Saoussen Ben Khedher

MEDIUM OPTIMIZATION OF ANTIFUNGAL ACTIVITY PRODUCTION BY Bacillus amyloliquefaciens USING STATISTICAL EXPERIMENTAL DESIGN

In order to overproduce biofungicides agents by Bacillus amyloliquefaciens BLB371, a suitable culture medium was optimized using response surface methodology. Plackett–Burman design and central composite design were employed for experimental design and analysis of the results. Peptone, sucrose, and yeast extract were found to significantly influence antifungal activity production and their optimal concentrations were, respectively, 20 g/L, 25 g/L, and 4.5 g/L. The corresponding biofungicide production was 250 AU/mL, corresponding to 56% improvement in antifungal components production over a previously used medium (160 AU/mL). Moreover, our results indicated that a deficiency of the minerals CuSO4, FeCl3 · 6H2O, Na2MoO4, KI, ZnSO4 · 7H2O, H3BO3, and C6H8O7 in the optimized culture medium was not crucial for biofungicides production by Bacillus amyloliquefaciens BLB371, which is interesting from a practical point of view, particularly for low-cost production and use of the biofungicide for the control of agricultural fungal pests.

Improvement of Bacillus thuringiensis bioinsecticide production by sporeless and sporulating strains using response surface methodology.

Statistical experimental designs, involving a Plackett-Burman design followed by a rotatable central composite design were used to optimize the culture medium constituents for Bacillus thuringiensis bioinsecticide production. This was carried out by using firstly an asporogenic strain and extrapolated to some sporeless and sporulating strains. Initial screening of production parameters was performed and the variables with statistically significant effects on delta-endotoxin production were identified: glucose, glycerol, yeast extract and MnSO(4). These variables were selected for further optimization by response surface methodology. The obtained results revealed that the optimum culture medium for delta-endotoxin production consists of 22.5 g/l of glucose, 4.8g/l of glycerol, 5.8 g/l of yeast extract and 0.008 g/l of MnSO(4). Under these conditions, delta-endotoxin production was 2,130 and 2,260 mg/l into 250 and 1,000 ml flask respectively, which represent more than 38% improvement in toxin production over the basal medium (1,636 mg/l). Such medium composition was shown to be suitable for overproducing delta-endotoxins by sporeless and sporulating strains.

Bacillus amyloliquefaciens AG1 biosurfactant: Putative receptor diversity and histopathological effects on Tuta absoluta midgut

The use of biosurfactant in pest management has received much attention for the control of plant pathogens, but few studies reported their insecticidal activity. The present study describes the insecticidal activity of biosurfactant extracted from Bacillus amyloliquefaciens strain AG1. This strain produces a lipopeptide biosurfactant exhibiting an LC50 of about 180ng/cm(2) against Tuta absoluta larvae. Accordingly, the histopathologic effect of this biosurfactant on T. absoluta larvae showed serious damages of the midgut tissues including rupture and disintegration of epithelial layer and cellular vacuolization. By PCR, we showed that this biosurfactant could be formed by several lipopeptides and polyketides including iturin, fengycin, surfactin, bacyllomicin, bacillaene, macrolactin and difficidin. Binding experiment revealed that it recognized five putative receptors located in the BBMV of T. absoluta with sizes of 68, 63, 44, 30 and 19kDa. Therefore, biosurfactant AG1 hold potential for use as an environmentally friendly agent to control the tomato leaf miner.

Overproduction of the Bacillus thuringiensis Vip3Aa16 toxin and study of its insecticidal activity against the carob moth Ectomyelois ceratoniae.

The vip3Aa16 gene of Bacillus thuringiensis strain BUPM95 was cloned and expressed in Escherichia coli. Optimization of Vip3A16 protein expression was conducted using Plackett-Burman design and response surface methodology. Accordingly, the optimum Vip3A16 toxin production was 170μg/ml at 18h post-induction time and 39°C post-induction temperature. This corresponds to an improvement of 21times compared to the starting conditions. The insecticidal activity, evaluated against Ectomyelois ceratoniae, displayed an LC50 value of 40ng/cm(2) and the midgut histopathology of Vip3Aa16 fed larvae showed vacuolization of the cytoplasm, brush border membrane destruction, vesicle formation in the apical region and cellular disintegration.

