00
Content
Egyptian Journal of Natural Toxins, Vol. 6(1): 83-93, January 2009
THE EFFECT OF ENDOTOXIN PRODUCED BY BACILLUS THURINGIENSIS (BT.) AGAINST MELOIDOGYNE INCOGNITA
Tamer S. Abd El-Moneim, and Samia I. Massoud
Suez Canal University, Faculty of Agriculture, Department of Agricultural Botany, Ismailia, Egypt
Received 21/10/2008 Accepted 22/12/2008
ABSTRACT
isolated from soil F our isolates of Bacillus thuringiensis were plant collected20from samples of the rhizosphere of Alfa alfa four locations in Ismailia governorate. The colonies on tryptone casein hydrolysate (TCH) medium were divided into four different morphological groups based on morphological characters of the colony and crystal shape within the isolate as observed by light microscopy. Two isolates (AI, AII) produced typical spherical crystals, one isolate (AIII) produced a pyramidal crystal and the fourth one (AIV) produced a bipyramidal crystal. The analysis of the bacterial endotoxin by SDS-PAGE showed that the crystals from the isolates AI, AII and AIII contained protein bands of 128,125 and 139 KDa respectively, while the isolate AIV contained a protein band of 143 KDa. The treatment of the second stage juvenile (J2) of Meloidogyne incognita with purified crystals toxin (1×108 crystal/1ml) showed the best result in the treatment with isolates AI and AII. Key words: Bacillus thuringiensis, endotoxin, parasitic nematode, SDS-PAGE.
INTRODUCTION
The effects of Bacillus thuringiensis (Bt.) as a nematocidal biocontrol agent has been investigated for free living nematodes, animal-parasitic nematodes, insect-parasitic nematodes and plantparasitic nematodes. The strains Bt. israelensis, Bt. kurstaki and Bt. morrisoni showed considerable variability with respect to lethality for animal-parasitic and free living nematodes (Bottjer et al., 1985).
* Corresponding author: Tamer S. Abd El-Moneim; [email protected]
The Effect of Endo Toxin Produced By Bacillus thuringiensis (Bt.) Against Meloidogyne incognita
Similarly, Osman et al. (1988) evaluated two commercially available strains of Bacillus thuringiensis (SAN 415 and Dipel), which had a biocontrol effect agents against Meloidogyne javanica and Tylenchulus semipenetrans. They found that SAN 415 strain was more effective than Dipel strain, and it was approximately similar to the standard nematicide named Nemacur. Moreover, Medina et al. (1990) found that B. thuringiensis had antagonistic action against phytophagus nematode and reduced its populations to levels of no economic importance. However, Devidas and Rehberger (1992) tested two formulations of exotoxin preparation from B. thuringiensis against M. incognita by applying them in cucumber seeds. They reported the absence of any nematicidal activity or any effect on egg hatching. Zuckerman et al. (1993) tested the efficacy of an isolate of B. thuringiensis in controlling plant parasitic nematode. The treatment with such isolate gave a significant reduction in galls due to Meloidogyne infestation on tomato than in the untreated controls. Carneiro et al. (1998) tested twenty-one strains belonging to Bacillus sp. against J2 of M. javanica in vitro and in greenhouse. They found that bacterial supernatant and whole culture of B. thuringiensis-brasiliensis and B. laterosporus killed freshly hatched J2 within 24 to 48 hours. Marroquin et al. (2000) studied the effect of protein toxins produced by B. thuringiensis on the nematode Caenorhabditis elegans to investigate the potential of these toxins to control parasitic nematodes. They found that the nematode undergoes extensive damage to the gut and decrease infertility followed by death consistent with toxin effects in insects. They concluded that the Bt. toxins hold promise as nematicides. Braun (2000) reported that the crystal proteins encoded by Cry genes have been classified as CryI to CryVI depending on a host specificity and amino acid homology. The homology group CryI to CryV specified to insects as a host and CryVI specified to nematode as a host. El-Nagdi and Yossef (2004) tested the soaking of faba beans seeds or treating their soil with the bio-agent (strain of B. thuringiensis) and compared the results with the nematicide Oxamyl. The biocontrol agent significantly reduced the population density of M. incognita with increasing the measured plant growth. On this basis, seed soaking in such biocontrol agent was recommended as an economical method for managing M. incognita.
84
Abd El-Moneim, T.S. and Massoud, S.I.
The aim of the present study is to investigate the effect of the endotoxin produced by Bacillus thuringiensis against root knot nematode (Meloidogyne incognita) as a biological agent, and comparing between bacterial isolates based on crystal toxin shape as well as toxin protein profile using SDS-PAGE technique.
