Biocontrol efficiency of Bacillus thuringiensis toxins against root-knot ...
A Mehmet Journal of Baki Cell Yokeş and Molecular Biology 7(1): 57-66, 2008
Haliç University, Printed in Turkey.
http://jcmb.halic.edu.tr
Biocontrol efficiency of Bacillus thuringiensis toxins against
root-knot nematode, Meloidogyne incognita
S. H. Mohammed 1,* , M. Anwer El Saedy 2 , Mohamed R. Enan 1 , Nasser. E. Ibrahim 3 , A.
Ghareeb 4 and Salah. A. Moustafa 1
1
Agriculture Genetic Engineering Research institute (AGERI), Agricultural Research Center (ARC),
Giza 12619
2
Department of Plant Pathology, Faculty of Agriculture, university of Alexandria, Alexandria, Egypt
3
Department. of Bioinformatics, Genetic Engineering and Biotechnology Res. Inst., Minufiya university,
Minufiya, Egypt
4
Department of Plant, Faculty of Science, university of Zagazig, Zagazig, Egypt.
(*author for correspondence; mohamed.enan@uaeu.ac.ae)
Received 18 December 2007; Accepted 30 May 2008
_______________________________________________________________________________________
Abstract
The toxin proteins produced by Bacillus thuringiensis (Bt) are the most broadly used natural insecticides in
agriculture. To investigate the potential use of vegetative and crystal toxins to control parasitic nematodes,
we studied the nematicidal effect of Bt toxins against root-knot nematode. Nematicidal effects of
spore/crystal proteins (SCP) of ten Bt isolates were studied in vitro against Meloidogyne incognita nematode.
The spore/crystal proteins of isolates Bt7N, BtDen, Bt18, BtK73, BtSoto and Bt7 showed the highest
nematicidal activities, with the mortality range of 86-100%. In addition, ammonium sulfate cut-off fraction
of vegetative cultures of the most potent isolates (Bt7, Bt7N, BtSoto and BtDen) was examined in vitro for
their nematicidal effects. The observed mortalities of Bt7N and Bt7were 100 and 89.4% for 80% ammonium
sulfate cut-off respectively. The culture fluid (CF), cell-free supernatant (CFS) and cell-pelleted residues
(CP) of each of the four isolates (Bt7, Bt7N, BtSoto and BtDen) were evaluated for their nematicidal activities
in vivo, using tomato plants as a host. The results demonstrate that both crude suspension (CS) and cellfree
supernatant (CFS) of isolate Bt7N reduced the number of egg masses by 78% and 77% respectively, and
number of eggs by 84% and 76% compared to control.
Key Words: Bacillus thuringiensis, biological control, Meloidogyne incognita, root-knot nematode
Bacillus thuringiensis toksinlerinin kök-budak nematodu Meloidogyne incognita’ya
karşı biyokontrol etkinliği
Özet
Review Article 57
Bacillus thuringiensis (Bt) tarafından üretilen toksin proteinleri tarımda en yaygın olarak kullanılan doğal
böcek öldürücülerdendir. Vejetatif ve kristal toksinlerin parazitik nematodları kontrol etmek için potansiyel
kullanımını araştırmak amacıyla, Bt toksinlerinin nematisidal etkilerini kök-budak nematoduna
karşılaştırdık. Bt izolatının spor/kristal proteinlerinin (SCP) nematisidal etkileri Meloidogyne incognita nematoduna
karşı in vitro olarak araştırılmıştır. Bt7N, BtDen, Bt18, BtK73, BtSotoveBt7 izolatlarının
spor/kristal proteinleri %86-100 mortalite aralığıyla en yüksek nematisidal aktiviteyi göstermiştir. Ek olarak,
en etkili izolatların (Bt7, Bt7N, BtSoto ve BtDen) amonyum sülfat eşik değeri fraksiyonu vejetatif kültürlerininin
in vitro nematisidal etkileri araştırılmıştır. Bt7N ve Bt7’nin gözlenen mortaliteleri %80 amonyum
sülfat eşik değeri için sırasıyla %100 ve %89.4 bulunmuştur. Her 4 izolatın kültür sıvısı, hücresiz
