Estimation of the Efficiency of Bacterial Isolate Compared to Commercial Formulated Bioinsecticides Against Spodoptera littoralis (Boisduval) in Vitro

The journal of Toxicology and pest control is one of the series issued twice by the Egyptian Academic Journal of Biological Sciences, and is devoted to publication of original papers related to the interaction between insects and their environment. The goal of the journal is to advance the scientific understanding of mechanisms of toxicity. Emphasis will be placed on toxic effects observed at relevant exposures, which have direct impact on safety evaluation and risk assessment. The journal therefore welcomes papers on biology ranging from molecular and cell biology, biochemistry and physiology to ecology and environment, also systematics, microbiology, toxicology, hydrobiology, radiobiology and biotechnology. www.eajbs.eg.net Provided for non-commercial research and education use. Not for reproduction, distribution or commercial use.


INTRODUCTION
In Egypt, the cotton leafworm Spodoptera littoralis (Boisduval) (Lepidoptera: Noctuidae) is destructive phytophagous lepidopterous pest causing various ravages not only for cotton plants (Ahmad, 1988 andHatem et al., 2009) but also for other field crops, vegetables (Hosny et al., 1986), ornamentals and orchard trees (Domínguez, 1993 andBelda et al., 1994) all over the year in Egypt (Hamouda and Dahi, 2008).Development of an effective control method against this pest is urgently needed since it does serious damage to many important agricultural crops in Egypt.There is a serious interest in the use of microbial insecticides for biological control of the cotton leafworm, as alternatives to chemical control, since they neither leave toxic chemical residues in the environment nor do they develop resistance in their insect hosts.And hence, the public awareness and concern for environmental quality, has led to more focused attention on research aiming at developing biological agents.A promising strategy with good potential to control insect pests and, at the same time, to minimize the adverse effects of chemical insecticides is the use of microbial agents (Ahmed and El-Katatny, 2007).For the purpose of evaluating the different biological and biochemical effects of commercially formulated Bacillus thuringiensis kurstaki (BTK) and a locally isolated BTK, the present study was conducted under laboratory conditions.

Insects used
A laboratory susceptible strain of the cotton leaf worm Spodopter littoralis (Boisd.)(Lepidoptera: Noctuidae), reared for more than 10 generations was obtained from the Research Division of the cotton leaf worm, Plant Protection Research Institute.Insects were reared under controlled conditions in an incubator at 26 ± 2º C, of 65 ± 10% R. H., and 8:16 L: D photoperiod at the Plant Protection Research Institute, Dokki-Giza, Egypt (El-Sawaf, 1971).

Bacterial isolation:
Soil samples from maize field at El-Dakhaly village, the western side of Rashid branch, Kom Hamada Center, Beheira Governorate were collected in glass flasks.About 1g of soil was cultured in 10mL of Nutrient Broth for 24h at 30°C with orbital shaking at 150rpm.From the culture, 2mL was taken and heat-shocked in water bath at 60°C for 1h.Then the samples were spread on Nutrient Agar plates (0.5% Peptone, 0.3% beef extract, 0.5% NaCl and 1.5% agar), incubated at 30°C for 24h and checked for the presence characteristic colonies of B. thuringiensis phenotype (matte white colour, flat, dry and uneven borders).Each sporulated culture was then heat fixed and stained (0,133 % Coomassie Blue stain in 50 % acetic acid) in a glass microscope slide (Ammons et al., 2002).The Bt-like cells identification was performed by microscopic examination using a Leyca DM 750 bright field microscope (Leyca Microsystems).

Molecular characterization of Bacillus thuringeinsis kurstaki:
The locally isolated B. thuringiensis strains were grown for 24 h on LB broth medium at 30°C as follows: 1) Pellets of 5 × 10 6 cells were grown in suspension.This was done by spinning at 1200xg in a centrifuge tube.The supernatant was discarded.Then the cells was washed once with PBS and resuspended with 200 μl cold (4°C) PBS; 2) 25 μl of OB Protease (D3496) or Proteinase K (D3495) was added at 20 mg/ml solution; it was vortexed to mix well, and incubated at 65 °C for 5 min to complete lysis; 3) 220 μl Buffer BL was added and vortexed to mix well.It was incubated at 70 °C for 10 min.4) The column was placed in a second 2 ml tube and washed by pipetting 750 μl of wash Buffer diluted with ethanol; it was centrifuged at 8,000xg for 1 min, and collection tube and flow-through liquid were disposed; 5) A new collection tube was used, the column was washed with a second 750 μl of wash Buffer and centrifuged as above.Discard flow through; 6) the same collection tube was used, centrifuged at highest speed (10,000 x g) for 2 min, to dry the column.7) The column was placed in a sterile 1.5 ml microfuge tube and 200 μl of preheated (70°C) Elution Buffer was added.Tubes were allowed to sit for 3 min, at room temperature.Incubating at 70°C rather than at room temperature will give a modest increase in DNA yield per elution.Alternatively, the second elution may be performed using the first eluate.The expected yield from a 30 mg sample is 8-35 μg genomic DNA, depending on type of tissue.

