Volume 03 Issue 07-2023
23
American Journal Of Applied Science And Technology
(ISSN
–
2771-2745)
VOLUME
03
ISSUE
07
Pages:
23-31
SJIF
I
MPACT
FACTOR
(2021:
5.
705
)
(2022:
5.
705
)
(2023:
7.063
)
OCLC
–
1121105677
Publisher:
Oscar Publishing Services
Servi
ABSTRACT
Nematodes, which are the subject of this study, are the 3rd most widely used bioagent after Trichogramma and
Bacillus thuringiensis for biological control purposes. To collect entomopathogenic nematodes, they are placed in a
"White trap". The research was conducted on a potato farm in Surkhandarya, southern region of Uzbekistan. The main
tasks were defined as: potato agrobiocenosis of entomopathogenic nematodes determine species composition;
reveal the bioecology of entomopathogenic nematodes ; It is to carry out a molecular-genetic analysis of
entomopathogenic nematode species identified for the first time in potato agrobiocenoses in the conditions of
Uzbekistan.
KEYWORDS
Uzbekistan, Surkhandarya, entomopathogenic nematodes, Steinernema spp, Heterorhabditis spp, in vivo, potato.
INTRODUCTION
It is known that the global bioproducts market will
reach $4,852 million in 2021 and reach $13,634 million by
2030, with a CAGR of 12.2% during 2021-2030 is
expected to grow. In this case, the increase in the use
of biological products in relation to chemical plant
protection means has ensured the growth of the
corresponding bioproducts market [26]. Therefore,
biological control agents are environmentally friendly,
do not have a harmful effect on the human div, and
do not cause genetic resistance in pests.
Currently, the major drivers of the biocontrol agents
market are increasing global demand for organic
farming, stringent regulations against chemicals, and
positive consumer attitudes toward biocontrol agents.
The reason is that Europe aims to reduce the use of
dangerous pesticides by 50% by 2030 [3].
According to the FAO, potatoes are grown in 150
countries of the world on more than 20 million
hectares. World production is 359 million tons. This
Research Article
CAN ENTOMOPATHOGENIC NEMATODES BE THE BEST MEASURE FOR
GROWING ECO-FRIENDLY AGRICULTURAL PRODUCTS?
Submission Date:
July 20, 2023,
Accepted Date:
July 25, 2023,
Published Date:
July 30, 2023
Crossref doi:
https://doi.org/10.37547/ajast/Volume03Issue07-06
Nafosat Kurbonova Sattor Kizi
Research Scientific Institute Of Plant Protection And Quarantine, Uzbekistan
Journal
Website:
https://theusajournals.
com/index.php/ajast
Copyright:
Original
content from this work
may be used under the
terms of the creative
commons
attributes
4.0 licence.
Volume 03 Issue 07-2023
24
American Journal Of Applied Science And Technology
(ISSN
–
2771-2745)
VOLUME
03
ISSUE
07
Pages:
23-31
SJIF
I
MPACT
FACTOR
(2021:
5.
705
)
(2022:
5.
705
)
(2023:
7.063
)
OCLC
–
1121105677
Publisher:
Oscar Publishing Services
Servi
indicator may increase to 500 million tons in 2025, and
to 750 million tons in 2030 [26].
While resistant cultivars are considered a good method
of disease management, this trait declines over the
years, and there are no cultivars that are completely
resistant to some pests. And chemicals can cause
environmental pollution and food poisoning.
Therefore, the use of bioagents for biological control
of potato crop pests is an urgent research area. In
particular, entomopathogenic nematodes, which are
the object of this research work, are the 3 most widely
used bioagents after Trichogramma and Bacillus
thuringiensis [24] .
Based on these tasks, including our country
determining the fauna and species composition of
entomopathogenic nematodes distributed in potato
agrobiocenoses,
systematic
analysis,
their
morphology,
ecology,
economic
importance,
development of measures for sustainable use for the
purpose of biological pest control is of great scientific
and practical importance.