A 58
S. H. Mohammed et al.
süpernatantı ve hücre-pellet kalıntıları (Bt7, Bt7N, BtSoto ve BtDen) in vivo nematisidal aktiviteleri için
domates bitkileri konak olarak kullanılarak değerlendirilmiştir. Sonuçlar Bt7N izolatının ham süspansiyonunun
(CS) ve hücresiz süpernatantının (CFS) kontrolle karşılaştırıldığında yumurta kitlelerini sırasıyla %78
ve %77 ve yumurta sayısını da sırasıyla %84 ve %76 azalttığını göstermiştir.
Anahtar Sözcükler: Bacillus thuringiensis, biyolojik kontrol, Meloidogyne incognita, kök-budak nematodu
______________________________________________________________________________________
Introduction
Nematodes are the most abundant multicellular
animals on the face of earth. Several hundreds
species of nematodes are known to feed on living
plants and cause a variety of plant diseases
worldwide. Root-knot nematodes are capable of
harshly damaging a broad range of crops, in particular
vegetables, causing dramatic yield losses
mainly in tropical and sub-tropical agriculture
(Sikora and Fernandez, 2005). During last decades,
intensive studies of nematicidal effects of Bt
have also been carried out, mainly aimed at development
of bacterial preparations effective
against economically important phyto-parasitic
nematodes such as Globodera pallida (Racke and
Sikora, 1992a, b) and M. incognita (Deviddas and
Siddiqui Rehberger, 1992; Mahmood, 1995). The
use of biological insecticides is one effective way
of coping with insect pests. There are predictions
of an annual increase of biological pesticide production
of 10-15%, in comparison with the increase
in chemical pesticide production of 1-2%
(Menn, 1996). Strains of Bt can produce toxic
compounds of various chemical structures and
properties. Most studies confirmed that δendotoxin
acts selectively against the larvae of
some target insects (Stepanova et al., 1996). Toxicity
of Bt towards several groups of soil invertebrates
other than pterygota e.g. Acarin, Nematoda,
Collembola, Amelida has also been demonstrated
(Addison, 1993). The extensive variety of
Bt strains and the toxins that they produce permit
the production of bioinsecticides using the bacteria
themselves and also allows use of the toxin
genes in the development of transgenic plants
(Romeis et al., 2006). The aim of the present
study was to identify the isolates of Bacillus thuringiensis
showing toxic activity to root-knot nematode,
the nematicidal actions of the spore/crystal
proteins and vegetative protein fractions of the
most potent isolates were studied in vitro and under
greenhouse conditions.
Materials and methods
Plant growth
Tomato seeds (Solanum Lycopersicon L. cv. Castlerock
II PVP) were obtained from agricultural
genetic engineering research institute (AGERI),
agricultural research center (ARC), ministry of
agriculture, Giza, Egypt. Seeds of tomato were
surface disinfected for 1 min with 70% ethanol,
rinsed five times with sterile distilled water and
then disinfected again with 0.5% sodium hypochloride.
The seeds were germinated as described by
Asaka and Shoda (1996). After four weeks the
seedlings were utilized for greenhouse experiment.
Bacterial strains and root-knot nematode
Bacillus thuringiensis isolates used in this study
were previously identified by their morphological,
biochemical and molecular features at microbial
molecular genetics laboratory, agricultural genetic
engineering research institute (AGERI), agricultural
research center (ARC), ministry of agriculture,
Giza, Egypt. The root-knot nematode, M. incognita
used in this study was provided from Plant Pathology
Department, Faculty of Agriculture, Alexandria
University. The root-knot nematode was reared
as definite population on tomato plants cv. Castlerock
II PVP grown in sandy clay soil.
Sample preparation
Root-knot nematode preparation
Inocula of M. incognita were prepared as described
by Hussey and Barker (1973) by extracting
nematode eggs from eight-week-old, nematode-infected
tomato roots. Active juveniles (J2)
of M. incognita were obtained by using Baermann
plate technique (Ayoub, 1980).