MATERIAL AND METHOD
Isolation of Bacillus thuringiensis from soil Twenty soil samples were collected from rhizosphere of Alfa alfa plant from four locations in Ismailia governorate. Soil samples (1 g) were added to L-broth medium (20 ml) supplemented with sodium acetate (0.25 M, pH 6.8) in Erlenmeyer flask (125 ml). The mixture was shaken at 1,000 × g for 4 h at 30ºC. Samples of about 0.5 ml in 10 ml test tubes were heated for 5 min in a water bath at 80ºC to kill vegetative cells and nonspore-forming bacteria, and used to inoculate L-agar medium plates, without adding sodium acetate to the medium. Colonies formed after overnight growth at 30ºC were transferred onto TCHA medium (tryptone, 5 g/liter; casein hydroly-sate, 2 g/liter; K2HPO4, 12.5 mM; MgSO4, 12.5 mM; MnSO4, 0.05 mM; ZnSO4, 1.2 mM; Fe2(SO4)3, 1.2 mM; H2SO4, 0.5%; CaCl2, 25 mM and Agar 15 gm/liter) complemented with 0.3% glucose. Cultures were allowed to grow and sporulate for 40 h at 30ºC then examined by light microscope based on colony morphology and crystal shape (Braun, 2000). Mass culturing of isolated colonies Colonies formed on TCHA after incubation at 30ºC were selected and removed individually in 2 ml of TCH (without agar) medium complemented with 0.3% glucose and allowed to grow at 30°C for 8 h with vigorous shaking (300×g). The culture was then added to 100 ml of the same medium in 500-ml flasks and allowed to grow under the same conditions, as the pre-culture, until sporulation and lysis were completed. Five replicas were prepared for each isolate. Cultures were checked for the presence of parasporal crystals by light microscope. Purification of the crystals toxin Crystals toxin and spores were harvested by centrifugation at 4,000 × g at 4°C for 15 min. The pellet was resuspended in 250 ml of 1 M NaCl and vigorously shaken until formation of spore rich foam, which
85
The Effect of Endo Toxin Produced By Bacillus thuringiensis (Bt.) Against Meloidogyne incognita
was then removed with a spatula. The suspension was centrifuged again as mentioned above. The pellet was resuspended in 10 ml of sterile distilled water containing 1 mM phenylmethyl-sulfonyl fluoride and stored on ice prior to sonication at 100 W for 30 s to disrupt aggregates of crystals, and then centrifuged at 16,000 × g for 90 min at 4°C. The band containing purified crystals was collected, diluted fivefold in distilled water, and centrifuged at 20,000 × g for 30 min at 4°C. The last step was repeated three times. The remained pellet was resuspended in 1 ml of sterile distilled water containing 1 mM phenylmethyl-sulfonyl fluoride. The purity of the sample, (presence of more than 20 crystals for one spore) was checked by light microscopy. Then samples were partitioned and stored at -20°C. Comparing between isolates based on crystals toxin by SDS-PAGE Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate (SDS-PAGE) was prepared using a 10% (w/v) separating gel (pH 8.8) and 5% (w/v) stacking gel (pH 6.8) as described by Laemmli (1970). Purified crystals were mixed with equal volume of electrophoresis sample buffer (0.06 M Tris-HCl [pH 6.8], 2% SDS, 10% glycerol, 5% 2-mercaptoethanol, 0.01% bromophenol blue). The obtained sample was incubated in a water bath (80-90ºC) for 5 min and then immediately cooled on ice. Twenty microliter of each protein sample of the obtained isolates were loaded on adjacent lane. Electrophoresis device was adjusted at 100 volt and 80 mA until the tracking dye reached to the bottom of the gel. The gel was stained with 0.1% Coomassie blue R-250 in 7% (v/v) acetic acid containing 50% methanol. The protein molecular marker was purchased from Sigma (St. Louis, Mo, USA). Determination of toxicity Toxicity of toxin crystals was determined through bioassay against plant parasitic nematode (Meloidogyne incognita) second stage juvenile (J2) by using three dilutions of purified crystal toxin PCT (app. 1×105, 1×106 and 1×108 Cry/1ml) for the tested isolates of Bt. (AI, AII, AIII,AIV). The twelve treatments plus the untreated one were replicated three times as follows: 1 ml from the first dilution of PCT from isolate AI was added into sterilize Ependorf tube 2 ml in size, which contained 500 freshly hatching J2 of M. incognita in 1 ml of distilled water. After 30 min from PCT addition, the treated J2 were
86
Abd El-Moneim, T.S. and Massoud, S.I.
added to susceptible tomato seedling (Lycopersicon esculantum Mill cv. Rutgers) grown in the pots 25 cm in diameter at 2 cm depth trench around the stem-base and was covered with soil after inoculations. The same treatment was repeated with another PCT dilution (1×106 and 1×108 Cry/1ml) as was done in isolate AI, but with change of Bt. isolates and PCT dilutions. In addition, seedling inoculated with J2 and left free from treatment with PCT to serve as a check. Forty five days, at the end of the experiment, tomato plants were removed from the soil to determine J2/root system, galls/root system and the plant performance such as shoot dry weight (g) and plant height (cm).
