Biocontrol efficiency of Bacillus thuringiensis toxins against root-knot ...

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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.

References

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Bacillus thuringiensis in soil: a review. Can.

J. For. Res. 23: 2329-2342, 1993.

Asaka O and Shoda M. Biocontrol of Rhizoctonia

solani Damping-off of tomato with Bacillus

subtilis RB14. Appl. Environ. Microbiol.

62:4081-4085, 1996.

Ayoub SM. Plant nematology, an agriculture

training aid. Nema. Aid. Publications, Sacramento,

California. USA, pp195, 1980.

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