Pak. J. Phytopathol.,
Vol. 21 (1): 71-76, 2009.
EFFECT OF ENTOMOPATHOGENIC NEMATODES ON THE INVASION AND
DEVELOPMENT OF MELOIDOGYNE INCOGNITA IN TOMATO
Sajid Aleem Khan, Nazir Javed, M. Aslam Khan and M. Mumtaz Khan*
Department of Plant Pathology, University of Agriculture, Faisalabad
*Institute of Horticultural Sciences, University of Agriculture, Faisalabad
ABSTRACT
Steinernema asiaticum, Steinernema glaseri, Heterorhabditis indica and Heterorhabditis bacteriophora
were investigated for invasion and development of root knot nematodes Meloidogyne incognita in tomato
roots. Harvesting of treatments was done at various time intervals over a period of 28 days. Reduction of J2s
was recorded in EPN treatments along with reduced number of females in EPN treated roots S.glaseri and S.
asiaticum were the most successful in suppressing the invasion and development of root knot nematodes in
tomato while H.indica and H.bacteriophura also suppressed the root knot nematode development. Maximum
invasion and development was observed in control.
Key words: Tomato, invasion and development, Meloidogyne incognita, entomopathogenic nematodes,
tomato due to root knot nematode (Shahid et al.,
2007).
Continuing environmental problems associated
with the use of nematicides have resulted in a
sense of urgency regarding the search for
alternative nematode management strategies
(Jairajpuri et al., 1990; Kerry, 1990; Veremis and
Roberts, 1996). Entomopathogenic nematodes are
effective for the management of root knot
nematodes (Grossman, 1997) and used as
bioinsecticides against soil pests (Klein 1990,
Georgis
and
Manweiler,
1994).
Entomopathogenic nematodes can provide better
management for plant-parasitic nematodes due to
environmentally safe and their non target effect
without affecting the free living nematodes that
play important role in nutrient cycling (Raichon
et al. 1994; Bonning and Hammock, 1996;
Somasekhar et al., 2002). Suppression of plantparasitic nematodes has been recorded earlier by
using live entomopathogenic nematodes (Bird
and Bird, 1986; Ishibashi and Kondo, 1986;
Ishibashi and Choi, 1991; Smitley et al., 1992;
Gouge et al., 1994; Grewal et al., 1997; Perry et
al., 1998; Grewal et al., 1999; Somasekhar et al.,
2000; Lewis et al., 2001) and dead EPN (Grewal
et al., 1999; Jagdale et al., 2002) but little
information was available on the effect of
entomopathogenic nematodes on life cycle of M.
incognita. Objective of study was to assess the
effect of entomopathogenic nematodes on life
cycle of root knot nematodes.
INTRODUCTION
Tomato (Lycopersicon esculentum Mill.) belongs
to solanaceae family and originated from the
highlands of the West coast of South America
(Smith, 1994). It is an important vegetable crop
and plays a vital role in maintaining health
(Myers and Croll, 1921; Saywell and Lane, 1933;
Conn and Stumpy., 1970). In Pakistan it is
cultivated over 46.23 thousands hectares with the
annual production of about 468.14 thousands
tonnes (Anonymous, 2007). Punjab, Sindh,
NWFP and Balochistan have 14%, 10%, 35% and
45% share respectively in tomato production
(Anonymous, 2007). Among the biotic factors
(fungi, bacteria, viruses and nematodes) that are
obstacles in getting the high yield, root knot
nematode Meloidogyne incognita (Kofoid and
white, 1919; Chitwood, 1949) is widespread and
the most destructive pathogen of tomato (Sasser,
1980; Jones et al., 1991; Fourie and McDonald,
2000). Root-knot nematodes tremendously reduce
quality and quantity of fruit. Root knot
nematodes are obligate sedentary endoparasites
with wide host range encompasses more than
2000-3000 plant species (Hussey, 1985; Abad et
al., 2003; Agrios, 2005). In tomato, yield losses
by Meloidogyne spp. has been estimated from 20
% to 33% (Sasser, 1979; Sasser and Carter, 1982;
Upadhyay and Dwivedi, 1987; Sasser, 1989).
Nagnathan (1984) reported 61% yield loss in
tomato due to M. incognita and 39% by Reddy
(1985) at initial population of 20 J2/g soil.
Diseases caused by nematodes in Pakistan are
important because the climate and sandy warm
soil are favorable for the development and
activity of nematodes (Brown, 1963). In Punjab,
75-100% disease incidence was reported on
MATERIALS AND METHODS
The Greater wax moth Gallaria mellonella (L.)
were obtained from bee hives infected with
Galleria mellonella. Last instars larvae of G.
71
mellonela were separated for nematode culture,
leaving small sized larvae for moth emergence
and egg laying. Fresh laid eggs were transferred
to modified artificial diet prepared by mixing oat,
wheat, rice and maize porridge (20 g), yeast
granules (50 g) in solution of 80 ml warm honey
and (100 g) glycerol (Alrubei and Al-Izzim,
1986). Diet with galleria was then kept at 27 ºC
in an incubator. After reaching last instars, they
were taken out from the diet and used for storage
and nematode isolation/multiplication. Both live
and dead Steinernema glaseri, Steinernema
asiatica,
Heterorhabditis
indica
and
Heterorhabditis bacteriophora were evaluated
against
Meloidogyne
incognita.