Combinatorial effect of Bacillus thuringiensis kurstaki and Photorhabdus luminescens against Spodoptera littoralis (Lepidoptera: Noctuidae)

Spodoptera littoralis, one of the major pests of many important crop plants, is more susceptible to Bacillus thuringiensis aizawai delta‐endotoxins than to those of Bacillus thuringiensis kurstaki. Within the framework of the development of efficient bioinsecticides and the prevention against insect resistance, we tested the effect of mixing B. thuringiensis kurstaki delta‐endotoxins and Photorhabdus luminescens cells on S. littoralis growth. The obtained results showed that the growth inhibition of this insect was more effective when B. thuringiensis kurstaki spore‐crystal mixture and Photorhabdus luminescens cells were used in combination. Furthermore, this synergism is mainly due to the presence of Cry1Ac, which is one of the three delta‐endotoxins that form the crystal of B. thuringiensis kurstaki strain BNS3 in addition to Cry1Aa and Cry2Aa. This work shows a possibility to use B. thuringiensis as a delivery means for Photorhabdus bacteria in order to infect the insect hemocoel and to reduce the risk of developing resistance in the target organism.

Efficacy of Bacillus subtilis V26 as a biological control agent against Rhizoctonia solani on potato.

The aim of this study is to evaluate the efficacy of the strain Bacillus subtilis V26, a local isolate from the Tunisian soil, to control potato black scurf caused by Rhizoctonia solani. The in vitro antifungal activity of V26 significantly inhibited R. solani growth compared to the untreated control. Microscopic observations revealed that V26 caused considerable morphological deformations of the fungal hyphae such as vacuolation, protoplast leakage and mycelia crack. The most effective control was achieved when strain V26 was applied 24h prior to inoculation (protective activity) in potato slices. The antagonistic bacterium V26 induced significant suppression of root canker and black scurf tuber colonization compared to untreated controls with a decrease in incidence disease of 63% and 81%, respectively, and promoted plant growth under greenhouse conditions on potato plants. Therefore, B. subtilis V26 has a great potential to be commercialized as a biocontrol agent against R. solani on potato crops.

Response of larval Ephestia kuehniella (Lepidoptera: Pyralidae) to individual Bacillus thuringiensis kurstaki toxins mixed with Xenorhabdus nematophila.

Bacillus thuringiensis kurstaki strain BNS3 produces parasporal crystals formed by Cry1Aa, Cry1Ac and Cry2Aa delta-endotoxins. In a previous work, we showed that the latter exhibited individually, a weak insecticidal activity against Ephestia kuehniella. In order to improve their toxicities, we studied the combined effect of each delta-endotoxin with X. nematophila cells on E. kuehniella larvae growth. Xenorhabdus cells were used in combination with spore crystal mixture of the wild strain BNS3, known to be active against E. kuehniella, but no improvement in toxicity was observed. This could be due to the high efficiency of BNS3 crystals against this insect. However, when X. nematophila was combined with each of Cry1Aa, Cry1Ac and Cry2Aa, improvement of toxicity was noticed. The best improvements were obtained with Cry1Ac and Cry2Aa, which are more toxic to E. kuehniella than Cry1Aa. The difference in toxicity improvement was attributed to the low affinity of Cry1Aa to BBMV receptors, compared to those of Cry1Ac and Cry2Aa. This synergism between Cry toxins and Xenorhabdus cells could be exploited on control target insect, particularly in case of resistance to Cry toxins.

