ISOLATION AND IDENTIFICATION OF NOVEL LOCAL ISOLATES OF Bacillus thuringiensis ACTIVE AGAINST RED PALM WEEVIL ( RPW )

he gram-positive bacterium B. thuringiensis is well-known as sources of insecticidal proteins, most of which accumulate in crystalline inclusions during sporulation, these parasporal inclusions contain various types of insecticidal crystal protein (ICP). The crystal proteins from B. thuringiensis are among the most successful biological control agents for the suppression of agriculturally and medically important insect pests (Roh et al., 2007), being toxic to the larvae of lepidopteron, dipteran, and coleopteran insects (Johnson et al., 1998), as well as certain hymenoptera, homoptera, and mallophaga, in addition to many nematodes, flatworms, and Sarcomastigophora (Walters and English, 1995; Horak et al., 1996).

he gram-positive bacterium B. thuringiensis is well-known as sources of insecticidal proteins, most of which accumulate in crystalline inclusions during sporulation, these parasporal inclusions contain various types of insecticidal crystal protein (ICP).The crystal proteins from B. thuringiensis are among the most successful biological control agents for the suppression of agriculturally and medically important insect pests (Roh et al., 2007), being toxic to the larvae of lepidopteron, dipteran, and coleopteran insects (Johnson et al., 1998), as well as certain hymenoptera, homoptera, and mallophaga, in addition to many nematodes, flatworms, and Sarcomastigophora (Walters and English, 1995;Horak et al., 1996).
At present, more than 130 B. thuringiensis crystal proteins have been described based on their gene sequences and amino acid homologies (Crickmore et al., 1998).Recently, all crystal proteins show some relatedness is grouped into four main groups.B. thuringiensis isolates are distributed worldwide, and more than 60,000 have already been collected by various industries in an effort to obtain novel crystal proteins (Martin and Travers 1989;Li et al., 2007).In addition, B. thuringiensis isolates showing different crystal protein gene patterns from the reference strains have also been reported (Li et al., 2002).
The red palm weevil (RPW) Rhynchophorus ferrugineus (Olivier), a concealed tissue borer, is a lethal pest of palms and is reported to attack 17 palm species worldwide.Although the weevil was first reported on coconut (Cocos nucifera) in South Asia during the last two decades it has gained a foothold on date palm Phoenix dactylifera in several Middle Eastern countries from where it has moved to Africa and Europe, mainly due to the movement of infested planting material.In the Mediterranean region, RPW also severely damages Phoenix canariensis.Infested palms, if not early detected and treated, often declined and die.However, palms in the early stages of infestation usually respond to chemical insecticide treatments.RPW has been managed in several countries through an integrated pest management (IPM) pro-T gram (Faleiro, 2006).The search for effective natural enemies (biological control) for RPW still continues.
Bacillus thuringiensis soil isolates A21, A51 and C17 isolated from different regions of the Cuban archipelago showed higher larvicide activity than Bactivec's isolated reference strain, against both Aedes aegypti and Culex quinquefasciatus (González et al., 2011).Accordingly, This study describe the isolation and identification of novel isolates of B. thuringiensis, in order to study insecticidal activity of isolates produced proteins against Red Palm Weevil insect.

Soil Samples and microbial strains
Soil samples were collected from different locations in the Governorate of Sharkia, Egypt.For the isolation and characterization of B. thuringiensis local isolates, bacterial reference strain; B. thuringiensis var.kurstaki was provided kindly from Microbial Biology Molecular Biotechnology Laboratory, Agricultural Genetic Engineering Research Institute, Giza, Egypt.

Bacterium isolation and morphological characterization
Soil suspensions were made by adding 5 g of soil to 50 ml sterile basic salt solution.Ten fold dilutions of these suspensions were plated on Luria-Bertani (LB) agar.Only colonies from the highest dilution of the soil suspensions were selected for the isolation and identification of bacteria.Standard physiological and biochemical identification analyses were carried out as described in Bergey's Manual of Systematic Bacteriology (Sneath, 1986).The candidate isolates were examined for the distinct crystal morphology by using phase-contrast microscopy.

Protein and DNA extraction
The sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) was performed using the method of (Li et al., 2002).The DNA was extracted from the bacterial cells by boiling the bacterial growth in a water bath for 5 min to lyses the cells and then the tubes were spun briefly to collect the condensate cells (Carozzi et al., 1991).

