Int.J.Curr.Microbiol.App.

Sci (2014) 3(10) 693-699

ISSN: 2319-7706 Volume 3 Number 10 (2014) pp. 693-699

http://www.ijcmas.com

Original Research Article

Isolation and Identification of Entomopathogenic Nematodes of
Kodaikanal Hills of South India

M.Razia1* and S.Sivaramakrishnan2

1
Department of Biotechnology, Mother Teresa Women s University, Kodaikanal, India
2
Bharathidasan University, Tiruchirapppalli, TamilNadu, India
*Corresponding author

ABSTRACT

Isolation and identification of indigenous species of Entomopathogenic nematodes

are necessary for successful control of crop pest in vegetables. In the present study,
Keywords
different crop soils were collected and screened for occurrence of nematodes. A
Entomo- survey was conducted and totally 200 samples were collected in Kondikanal
pathogenic regions of Dindigul district of TamilNadu. Galleria bait method is used for
nematode, isolation of nematode was performed. 7 samples were positive for
Steinernema, entomopathogenic nematodes, with 3 containing Heterorhabditis and 5
Heterorhabditis, Steinernema isolates. Morphological and molecular studies were carried out to
Galleria, characterize isolates. The results of morphological and molecular characterizations
Insect pest of isolated nematodes represent Steinernema species and identified as S. siamkayai
was the most common species, which was isolated from three provinces and one
Heterorhabditis species, H. indica respectively.

Introduction

Entomopathogenic nematodes are parasites own associated bacterial symbionts, which is

and ubiquitous in distribution throughout the present in their intestinal space (Laznik et
world (Hominick, 2002) except Arctic and al., 2011). The bacteria multiply inside the
Antarctica. It search the suitable insect host host and release a number of virulence
with the help of leakage of root wounded factors, including complexes of toxins,
plant compounds, carbon dioxide etc. hydrolytic enzymes, hemolysins, and
Nematodes act as a biological control agents antimicrobial compounds (Eleftherianos et
and the non-feeding infective juvenile al., 2010), thus providing nutrients for the
stage (IJs) kill the insects (different stages of nematodes development and reproduction
larva, pupa, and adult) depend upon the within the insect cadaver.
species of nematodes and insects; the
nematode penetrates into the insect body, Entomopathogenic nematode belongs to the
usually through natural body openings genera Steinernema and Heterorhabditis and
(mouth, anus and spiracles) or areas of thin their symbiotic bacteria in the genera
cuticle within 24-72 h with the help of their Xenorhabdus Poinar and Photorhabdus

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respectively. In the last two decades, survey Materials and Methods

has been carried out and several species of
nematode species isolated (Rosa et al., Soil Collection
2000). Environmental factors influence the
nematode occurrences and distribution and The occurrence and distribution of EPNs
their survival. Biotic and abiotic factors was surveyed in crop fields and forest area
cause the distribution EPN to differ across of Kodaikanal, Dindigul district of Tamil
different regions (KarthikRaja et al., 2011). Nadu. Study area has been segregated into
four areas and the soil samples were
Major factors namely temperature and host collected. The study sites namely Combai,
availability are thought to be important in ThandiKudi, Pumbarai, Manavannur
determining their distribution. Since, few respectively. EPNs distribution in relation
commercial strains of EPN species available to environmental changeable and soil
and isolated from North America or Europe physical chemical characteristic from each
are used worldwide (Susurluk, 2011). locality soil samples were taken from
Nowadays, application of EPNs is vegetable crops include cabbage,
developed in large scale liquid culture, cauliflower, garlic, carrot and sampling
production costs compact and their usage in interval was about 5 km. Totally 500 soil
particularly horticulture, agriculture, and samples were collected in the polythene bag
forestry. Therefore, these strains may from the depth of 10-15cm and transported
perhaps not be well adapted to local climates to laboratory for further processing.
and their value might be reduced. Several
surveys have searched for new EPN species Isolation and Propagation of nematodes
with the intent to control important (Galleria Trap Method)
agricultural and horticultural pests under
specific conditions. In the laboratory, soil samples was
processed with the insect-baiting (Galleria
Exotic EPN displace native nematodes, trap) method (Bedding & Akhurst,1975).
effects on non-target organisms (Ehlers, 250g soil sample was placed in a plastic box
2005). Isolates of EPNs are usually found in and baited with five larvae of G. mellonella.
a variety of habitats, exhibit considerable The boxes were stored at 25ºC for five days;
variation in their respective studies such as the dead larvae were collected and
host range, reproduction, infectivity, and transferred to a white trap (White, 1927) to
survival (Laznik and Trdan, 2012). collect the infective juveniles (IJs) of
nematodes. All the soil samples were baited
Therefore EPNs are considered as one of the three times with larvae to get the maximum
most relevant non-chemical alternatives to number of positive soil samples. The
insect pest control due to their high collected IJs were checked for their
reproductive potential, ease of mass pathogenicity against Galleria larvae
production and their harmlessness to (Pelezar and Reid, 1972).
microbes, animals, humans and plants. In
this study, a survey was conducted to isolate Nematode propagation was performed by
and identify EPNs from disturbed habitats in applied IJs to a 4.5 cm diam Petri dish lined
the Kodaikanal area of Dindigul region. with Whatman No.5 filter paper containing
Morphology, morphometrics and ITS region five G. mellonella larvae. The Petri dishes
of rRNA of nematodes was studied. were incubated at room temperature until IJs