Correlation between delta-endotoxin and proteolytic activities produced by Bacillus thuringiensis var. kurstaki growing in an economic production medium

Abstract Bacillus thuringiensis is a Gram positive bacterium that produces an insecticidal crystalline protein making it one of the most important biocontrol agents for pest management. Bioinsecticides based on B. thuringiensis were produced by fermentation processes in liquid media. Cultural conditions controlling proteolytic activities in different culture media were investigated to study the possible correlations between B. thuringiensis production of proteases and delta-endotoxins in a low-cost complex medium. Aeration appeared to play an important role in delta-endotoxin production. The correlation between proteolytic activity and aeration does not seem to be reliable. A negative correlation (correlation coefficient =− 0.774) was established between protease activity and delta-endotoxin production. In order to prove this correlation, protease hypo-producing and overproducing mutants were isolated through random mutagenesis of two wild strains, BUPM13 and BUPM5, by using nitrous acid. Interestingly, delta-endotoxin production of BUPM13-1, BUPM13-2 and BUPM13-3 was markedly improved when compared to the wild strain BUPM 13, reaching 2.1-fold, 3.69-fold and 8.13-fold, respectively. Maximal protease activity (540-2468 UI) obtained by BUPM5-1 and BUPM5-2 was 2.34-fold and 10.7-fold, respectively, more than that obtained by the wild strain BUPM5 with a drastic decrease of their delta-endotoxin production. Study of delta-endotoxin production by the selected mutants confirmed that insecticidal crystal protein stability in the culture strongly depends on the level of endogenous protease activity. This was also confirmed by bioassays measuring the LC50 using larvae of Ephestia kuehniella. Determining protease activity in fermentation culture could be useful in indirectly predicting the potency of B. thuringiensis strains with high insecticidal activities. This would allow low-cost selection of overproducing wild isolates or mutants in the screening programmes for the reduction of production cost, which is important from a practical point of view.

Improvement of Bioinsecticides Production by Sporeless Bacillus thuringiensis Strains in Response to Various Stresses in Low Cost Medium

The use of bioinsecticides, particularly those produced by sporeless Bacillus thuringiensis strains, has been shown to be a good alternative in pest management. Two types of sporeless mutants were distinguished. The asporogenic mutants which completely lack spores produce a regular bipyramidal crystal inclusion. The oligosporogenic mutants kept the ability to produce insecticidal crystal proteins. However, sporulation in such mutants was not totally blocked and very few of them could still produce spores. In order to improve bioinsecticides production, adaptation of sporeless strains to heat shock and osmotic stress was investigated. Delta-endotoxin production by 78% of sporeless mutants was significantly improved by osmotic stress with an overproduction of about 17%, compared to the wild strain BNS3. However, toxin production was improved by only 21% of mutants after heat shock, in low cost medium. The statistical analysis proved that delta-endotoxin production, cell growth, and spore formation of asporogenic and oligosporogenic mutants depended on the type of applied stress. Each strain has an important potential when applying the adequate stress. Moreover, adaptation of sporeless mutants to NaCl may allow the substitution of all minerals of the medium by diluted sea water which appeared to be a good alternative for the economic production of bioinsecticides at industrial scale which is of great importance from the practical point of view.

Quantification of Bacillus thuringiensis Vip3Aa16 Entomopathogenic Toxin Using Its Hemolytic Activity

Vegetative insecticidal proteins produced by some Bacillus thuringiensis strains are specifically toxic to different agricultural pests such as the polyphagous Spodoptera and several other Lepidopteran insects, but one of the major problems found in the use of these biopesticides was the lack of an easy and credible method of quantification of such secreted toxins. Heterologous expression of B. thuringiensis Vip3Aa16 toxin was performed in Escherichia coli then the protein was purified by chromatography. Using blood agar as well as blood agar overlay (zymogram assay), we reported, for the first time, the capacity of Vip3Aa16 to induce hemolysis. The hemolytic activity of this protein was shown to be relatively stable after treatment at 40 °C and at a range of pH between 6.5 and 9. Moreover, a linear relationship was shown between hemolysis levels and Vip3Aa16 concentrations. The model established in the present study could quantify Vip3A toxin as a function of hemolytic activity and the assay proposed showed to be a simple and low-cost method to readily assess Vip3A toxins in liquid cultures and facilitate the use of this kind of bioinsecticides in pest management programs.