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volume 4, issue 3, 2025
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ENTOMOPATHOGENIC BACILLUS THURINGIENSIS TO ISOLATE BACTERIAL
STRAINS AND APPLY THEM AGAINST GREENHOUSE MITES
To‘xtamishyeva Nilufar Alisher kizi
Master of Tashkent State Agrarian University
Jumanazarov G‘ayrat Xusanovich
Associate professor of Tashkent State Agrarian University
Abstract:
The article presents experimental data on methods for isolating Bacillus thuringiensis
bacterial strains from dead greenhouse aphids and soil, and on the use of different concentrations
against greenhouse aphids that damage tomato plants.
Keywords:
Bacillus thuringiensis, bacteria, strain, culture medium, tomato, pest, greenhouse
aphid, nutrient medium, entomopathogen, soil.
Introduction. The eve of the 21st century is characterized by an unprecedented scale of conscious
use of microorganisms in agricultural production. In all areas of the agro-industrial complex, the
need for the use of microorganisms and their metabolites was recognized, and, accordingly, the
development and use of biopreparations for the needs of Plant Science, Livestock, Veterinary
and feed production is being established.
There is also an increasing need for microbial preparations in the field of protecting plants and
agricultural animals from pests and parasites. As a competitor to the chemical means of
controlling insects, rodents and phytopathogens, biopesticide is becoming an attribute of
advanced agricultural systems. The need for them is associated with the size of man-made loads
on the environment, the inefficiency of chemical pesticides, the risk of toxic, allergic and even
genetic adverse effects with their widespread use and the development of resistance in pests, the
elimination of natural mechanisms for regulating their number and, finally, the pollution of
landscapes, water resources and food products.
Scientific sources know that in later times the microbiological method of combating insects is
widely used in the agrarian industry. Therefore, the production of environmentally friendly
microbial pesticide biopreparations is of great scientific theoretical and practical importance.
Research methods. The distribution of the main pests of tomato and cucumber plants in the
greenhouse was examined by taking samples diagonally or chess. In the course of the survey,
samples of pest insects that had died were collected in sterilized paper bags, which were prepared
in advance. Each sample was stored in refrigerators until it was examined in the laboratory.
In order to extract entomopathogenic fungi from pests of tomato and cucumber plants, insect
specimens that died due to the disease were cleaned of microorganisms, that is, their outer part
was sterilized. The pest samples that were being tested were soaked in a 0.5-1% sodium
hypochloride (NaOCl) solution for 30 seconds and thoroughly washed out of the solution and
rinsed once again in another container of sterilized water, then the pest samples were picked up
in front of an alcohol lamp flame on the surface of a solid food medium placed on sterilized Petri
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saucers. Each specimen was placed in a separate petri saucer with a pest sample of 10-15 pieces.
Petri was stored in thermostats with a temperature of 24-260 C. The growth of bacteia in petri
lycobchas was observed for 15 days from the third day. Then the fungal mycelium grown from
the specimens planted in the artificial food environment in Petri's saucers was planted in test
tubes with a sterilized microbiological loop in front of an alcohol lamp flame. In order to extract
disease-causing bacteria from insects, the composition was used from different food
environments (Agar Chapek, agaric beer suslosi, agaric potatoes). The pure culture of the
bacteias was assigned to the solid food environments in the test tubes.
To isolate the isolates of Bacillus thuringiensis, extinct effluent samples collected from the field
were used. Bacterial extraction was conducted according to the method described by Gonzalez et
al (2011). The collected effluents were previously crushed in an 80 oC water bath for 10 minutes
to kill vegetative cells and non-spore-forming bacteria. This ground oocyanote was then grown
in a GPA nutrient medium. Colonies similar to those of Bacillus have been isolated and
examined for the presence of papasporal crystals. Then B.thuringiensis colonies were identified
and isolated. Here are 3 B.isolated thuringiensis isolates (BTE-1, bte-2, bte-3). They were found
to differ from each other in crystal shape and morphology of their colony.
Also isolated from the soil were the isolates Bacillus thuringiensis. To do this, 2-3 cm of the top
layer was removed. Then a 20 gr soil sample was placed in special bags. The special bags were
kept at 4 oC until laboratory work began. To extract bacteria from soil samples, Song et al. (2008)
through the method described. 1 g of soil sample 10 ml 0.85% NaCl was suspended. Then heated
in a vibrating thermostat at 70 °C for 10 minutes. The nutrient agar (0.5% pepton, 0.3% beef
extract, 0.5% NaCl and 1.5% agar) was suspended for 100 µl. This used 50 petri dishes, all
incubated at 30±2 °C for 48 hours. Bacterial colonies showing a phenotype similar to Bacillus
thuringiensis were isolated and moved to a new food environment. These colonies were reared a
second time. The cultures were stained with fuchsin and examined under a standard light
microscope for initial identification. From The Soil B.2 isolates similar to thuringiensis were
isolated (BTE-4, BTE-5).
Figure 1. Isolated B.colonies of thuringiensis isolate and its appearance under a microscope
Research results. The fact that the greenhouse leech, which is considered the most dangerous
pest insect in our God at the next time, has increased, causes enormous problems in growing
pamidors and cucumbers in the greenhouse. As a result of this, 25-30% of the crop extracted
from pamidor and cucumbers in the cultivated areas of the Republic is destroyed under the
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influence of harmful organisms.