Bacterial preparation
For vegetative state, Bt was grown in LB broth
medium (Miller, 1972) on a rotary shaker (200
rpm) at 30°C for 18 h. Then, the vegetative supernatant
was collected by centrifugation at 8000
rpm for 10 min. For sporulation, Bt was grown in
liquid T3 medium (Yamagata et al., 1987) on a
rotary shaker (200 rpm) at 30°C for 72 h. Then
the liquid cultures of Bt isolates were used to
obtain the following bacterial preparations for the
in vivo experiments. The culture fluid (CF) was
obtained by filtration the bacterial cultures
through a Millipore membrane filter (0.22µm).
The CFS, CP, SCP, were obtained by centrifugation
at 8000 rpm for 15 min, then the pellet was
washed with sterile distilled water three times and
Nematicidal activity of a novel Bt isolate 59
A
Table 1. The nematicidal effect of different concentrations (J 2 Mortality %*) of the purified crystal
proteins of B.t. isolates on root-knot nematode, M. incognita
(Bt)
isolates
finally re-suspended in sterile distilled water to
achieve the final volume.
Preparation of spore/crystal proteins
Fifty mg of spore/crystal proteins (SCP) was dissolved
in 20 ml of 100 mmol l -1 Na2CO3 (pH 9.5)
supplemented with 10 mmol l -1 DTT (Dithiothreitol)
and stirred for at least 2 h at room temperature
and centrifuged at 15000 rpm for 15 min. The supernatant
that contains the solublized crystal protein
(pro-toxin protein) was dialyzed overnight at
4ºC against 2 liters of 100 mmol l -1 NH4HCO3
containing 0.2% β-Mercaptoethanol (Hofmann et
al., 1988).
Preparation of supernatant protein fractions from
of vegetative cultures
The supernatant was precipitated by slowly adding
ammonium sulfate cut off to get 40%, 60% and
80% fractions, respectively (Englard and Seifter,
1990). The protein was collected by centrifugation
at 15000 rpm/ 4 o C for 25 min and the protein pellets
were dissolved in 100 mmol l -1 phosphate buffered
saline (PBS) pH 7.2, then dialyzed overnight
against 100 mmol l -1 PBS (pH 7.2) for 12 h at 4 o C.
Protein Concentration (µg/ml)
32 16 8 3.2 1.6
7N 100 100 93.6 68.8 44.4
Den 100 100 91.7 66.7 35.7
Soto 98 94.5 70.5 41 31.3
18 100 92.4 88.7 79 56.6
13 94 91 83 66 22
K73 100 94 78.4 75.1 49.7
Aiz 93 90 92.7 72.9 45.9
Ento 90 75.8 84.1 74.2 73.3
7 96 93.2 82 76.2 69.8
Ber 89 72 49 21 18
Control 11.4
*Mortality = [Number of dead juveniles J2/ Total number of J2] X100.
Data are average of three replicates
A 60
S. H. Mohammed et al.
SDS-PAGE gel electrophoresis
The total protein composition of the Bt isolates
was analyzed by sodium dodecyl sulphatepolyacrylamide
gel electrophoresis (SDS-PAGE).
SDS-PAGE was conducted as described by
Laemmli (1970) with a 4% stacking gel and 10 %
separating gel. The spore/crystal and vegetative
protein fractions were extracted from the cultures
(Lecadet et al., 1991). 100 µg of proteins were
taken and mixed with 10 µl of sample buffer in
microfuge tubes and denatured by boiling for five
minutes. The samples containing equal amounts
of proteins were loaded into the wells of gels.
After electrophoresis, the gels were stained and
fixed in 40% methanol, 10% acetic acid and
coomassie blue (0.1%) for about 12 h, and destained
in 40 % methanol and 10 % acetic acid for
2 h with agitation.