Amplification of 16S rRNA gene
16SrRNA analysis was carried out using specific universal primers for 16SrRNA, forward primer 5'-CAGGCCTAACACATGCAAGTC-3' and reverse primer 5'-GGGCGGTGTGTACAAGGC-3' (Yoon et al., 1998).Isolated DNA from pure bacterial culture was used as template.PCR was performed with a 50 μl reaction mixture containing 16S gene primer, DNA template buffer, MgCl 2 , dNTPs and Taq polymerase.PCR program was carried out in Bio-RAD i-cycler which comprises of three steps; 1) Denaturation at 94ºC for 40 seconds; 2) Annealing at 51ºC for 50 seconds; 3) Extension at 72ºC for 1.5 minute all running for 35 cycle, with initial denaturation 94ºC for 4 minute and final extension 72ºC for 7 minute.PCR Products were analyzed by electrophoresis in 1.5% agarose gel then one of them is gelpurified using (Promega Wizard SV Gel and PCR Clean Up-system Kit cat.# A9282).After cloning PCR product into pGEM-easy vector (Promega, Madison, USA), 4 μL of the purified DNA containing cloned PCR product was sequenced using ABI PRISM (310 Genetic Analyzer).Obtained sequence was edited to exclude the PCR primer binding sites and manually corrected with Chromas 2.3 (Chromas version 2.3; www.technelysium.com.au.chromas.html).

Analysis of 16SrRNA gene sequence
16SrRNA gene sequence compared with already submitted in database using the standard nucleotide-nucleotide BLAST algorithm (Altschul et al., 1990).Further, most similar sequences were aligned by ClustalX2 version 2.0.8 soft-ware (Thompson et al.,1997), using the same software phylogenetic tree was constructed to analyze evolutionary relationships among sequence of isolated microorganism and closest relatives using the neighbor-joining (NJ) method (Saitou and Nei, 1987) with parsimony and heuristic search criteria and 1000 bootstrap replications to assess branching confidence.The identities of the relatives were determined on the basis of the highest GenBank accession numbers score.Phylogenetic tree was visualized using Dendroscope 2.6 software (Huson et al., 2007).

Preparation of crystal proteins for insect bioassay
To evaluate crystal protein production, wild-type isolate and reference strain of B. thuringiensis were typically grown in T3 sporulation medium (Donovan et al., 1988), at 25-30°C for 3-4 days, cultures that fully sporulated and lysed were harvested by centrifugation, washed once or twice in an equal volume of wash buffer (10 mM Tris-HCl [pH 7.5]-0.005%Triton X-100 or 0.005% Triton X-100 alone), and suspended at 1/10 of the original volume in the washing buffer.This 10X spore-crystal concentrate was used directly in bioassays.Sucrose step gradients (7.5 ml each of 79, 72, 68 and 55% sucrose in wash buffer) were prepared in 25-by 89-mm Ultra-Clear centrifuge tubes (Beckman Instruments, Inc.).Five ml of Spore-crystal suspensions were layered on top of the gradients.The gradients were centrifuged at 18,000 rpm at 4ºC in an L8-70 M ultracentrifuge (Beckman Instruments, Inc.) for overnight period.The protein crystals of EG11529 separated into two distinct bands, one at the 68-72% interface, and the other at the 72-79% interface.The bands were recovered from the gradients by use of a pipette and the protein crystals were concentrated by centrifugation.The protein crystals were washed twice in buffer.Crystal proteins were quantified by SDS-PAGE and densitometry by using bovine serum albumin as a standard.

Determination of insecticide activity
Bioassay screenings were performed via surface inoculation of an artificial diet (Marrone et al., 1985), Sporecrystal suspensions or gradient-purified protein crystals were diluted in an aqueous solution of 0.005% Triton X-100 and applied to the surface of the diet.After the solution had dried, first-instar larvae were placed on the diet and incubated at 28ºC.At least five concentrations and five replicated per dose were performed.Bioassays were replicated on at least three different times.Ten larvae were tested per concentration.Mortality was scored after 1-3 days by using the diluent-only treatment as the untreated check assay.All the tests were conducted at 27ºC in 60-70% humidity with a light 16 h: dark 8 h cycle and repeated three times.The number of surviving larvae per concentration as well as control was recorded at 24, 48 and 72 hours from the beginning of the experiment to estimate.The mortility percentages were plotted on Log-probit regression line.The median lethal concentra-tions (LC 50 ), standard error (SE) and the slop were determined from the regression line.The LC 50 were expressed in mg/l.A strain B. thuringiensis var.kurstaki was used as reference for comparing the results of bioassays.

Isolation and identification of local isolates
Twenty soil samples collected from different sites in Sharkia Governorate were used for the isolation of B. thuringiensis.Morphological comparison was employed between obtained bacterial colonies from soil suspensions and reference isolate colony in order to determinate candidate B. thuringiensis colony.