In this regard, recommendations on the optimal
methods and norms of using entomopathogenic
nematodes for the purpose of biological control
against harmful insects are the development of
practical experimental materials for farms, clusters and
all such agricultural crops growers in our republic is an
urgent task for the scientists of the field.
In Uzbekistan, no research has been carried out to
study the species composition, morpho-biology and
economic
importance
of
entomopathogenic
nematodes, and the fauna, biology and systematics of
entomopathogenic nematodes of Uzbekistan have not
been studied to date. Therefore, it is important to
study the species composition, distribution, morpho -
biologic
characteristics
of
entomopathogenic
nematodes, and to develop and implement methods of
biological control of agricultural crop pests.
MATERIALS AND METHODS
For in vivo propagation of entomopathogenic
nematodes, all debris is removed from soil samples
collected from the field. If necessary, sprinkle water
and improve soil moisture. Prepared soil samples are
placed in plastic containers with lids and ten instar
larvae of G. mellonella are placed on the surface of
each soil sample. Finally, cover the plastic containers
with lids and incubate upside down in a dark place at
55% relative humidity (RH) and 25°C [12].
After 7 days, dead larvae with specific signs of infection
with entomopathogenic nematodes are removed from
the soil and placed in a "White trap" [6] to collect
entomopathogenic nematodes in the third stage of
infection.
Identification
of
entomopathogenic
nematodes is determined by the color characteristics
of dead larvae. In this case, the larvae of the wax moth
turn gray if they are infected with entomopathogenic
nematodes of the Steinernema genus - the third
infection stage, and red-black if they are infected with
entomopathogenic nematodes of the third infection
stage
–
Heterorhabditis [15,16].
The "White trap" method helps to collect
entomopathogenic nematodes from infected wax
moth carcasses. In this case, a Petri dish is placed
upside down inside a large plastic container. Then 200
ml of sterile distilled water is poured into a plastic
container. A circular Whatman filter paper is placed in
a Petri dish. It is important to ensure that the perimeter
of the filter paper touches the water in the plastic
Volume 03 Issue 07-2023
25
American Journal Of Applied Science And Technology
(ISSN
–
2771-2745)
VOLUME
03
ISSUE
07
Pages:
23-31
SJIF
I
MPACT
FACTOR
(2021:
5.
705
)
(2022:
5.
705
)
(2023:
7.063
)
OCLC
–
1121105677
Publisher:
Oscar Publishing Services
Servi
container. Approximately 2-4 to 10 wax moth larvae per
trap are then placed on filter paper in a Petri dish. The
plastic container is closed with a lid and the container
is incubated at a temperature of 20-27°C. After 8 days,
new entomopathogenic nematodes develop from
dead wax moth larvae and are collected from the
water near the filter paper in the trap for 8-20 days.
Then put the collected water containing nematodes in
a 100 ml glass and let it sink to the bottom of the
container for 30 minutes. After the required time,
nematodes are washed by filling the container with
sterilized distilled water, this process is repeated 3-4
times until the suspension looks clear. At the end of the
procedure, the suspension is collected in a clean,
transparent tissue culture flask. The time and date of
collection and the type of nematode are recorded on
the label on the special tube. A special flask containing
a suspension of entomopathogenic nematodes is
stored in an incubator at a temperature of 10-15 °C
[2,8,9].
All insects suspected of having nematodes should be
dissected , requiring the use of saline (Ringer's solution
or 0.9% NaCl) to prevent nematode rupture as a result
of osmotic imbalance . It is necessary to examine
various tissues of the observed insect , including
hemolymph , digestive system, salivary ducts , muscles
, adipose tissue , glands, reproductive system .
Examination
samples
of
entomopathogenic
nematodes with caution should be prepared. Live
specimens should be heat-killed and placed directly
into the fixative to avoid damage to the div structure.