Nematicidal activity of the purified spore/crystal
proteins
Different concentrations of the soluble crystal/spore
protein (SCP) of ten B.t. isolates were
prepared by adding the appropriate volumes of
distilled water to the standard solution. Direct
contact assay was carried out in 24-well plate by
modification of the standard method described by
Prasad et al. (1972). One ml of each concentra-
tion was added to 50µl of nematode J2 suspension
(containing about 12 J2) in each well, and incubated
at room temperature. A solution containing 100
mmol l -1 Na2CO3 (pH 9.5) and 10 mmol l -1 DTT
was used as a control treatment. All treatments
were replicated three times. The numbers of active
and dead J2 were counted under compound microscope
24 h post incubation, then mortality percentages
was calculated. Lethal concentration (LC50) of
the soluble protoxin of each B.t. isolates was determined
by probit analysis (Bourgouin et al.,
1990).
Nematicidal activity of vegetative proteins
Various concentrations of each vegetative protein
fraction of Bt isolates were prepared. One ml of
each concentration was transferred to 24 well
plates, and then 50 µl of nematode J2 suspension
(15 J2) were added to each well, and incubated at
room temperature. Phosphate-buffered saline (100
mmol l -1 ) was used as a control treatment.
Greenhouse experiment
Nematicidal activity of Bt was tested against nematodes
inhabiting the rhizosphere of tomato plants.
Tomato seeds were planted in pots, each pot was
15 cm in diameter and 14 cm in depth. All pots
were filled with 1kg of autoclaved soil mixture;
Figure 1. SDS-PAGE of the purified soluble crystals proteins of the selected B.t. isolates. Lanes 1-5 represent
B.t. isolates 13, 18, Ber, Ento, and K73 (Gel A). Lanes 6-10 represent B.t. isolates 7, Den, Soto, 7N, and Aiz
(Gel B). Lane M represents pre-stained protein marker; the protein gel was stained by coomassie blue stain
clay: sand (1: 3, v: v). Three tomato seedlings
were transplanted in each pot. Nematode egg
suspensions were applied to all pots at transplanting;
the density of inoculums was adjusted to
13000 egg /pot. After nematode inoculation, 500
ml of each bacterial treatment were applied. Each
isolate of Bt was applied in three phases; CF,
CFS, and CP. Each phase was represented by five
replicates. Five pots were left without bacterial
treatment to serve as a control. Pots were arranged
in a complete randomized design. The
pots were maintained for two months in the
greenhouse at 25°C. The root systems were harvested
and assessed for galling (number of
galls/root system), and egg masses/root using an
aqueous solution of phloxine-B stain (0.15 gl -1
tap water) for 15-20 min, and then roots were
rinsed in running tap water to remove residual
stain (Ayoub, 1980).
Statistical analysis
The data were analyzed with one-way analysis of
variance (ANOVA) using SAS software (SAS
Institute, 1988) to calculate numbers of nematode
galls and egg-masses.
Nematicidal activity of a novel Bt isolate 61
A
Table 2. The Effect of different concentrations (J 2 Mortality*) of vegetative protein fractions of the isolates Bt7,
Bt7N, BtDen, and BtSoto on root-knot nematode, M. incognita
(Bt) isolates Protein fraction
Results
Nematicidal activity and purification of
spore/crystal proteins
The nematicidal activity of the spore/crystal proteins
of the B.t. isolates (ten isolates) was tested
against the J2 of the root-knot nematode, M. incognita
in order to screen the most potent isolates.