Candidate B. thuringiensis colonies
were tested for the presence of crystals and only those with crystals were considered for further examination.The type of crystal observed were rhomboidal and cuboidal but don't show bi-pyramidal, triangular and irregular shapes.Candidate

B. thuringiensis was designated BTRW1
and BTRW2 which was found to produce rhomboidal parasporal inclusions, while candidate BTRW3, BTRW4 and BTRW5 were found to produce cuboidal parasporal inclusions (Fig. 1), and significant differences were found in the shape and size of the vegetative cells, spores, and parasporal inclusions between BTRW isolates and reference strain B. thuringiensis subsp.var.kurstaki, which produces the bi-pyramidal crystal shape.

Isolation and sequence analysis of 16S rRNA gene
Amplification of new isolate 16SrRNA gene showed a candidate band about (1350 bp) in Fig.
(2).The purified candidate 16SrRNA fragment was ligated into pGEM-T easy vector and transformed into E. coli DH5α competent cells.Plasmids were isolated from candidate colonies obtained from transformation.Isolated fragment was sequenced using ABI PRISM (310 Genetic Analyzer).The sequence data (Fig. 3) was utilized to run a homology search using blast tool provided by NCBI, with specifying blast to the microbial 16SrRNA data.The results of the homology search revealed that the isolated fragment was designated as 16SrRNA of B. thuringiensis, Sequence showed high similarly to B. thuringiensis strain IAM 12077 16S ribosomal RNA and accession NR_043403.Blast also was performed with the sequence specifying only B. thuringiensis data, the sequence showed similarly to many B. thuringiensis accessions.Phylogenetic tree (Fig. 4) was drawn with both new isolate and its closest relative ( et al., 1992).

SDS-PAGE analysis of vegetative and sporulated cells
Protein SDS-PAGE analysis of vegetative and sporulated cells of the five isolates and reference strains revealed that the five isolates were different from each other in their banding pattern as shown in (Figs. 5 and 6).The protein contents of the sporulated cells (Fig. 5) showing two major bands (≈ 34-40 kDa).The higher mo-

Evaluation of Insecticidal activity
The insecticidal activities of the crystal proteins produced by five local isolates BTRW were evaluated against red palm weevil and cotton leaf worm In conclusion, we showed evidence of the potential of Egyptian Bt strains for insect pest control.Despite the concerns on the use of BTRW strains as a bioinsecticide, the capacity of these strains to control coleopteran pests is clear.We are also reporting a new strain with potential use for the control of coleopteran pests.These strains can be grown using inexpensive ingredients and similar fermentation technology for Bt commercial production at pilot scale.However, commercializing their use as bioinsecticides in field may be delay, in spite they have been shown to be an alternative for chemical insecticides in Egypt.

SUMMARY
lecular weight band of ≈ 130-140 KDa for reference strain B. thuringiensis subsp.var.kurstaki representing the bi-pyramidal crystal (lepidopteran toxin) and the lower band of ≈ 34-40 kDa for Bt isolates BTRWs representing the rhomboidal crystal for isolates BTRW1 and BTRW2, and cuboidal crystal for isolates BTRW3, BTRW4 and BTRW5 (Coleopteran toxin).
Bacillus thuringiensis, a gram positive bacterium known for it's widely usage to control insect pests represent a threat for agriculture.Five local isolates of Bacillus thuringiensis were found in soil samples collected from different areas in Sharkia governorate, Egypt.Morphological, Biochemical analysis and molecular biology methods were employed to identify these isolates.When crystal morphology of bacterial isolates was examined, isolates were found to produce distinct crystal proteins which were very similar to crystal proteins produced normally by Bacillus thuringiensis.Moreover, Identification was performed using 16S rRNA gene sequencing which revealed that the isolates were Bacillus thuringiensis.Protein SDS-PAGE analysis of vegetative and sporulated cells of the five isolates revealed that the protein banding pattern of the five isolates was different from each other.Insecticidal activity of the novel isolates have been tested against the red palm weevil (RPW), Rhynchophorus ferrugineus.The LC 50s of the local isolates BTRW1, BTRW2, BTRW3, BTRW4 and BTRW5 against RPW have been determined and were 0.062, 0.252, 0.178, 0.060 and 0.061 µg/ml, respectively.In the same time, no insecticidal activities have been detected for these isolates against cotton leaf worm.

Fig
Fig. (7): Slopes of the LC 50 (μg/ml) values of Bt local isolate soluble purified crystal which showed a different degrees of toxicity towards R. ferrugineus larvae.

Table ( 2
): Efficiency of local B. thuringiensis isolates and reference strain B. thuringiensis subsp.var.kurstaki crystal protein toxin against cotton leaf worm.

Table ( 3
): LC 50 and slope values for B. thuringiensis local isolates against red palm weevil larva, R. ferrugineus