RESULTS
Four isolates of Bacillus thuringiensis were isolated from soil samples. The obtained bacterial colonies on TCHA medium were divided into four different morphological groups based on colony morphology and crystal shape as observed by light microscopy (Fig. 1). Two isolates produced typical spherical crystals (AI, AII), one isolate produced a pyramidal crystal (AIII) and other produced a bipyramidal crystal (AIV). The analysis of the bacterial endotoxin by SDS-PAGE (Fig. 2) showed that the crystals of isolates AI, AII and AIII contained a single protein band 128,125 and 139 KDa respectively, while the isolate AIV contained a protein band 143 KDa. Data presented in table (1) and illustrated in figure (3) showed the influence of four isolates of Bacillus thuringiensis (AI, AII, AIII, AIV), by using three dilutions of purified crystal toxin PCT (app. 1×105, 1×106 and 1×108 cry/1ml) on the J2 of M. incognita infection and plant performance after 45 days from nematode infection. Results from table (1) indicate that all the treatments gave a significant reduction in nematode infection comparing with untreated plant. The highest reduction of nematode (173 J2/root system and 18 galls/root system) was recorded in case of treatment with bacterial isolates AII at 1×108 cry/ml (as a dilution of crystal toxin / one ml) comparing with untreated tomato plants (378.3 J2/root system and 197.0 galls/root system). Moreover, plant performance such as plant shoot dry weight (g) and plant height (cm), the highest plant parameter (532.0 g and 50 cm) were recorded in the same treatment (AII at1×108 cry/ml) comparing with untreated plant (321.0 g and 38 cm).
87
The Effect of Endo Toxin Produced By Bacillus thuringiensis (Bt.) Against Meloidogyne incognita
A
B
C
Figure (1): Light micrograph of purified crystal toxin of B. thuringiensis A: Spherical crystals toxin (Bt.AI, AII); B: Pyramidal crystal toxin (Bt.AIII); C: Bipyramidal crystal toxin (Bt.AIV).
M AI AII AIII AIV 200 97.4 68.0 M AI AII AIII AIV
43.0 29.0 15.0
Figure (2): Polypeptide pattern of bacterial crystal endotoxin by SDS-PAGE analysis. (M: marker)
88
Table (1): The influence of four isolates of Bacillus thuringiensis (Bt.) on the nematode infection and plant performance after 45 day from experiment
Abd El-Moneim, T.S. and Massoud, S.I.
-The mean sample size = 3.00 -Values in the same column followed by different letters are significantly different according to Duncan's multiple range test (P = 0.05).
89
The Effect of Endo Toxin Produced By Bacillus thuringiensis (Bt.) Against Meloidogyne incognita
J2/root system
Galls/root system
Untreated Isolate AIV
Untreated Isolate AIV Isolate AIII Isolate AII Isolate AI
0 50 100 150 200 250 300 350 400
1.00E+08 1.00E+06 1.00E+05 1.00E+08 1.00E+06 1.00E+05 1.00E+08 1.00E+06 1.00E+05
1.00E+08 1.00E+06 1.00E+05 1.00E+08 1.00E+06 1.00E+05 1.00E+08 1.00E+06 1.00E+05 1.00E+08 1.00E+06 1.00E+05 0 50 100 Plant height (cm) 150 200 250
Isolate AII Isolate AI
Isolate AIII
1.00E+08 1.00E+06 1.00E+05
Plant shoot dry weight (g)
Untreated Isolate AIV
Untreated Isolate AIV Isolate AIII Isolate AII Isolate AI
0 100 200 300 400 500 600
1.00E+08 1.00E+06 1.00E+05 1.00E+08 1.00E+06 1.00E+05 1.00E+08 1.00E+06 1.00E+05
1.00E+08 1.00E+06 1.00E+05 1.00E+08 1.00E+06 1.00E+05 1.00E+08 1.00E+06 1.00E+05 1.00E+08 1.00E+06 1.00E+05 0 10 20 30 40 50 60
Isolate AII Isolate AI
Isolat e AIII
1.00E+08 1.00E+06 1.00E+05
Figure (3): The influence of four isolates of Bacillus thuringiensis on the nematode infection and plant performance after 45 days from experiment.
90
Abd El-Moneim, T.S. and Massoud, S.I.
DISCUSSION
Very little work has been done to study the nematicidal activity of Bacillus thuringensis toxin against plant parasitic nematode generally and the root-knot nematode Meloidogyne spp. in particular. In the present study, the mode of action of bacterial toxin on J2 of M. incognita was due to bacterial endotoxin, the efficacy of endotoxin as a nematicidal effect based on the morphological structure of crystal toxin. The spherical crystal toxin gave the highest reduction in nematode population because they can easily pass through the nematode mouth part. These results are supported by the results obtained by Devidas and Rehberger (1992), who applied exotoxin prepared by B. thuringiensis against M. incognita and reported no nematicidal effect on egg hatching. The present results are in agreement with Osman et al. (1988), Zuckerman et al. (1993), Carneiro et al. (1998) and El-Nagdi and Yossef (2004), who used the whole culture or bacterial supernatant which killed freshly hatched J2. The toxin produced by the four studied isolates of B. thuringiensis were classified into three groups based on the toxin structure, which observed by light microscope. Two bacterial isolates (AI and AII) produced typical spherical crystal shape, and one (AIII) isolate produced a pyramidal crystal, while the other produced a bipyramidial crystal (AIV). These results were confirmed by using SDS-PAGE. The crystals from isolates AI and AII contained a protein bands very close to each other (128,125 KDa respectively). These two isolates gave highly significant reduction in J2 and galls on tomato plants comparing with the untreated plants. The other two isolates (AIII and AIV) recorded significant reduction in nematode population on tomato plants comparing with plant check, but in less values than those recorded in the presence of isolates AI and AII . The highly percent of increase in plant performance values (plant shoot dry weight (g) and plant height (cm) were found in the presence of bacterial isolates AII, AI then AIII and AIV at concentrated level of crystal toxin 1X108/ 1ml comparing with untreated plant.