Physiological solution at a temperature of 60 ° C is best
for living specimens. As fixatives , it is optimal to use
TAF fixative (97 ml 40% formalin, 2 ml of
triethanolamine and 91 ml of distilled water). If this
fixative is not available, 3% formalin solution can be
used instead. If formalin solution is not found, alcohol
solution (70%) can be used. After one week, the
samples placed in a small petri dish are free from the
fixative in order to do this, an equivalent container with
70 ml of 95% ethanol solution and a mixture of 5 ml of
glycerin and 25 ml of water is used. In this case, the
container is partially filled with this liquid and left for
one to two weeks depending on the room
temperature . This simple method is due to nematodes
removing water from the sample and evaporating it
helps to create quality material [2].
So,
1. Nematodes are transferred under a microscope to a
slide with a drop of glycerin (pereparoval nina).
2. Put three pieces of glass cotton fiber on the product
window and cover the window, then the windows are
sealed with nail polish or wax.
5. After 48 hours, the quality is checked and resealed if
necessary [22].
The following diagnostic key can be used to identify
types of entomopathogenic nematodes.
Dianostics of the family Steinernematidae
The female
1.
The div is large, variable in size. They have
smooth skin or lateral ring.
2.
Separating holes are clearly visible.
3.
The head is round, rarely flat. They have six
labia that are fully fused or sometimes
separated, with one labial papilla per lip, and
occasionally additional papilla-like structures
appear next to the labial papillae.
4.
The number of head papillae is 4. Has small
amphids. The stoma is usually short and in
Volume 03 Issue 07-2023
26
American Journal Of Applied Science And Technology
(ISSN
–
2771-2745)
VOLUME
03
ISSUE
07
Pages:
23-31
SJIF
I
MPACT
FACTOR
(2021:
5.
705
)
(2022:
5.
705
)
(2023:
7.063
)
OCLC
–
1121105677
Publisher:
Oscar Publishing Services
Servi
lateral view forms a ring resembling two large
sclerotized dots.
5.
Additional
elements
of
the
stoma
asymmetrically form a funnel with a thick
anterior end. Esophagus with metacorpus is
slightly swollen, short isthmus is surrounded by
nerve ring and large basal bulb with valve is
clearly visible.
6.
The esophageal valve is usually simple in
structure.
7.
The reproductive system is usually didelphous,
amphiphilous and reflexive. Vulva in the middle
of the div, with or without an epiptigma.
Male
1.
The div is smaller than the female.
2.
The anterior end usually consists of six labial
papillae, four large cephalic papillae, and usually a
perioral disc.
3.
The esophagus is similar to that of the female.
4.
Testicle is single, reflexed; spicules paired;
gubernaculum sometimes as long as spicule; bursa
is not available.
5.
The tip of the tail is rounded, with a muzzle.
There are 10-14 pairs of genital papillae.
Infectious larvae
1.
They usually do not have a stoma.
2.
Their bodies have extra skin (in exchange for
their second-stage larvae shedding their skin).
3.
The esophagus and intestine are shortened.
There are separate outlets.
4.
The tail is usually cone-shaped.
5.
Phasmid is not visible.
Key to identification of Steinernema species
Steinernema species can be recognized using the
following key, but identification can be confirmed by
analyzing its morphometry in parallel with data from
the original description.
YL = 3rd instar larva of infective juvenile nematode.
EP = distance from anterior end to excretory orifice.
ES = length of esophagus.
SP = spicule length.
T = tail length.
D% = EP/ES × 100.
E% = EP/ T × 100.
SW = spicule length divided by anal div width [28].
RESULTS
The structure of these entomopathogenic nematodes
belonging to the family Rhabditida (Oerley, 1880) [23]is
unique and is based on the following diagnostic
features: the number of lips varies from one to six, the
stoma is tubular, and the walls of the stoma are
covered with rhabdions. Esophagus Consists of parts
such as procorpus, metacarpus and isthmus and is in
the form of a basal bulb with valves. The separation
system consists of lateral channels , and the terminal
channel is the cuticle .
Females have one or two ovaries . The vulva is located
further back. There is a bursa [10,20].