Polymerase chain reaction (PCR)
PCR amplification of the CryI gene of the local B. thuringiensis strains was conducted using according to the procedure previously published by Ahmed et al. (2015).All molecular identification techniques were performed at the Central Laboratory, Faculty of Science, Ain Shams University.

Biological tests
The half of the recommended concentration of the product was obtained according to the recommendation of the Egyptian Ministry of Agriculture.0.125g of each commercial product was dissolved in a 100ml of distilled water.The isolated B. thuringeinsis was used at concentration 65×10 8 spores/ml.Newly ecdysed 2 nd and 4 th instar S. littoralis larvae (12-24 h old) were offered castor bean oil leaves treated with Protecto and Dipel DF at 0.125 gm/100 ml distilled water, and isolated B. thuringeinsis at concentration 65×10 8 spores/ml for 48h.After treatment time, larvae were offered untreated leaves.Treatment of larvae was conducted by the leaf dipping technique (Tabashnik et al., 1991).Two sets of five replicates each contain 10 newly molted 2 nd and 4 th instar larvae for each concentration of each tested product.The same numbers of larvae were considered as a control, these larvae were offered castor oil leaves immersed in distilled water.The following parameters were recorded: Larval instars duration, from the initial treated instar up to pupation.Percentage of pupation and mean pupal duration.Percentage adult emergence and mean adult longevity.

Biochemical tests
Larvae at 6 th instar larvae that survived treatment with tested bioagents at 4t instar larvae were selected.The larvae of each instar were anaesthetized and rinsed with 5 ml acetone to remove surface residues, the larvae were weighed then homogenized in phosphate buffer (pH7) using a Teflon tissue homogenizer surrounded by crushed ice.The homogenates were centrifuged at 8000 rpm for 20 min at 4º C and the supernatant was used directly for the determination of the following:

Enzymes assay
The following enzymes activity was determined: Non-specific α-and βesterase activity was measured as described by Van Asperen (1962) using α-naphthyl acetate and β-naphthyl acetate, respectively, as substrates.
Acid and alkaline phosphatase activity was measured from the larval heamolymph as described by Laufer and Schin (1971).

Statistical analysis:
Data were statistically analyzed by ANOVA procedure (Snedecor and Cochran, 1980) at P<0.05 using SPSS statistics 17.0 release 17.0.0software.When the ANOVA statistics were significant at (P ≤ 0.01), means were compared by the Duncan's multiple range test using SPSS 17.0 software.

Bacterial identification
Microscope examinations of the B. thuringiensis strain indicated that the bacteria were gram positive, rod-shape cells containing only one endospore.The polymerase chain reaction products for B. thuringiensis subsp.Kurstaki were 521 bp for Cry1A (Fig. 1).