REFERENCES
Bottjer, K.P.; Leon, W. and Sarjeet, S. (1985): Nematode: Susceptibility of the egg to Bacillus thuringiensis toxins. Exp. Parasitol., 60: 239-244.
91
The Effect of Endo Toxin Produced By Bacillus thuringiensis (Bt.) Against Meloidogyne incognita
Braun, S. (2000): Production of Bacillus thuringiensis insecticides for experimental uses.49-72 In: A. Navon and K.R.S. Ascher, "Bioassays of Entomopathogenic Microbes and Nematodes". CABI, London, UK. Carneiro, R.M.; De Souza, I.S. and Belarmino, L.C. (1998): Nematicidal activity of Bacillus spp. strains on juveniles of Meloidogyne javanica. Nematol. Mediterr, 22(1): 12-21. Devidas, P. and Rehberger, L.A. (1992): The effect of exotoxin thuringiensis from Bacillus thuringiensis on Meloidogyne incognita and Caenorhabditis elegans. Plant Soil, 145(1): 115-120. El-Nagdi, W.M. and Youssef, M.M. (2004): Soaking faba bean seed in some bio-agents as prophylactic treatment for controlling Meloidogyne incognita root-knot nematode infection. J. Pest Sci., 77(2): 75-78. Laemmli, U.K. (1970): Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London), 227: 680685. Marroquin, L.D.; Elyassnia, D.; Griffitts, J.S.; Feitelson, J.S. and Aroian, R.V. (2000): Bacillus thuringiensis (Bt.) toxin susceptibility and isolation of resistance mutants in the nematode Caenorhabditis elegans. Gen. Soc. Amer., 155: 1693-1699. Medina, J.R.; Torres, D.D.; Zorilla, R.A.; Davide, R.G. and Santiago, D.R. (1990): Promising biological control technologies for integrated pest management. College, Laguna, Philippines 14. Osman, G.Y.; Salem, F.M. and Ghattas, A. (1988): Bio-efficiency of two bacterial insecticide strains of Bacillus thuringinesis as a biological control agent in comparison with a nematocide, Phenamiphos, on certain parasitic nematodes. Sci. J. Fac. Sci. Menoufia Univ., 2: 17-25. Zuckerman, B.M. Dicklow, and Acosta, N. (1993): A strain of Bacillus thuringiensis for the control of plant parasitic nematodes. Biocontrol Sci. Techn., 3: 41-46.
92
.Abd El-Moneim, T.S. and Massoud, S.I
تأثير السم الداخلي المنتج بواسطة بكتيريا الباسيلس سيرونجنسيس ضد نيماتودا مليدوجينى انكوجنيتا
تامر شوقي عبد المنعم، سامية إبراھيم مسعود
قسم النبات الزراعي – كلية الزراعة - جامعة قناة السويس – اإلسماعيلية - مصر
تم عزل أربعة عزالت من بكتيريا باسيلس سيرونجنسيس من 02 عينة تربة تم تجميعھا من منطقة الريزوسفير لنباتات البرسيم الحجازي من أربعة أماكن بمحافظة اإلسماعيلية. قسمت المستعمرات النامية على بيئة TCHإلى أربعة مجموعات مورفولوجيا ً بناءاً على شكل المستعمرات النامية وشكل البلورات الداخلية بواسطة الفحص الميكروسكوبي. وقد احتوت عزلتان ) (AI , AIIعلى الشكل الكروي للبلورات و احتوت عزلة واحدة ) (AIIIعلى بلورات ذات شكل ھرمي، في حين إحتوت العزلة األخيرة ) (AIVعلى بلورات ذات شكل ھرمي مضاعف. وبتحليل التفريد الكھربي لبروتين السم الداخلي للعزالت األربعة اظھر اختالفات فيما بينھم فسجلت العزالت )128 (AI,AII, AIIIو 521و 931 كيلو دالتون على الترتيب. بينما كانت العزلة ) (AIVلھا حزمة بروتينية 341 كيلودالتون، وبمعاملة الطور اليرقى الثاني لنيماتودا تعقد الجذور بالبلورات الداخلية للبكتيريا أعطت أفضل النتائج عند تركيز 1 810 Xبلورة / 1ملى للعزالت كلھا وكانت أفضل النتائج في حالة استخدام الشكل البلورى الكروى في حالة العزلتين . AII,AI
39
THE EFFECT OF ENDOTOXIN PRODUCED BY BACILLUS THURINGIENSIS (BT.) AGAINST MELOIDOGYNE INCOGNITA
Tamer S. Abd El-Moneim, and Samia I. Massoud
Suez Canal University, Faculty of Agriculture, Department of Agricultural Botany, Ismailia, Egypt
Received 21/10/2008 Accepted 22/12/2008
ABSTRACT
isolated from soil F our isolates of Bacillus thuringiensis were plant collected20from samples of the rhizosphere of Alfa alfa four locations in Ismailia governorate. The colonies on tryptone casein hydrolysate (TCH) medium were divided into four different morphological groups based on morphological characters of the colony and crystal shape within the isolate as observed by light microscopy. Two isolates (AI, AII) produced typical spherical crystals, one isolate (AIII) produced a pyramidal crystal and the fourth one (AIV) produced a bipyramidal crystal. The analysis of the bacterial endotoxin by SDS-PAGE showed that the crystals from the isolates AI, AII and AIII contained protein bands of 128,125 and 139 KDa respectively, while the isolate AIV contained a protein band of 143 KDa. The treatment of the second stage juvenile (J2) of Meloidogyne incognita with purified crystals toxin (1×108 crystal/1ml) showed the best result in the treatment with isolates AI and AII. Key words: Bacillus thuringiensis, endotoxin, parasitic nematode, SDS-PAGE.