This family is divided into two subfamilies: Rhabditina
and Cephalobina. All species belonging to the family
Steinernematidae and Heterorhabditidae of the
suborder Rhabditina are pathogenic species .
Steinernem a carpocapsae (Weiser, 1955)
Volume 03 Issue 07-2023
27
American Journal Of Applied Science And Technology
(ISSN
–
2771-2745)
VOLUME
03
ISSUE
07
Pages:
23-31
SJIF
I
MPACT
FACTOR
(2021:
5.
705
)
(2022:
5.
705
)
(2023:
7.063
)
OCLC
–
1121105677
Publisher:
Oscar Publishing Services
Servi
Diagnostics . Men i: the head part of the div has six
labial and four circular head papillae . Release foramen
gi is usually located anterior to the nerve ring. D% 41 (27
- 55). The tail is curved. The cephalic part of the spicule
is longer, wider than the ventral part. Spicule length 66
(58 - 77) nm, ratio of spicule length to spicule width
approximately 5.2 (4.7
–
6.0). SW = 1.72 (1.40 - 2.0); GS
= 0.71 ( 0.59
–
0.88 ) , guberna c ulum in the ventral view
narrows towards the front ( Fig . 1 ) .
Infectious larvae : Average div length is 558 (438 -
650) n m. EP = 38 (30 -60) nm , E% about 60 (54 - 66). T
= 53 (46 - 61).
For molecular genetic studies, DNA was extracted from
3 male specimens of Steinernema carpocapsae [22].
Figure 1. Structure of male Steinernema carpocapsae (Weiser, 1955) .
1. Anterior region of the div, 2. Tail region, 3a,b -spicule comprehensive view.
Volume 03 Issue 07-2023
28
American Journal Of Applied Science And Technology
(ISSN
–
2771-2745)
VOLUME
03
ISSUE
07
Pages:
23-31
SJIF
I
MPACT
FACTOR
(2021:
5.
705
)
(2022:
5.
705
)
(2023:
7.063
)
OCLC
–
1121105677
Publisher:
Oscar Publishing Services
Servi
The results of the research show that 740 base pairs of nucleotides belonging to the ITS region of pDNA of 3 samples
of Steinernema carpocapsae species were isolated (Fig. 2).
Steinernema carpocapsae and Steinernema carpocapsae (accession number: MN044870) rDNA ITS region in the 5' to
3'-terminal direction, identical nucleotide bases are marked with dots.
Volume 03 Issue 07-2023
29
American Journal Of Applied Science And Technology
(ISSN
–
2771-2745)
VOLUME
03
ISSUE
07
Pages:
23-31
SJIF
I
MPACT
FACTOR
(2021:
5.
705
)
(2022:
5.
705
)
(2023:
7.063
)
OCLC
–
1121105677
Publisher:
Oscar Publishing Services
Servi
Figure 2 . Steinernema carpocapsae ( Weiser,1955) based on sequence material nucleotides of the ITS region of
pDNA of species belonging to the genus sequence comparison .
CONCLUSION
It is necessary to carry out many new studies on the
isolation
and
identification
of
these
entomopathogenic nematodes from the territory of
Uzbekistan and their biocontrol potential against
agricultural crops. In particular, the above-mentioned
species Steinernema carpocapsae (Weiser, 1955) is
used today against the main pests of many countries
[4,5,7,11], and good results are obtained. If the
researchers master the methods of reproduction of
such new species in vivo and test them against the
pests of potatoes, cotton, grain and orchards, in the
future, the environmentally friendly, inexpensive local
bioagent will be widely used [13,17,21] would have
contributed to it.
REFERENCES
1.
Blaxter, ML, De Ley, P., Garey, JR, Liu, LX,
Scheldeman, P., Vierstraete, A., Vanfleteren, JR,
Mackey, LY, Dorris, M., Frisse, LM, Vida, JT &
Thomas, WK (1998). A molecular framework for
the phylum Nematoda. Nature 392, 71-75.
2.
