Rhizobacterial inoculation to improve the responses of olive cultivars to drought stress.

Tunisia is known as an underprivileged country in water resources, and water scarcity is evident in certain regions. In the long term, this situation could become more exacerbated, considering the increased risk of aridity. In this context, this work was carried out to study and compare the ecophysiological behavior of five olive cultivars facing drought stress and to evaluate the contribution of rhizobacteria to mitigate the effects of drought stress on these cultivars. The results showed a significant decrease in the relative water content (RWC) with the lowest percentage recorded for the 'Jarboui' cultivar (RWC = 37%), and the highest percentage was registered for the 'Chemcheli' cultivar (RWC = 71%). In addition, the performance index (PI) decreased for all the five cultivars and it reached the lowest values for 'Jarboui' and 'Chetoui' with 1.51 and 1.57, respectively. For the SPAD index, a decrease was registered for all the cultivars, except 'Chemcheli' (SPAD index = 89). Furthermore, the bacterial inoculation treatment improved the responses of cultivars to water stress. In fact, for all of the studied parameters, it was found that rhizobacterial inoculation significantly attenuated the effects of drought stress with variability dependent on the level of tolerance of the tested cultivars. This response improvement was noted especially in susceptible cultivars like 'Chetoui' and 'Jarboui'.

Comparative analysis of the susceptibility/tolerance of Spodoptera littoralis to Vip3Aa, Vip3Ae, Vip3Ad and Vip3Af toxins of Bacillus thuringiensis.

The cotton leaf worm Spodoptera littoralis is known for causing serious damages to various crops. In this study, the susceptibility/tolerance of this larvae to four Vip3A (Vip3Aa, Vip3Ae, Vip3Ad and Vip3Af) toxins was investigated. UnlikeVip3Ad which showed no activity to S. littoralis, Vip3Aa, Vip3Ae and Vip3Af exhibited high toxicity to this larva with LC50 of 228.42 ng/cm2, 65.71 ng/cm2, and 388.90 ng/cm2, respectively. Activation of the 90 kDa Vip3A proteins by S. littoralis larvae juice generated four major bands of sizes 62, 45, 33 and 22 kDa. Binding experiments between biotinylated Vip3A toxins and the brush border membrane vesicles (BBMV) revealed two binding proteins of 55 and 100 kDa with Vip3Aa. Vip3Ae and Vip3Af recognized one single putative receptor of 65 kDa, whereas Vip3Ad did not bind to S. littoralis BBMV. In histopathological observations, Vip3Aa, Vip3Ae and Vip3Af toxins showed approximately similar damages on S. littoralis midgut including rupture and disintegration of epithelial layer and cellular vacuolization. These findings showed that Vip3Aa, Vip3Ae and Vip3Af might be useful for controlling S. littoralis.

Improvement of Vip3Aa16 Toxin Production and Efficiency Through Nitrous Acid and UV Mutagenesis of Bacillus thuringiensis (Bacillales: Bacillaceae).

Bacillus thuringiensis Berliner (Bacillales: Bacillaceae) strain BUPM95 was known by the efficiency of its vegetative insecticidal protein (Vip3Aa16) against different Lepidoptera such as Spodoptera littoralis (Lepidoptera: Noctuidae). To overcome the problem of the low quantities of Vip3 proteins secreted by B. thuringiensis strains in the culture supernatant, classical mutagenesis of vegetative cells of BUPM95 strain was operated using nitrous acid and UV rays. The survivors were screened on the basis of their hemolytic activity and classified in three groups: unaffected, overproducing, and hypo-producing mutants. Using different mutants improved in their hemolytic activity, the supernatants showed an improved toxicity toward S. littoralis larvae (83.33-100% of mortality) compared with the wild-type supernatant (76%). After Vip3 protein quantification in the different supernatants, bioassays against S. littoralis larvae demonstrated that mutants M62, M43, and M76 were improved in the efficiency of their toxin as demonstrated by the lower values of LC50 and LC90 compared with the wild-type Vip3Aa16 protein. However, M26 and M73 mutants were improved in the toxin quantities produced in the supernatant. The improvement of the production and the efficiency of B. thuringiensis Vip3 toxins should contribute to a significant reduction of the production costs of these very interesting B. thuringineis proteins and facilitate the use of these toxins in the pest control management.