INTRODUCTION
The effects of Bacillus thuringiensis (Bt.) as a nematocidal biocontrol agent has been investigated for free living nematodes, animal-parasitic nematodes, insect-parasitic nematodes and plantparasitic nematodes. The strains Bt. israelensis, Bt. kurstaki and Bt. morrisoni showed considerable variability with respect to lethality for animal-parasitic and free living nematodes (Bottjer et al., 1985).
* Corresponding author: Tamer S. Abd El-Moneim; [email protected]
The Effect of Endo Toxin Produced By Bacillus thuringiensis (Bt.) Against Meloidogyne incognita
Similarly, Osman et al. (1988) evaluated two commercially available strains of Bacillus thuringiensis (SAN 415 and Dipel), which had a biocontrol effect agents against Meloidogyne javanica and Tylenchulus semipenetrans. They found that SAN 415 strain was more effective than Dipel strain, and it was approximately similar to the standard nematicide named Nemacur. Moreover, Medina et al. (1990) found that B. thuringiensis had antagonistic action against phytophagus nematode and reduced its populations to levels of no economic importance. However, Devidas and Rehberger (1992) tested two formulations of exotoxin preparation from B. thuringiensis against M. incognita by applying them in cucumber seeds. They reported the absence of any nematicidal activity or any effect on egg hatching. Zuckerman et al. (1993) tested the efficacy of an isolate of B. thuringiensis in controlling plant parasitic nematode. The treatment with such isolate gave a significant reduction in galls due to Meloidogyne infestation on tomato than in the untreated controls. Carneiro et al. (1998) tested twenty-one strains belonging to Bacillus sp. against J2 of M. javanica in vitro and in greenhouse. They found that bacterial supernatant and whole culture of B. thuringiensis-brasiliensis and B. laterosporus killed freshly hatched J2 within 24 to 48 hours. Marroquin et al. (2000) studied the effect of protein toxins produced by B. thuringiensis on the nematode Caenorhabditis elegans to investigate the potential of these toxins to control parasitic nematodes. They found that the nematode undergoes extensive damage to the gut and decrease infertility followed by death consistent with toxin effects in insects. They concluded that the Bt. toxins hold promise as nematicides. Braun (2000) reported that the crystal proteins encoded by Cry genes have been classified as CryI to CryVI depending on a host specificity and amino acid homology. The homology group CryI to CryV specified to insects as a host and CryVI specified to nematode as a host. El-Nagdi and Yossef (2004) tested the soaking of faba beans seeds or treating their soil with the bio-agent (strain of B. thuringiensis) and compared the results with the nematicide Oxamyl. The biocontrol agent significantly reduced the population density of M. incognita with increasing the measured plant growth. On this basis, seed soaking in such biocontrol agent was recommended as an economical method for managing M. incognita.
84
Abd El-Moneim, T.S. and Massoud, S.I.
The aim of the present study is to investigate the effect of the endotoxin produced by Bacillus thuringiensis against root knot nematode (Meloidogyne incognita) as a biological agent, and comparing between bacterial isolates based on crystal toxin shape as well as toxin protein profile using SDS-PAGE technique.