Data in Table 1 showed the effect of different concentrations
of the solubilized crystal proteins of the
isolates. The isolates Bt7N, BtDen, Bt18, BtK73,
BtSoto, and Bt7 showed the highest nematicidal
activity (100, 100, 100, 100, 98 and 96 % mortality),
respectively at concentration 32 µg/ml and
their (LC50s) were (2.22, 2.56, 4.30, 1.28, 3.52, 1.82
µg/ml) after 24 h of incubation, respectively. The
B.t. isolates were grown for sporulation and the
bacterial culture was collected by centrifugation,
the spores/crystals mixtures of the ten Bt isolates
were resolved on SDS-PAGE. The electrophoretic
profile under denaturing conditions showed a very
specific banding pattern for each isolate, the protein
content from ten isolates of Bt are quantitatively
and qualitatively different. The banding pattern
reported major soluble crystal proteins with mole-
Protein concentration (µl/ml)
60 30 15 6 3
Bt 7 40% fraction 35.5 28.2 18.8 14.4 8
60% fraction 71.1 30.9 18.6 10.2 3.5
80% fraction 89.4 21.3 8.5 5.8 2
Bt 7N 40% fraction 72 59.2 20.6 10 4.8
60% fraction 34 20 10.6 9 7
80% fraction 100 77.8 67.3 36.6 24.6
Bt Den 40% fraction 70 62 23 10 7
60% fraction 50 28 15 11 3
80% fraction 55 21 18 8 5
Bt Soto 40% fraction 69 53 19 12 8
60% fraction 68 55 19 9 4
80% fraction 26 11 9 5 3
Control 3.5
*Mortality = [Number of dead juveniles J2/ Total number of juveniles J2] X 100
• Data are average of three replicates
62
A S. H. Mohammed et al.
cular mass ∼130 kDa are in all tested isolates, in
addition to other polypeptides with molecular
weight ranging from 30 to 120 kDa, while this
polypeptide is (130 kDa) not stated in isolate Bt7
(Figure 1 A and B).
Nematicidal activity and fractionation of vegetative
proteins
The nematicidal activity of the supernatant of
vegetative cultures (exo-secreted proteins) of the
most potent isolates (Bt7, Bt7N, BtSoto, and
BtDen) was tested in controlling the J2 of M.
incognita. Data in Table 2 showed the effect of
different concentrations of the supernatant protein
fractions (40, 60, and 80% ammonium sulfate
cut-off) on nematode. Protein fraction 80% of Bt7
and Bt7N achieved the highest mortality (89.4
and 100%, respectively) at concentration of 60
µg/ml while the protein fraction 40% of isolates
BtSoto and BtDen achieved 69 and 70 % mortality,
respectively, by the same concentration. The
LC50 of the vegetative supernatant protein fractions
40, 60, and 80% of isolate Bt7 were 176.1,
41.7, and 37.9 µg/ml and in case isolate Bt7N
were 30.8, 206.5, and 9.6 µg/ml, 24 h after incubation.
While, the LC50 of protein fractions 40,
60, and 80% of BtSoto and BtDen isolates were
(34.4, 34.30, and 216.1 µg/ml) and (30.1, 74.1, and
67.1 µg/ml), after 24 h of incubation, respectively.
The vegetative proteins of the most potent Bt isolates
(Bt7, Bt7N, BtSoto, and BtDen) have been
fractionated using ammonium sulfate cut off. The
qualitative and quantitative profiles of the vegetative
protein fractions 40, 60, and 80% of isolates
Bt7, Bt7N, BtDen and BtSoto were assessed
through SDS-PAGE analysis as shown in Figures 2
A and B. The vegetative protein fractions were
resolved by SDS-PAGE showed that heterogeneous
protein profiles. The results analysis of Bt7N fractions
40 % and 80 % gave distinctive polypeptide
with molecular mass ∼35 kDa, whereas this polypeptide
was not reported in protein fractions of
isolate Bt7 as indicated in Figure 2A. The 35 kDa
polypeptide of isolate BtDen was stated in the
protein fractions 40% (lFigure2B), and not reported
in the protein fractions 60 and 80 % (Figure2B).
Smilarly, this characteristic 35 KDa polypeptide
was not revealed in the protein fractions of the
isolate BtSoto. Therefore, based on the presence
and absence of major distinctive polypeptide, the
protein fractions 40 and 80% of the isolate Bt7N
have been selected for the greenhouse experiment.