Biological studies:
Larvae that treated as 2 nd and 4 th instar larvae, and survived treatment with half recommended concentrations of Protecto ® and Dipel DF ® (0.125g/100ml), and isolated B. thuringeinsis (at concentration 65×10 8 spores/ml) showed that the duration of those instars and their subsequent instars up to pupation was affected (Table 1).
Obtained results showed that the mean larval durations of treated 2 nd and 4 th instar larvae with tested agents were decreased compared to control.Results also showed that there was a low significant difference obtained in case of Protecto ® compared to control, while no significant difference obtained when both Dipel DF ® and isolated BTK.Results also revealed that the pupation percentage of 2 nd and 4 th instar larvae survived treatment with tested bioagents decreased to approximately half.Obtained results showed that treatment with tested bioagents shortened the mean pupal duration of survived pupae that treated as 2 nd and 4 th instar larvae.Low significant difference was obtained in case of Protecto ® and Dipel DF ® .No significant difference was observed in case of treatment with BTK in survived pupae that treated as 2 nd and 4 th instar larvae.2) showed the latent effect of tested bioinsecticides on adult emergence rate and mean adult longevity for adults treated as 2 nd and 4 th larval instars.Results showed that Treatment of 2 nd and 4 th instar larvae with tested bioagents reduced the adult emergence rate.In addition, results showed that the smallest adult emergence percentage was observed when the BTK isolate was applied.Furthermore, obtained data revealed shortening in mean male adult longevity for all treatments, although no significant difference was detected.On the other hand, the mean female adult longevity was increased and no significant difference was observed.These results agreed with previously published researches by Dutton et al. (2003);Gamil (2004); Abdel-Aziz (2007); and Abdel-Salam et al. (2013) found that the development time of larvae and pupae were extended as well as adult emergence after treatment with bacterial agents.Results also agreed with Hegab and Zaki (2012) who found the same effects when treated both the pink bollworm, Pectinophora gossypiella and the spiny bollworm, Earias insulana larvae, with biocide Btk (Dipel 2×).Abedi et al. (2014) reported similar delay in mean larval duration, mean pupal duration, and mean adult longevity.

Biochemical studies: Effect of tested bioinsecticides on αand β-esterases activity:
Data presented in Table (3) showed the effect of treatment of 4 th instar larvae with tested bioagents on the nonspecific esterases (α-and βesterases) in late 6 th instar larvae.Results showed that treatment with Protecto ® and Dipel DF ® decreased the β-esterase activity compared to control.However, treatment with BTK isolate increased the β-esterase activity.
Results also showed that the αesterase activity was highly increased due to treatment with all tested bioagents compared to control.BTK treatment has the highest enzyme activity.

Effect of tested bioinsecticides on Acid Phosphatase (ACP) and Alkaline Phosphatase (ALP) activity:
Effect of treatment of the 4 th larval instar with tested bioagents on the detoxification enzymes, Acid phosphatase (ACP) and Alkaline phosphatase (ALP), in the late 6 th instar larvae of S. littoralis is shown in Table (4).ACP activity decreased significantly in all treated groups comparing to the control.In addition, treatment with tested bioagents decreased the ALP activity compared to untreated larvae.Generally speaking, increase of activity of detoxification enzymes is the most universal resistant mechanism in insects.
As a result of infection with B. thuringiensis kurstaki the epithelial cells undergo an excessive oxidation due to bacterial endotoxin, viral replication and their combined action.This may be caused a stress on the lysosome, leading to their rupture, and finally the release of the acid phosphatase enzyme.It has been reported that substances and bioagents can increase the number of lysosomes inside the cell as defensive mechanism against the attack of these substances (Assar et al., 2010;Gaikwad et al., 2010;and Mohamed and Mohamady, 2010).The levels of acid phosphatase can be used as a marker on the cell death (Mohamed and Mohamady, 2010).

Table 1 :
Effect of Protecto ® , Dipel DF ® , and isolated Bucillus thuringiensis kurstaki (BTK) on mean larval duration, mean pupal duration, and pupation percentage of Spodoptera littoralis treated as 2 nd and 4 th instar larvae Means followed by the same letter in a column are not significantly different at the 5% level of probability (Duncan's Multiple Range Test).

Table 2 :
Effect of Protecto ® , Dipel DF ® , and isolated Bucillus thuringiensis kurstaki (BTK) on adult emergence percentage and mean adult longevity of Spodoptera littoralis treated as 2 nd and 4 th Means followed by the same letter in a column are not significantly different at the 5% level of probability (Duncan's Multiple Range Test).

Table 3 :
Effect of Protecto ® , Dipel DF ® , and isolated Bucillus thuringiensis kurstaki (BTK) on αesterase and β-esterase activity in the late 6 th instar larvae of Spodoptera littoralis treated as 4 th Means followed by the same letter in a column are not significantly different at the 5% level of probability (Duncan's Multiple Range Test).

Table 4 :
Effect of Protecto ® , Dipel DF ® , and isolated Bucillus thuringiensis kurstaki (BTK) on Acid Phosphatase (ACP) and Alkaline Phosphatase (ALP) activity in the late 6 th instar larvae of S. littoralis treated as 4 th instar larvae Treatment ACP activity (U×10 3 ml/gm per body weight) (Mean± S.E.) Means followed by the same letter in a column are not significantly different at the 5% level of probability (Duncan's Multiple Range Test).