MATERIAL AND METHOD
Isolation of Bacillus thuringiensis from soil Twenty soil samples were collected from rhizosphere of Alfa alfa plant from four locations in Ismailia governorate. Soil samples (1 g) were added to L-broth medium (20 ml) supplemented with sodium acetate (0.25 M, pH 6.8) in Erlenmeyer flask (125 ml). The mixture was shaken at 1,000 × g for 4 h at 30ºC. Samples of about 0.5 ml in 10 ml test tubes were heated for 5 min in a water bath at 80ºC to kill vegetative cells and nonspore-forming bacteria, and used to inoculate L-agar medium plates, without adding sodium acetate to the medium. Colonies formed after overnight growth at 30ºC were transferred onto TCHA medium (tryptone, 5 g/liter; casein hydroly-sate, 2 g/liter; K2HPO4, 12.5 mM; MgSO4, 12.5 mM; MnSO4, 0.05 mM; ZnSO4, 1.2 mM; Fe2(SO4)3, 1.2 mM; H2SO4, 0.5%; CaCl2, 25 mM and Agar 15 gm/liter) complemented with 0.3% glucose. Cultures were allowed to grow and sporulate for 40 h at 30ºC then examined by light microscope based on colony morphology and crystal shape (Braun, 2000). Mass culturing of isolated colonies Colonies formed on TCHA after incubation at 30ºC were selected and removed individually in 2 ml of TCH (without agar) medium complemented with 0.3% glucose and allowed to grow at 30°C for 8 h with vigorous shaking (300×g). The culture was then added to 100 ml of the same medium in 500-ml flasks and allowed to grow under the same conditions, as the pre-culture, until sporulation and lysis were completed. Five replicas were prepared for each isolate. Cultures were checked for the presence of parasporal crystals by light microscope. Purification of the crystals toxin Crystals toxin and spores were harvested by centrifugation at 4,000 × g at 4°C for 15 min. The pellet was resuspended in 250 ml of 1 M NaCl and vigorously shaken until formation of spore rich foam, which
85
The Effect of Endo Toxin Produced By Bacillus thuringiensis (Bt.) Against Meloidogyne incognita
was then removed with a spatula. The suspension was centrifuged again as mentioned above. The pellet was resuspended in 10 ml of sterile distilled water containing 1 mM phenylmethyl-sulfonyl fluoride and stored on ice prior to sonication at 100 W for 30 s to disrupt aggregates of crystals, and then centrifuged at 16,000 × g for 90 min at 4°C. The band containing purified crystals was collected, diluted fivefold in distilled water, and centrifuged at 20,000 × g for 30 min at 4°C. The last step was repeated three times. The remained pellet was resuspended in 1 ml of sterile distilled water containing 1 mM phenylmethyl-sulfonyl fluoride. The purity of the sample, (presence of more than 20 crystals for one spore) was checked by light microscopy. Then samples were partitioned and stored at -20°C. Comparing between isolates based on crystals toxin by SDS-PAGE Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate (SDS-PAGE) was prepared using a 10% (w/v) separating gel (pH 8.8) and 5% (w/v) stacking gel (pH 6.8) as described by Laemmli (1970). Purified crystals were mixed with equal volume of electrophoresis sample buffer (0.06 M Tris-HCl [pH 6.8], 2% SDS, 10% glycerol, 5% 2-mercaptoethanol, 0.01% bromophenol blue). The obtained sample was incubated in a water bath (80-90ºC) for 5 min and then immediately cooled on ice. Twenty microliter of each protein sample of the obtained isolates were loaded on adjacent lane. Electrophoresis device was adjusted at 100 volt and 80 mA until the tracking dye reached to the bottom of the gel. The gel was stained with 0.1% Coomassie blue R-250 in 7% (v/v) acetic acid containing 50% methanol. The protein molecular marker was purchased from Sigma (St. Louis, Mo, USA). Determination of toxicity Toxicity of toxin crystals was determined through bioassay against plant parasitic nematode (Meloidogyne incognita) second stage juvenile (J2) by using three dilutions of purified crystal toxin PCT (app. 1×105, 1×106 and 1×108 Cry/1ml) for the tested isolates of Bt. (AI, AII, AIII,AIV). The twelve treatments plus the untreated one were replicated three times as follows: 1 ml from the first dilution of PCT from isolate AI was added into sterilize Ependorf tube 2 ml in size, which contained 500 freshly hatching J2 of M. incognita in 1 ml of distilled water. After 30 min from PCT addition, the treated J2 were
86
Abd El-Moneim, T.S. and Massoud, S.I.
added to susceptible tomato seedling (Lycopersicon esculantum Mill cv. Rutgers) grown in the pots 25 cm in diameter at 2 cm depth trench around the stem-base and was covered with soil after inoculations. The same treatment was repeated with another PCT dilution (1×106 and 1×108 Cry/1ml) as was done in isolate AI, but with change of Bt. isolates and PCT dilutions. In addition, seedling inoculated with J2 and left free from treatment with PCT to serve as a check. Forty five days, at the end of the experiment, tomato plants were removed from the soil to determine J2/root system, galls/root system and the plant performance such as shoot dry weight (g) and plant height (cm).