All
entomopathogenic nematode species used could
be reared on the late instar larvae of G.
mellonella, the greater wax moth. The in vivo
production of entomopathogenic nematodes was
slightly modified from the basic methods
described by Poinar (1979) and summarized by
Woodring and Kaya (1988). Larvae were kept at
15 °C. Entomopathogenic nematodes were
collected from dead G. mellonella larvae by
modified White trap (White, 1927) and then
stored at about 10 -15 ºC. Live infective juveniles
were used within fifteen days of emergence from
the cadavers of insect host. Culture of M.
incognita was maintained on the roots of
susceptible tomato variety ‘Moneymaker’ in
green house. Juveniles were isolated from
infested roots by modified Whitehead and
Hemming tray method (White-head and
Hemming, 1965). Only freshly hatched second
stage juveniles (24-48 hours old) were used.
Three weeks old tomato nursery ‘Moneymaker’
was planted in small pots containing 240 ml
formalin sterilized soil ((72% sand, 17% silt and
8% clay). The sterilization of soil was
accomplished by applying formalin. Diluted
formalin (1:320) was poured in the small heap of
soil and covered with polythene sheet to avoid
the evaporation completely. This process
continued for a week. After a week the soil was
exposed to get rid from residual formalin, mixed
the soil thoroughly and then filled the pots.
After two weeks when the plants established their
root system, EPN and M. incognita were applied
simultaneously in separate 5 ml water
suspensions in the rhizosphere by making 3-4
holes near the base of plant with sharp wooden
needle (Campos and Campos, 2005) and filled
with soil to prevent drying. Application rate for
M. incognita was 500/plant and 5,000/plant for
entomopathogenic nematodes. The un inoculated
plants served as control. Treatments were
replicated five fold and arranged in completely
randomized design in green house. Plants were
harvested after 7, 14, 21 and 28 days. At harvest,
plant height and root fresh weight were recorded
and roots were stained in 0.1% acid fuchsin and
macerated (Bridge et al., 1981). M. incognita at
different developmental stages in roots was
estimated under a stereomicroscope (Olympus SZ
61) at 3.5X magnification. To facilitate counting
of egg masses, the washed roots were stained
with phloxine B (Southey, 1986). Statistical
analysis of data was made at the end of
experiment using Dunnett’s test.
RESULTS
At the first harvest after seven days of application
of M. incognita (J2s) and entomopathogenic
nematodes, number of J2s in roots were
significantly (p<0.01) lower in S. asiatica and S.
glaseri (not different significantly) while the
number of root knot nematodes in root in
treatments of H. indica and H. bacteriophora
were not significantly different. Root knot
nematodes numbers in all the treatments were
significantly lower in tomato roots as compared
to control, dJ2s were also lower significantly in
all the treatments as compared to control. There
was lower number of dJ2s in S. asiaticum
followed by H. indica, S. glaseri and H.
bacteriophora. J3 were higher in H. indica and
control. All the treatments varied significantly.
At second harvest (after 14 days) number of J2s
was significantly lower in S. glaseri and S.
asiaticum as compared to control. J2s in
treatments of H. indica and H. bacteriophora
were not significantly different from each other.
Number of dJ2s was different significantly
(p<0.01) in all the treatments as compared to
control. There was low number of dJ2s in S.
asiaticum followed by S. glaseri (not different
significantly), H. indica and H. bacteriophora as
compared with control. J3 were lower in H.
bacteriophora followed by S. glaseri and H.
indica. J4 were also lower significantly from
control. Lower number of J4 was recorded in S.
asiaticum followed by S. glaseri, H. indica and
H. bacteriophora while maximum number was
recorded in control. Number of females was
lower in all treatments and were similar and
different from control statistically.
At the 3rd harvest after 21 days, dJ2s were
significantly lower in S. glaseri followed by S.
asiaticum, H. indica and H. bacteriophora while
maximum numbers were recorded in control
where only M. incognita was applied. Less
number of J3 were recorded in S. asiaticum
followed by S. glaseri, H. bacteriophora, H.
indica and control and were statistically similar
(p<0.01). Maximum number of females was
recorded in control while minimum in H. indica.