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emerged from the cadavers and were for 10minutes. The upper layer was removed
collected in a White trap. The IJs were then and to which add chloroform: isoamylalchol
stored for less than three weeks at 15°C in in the ratio of 24:1, mixed and centrifuge,
distilled water until further testing. The IJs 12,000rpm for 10minutes. The upper phase
were stored at a concentration of 1000 IJs/ was separated and 3M ammomium
ml in distilled water with 0.1% formalin in precipitate was added for better DNA
tissue culture flask, were stored at 19°C in precipitation. The suspensions freeze for 2
B.O.D incubator. Nematodes were routinely hours and centrifuge for 30 minutes at
cultured in G. mellonella larvae (Woodring 12,000 rpm. DNA was precipitated from
and Kaya, 1988). aqueous phase and after drying resuspended
in TE buffer. The DNA sample was run by
Nematode Identification agarose gel electrophoresis using 1kb
markers of lambda DNA as standard. For
Morphological characterization polymerase chain reaction (PCR)
amplification and sequencing of the ITS,
For morphological characterization of the primers were designed by the method of
isolate, nematodes examined heat-killed in Joyce et al. (1994).
60°C Ringer s solution. The heat-killed
nematodes place in triethanolamine Results and Discussion
formalin (TAF) fixative and processed to
anhydrous glycerine for mounting. The Isolation of Entomopathogenic nematodes
morphological features of males and IJs and
hermaphroditic female randomly selected A total of 500 soil samples from the
from different G. mellonella under light Kodaikanal region of Dindigul district were
microscopy according to procedures surveyed for EPNs, of which 7 resulted in
described by Seinhorst method (1959). The positive nematode isolates (Table 1). Two of
identity was verified by comparing its the samples were positive for
morphometrics with the data from original Heterorhabditis species and 5 were positive
descriptions. A morphological feature of a for Steinernema species. Compare to forest
representative isolate was examined using area, occurrence of EPNs was present in
scanning electron microscopy (SEM). crop lands and absence in forest area. This
was due to presence of insect pest was rare
Molecular Characterization in forest, because only found long tree
trunks only seen, low number of weed plants
The isolates were molecularly characterized there(Razia et al., 2011).
by analysis of the internal transcribed spacer
region (ITS) ribosomal DNA sequences. Morphological Characterization
DNA extraction was performed according to
Stock et al. (2001). The IJs were crushed The morphometric measurements of
using extraction buffer (0.05M EDTA, Heterorhabditis sp. collected from two
1%SDS, 400µl Proteinase K) and places was identified as H. indica based on
centrifuged for 10-15 minutes for 12,000 the length of IJs less than 600µm, Head to
rpm. The supernant was collected and add excretory pore 98µm and Tail length 101µm
equal volume of phenol: chloroform: (Table 2). The male and IJs coincide well
isoamylalchol at the ratio 25:24:1. Then the with the description given by Poinar et al.
tubes mixed well and centrifuge 12,000 rpm (1992).

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Table.1 Occurrence of EPNs in different agroecosystem of Kodaikanal Region of Dindigul

Sampling Area Steinernema Heterorhabditis

spp. spp.
Combai 2 1
ThandiKudi 1 -
Pumbarai 1 1
Mannavanur 1 1

Table 2. Morphomertric characters of infective juveniles of H. indica (n=25)

(Mean and Range, all measurements in µm)

Character MTU06 MTU07 Poinar et al. (1992)

Total Length (L) 512.4 510.7 528

(500 - 560) (480 - 570) (479 - 573)
Greatest Width (W) 19.9 19.7 20
(19 - 21) (19 - 20) (19 - 22)
Anterior end to 93.2 97.3 90
Excretory pore (EP) (90 -100) (93 -105) (88 - 107)
Esophagus (ES) length 105.8 104.3 115
(100 - 119) (100 - 120) (109 -123)
Tail length (L) 98.5 99.0 101
(95 -108 ) (95 -106) (93 - 109)

