time is spent on 1 sample), and the
absence of use of toxic (poisonous) reagents. The
mechanism of action is based on the use of a
lysing reagent with guanidinethiocyanate, which
leads to cell lysis, cell solubilization, and cell
nuclease denaturation. In the presence of a lysing
(decomposing) reagent, DNA is actively absorbed
in the NucleosTM sorbent kit, then it is easily
washed from proteins and salts in an alcohol
solution [27]. DNA separated from the sorbent
can be used in PCR [4].
The composition of the kit: disintegration
reagent,
saline
buffer
Nucleos
sorbent
suspension, "Extra Gen" ion exchange mixture
suspension.
The method of DNA extraction from nematode
tissues using the Diatom DNA Prep 200 reagent
kit includes the following steps [14].
1. A buffer working solution is prepared
according to the instructions.
2. A piece of tissue weighing 0.05 g is cut from the
div of the nematode in a 1.5 ml test tube (it is
better to take the head part of the nematode,
because the tail part is important for
morphological identification of the species) and
grind it with 800 μl of the disintegration reagent
and use the hand for 5-10 mixed once.
3. The mixture is placed in a thermostat with a
temperature of 65°C for 5-7 minutes.
4. The mixture in the test tube is centrifuged for
10 seconds at a speed of 5000 rpm.
5. The supernatant is taken into a clean test tube,
and the pre-homogenized Nucleos suspension is
added to it in the amount of 20-
40 μl.
6. Mix the tube with a rotator at 10
–
20 rpm or by
hand, then spin at 5000 rpm for 10 sec.
centrifuged and the supernatant removed.
7. 400 μl of dissolution reagent is added to the
precipitate and vigorously mixed with a vortex
until homogenous.
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8. Add 1 ml working solution of saline buffer to
the mixture and mix 5-10 times, 5000 rpm.
centrifuged at 10 s for 10 sec, then the
supernatant is removed.
9. Step 7 is repeated.
10. The precipitate is dried at a temperature of
65°C for 4-5 minutes.
11. "Extra Gen" suspension is poured into this test
tube in the amount of 50-
100 μl and mixed until it
reaches a homogeneous state, then it is placed in
a thermostat at a temperature of 65°С for 5
minutes and mixed again.
12. Then 10000 rpm for 1 minute. centrifuged at
high speed.
13. The supernatant is taken into a clean test tube
and stored at -20°C.
The concentration of isolated DNA is 0.12-0.17
µg/µl.
DNA extracted from the "fast" FX method must be
cleaned of protein and carbohydrate residues in
most cases. This can be cleaned using the Nucleos
suspension in the Diatom reagent kit, starting
from step 5 of the method (Figure 1).
Figure 1. DNA isolation procedure (diagram)
PCR-amplification method. In order to study the nucleotide sequence of the COI region of the chromosome,
the genomic DNA of the insects isolated for amplification was amplified using the reagents of the "Silex"
company - sterilized water, 10x PCR buffer, dNTP solution, Taq-polymerase and the following primers used
in molecular taxonomy (Table 1) .
Table 1
Primers used in Taq polymerase and molecular taxonomy
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No Primer Name
(Correct Primer)
Primer Name
(Correct Primer)
1.
FWD_533:
5
′
-
CAAGTCTTATCGGTGGATCAC 3
′
REV_534
5′
-
GCAATTCACGCCAAATAACGG-
3′
Polymerase chain reaction (PCR) was performed using a programmable autoamplifier (Touchgene
Gradient, UK). Master-mix was prepared from the following reagents based on the company's instructions
(Table 2).
Table 2
List of reactants for master mix
PCR was carried out according to the following scheme: 1 - step - denaturation of DNA at 95°C for 3 minutes,
2 - step - denaturation of DNA at 93°C for 20 seconds, 3 - step - binding of primers to DNA at 55°C for 30
seconds , step 4
–
elongation at 72°C for 2 minutes, step 5
–
chain elongation at 72°C for 10 minutes. From
the second to the fourth step, the process was repeated up to 35 times in a loop form (Table 3).
Table 3
PCR temperature and time
Water (ster.)
13.8 μl
10x PCR buffer
2 μl
dNTP
0.6 μl
From each primer
1.5+1.5 μl
Taq polymerase
0.6 μl
Total:
20 μl
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Reaction
(Program)
Stage
Temperature (°C), stage cycle
Time
I
Initial denaturation
95
3 minutes
Denaturation
93
45 cycles
20 seconds
Finish
48
30 seconds
Elongation
72
2 minutes
Finish the chain
72
10 minutes
Agarose gel electrophoresis method. After
completion of the polymerase chain reaction, gel
electrophoresis was used. This method is an
analytical method used to isolate, align, and
purify DNA fragments. DNA electrophoresis is
carried out in the horizontal direction [8].
