Biocontrol efficiency of Bacillus thuringiensis toxins ... - CiteSeerX

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Mehmet Baki Journal of CellYokeş and Molecular Biology 7(1): 57-66, 2008 Haliç University, Printed in Turkey. http://jcmb.halic.edu.tr

Review Article

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Biocontrol efficiency of Bacillus thuringiensis toxins against root-knot nematode, Meloidogyne incognita S. H. Mohammed1,*, M. Anwer El Saedy2, Mohamed R. Enan1, Nasser. E. Ibrahim3, A. Ghareeb 4 and Salah. A. Moustafa1 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; [email protected]) 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 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

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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.

Nematicidal activity of a novel Bt isolate

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Sample preparation Root-knot nematode preparation

finally re-suspended in sterile distilled water to achieve the final volume.

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).

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).

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

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/ 4oC 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 4oC.

Table 1. The nematicidal effect of different concentrations (J2 Mortality %*) of the purified crystal proteins of B.t. isolates on root-knot nematode, M. incognita

(Bt) isolates

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

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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.

Results Nematicidal activity spore/crystal proteins

and

purification

Protein fraction

Protein concentration (µl/ml) 6

3

28.2 18.8 14.4 30.9 18.6 10.2 21.3 8.5 5.8 Bt 7N 59.2 20.6 10 20 10.6 9 77.8 67.3 36.6 Bt Den 62 23 10 28 15 11 21 18 8 Bt Soto 53 19 12 55 19 9 11 9 5 Control 3.5 *Mortality = [Number of dead juveniles J2/ Total number of juveniles J2] X 100 • Data are average of three replicates

8 3.5 2 4.8 7 24.6 7 3 5 8 4 3

Bt 7

40% fraction 60% fraction 80% fraction 40% fraction 60% fraction 80% fraction 40% fraction 60% fraction 80% fraction 40% fraction 60% fraction 80% fraction

of

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-

Table 2. The Effect of different concentrations (J2 Mortality*) of vegetative protein fractions of the isolates Bt7, Bt7N, BtDen, and BtSoto on root-knot nematode, M. incognita

(Bt) isolates

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60 35.5 71.1 89.4 72 34 100 70 50 55 69 68 26

30

15

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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

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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 2nd 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.

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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

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

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

Root fresh wt. (g)

No. of Galls

*Reduction %

No. of Egg masses

*Reduction %

No. of eggs

*Redu ction %

Control

33

470

-

414

-

135

-

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

48

450

4

91

78

22

84

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

47

429

9

159

62

58

57

44

382

19

127

69

47

65

63

474

-

270

35

87

35

Bt isolates

Bt 7N Culture fluid Supernatant

Bt Soto Culture fluid Supernatant Cell Pellet


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 Addison JA. Persistence and non-target effects of 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. Borgonie G, Claeys M, Leyns F, Arnaut G and De Waele D. Effect of a nematicidal Bacillus thuringiensis strain on free-living nematodes. Characterization of the intoxication process. Fundam. Appl. Nematol. 19:523-528, 1996. Bourgouin C, Delecluse A, Torre F and Szulmajster J. Transfer of the toxin protein genes of Bacillus sphaericus into Bacillus thuringiensis subsp. israelensis and their expression. Appl. Environ. Microbiol. 56:340-344, 1990.

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