At the 4th harvest after 28 days though J3 were
less in H. indica, all the treatments were
statistically similar. Maximum J4 was recorded in
72
Table: Effect of entomopathogenic nematodes on the invasion and development of M. incognita in tomato
Harves
t
interval
7 Days
Treatment
Plant
Height
Root fresh
weight
J2S
dj2s
J3
J4
Females
S. glaseri
S. asiaticum
15.23a
4.5025a
15.20a
4.53a
127.3b
113.8 b
23.25 b
15.75 b
4.000 c
4.750bc
0.00 a
0.00 a
0.00 a
0.00 a
15.27a
4.57a
143.0ab
21.50 b
0.00 a
0.00a
27.50 b
0.00 a
4.537a
142.3ab
0.00 a
15.20a
15.23a
4.547a
164.5a
75.00 a
9.500a
6.500ab
c
7.500ab
0.00 a
0.00 a
31.77
13.32
3.11
H. indica
H. bacteriophora
Control
LSD
14
days
S. glaseri
21.500a
6.272a
14.75 c
35.50c
69.00 b
70.75 b
3.250 b
S. asiaticum
20.100a
6.235a
17.25c
32.50 c
70.75 b
45.50 c
2.500 b
H. indica
20.800a
21.575a
6.115a
6.142a
45.50 b
43.75b
55.25 b
67.00ab
80.75 b
66.00 b
78.25 b
88.50 b
1.250 b
5.000 b
22.800a
6.645a
62.00a
72.2 a
139.0 a
143.3 a
10.50 a
14.16
13.02
16.30
18.96
4.46
H. bacteriophora
Control
LSD
21
days
S. glaseri
22.400a
8.420a
0a
6.250d
52.750a
64.75 c
77.25 b
S. asiaticum
23.250a
8.060a
0a
7.50cd
39.750a
53.75 c
77.00 b
8.495a
0a
13.50bc
50.750a
98.50 a
73.50 b
20.00 b
44.250a
81.0 b
95.00 b
32.75a
43.750a
98.25 a
139.8 a
13.24
26.63
H. indica
21.825a
H. bacteriophora
21.975a
8.472a
0a
Control
23.475a
9.162a
0a
LSD
S. glaseri
28
days
7.06
25.475a
9.173b
0a
0a
8.000a
16.00 b
178.5 bc
0a
8.000a
11.00 b
161.5 c
S. asiaticum
26.300a
8.50b
0a
H. indica
26.625a
9.427ab
0a
0a
4.000a
12.00 b
190.5 b
9.410ab
0a
0a
8.500a
10.25 b
186.3 b
10.35a
0a
0a
7.500a
24.00 a
264.5 a
7.33
21.23
H. bacteriophora
Control
LSD
26.900a
25.550a
0.99
*Numbers followed by different letters in the same columns are significantly different from each other at 1%
probability level. Data is mean of five replications.
control and were significantly similar in all the
other treatments. Maximum number of J4 was
recorded in control while minimum was in S.
asiaticum. Number of females was significantly
lower in all the treatments as compared to the
control where only the root knot nematodes were
applied. There was maximum number of females
in control while minimum in S. asiaticum, H.
bacteriophora and H. indica. At the final harvest
after 28 days, plant height was different in each
treatment but it was statistically similar (p<0.01).
Similarly after 28 days, root weight was higher in
control and there was a difference. It was lower in
S. asiaticum and higher in H. bacteriophora.
DISCUSSION
The role of S. asiaticum, S. glaseri, H. indica and
H. bacteriophora was investigated on invasion
and development of root knot nematodes in
tomato roots. Harvesting of treatments was done at
various time intervals over a period of 28 days.
Reduction of root knot nematodes was recorded in
EPN treatments along with reduced number of
73
females in EPN treated roots. It can be concluded
that it was due to a delayed development/
maturation effect of EPN on the maturity of root
knot nematodes.
Different factors are responsible for the
suppressive
effects
of
entomopathogenic
nematodes on plant-parasitic nematodes as
competition between the nematode groups for
space in rhizosphere (Bird and Bird, 1986; Tsai
and Yeh, 1995), attraction towards the CO2 and
other root exudates (Robinson, 1995), increased
density of predators resulting from the application
of nematode biomass to the soil (Ishibashi and
Kondo, 1986), behavioral response and increased
natural enemies (Grewal et al., 1999) and
production
of
allelochemicals
by
the
entomopathogenic nematode symbiotic bacteria
complex (Grewal et al., 1999; Hu et al., 1999;
Samaliev et al., 2000; Lewis et al., 2001).
Nematicidal properties of metabolites of symbiotic
bacteria Xenorhabdus spp. associated with
Steinernema spp. (Grewal et al., 1999; Hu et al.,
1999; Samaliev et al., 2000) and P. temperate and
P. luminescens with H. megidis and H.
bacteriophora (Boemare, 2002) might be
esponsible for the suppressive effect of
entomopathogenic nematodes on root knot
nematodes. The difference in the suppressive
effect might due to the difference of the associated
bacteria and its toxic metabolites. Cell-free
extracts of Enorhabdus spp. were found to be
toxic and repellent to M. incognita juveniles and
inhibited its egg hatch (Grewal et al., 1999).
Entomopathogenic nematodes belonging to
Steinernematids were found in tomato roots.
Steinernema spp. has ability to enter in roots by
following infecting root-knot nematodes. M.
incognita suppression using Heterorhabditids was
less consistent than steinernematids (Fallon et al.,
2002). It can be concluded that the Steinernema
spp. were more efficient in suppressing M.
incognita due to their ability to enter the roots and
release associated bacteria inside the roots. The
bacteria inside the root tissue release
allelochemicals those are toxic and repellent to
root knot nematodes (Grewal et al., 1999; Fallon
et al., 2002).
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