The gel contains: 1X TAE (pN 8.1), agarose,
ethidium bromide. Preparation of agarose gel and
electrophoresis of PCR products were carried out
in the following sequence.
Before placing the gel in the electrophoresis bath,
wells were made by installing plate-mirror combs
to insert the samples. The lower teeth of the
combs were placed at a distance of 2 mm from the
base of the gel with a total volume of 50 ml
(placed at a distance of 1 mm from the base of the
gel with a total volume of 150 ml).
50 ml of 1X TAE and 1 g of agarose were added to
prepare 50 ml of 2% agarose gel [9]. A starting
concentration of 1X TAE is prepared from a 50X
TAE solution (Tris, 0.5M EDTA pH8.0, glacial
acetic acid). The mixture of TAE with agarose
placed in the flask is heated until it reaches a
homogeneous state (there should be no
undissolved particles of agarose in the solution).
After this process, it was cooled to 50°C and 0.5 μl
of ethidium bromide was added. The entire gel
volume was poured into the electrophoresis bath.
After the gel had cooled (30
–
45 min at room
temperature), the combs were slowly removed
and 1X TAE buffer was poured into the
electrophoresis bath until the gel was completely
covered. After 10-15 minutes, 2.5 µl DNA marker
DNA Ladder 100pb (Promega) was added to one
of the wells [22].
For DNA separation, the voltage should not
exceed 5 volts per centimeter of gel. After 40-45
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minutes, the gel was viewed and photographed
under ultraviolet and transilluminator light, and
the results were recorded [6] .
DNA purification. The desired fragments
resulting from electrophoresis were cut from the
gel using a scalpel and placed in a 1.5 ml
eppindorf tube. A kit of reagents produced by
Sileks M (Moscow, Russia) was used to extract
DNA from the gel, following the manufacturer's
instructions [5,8].
Sequencing - Determining the Nucleotide
Sequence of DNA - When sequencing gel-purified
PCR products, gel-purified DNA concentrations
were measured and sequenced using PCR
primers.
In order to correct the errors of the data obtained
from the sequence, the results of the sequence,
which were trained with the help of forward and
reverse primers, were converted into FASTA-
format. Then, to combine the results of two
chromatography, it was done using the program
"Clustal X version 1.81" [Swofford D.L., 1998].
"Gendoc version 2.5.000" [McCarthy; URL: www.
Chris. com.] program, unnecessary nucleotides
are removed. "ForCon version 1.0 for Windows"
[Raes J., 1999] program was used to convert to
Nexus-format [7].
R
ESULTS AND DISCUSSION
Steinernemu feltiae (Filipjev, 1934)
Diagnostics. Males: Males of this species also have
six labial and four circular cephalic papillae on the
head. The exit opening is located in front of the
nerve ring. D% - 60 (51 - 64). It has a long beaked
tail. The head part of the spicule is elongated, the
ratio of the length and width of the spicule is
about 1.5 - 2.0 nm. A spicule shaft is present,
without a beak [4,5].
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2- rasm. Steinemema feltiae
1,2 - the front and rear ends of the male div of the first generation; 3,4 - the front and back ends of the
div of the infectious larva; 5 - the lateral part of the div of the infectious larva
The length of the spicule is 70 (65 - 77) nm, the ratio of the length of the spicule to the width is 6.0 (5.8-
6.2). SW = l,l (1.0 - 1.3); GS = 0.59 (0.52
–
0.61). The gubernaculum is laterally boat-shaped, with a narrow
neck area. The stem is short, Y-shaped [23, 24]. Infectious larvae: Average div length 849 (736 - 950) nm.
EP = 62 (53 -67) m, E% = 78 (69 - 86). T = 81 (70 - 92) nm.
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Figure 2. Comparison of the nucleotide sequence of the pDNA ITS region of species belonging to
the genus Steinernema Travassos, 1927, based on the sequence material.
Izox: Steinernema feltiae and Steinernema feltiae
(accession number: MN044870) species rDNA
ITS region in the 5' to 3'-end direction, identical
nucleotide bases marked with dots.
C
ONCLUSION
As can be seen from the table above, 99.6%
nucleotide similarity with Steinernema feltiae
(accession number: MN044870) obtained from
the international bioinformatics gene database
was found, and the studied species from the
molecular genetic point of view is Steinernema
feltiae. found its proof.
There are 3 nucleotide differences between these
species, which are T-thymine instead of G-
guanine at 36 nucleotides, C-cytosine instead of
A-adenine at 457 nucleotides, and T-thymine
instead of C-cytosine at 710 nucleotides. It was
explained by the exchange of nucleotides.
These obtained data were placed in the Genbank
database of the National Center for Biotechnology
Information (Genebank, NCBI) and the accession
number (OP380866) was obtained.
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