Table 3. Morphomertric characters of first generation male of H.indica (n=25)

(Mean and Range, all measurements in µm)
Character MTU06 MTU07 Poinar et al. (1992)
Total Length (L) 582.6 591.3 721
(575 - 685) (580 -780) (573 -788)
Greatest Width (W) 39.9 41.7 42
(36 - 45) (38 - 45) (35 - 46)
Excretory pore (EP) 110.5 119.7 123
(110 -130) (115 -135) (109 - 138)
Esophagus (ES) 96.2 97.8 101
(93 - 100) (95 -105) (93 - 109)
Tail length (L) 24.6 26.8 28
(24 - 30) (25 - 31 ) (24 -32)
Spicule Length (SpL) 36.9 37.9 43
(35 - 45) (36 - 46) (35 - 48)
Gubernaculum length 18.5 19.6 21
(GuL) (18 - 21) (18 - 20) (18 - 23)

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Table 4. Morphometric characters of infective juveniles of S.siamkayai (n=25)

(Mean and Range, all measurements in µm)

Nematode Total Greatest Excretory Esophagus Tail length

Isolates Length Width pore length (ES) (TL)
(L) (W) (EP)
MTU01 459.9 21.2 30.8 97.9 35.9
(447- 470) (18 - 23) (30 - 37) (94 -100) (31 - 41)
MTU02 473.1 23.2 38.3 106.2 41.3
(435 - 488) (19 - 24) (31 - 38) (92 - 107) (30 - 40)
MTU03 486.8 21.5 35.2 101.5 40.7
(474 - 492) (18 - 23) (32 - 38) (89 - 105) (35 - 41)
MTU04 489.6 20.6 36.7 105.6 41.9
(456 - 490) (19 - 24) (34 - 38) (90 - 107) (31 - 41)
MTU05 469.7 20.9 34.6 102.5 42.3
(428 -483) (19 - 23) (32 - 36) (90 -105) (31 - 40)
S. 446 21 35 94.5 35.5
siamkayai (398 - 495) (18 - 24) (29 - 38) (80 - 107) (31 - 41)

Table 5. Morphometric characters of first generation male of S.siamkayai (n=25) (Mean and
Range, all measurements in µm)

Isolates Total Greatest Excretory Esophagus Tail Spicule Gubernaculum

Length(L) Width (W) pore(EP) length (ES) length length (GuL)
(TL) (SpL)
MTU01 1141.3 123.8 55.6 135.2 24.1 76.9 53.8
(1100 -1190) (110 -150) (48 - 61) (128 -140) (22 - 30) (75- 80) (48 - 60)
MTU02 1133.5 123.5 56.8 138.1 24.8 75.8 55.2
(1130 - 1210) (121 - 155) (49 - 63) (130 - 140) (27 - 31) (75 - 79) (50 -65)
MTU03 1140.4 123.8 57.9 136.5 25.2 74.9 52.8
(1095 - 1195) (120 - 148) (48 - 59) (129 - 141) (22 - 30) (75 -79) (50 - 61)
MTU04 1131.3 128.9 59.1 138.2 24.8 75.2 54.6
(1092 - 1188) (110- 145) (49 - 61) (130 - 140) (22 - 32) (75 -78) (49 - 62)
MTU05 1143.8 124.8 55.4 136.5 23.8 74.1 55.1
(1092 - 1188) (110- 145) (49 - 61) (130-140) (22 - 30) (75 - 79) (50 - 61)
S. 1035 -1278 107-159 47.5 - 67 123 -141 22-32 75 - 80 47 65
siamkayai (1135) (139.5) (57) (134) (27.5) (77.5) (53.5)

In male, first generation male average length the basal bulb. Spicules paired and separate,
was 721µm whereas in the 596µm in the with pointed tips. Gubernaculum flat,
present study, spicule 40µm and narrow, approximately half the spicule
gubernaculum 20µm (Table 3). Head length.
truncate to slightly round. Nerve ring near

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Among Steinernema spp. five was identified strains observed. Further, molecular
as S. siamkayai based on their length of IJs characterization was performed by using
was less than 600µm, 486µm in the present ITS region of rDNA
study, Head to excretory pore 35µm and
Tail length 40µm (Table 4). The third stage Acknowledgement
of Juveniles is slender, tapering regularly
from base of oesophagus to anterior end and The authors would like to acknowledge the
from anus to terminus. Oesophagus is long, UGC, NewDelhi for providing Fund for this
and narrow. The first generation of male research work.
curved posteriorly, J -shaped when heat-
killed. The first generation of male, total References
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