Courtney, WD, Polley, D. & Miller, VL (1955). Taf, an
improved. xative in nematode techniques. Plant
Disease Reporter 39, 570-571.
3.
Ebssa, L., Borgemeister, C., Berndt, O. & Poehling,
HM
(2001).
Impact
of
entomopathogenicnematodes on different soil-
dwelling stages of western ower thrips,
Frankliniella
occidentalis
(Thysanoptera:
Thripidae), in the laboratory and under semi-field
conditions. Biocontrol Science and Technology 4,
515-525.
4.
English-Loeb, G., Villani, MG, Martinson, T.,
Forsline, A. & Consolie, N. (1999). Use of
entomopathogenic nematodes for control of
grape phylloxera (Homoptera: Phylloxeridae): a
laboratory evaluation. Environmental Entomology
2, 889-894.
5.
Feaster, MA & Steinkraus, DC (1996). Inundative
biological control of Helicoverpa zea (Lepidoptera:
Noctuidae) with the entomopathogenic nematode
Steinernema
riobravis
(Rhabditida:
Steinernematidae). Biological Control 7, 38-43.
6.
Filipjev, IN (1934). Miscellanea Nematologica 1. Eine
neue Art der Gattung Neoaplectana Steiner nebst
Bermerkungen über die systematicische Stellung
der letzteren. Magazine de Parasitologie du Musée
Zoologique de l'Académie des Sciences de l'USSR
4, 229-240.
7.
GLASER, RW (1932). Studies on Neoaplectana
glaseri , a nematode parasite of the Japanese
beetle ( Popillia japonica ). New Jersey Department
of Agriculture Circular 211.
8.
Glaser, RW & Farrell, CC (1935). Field experiments
with the Japanese beetle and its nematode
parasite. Journal of the New York Entomological
Society 43, 345-371.
9.
Glaser, RW & Fox, H. (1930). A nematode parasite
of the Japanese beetle ( Popillia japonica Newm.).
Science 71, 16-17.
10.
Hominick, WM, Briscoe, BR, Del Pino, FG, Heng, JA,
Hunt, DJ, Kozodoy, E., Mráïc Ek, Z., Nguyen, KB,
Reid, AP, Spiridonov, SE, Stock, P. , Sturhan , D. ,
Waturu , C. & Yoshida , M. ( 1997 ). Biosystematics
of entomopathogenic nematodes: current status,
protocols and de. nitions. Journal of Helminthology
71, 271-298.
11.
Journey, AM & Ostlie, KR (2000). Biological control
of the western corn rootworm (Coleoptera:
Chrysomelidae) using the entomopathogenic
Volume 03 Issue 07-2023
30
American Journal Of Applied Science And Technology
(ISSN
–
2771-2745)
VOLUME
03
ISSUE
07
Pages:
23-31
SJIF
I
MPACT
FACTOR
(2021:
5.
705
)
(2022:
5.
705
)
(2023:
7.063
)
OCLC
–
1121105677
Publisher:
Oscar Publishing Services
Servi
nematode,
Steinernema
carpocapsae
.
Environmental Entomology 29, 822-831.
12.
Kaya, HK & Stock, SP (1997). Techniques in insect
nematology. In: Lacey, LA (Ed.) Techniques in
insects pathology . London, Academic Press, pp.
281-324.
13.
Lacey, LA & Chauvin, RL (1999). Entomopathogenic
nematodes for control of diapausing codling moth
(Lepidoptera: Tortricidae) in fruit bins. Journal of
Economic Entomology 92, 104-109.
14.
Nadler, SA, Adams, BJ, Lyons, ET, Delong, RL &
Melin, SR (2000). Molecular and morphometric
evidence for separate species of Uncinaria
(Nematoda: Ancylostomatidae) in California sea
lions and Northern fur seals: hypothesis testing
supplants veri. cation. Journal of Parasitology 86,
1099-1106.
15.
Nguyen,
KB
&
Smart,
Jr,
GC
(1994).
Neosteinernema longicurvicauda n. gen., n. sp.