Figure 2. The SDS-PAGE of the vegetative proteins from the selected B.t. isolates. Lanes 1-3: represent Bt7N
(fractions; 40, 60, and 80%), respectively, Lanes 4-6: represent Bt7 (fractions; 40, 60, and 80%), respectively, (Gel
A). Lanes 1-3: represent BtDen (fractions; 40, 60, and 80 %), respectively, Lanes 4-6 represent BtSoto (fractions;
40, 60, and 80%), respectively, (Gel B). Lane M: represents pre-stained protein marker. The protein gel was stained
by coomassie blue stain.
Greenhouse experiment
The nematicidal activity of the three phases, CF,
cell-free supernatant CFS, CP, of the four selected
B.t. isolates (Bt7, Bt7N, BtDen, and Bt
soto) were performed under green house conditions.
The nematicidal effects of the most active
B.t. isolates showed that all treatments increased
the root fresh weight in compared to control (Table
3). CFS Bt7N reduced root galling by 52%,
number of egg masses by 77%, and number of
eggs by 76%. In contrast, the CFS of Bt7, BtDen
and B.t. soto reduced number of egg masses by
36, 56, and 69% respectively. In the same trend,
CF of Bt7N reduced number of egg masses by
78% and number of eggs by 84%. The effect of
the culture fluid of Bt7N on tomato is clearly
apparent as indicated in Figures 3 A and 3B.
Whereas, CF of the isolates Bt7, BtDen and Btsoto
resulted in decrease in the number of egg
masses by 70, 68, and 62% respectively.
Discussion
The most destructive diseases that destroy our
crops are caused by many plant pathogenic organisms,
of these diseases are those caused by
plant pathogenic nematodes. The crystal proteins
made by the bacterium Bt are pore-forming toxins
that specifically target insects and nematodes are
used around the world to eradicate insect pests.
The first step in this study was targeted to evaluate
the nematicidal activity of ten Bt isolates,
and testing the ability of their soluble crystal
proteins in controlling root-knot nematode. The
Nematicidal activity of a novel Bt isolate 63A
toxicity varied between the isolates, it is wellknown
that Bt can produce a number of toxins of
different structure and mode of action. The
spore/crystal mixture analyzed by SDS-PAGE
showed a major polypeptide of ~130 kDa, corresponding
to Cry1 toxins. The results of SDS-PAGE
analysis of tested Bt isolates revealed heterogeneous
profiles, this may be due to the genetic dissimilarity
among them. The highest nematicidal toxicity
of the tested isolates is likely to be correlated to the
presence of high concentration of the major polypeptide.
Our results are in agreement with the previous
results obtained by Yamamoto and Powell
(1993). The effect of the soluble spore/crystal proteins
on the mortality of 2 nd stage juveniles (J2) of
M. incognita showed that both isolate Bt7N and
BtDen are the most efficient isolates in vitro. They
achieved the maximum J2 mortality (100%). The
lethal concentration (LC50) of Bt7N and BtDen
soluble crystal proteins were 2.2 and 2.6 µg/ml,
respectively. The results of the current research are
in agreement with several numbers of earlier studies
(Kotz, et al., 2005; Huffman et al., 2004; Wei
et al., 2003; Griffitts et al., 2003; Hala et al., 2003;
Mozgovaya et al., 2002; Lopez-Arellano et al.,
2002; Griffitts et al., 2001). Both the genetic constitution
and the bioassay results are the determining
factors in selecting the most potent isolates
(Bt7, Bt7N, BtSoto, and BtDen). Examination of
vegetative protein fractions by SDS-PAGE showed
that the differences in the number and intensity of
protein banding profiles among supernatant. This
difference in intensity is probably caused by a
higher and lower secretion of vegetative protein.
Polypeptide with molecular mass ~ 35 kDa is the
Figure 3. The nematicidal effect of Bt7N culture fluid on tomato plants in comparison with control (A). The nematicidal
effect of Bt7N supernatant on tomato plant roots in comparison with control (B). Letter C, represents M.
incognita infected control pot. Letter T, represents Bt treated pot.