RESULTS
Four isolates of Bacillus thuringiensis were isolated from soil samples. The obtained bacterial colonies on TCHA medium were divided into four different morphological groups based on colony morphology and crystal shape as observed by light microscopy (Fig. 1). Two isolates produced typical spherical crystals (AI, AII), one isolate produced a pyramidal crystal (AIII) and other produced a bipyramidal crystal (AIV). The analysis of the bacterial endotoxin by SDS-PAGE (Fig. 2) showed that the crystals of isolates AI, AII and AIII contained a single protein band 128,125 and 139 KDa respectively, while the isolate AIV contained a protein band 143 KDa. Data presented in table (1) and illustrated in figure (3) showed the influence of four isolates of Bacillus thuringiensis (AI, AII, AIII, AIV), by using three dilutions of purified crystal toxin PCT (app. 1×105, 1×106 and 1×108 cry/1ml) on the J2 of M. incognita infection and plant performance after 45 days from nematode infection. Results from table (1) indicate that all the treatments gave a significant reduction in nematode infection comparing with untreated plant. The highest reduction of nematode (173 J2/root system and 18 galls/root system) was recorded in case of treatment with bacterial isolates AII at 1×108 cry/ml (as a dilution of crystal toxin / one ml) comparing with untreated tomato plants (378.3 J2/root system and 197.0 galls/root system). Moreover, plant performance such as plant shoot dry weight (g) and plant height (cm), the highest plant parameter (532.0 g and 50 cm) were recorded in the same treatment (AII at1×108 cry/ml) comparing with untreated plant (321.0 g and 38 cm).
87
The Effect of Endo Toxin Produced By Bacillus thuringiensis (Bt.) Against Meloidogyne incognita
A
B
C
Figure (1): Light micrograph of purified crystal toxin of B. thuringiensis A: Spherical crystals toxin (Bt.AI, AII); B: Pyramidal crystal toxin (Bt.AIII); C: Bipyramidal crystal toxin (Bt.AIV).
M AI AII AIII AIV 200 97.4 68.0 M AI AII AIII AIV
43.0 29.0 15.0
Figure (2): Polypeptide pattern of bacterial crystal endotoxin by SDS-PAGE analysis. (M: marker)
88
Table (1): The influence of four isolates of Bacillus thuringiensis (Bt.) on the nematode infection and plant performance after 45 day from experiment
Abd El-Moneim, T.S. and Massoud, S.I.
-The mean sample size = 3.00 -Values in the same column followed by different letters are significantly different according to Duncan's multiple range test (P = 0.05).
89
The Effect of Endo Toxin Produced By Bacillus thuringiensis (Bt.) Against Meloidogyne incognita
J2/root system
Galls/root system
Untreated Isolate AIV
Untreated Isolate AIV Isolate AIII Isolate AII Isolate AI
0 50 100 150 200 250 300 350 400
1.00E+08 1.00E+06 1.00E+05 1.00E+08 1.00E+06 1.00E+05 1.00E+08 1.00E+06 1.00E+05
1.00E+08 1.00E+06 1.00E+05 1.00E+08 1.00E+06 1.00E+05 1.00E+08 1.00E+06 1.00E+05 1.00E+08 1.00E+06 1.00E+05 0 50 100 Plant height (cm) 150 200 250
Isolate AII Isolate AI
Isolate AIII
1.00E+08 1.00E+06 1.00E+05
Plant shoot dry weight (g)
Untreated Isolate AIV
Untreated Isolate AIV Isolate AIII Isolate AII Isolate AI
0 100 200 300 400 500 600
1.00E+08 1.00E+06 1.00E+05 1.00E+08 1.00E+06 1.00E+05 1.00E+08 1.00E+06 1.00E+05
1.00E+08 1.00E+06 1.00E+05 1.00E+08 1.00E+06 1.00E+05 1.00E+08 1.00E+06 1.00E+05 1.00E+08 1.00E+06 1.00E+05 0 10 20 30 40 50 60
Isolate AII Isolate AI
Isolat e AIII
1.00E+08 1.00E+06 1.00E+05
Figure (3): The influence of four isolates of Bacillus thuringiensis on the nematode infection and plant performance after 45 days from experiment.
90
Abd El-Moneim, T.S. and Massoud, S.I.
DISCUSSION
Very little work has been done to study the nematicidal activity of Bacillus thuringensis toxin against plant parasitic nematode generally and the root-knot nematode Meloidogyne spp. in particular. In the present study, the mode of action of bacterial toxin on J2 of M. incognita was due to bacterial endotoxin, the efficacy of endotoxin as a nematicidal effect based on the morphological structure of crystal toxin. The spherical crystal toxin gave the highest reduction in nematode population because they can easily pass through the nematode mouth part. These results are supported by the results obtained by Devidas and Rehberger (1992), who applied exotoxin prepared by B. thuringiensis against M. incognita and reported no nematicidal effect on egg hatching. The present results are in agreement with Osman et al. (1988), Zuckerman et al. (1993), Carneiro et al. (1998) and El-Nagdi and Yossef (2004), who used the whole culture or bacterial supernatant which killed freshly hatched J2. The toxin produced by the four studied isolates of B. thuringiensis were classified into three groups based on the toxin structure, which observed by light microscope. Two bacterial isolates (AI and AII) produced typical spherical crystal shape, and one (AIII) isolate produced a pyramidal crystal, while the other produced a bipyramidial crystal (AIV). These results were confirmed by using SDS-PAGE. The crystals from isolates AI and AII contained a protein bands very close to each other (128,125 KDa respectively). These two isolates gave highly significant reduction in J2 and galls on tomato plants comparing with the untreated plants. The other two isolates (AIII and AIV) recorded significant reduction in nematode population on tomato plants comparing with plant check, but in less values than those recorded in the presence of isolates AI and AII . The highly percent of increase in plant performance values (plant shoot dry weight (g) and plant height (cm) were found in the presence of bacterial isolates AII, AI then AIII and AIV at concentrated level of crystal toxin 1X108/ 1ml comparing with untreated plant.