(Rhabditia: Steinernematidae), a parasite of the
termite Reticulitermes avipes (Koller). Journal of
Nematology 26, 162-174.
16.
Nguyen, KB, Maruniak, J. & Adams, JB (2001).
Diagnostic and phylogenetic utility of the rDNA
internal
transcribed
spacer
sequences
of
Steinernema. Journal of Nematology 33, 73-82.
17.
Poinar, Jr, GO (1986). Recognition of Neoaplectana
species. Proceedings of the Helminthological
Society of Washington 53, 121-129.
18.
Poinar, Jr, GO (1990). Taxonomy and biology of
Steinernematidae and Heterorhabditidae. In:
Gaugler, R. & Kaya, HK (Eds). Entomopathogenic
nematodes in biological control. Boca Raton,
Florida, USA, CRC Press, pp. 23-60.
19.
Powers, TO, Todd, TC, Burnell, AM, Murray, PCB,
Zsalanski, AL, Adams, BJ & Harris, TS (1997). The
rDNA internal transcribed spacer region as a
taxonomic marker for nematodes. Journal of
Nematology 29, 2441-2450.
20.
Reid, AP, Hominick, WM & Briscoe, BR (1997).
Molecular
taxonomy
and
phylogeny
of
entomopathogenic nematode species (Rhabditida:
Steinernematidae) by RFLP analysis of the ITS
region of ribosomal DNA repeat unit. Systematic
Parasitology 37, 187-193.
21.
Román, J. & Figueroa,W. (1994). Steinernema
puertoricensis
n.
sp.
(Rhabditida:
Steinernematidae)a
new
entomopathogenic
nematode from Puerto Rico. Journal of
Agriculture, University of Puerto Rico 78, 167-175.
22.
Seinhorst, JW (1959). A rapid method for the
transfer of nematodes from. xative to anhydrous
glycerin. Nematologica 4, 67-69.
23.
Steiner, G. (1923). Aplectana kraussei n. sp., eine in
der Blattwespe Lyda sp. parasitic Nematodenform,
nebst Bemerkungen über das Seitenorgan der
parasiticen
Nematoden.
Zentralblatt
für
Bakteriologie,
Parasitenkunde,
Infektions
Krankheiten und Hygiene, Abteilung II 59, 14-18.
24.
Steiner, G. (1929). Neoaplectana glaseri ngn sp.
(Oxyuridae), a new nemesis parasite of the
Japanese beetle ( Popillia japonica Newm.).
Journal of the Washington Academy of Science 19,
436-440.
25.
Stock, SP, Campbell, JF & Nadler, SA (2001).
Phylogeny of Steinernema Travassos, 1927
(Cephalobina: Steinernematidae) inferred from
ribosomal DNA sequences and morphological
characters. Journal of Parasitology 87, 877-889.
26.
Stock, SP, Heng, J., Hunt, DJ, Reid, AP, Shen, X. &
Choo, HY (2001). Redescription of Steinernema
longicaudum
Shen
&
Wang
(Nematoda:
Steinernematidae); geographic distribution and
phenotypic
variation
between
allopatric
populations. Journal of Helminthology 75, 81-92.
27.
Szalanski, AL, Taylor, DB & Mullin, PG (2000).
Assessing nuclear and mitochondrial DNA
sequence
variation
within
Steinernema
Volume 03 Issue 07-2023
31
American Journal Of Applied Science And Technology
(ISSN
–
2771-2745)
VOLUME
03
ISSUE
07
Pages:
23-31
SJIF
I
MPACT
FACTOR
(2021:
5.
705
)
(2022:
5.
705
)
(2023:
7.063
)
OCLC
–
1121105677
Publisher:
Oscar Publishing Services
Servi
(Rhabditida:
Steinernematidae).
Journal
of
Nematology 32, 229-233.
28.
Travassos, L. (1927). Sobre o genero Oxysomatium
. Boletim Biologico, Sao Paulo 5, 20-21.