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.

Vegetative insecticidal protein of Bacillus thuringiensis BLB459 and its efficiency against Lepidoptera.

Bacillus thuringiensis strain BLB459 supernatant showed a promising activity against Lepidopteran pests with extremely damages in the larvae midgut. Investigations of the genes that encode secreted toxin demonstrated that this strain harbored a vip3-type gene named vip3(459). Based on its original nucleotide and amino acid sequences, this gene was cloned into pET-14b vector and overexpressed in Escherichia coli. The expressed protein was purified and tested against different insects and interestingly the novel toxin demonstrated a remarkable activity against the stored products pest Ephestia kuehniella and the polyphagous insects Spodoptera littoralis and Agrotis segetum. As demonstrated, the acute activity of Vip3(459) protein against A. segetum can be due to its original amino acids sequence and the putative receptors of this toxin in the larvae midgut. These results demonstrated that this Vip3 toxin showed a wide spectrum of activity against Lepidoptera and support its use as a biological control agent.

Combinatorial effect of mutagenesis and medium component optimization on Bacillus amyloliquefaciens antifungal activity and efficacy in eradicating Botrytis cinerea.

This work is directed towards Bacillus amyloliquefaciens strain BLB371 metabolite production for biocontrol of fungal phytopathogens. In order to maximise antifungal metabolite production by this strain, two approaches were combined: random mutagenesis and medium component optimization. After three rounds of mutagenesis, a hyper active mutant, named M3-7, was obtained. It produces 7 fold more antifungal metabolites (1800AU/mL) than the wild strain in MC medium. A hybrid design was applied to optimise a new medium to enhance antifungal metabolite production by M3-7. The new optimized medium (35g/L of peptone, 32.5g/L of sucrose, 10.5g/L of yeast extract, 2.4g/L of KH2PO4, 1.3g/L of MgSO4 and 23mg/L of MnSO4) achieved 1.62 fold enhancement in antifungal compound production (3000AU/mL) by this mutant, compared to that achieved in MC medium. Therefore, combinatory effect of these two approaches (mutagenesis and medium component optimization) allowed 12 fold improvement in antifungal activity (from 250UA/mL to 3000UA/mL). This improvement was confirmed against several phytopathogenic fungi with an increase of MIC and MFC over than 50%. More interestingly, a total eradication of gray mold was obtained on tomato fruits infected by Botrytis cinerea and treated by M3-7, compared to those treated by BLB371. From the practical point of view, combining random mutagenesis and medium optimization could be considered as an excellent tool for obtaining promising biological products useful against phytopathogenic fungi.

Combinatorial effect of Bacillus amyloliquefaciens AG1 biosurfactant and Bacillus thuringiensis Vip3Aa16 toxin on Spodoptera littoralis larvae.

Spodoptera littoralis, one of the most serious and destructive agricultural pests in the world, is very susceptible to Vip3 toxin. In order to develop a new efficient bioinsecticide and to prevent the development of resistance by the target pest, insecticidal activity of biosurfactant produced by Bacillus amyloliquefaciens AG1 was evaluated against S. littoralis. Bioassays revealed the susceptibility of the first instar larvae of this pest to AG1 biosurfactant with an LC50 of 245ng/cm2. Moreover, the histopathology examination of the larval midgut treated by AG1 biosurfactant showed vacuolization, necrosis and disintegration of the basement membrane. Binding experiments revealed that the AG1 biosurfactant recognized three putative receptors located in the brush border membrane vesicles of S. littoralis with sizes of 91, 72 and 64kDa. Competition assays using biotinylated metabolites indicated that AG1 biosurfactant and Vip3Aa16 toxin did not compete for the same S. littoralis receptors. When combined, AG1 biosurfactant and Vip3Aa16 showed an additive effect against S. littoralis larvae. These findings suggested that B. amyloliquefaciens AG1 biosurfactant could be a promising biocontrol agent to eradicate S. littoralis and to prevent resistance development by this pest.

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.

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.

Overcome of Carbon Catabolite Repression of Bioinsecticides Production by Sporeless Bacillus thuringiensis through Adequate Fermentation Technology.