64
A S. H. Mohammed et al.
expected for nematicidal activity of vegetative
proteins. In vitro study of nematicidal activities of
the vegetative protein fractions 40%, 60%, and
80% of the four isolates indicated that, the protein
fraction of 80% gave the highest mortality in case
of Bt7 and Bt7N isolates, while the protein fraction
40% gave the highest mortality in case of
BtSoto and BtDen isolates. The results also
showed that the rate of mortality increased with
increasing fraction concentrations (concentrationdependent).
Since the highest nematicidal activity
was detected in both fractions 40% and 80% of
the isolate Bt7N. We tested the biocontrol activity
of four Bt isolates in controlling root-knot nematode,
M. incognita in greenhouse experiment on
tomato plants. The results of greenhouse experiment
indicated that the culture fluid, cell-free
suspension, and cell pelleted residues of the four
selected Bt isolates clearly showed a suppressive
effect on the occurrence of root galling of M.
incognita. The data indicated that both crude
suspension and cell-free supernatant of the isolate
Table 3. The effect of Bt7, Bt7N, Bt Den, and Bt Soto isolates on root-knot nematode, M. incognita on tomato plants.
Reduction% = [(Control-Treatment) / Control] X 100
Bt isolates
Control
Root
fresh wt.
(g)
No.
of
Galls
*Reduction
%
Bt7N were the most active in reducing both the
nematode egg masses and number of eggs. In contrast,
the cell-free supernatant of isolate Bt7N gave
the highest reduction in number of nematode galls
(Table 3). The results also showed that all treatments
increase the root fresh weight in comparison
with the control. The CFS of Bt7N caused the significant
reduction in the galling compared to control
plants. It is evident that both CFS and CF of
the Bt7N isolate were the most active fractions in
reducing both number of egg masses and number
of eggs. Previous studies have already shown that
B.t. strain CR-371 lead to a 53% reduction of tomato
root galling caused by M. incognita (Zuckermann
et al., 1993 and Rehberger, 1992). The reduction
of egg masses and number of eggs was reached
to maximum when Bt7N was applied. Although in
a previous study it was suggested that nematicidal
action of Bt toxins do not hold promise as biological
control agents (Borgonie et al., 1996), our outcome
in this investigation demonstrates that toxin
protein of Bt7N isolate which was not available in
No. of Egg
masses
*Reduction
%
No. of
eggs
33 470 - 414 - 135 -
*Redu
ction
%
Bt 7
Culture fluid 41 252 46 125 70 43 68
Supernatant
59 347 26 265 36 87 36
Cell Pellet
44 368 22 217 48 68 50
Bt 7N
Culture fluid
48 450 4 91 78 22 84
Supernatant
45 227 52 95 77 33 76
Cell Pellet
57 401 15 174 58 51 62
Bt Den
Culture fluid 46 309 34 132 68 34 75
Supernatant
46 358 24 183 56 48 64
Cell Pellet 63 400 15 262 37 77 43
Bt Soto
Culture fluid
47 429 9 159 62 58 57
Supernatant
44 382 19 127 69 47 65
Cell Pellet
63 474 - 270 35 87 35
any previous studies does hold such promise.
Nematicidal activity of Bt toxins might provide
an effective strategy to control plant-parasitic
nematodes. The importance of this goal is underscored
by the fact that methyl bromide is
mandated by the Montreal protocol to be phased
out as one of the most extensively used nematicidal
agent in agricultural. The data suggest the
feasibility and usefulness of searching for protein-derived
(vegetative protein) nematicidal
fraction in Bt supernatant as mean of developing
specific and efficient alternatives of biological
control to be engaged in integrated pest management
programs of nematodes.
Conclusion
Applications of formulated Bt are not toxic to
bird, fish, and most beneficial or predator insects;
and there is no evidence that Bt causes teratogenic
effects in mammals (PIP, 1996). This study
reports an alternative nematicidal protein from
Bt7N that could be providing an effective policy
for the biological control of nematodes. However,
additional research is required to identify the
active principles present in the toxin proteins of
Bt7N, this would help in increasing our weapon
store to overcome nematodes through the development
of the proper formulations.
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