REFERENCES
Bottjer, K.P.; Leon, W. and Sarjeet, S. (1985): Nematode: Susceptibility of the egg to Bacillus thuringiensis toxins. Exp. Parasitol., 60: 239-244.
91
The Effect of Endo Toxin Produced By Bacillus thuringiensis (Bt.) Against Meloidogyne incognita
Braun, S. (2000): Production of Bacillus thuringiensis insecticides for experimental uses.49-72 In: A. Navon and K.R.S. Ascher, "Bioassays of Entomopathogenic Microbes and Nematodes". CABI, London, UK. Carneiro, R.M.; De Souza, I.S. and Belarmino, L.C. (1998): Nematicidal activity of Bacillus spp. strains on juveniles of Meloidogyne javanica. Nematol. Mediterr, 22(1): 12-21. Devidas, P. and Rehberger, L.A. (1992): The effect of exotoxin thuringiensis from Bacillus thuringiensis on Meloidogyne incognita and Caenorhabditis elegans. Plant Soil, 145(1): 115-120. El-Nagdi, W.M. and Youssef, M.M. (2004): Soaking faba bean seed in some bio-agents as prophylactic treatment for controlling Meloidogyne incognita root-knot nematode infection. J. Pest Sci., 77(2): 75-78. Laemmli, U.K. (1970): Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London), 227: 680685. Marroquin, L.D.; Elyassnia, D.; Griffitts, J.S.; Feitelson, J.S. and Aroian, R.V. (2000): Bacillus thuringiensis (Bt.) toxin susceptibility and isolation of resistance mutants in the nematode Caenorhabditis elegans. Gen. Soc. Amer., 155: 1693-1699. Medina, J.R.; Torres, D.D.; Zorilla, R.A.; Davide, R.G. and Santiago, D.R. (1990): Promising biological control technologies for integrated pest management. College, Laguna, Philippines 14. Osman, G.Y.; Salem, F.M. and Ghattas, A. (1988): Bio-efficiency of two bacterial insecticide strains of Bacillus thuringinesis as a biological control agent in comparison with a nematocide, Phenamiphos, on certain parasitic nematodes. Sci. J. Fac. Sci. Menoufia Univ., 2: 17-25. Zuckerman, B.M. Dicklow, and Acosta, N. (1993): A strain of Bacillus thuringiensis for the control of plant parasitic nematodes. Biocontrol Sci. Techn., 3: 41-46.
92
.Abd El-Moneim, T.S. and Massoud, S.I
تأثير السم الداخلي المنتج بواسطة بكتيريا الباسيلس سيرونجنسيس ضد نيماتودا مليدوجينى انكوجنيتا
تامر شوقي عبد المنعم، سامية إبراھيم مسعود
قسم النبات الزراعي – كلية الزراعة - جامعة قناة السويس – اإلسماعيلية - مصر
تم عزل أربعة عزالت من بكتيريا باسيلس سيرونجنسيس من 02 عينة تربة تم تجميعھا من منطقة الريزوسفير لنباتات البرسيم الحجازي من أربعة أماكن بمحافظة اإلسماعيلية. قسمت المستعمرات النامية على بيئة TCHإلى أربعة مجموعات مورفولوجيا ً بناءاً على شكل المستعمرات النامية وشكل البلورات الداخلية بواسطة الفحص الميكروسكوبي. وقد احتوت عزلتان ) (AI , AIIعلى الشكل الكروي للبلورات و احتوت عزلة واحدة ) (AIIIعلى بلورات ذات شكل ھرمي، في حين إحتوت العزلة األخيرة ) (AIVعلى بلورات ذات شكل ھرمي مضاعف. وبتحليل التفريد الكھربي لبروتين السم الداخلي للعزالت األربعة اظھر اختالفات فيما بينھم فسجلت العزالت )128 (AI,AII, AIIIو 521و 931 كيلو دالتون على الترتيب. بينما كانت العزلة ) (AIVلھا حزمة بروتينية 341 كيلودالتون، وبمعاملة الطور اليرقى الثاني لنيماتودا تعقد الجذور بالبلورات الداخلية للبكتيريا أعطت أفضل النتائج عند تركيز 1 810 Xبلورة / 1ملى للعزالت كلھا وكانت أفضل النتائج في حالة استخدام الشكل البلورى الكروى في حالة العزلتين . AII,AI
39