The overcoming of catabolite repression, in bioinsecticides production by sporeless Bacillus thuringiensis strain S22 was investigated into fully controlled 3 L fermenter, using glucose based medium. When applying adequate oxygen profile throughout the fermentation period (75% oxygen saturation), it was possible to partially overcome the catabolite repression, normally occurring at high initial glucose concentrations (30 and 40 g/L glucose). Moreover, toxin production yield by sporeless strain S22 was markedly improved by the adoption of the fed-batch intermittent cultures technology. With 22.5 g/L glucose used into culture medium, toxin production was improved by about 36% when applying fed-batch culture compared to one batch. Consequently, the proposed fed-batch strategy was efficient for the overcome of the carbon catabolite repression. So, it was possible to overproduce insecticidal crystal proteins into highly concentrated medium.

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.

Application of statistical experimental design for optimisation of bioinsecticides production by sporeless Bacillus thuringiensis strain on cheap medium

In order to overproduce bioinsecticides production by a sporeless Bacillus thuringiensis strain, an optimal composition of a cheap medium was defined using a response surface methodology. In a first step, a Plackett-Burman design used to evaluate the effects of eight medium components on delta-endotoxin production showed that starch, soya bean and sodium chloride exhibited significant effects on bioinsecticides production. In a second step, these parameters were selected for further optimisation by central composite design. The obtained results revealed that the optimum culture medium for delta-endotoxin production consists of 30 g L-1 starch, 30 g L-1 soya bean and 9g L-1 sodium chloride. When compared to the basal production medium, an improvement in delta-endotoxin production up to 50% was noted. Moreover, relative toxin yield of sporeless Bacillus thuringiensis S22 was improved markedly by using optimised cheap medium (148.5 mg delta-endotoxins per g starch) when compared to the yield obtained in the basal medium (94.46 mg delta-endotoxins per g starch). Therefore, the use of optimised culture cheap medium appeared to be a good alternative for a low cost production of sporeless Bacillus thuringiensis bioinsecticides at industrial scale which is of great importance in practical point of view.

Application of statistical experimental design for optimisation of bioinsecticides production by sporeless Bacillus thuringiensis strain on cheap medium.

In order to overproduce bioinsecticides production by a sporeless Bacillus thuringiensis strain, an optimal composition of a cheap medium was defined using a response surface methodology. In a first step, a Plackett-Burman design used to evaluate the effects of eight medium components on delta-endotoxin production showed that starch, soya bean and sodium chloride exhibited significant effects on bioinsecticides production. In a second step, these parameters were selected for further optimisation by central composite design. The obtained results revealed that the optimum culture medium for delta-endotoxin production consists of 30 g L(-1) starch, 30 g L(-1) soya bean and 9 g L(-1) sodium chloride. When compared to the basal production medium, an improvement in delta-endotoxin production up to 50% was noted. Moreover, relative toxin yield of sporeless Bacillus thuringiensis S22 was improved markedly by using optimised cheap medium (148.5 mg delta-endotoxins per g starch) when compared to the yield obtained in the basal medium (94.46 mg delta-endotoxins per g starch). Therefore, the use of optimised culture cheap medium appeared to be a good alternative for a low cost production of sporeless Bacillus thuringiensis bioinsecticides at industrial scale which is of great importance in practical point of view.

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.

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.

Overproduction of delta-endotoxins by sporeless Bacillus thuringiensis mutants obtained by nitrous acid mutagenesis.

Asporogenic and oligosporogenic Bacillus thuringiensis mutants having the ability to overproduce insecticidal crystal protein were generated by using nitrous acid (50 mg/ml), as chemical mutagenic agent. Insecticidal crystal proteins produced by asporogenic mutants remained encapsulated within the cells. Delta-endotoxin production by most of mutants was improved compared to the corresponding wild strains BNS3 and a mutant M26. The overproduction by asporogenic and oligosporogenic mutants was attributed to defect in genes involved in sporulation and to random mutations affecting cell metabolism at different pathways and delta-endotoxin synthesis. Sporeless bioinsecticides could be developed based on stable and environmentally safe Bacillus thuringiensis mutants.