In solving this problem, in order to obtain biopreparation in our work, Bacillus thuringiensis was
tested in various concentrations against the most economically damaging greenhouse leachate in
the greenhouse industry by preparing a cultured liquid as a starting material from bacterial strains.
To study the entomopathogenic activity of the resulting strains, it was studied in relation to
ethanol in small field conditions. Our experiment was carried out as part of the study of the
entomopathogenic effects of Bacillus thuringiensis bacterium 5-endotoxin on greenhouse
effluents.
Table 1 below records the data obtained from the study of the effects of greenhouse effluents of
0.3% and 0.5% solution of isolated Bacillus thuringiensis bacterial cultures. From bacterial
strains Bacillus thuringiensis, a cultured liquid was prepared as the starting material. The
experiments were carried out in a greenhouse that harms the pamidor plant planted in the
experimental Agricultural Greenhouse of the Tashkent State Agrarian University in the Qibray
District of the Tashkent region. Before processing with a cultured liquid made from bacterial
strains, the damage to the greenhouse plant was monitored, that is, the number of pests was
calculated. In the experiment, 10 pamidor plants were selected in one row for each option, and
the number of pests on the plant was taken into account (Table 1).
Table 1.
Bacillus thuringiensis application of different concentrations of cultural fluidity of bacterial
strains against greenhouse fluidity
№
Ty
pe
s
Co
nc
en
tra
tio
n
of
cu
ltu
ra
l
flu
id
,%
Number
of living
greenho
use
leaks
until
processi
ng,
donado
na
Number of pests that die, %
Days of receipt of the chisobi after processing
3-day
6-day
9-day
12-day
1
BTE-1
0,3
82,7
9
20
36
49
0,5
47,4
14
27
41
54
2
BTE-2
0,3
65,3
11
23
39
51
0,5
72,5
17
32
53
68
3
BTE-3
0,3
49,7
-
-
15
27
0,5
56,4
-
9
18
31
4
BTE-4
0,3
67,2
-
-
-
-
0,5
58,6
-
-
-
17
5
BTE-5
0,3
77,8
-
-
13
26
0,5
81,1
-
7
16
29
6
Control (water)
48,9
-
-
-
-
During the experiment, the number of pests killed on 3, 6, 9 and 12 days after treatment with a
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cultured liquid made from bacterial strains was calculated. The best in the experiment showed
the result at a concentration of 0.5% of the BTE-2 strain. According to this, when the account of
the 3rd day after processing was taken, 17% recorded the death of the pest, by the 12th it was
found that 68% of greenhouse leeches had died. In our experiment, no pests that died in our
variant, which used a concentration of 0.3% of the BTE-4 strain, were observed.
Conclusion. Based on the experience carried out, it is possible to draw conclusions below. The
entomopathogenic activity of isolated BTE-1, bte-2, bte-3, bte-4, and bte-5 isolates against
greenhouse leaching has been studied. In doing so, isolated BTE-2 and bte-1 isolates from the
effluent itself were found to be most active against greenhouse effluents. And a 0.5% solution of
the cultured liquid obtained from them was recommended for use against greenhouse leaks.
Useful literature.
1.
Baxronova Z.F., Jumanazarov G‘.X.
Bacillus subtillus
bakteriya shtammini mosh
o‘simligining o‘sib rivojlanishiga va ildiz hosil qilish xususiyatlariga ta’siri.
Международный
научно-практический
электронный
журнал
«МОЯ
ПРОФЕССИОНАЛЬНАЯ КАРЬЕРА». Выпуск №52 (том 1) (сентябрь, 2023). 100-108 b.
2.
Baxronova Z.F., Jumanazarov G‘.X. Mosh o‘simligining fuzarioz kasalligiga qarshi
bacillus
subtillus
bakteriya shtammini antagonistik xususiyati. Международный научно-
практический электронный журнал «МОЯ ПРОФЕССИОНАЛЬНАЯ КАРЬЕРА».
Выпуск №52 (том 1) (сентябрь, 2023). 93-99 b.
3.
González A., Díaz R., Díaz M., Borrero Y., Bruzón R. Y., Carreras B., Gato, R. (2011).
Characterization of
Bacillus thuringiensis
soil isolates from Cuba, with insecticidal activity
against mosquitoes. Revista de biologia tropical, 59(3), 1007-1016.
4.
Jumanazarov G‘.X., Omonlikov A.U.
Bacillus subtilis
bakteriya shtammining kultural
suyuqligini pomidor o‘simligining o‘sishiga ta’siri. “Challenges of storage and processing of
agricultural products and their solutions based on modern technologies” international
scientific
and
practical
conference
october
22,
2024.
226-230
p.
https://doi.org/10.5281/zenodo.13954765
5.
Mamatolibov А.Е., Jumanazarov G‘.X.
Мarokash chigirtkasiga qarshi
(
dociostaurus
maroccanus
)
bacillus thuringiensis
bakteriya shtammining kultural suyuqligini qullash
samaradorligi. “Agrokimyohimoya va o‘simliklar karantini jurnali. Toshkent 2024. 3-son,
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