Isolation and characterization of a new Bacillus thuringiensis strain Lip harboring a new cry1Aa gene highly toxic to Ephestia kuehniella (Lepidoptera: Pyralidae) larvae.

The aim of this study was to characterize new Bacillus thuringiensis strains that have a potent insecticidal activity against Ephestia kuehniella larvae. Strains harboring cry1A genes were tested for their toxicity, and the Lip strain showed a higher insecticidal activity compared to that of the reference strain HD1 (LC50 of Lip and HD1 were 33.27 and 128.61 µg toxin/g semolina, respectively). B. thuringiensis Lip harbors and expresses cry1Aa, cry1Ab, cry1Ac, cry1Ad and cry2A. DNA sequencing revealed several polymorphisms in Lip Cry1Aa and Cry1Ac compared to the corresponding proteins of HD1. The activation process using Ephestia kuehniella midgut juice showed that Lip Cry1A proteins were more stable in the presence of larval proteases. Moreover, LipCry1A proteins exhibited higher insecticidal activity against these larvae. These results indicate that Lip is an interesting strain that could be used as an alternative to the worldwide used strain HD1.

Histopathological effects and determination of the putative receptor of Bacillus thuringiensis Cry1Da toxin in Spodoptera littoralis midgut.

Bacillus thuringiensis subsp. aizawai strain HD133, known by its effectiveness against Spodoptera species, produces many insecticidal proteins including Cry1Ab, Cry1Ca and Cry1Da. In the present study, the insecticidal activity of Cry1Da against Spodoptera littoralis was investigated. It showed toxicity with an LC(50) of 224.4 ng/cm(2) with 95% confidence limits of (178.61-270.19) and an LC(90) of 467.77 ng/cm(2) with 95% confidence limits of (392.89-542.65). The midgut histopathology of Cry1Da fed larvae showed vesicle formation in the apical region, vacuolization and destruction of epithelial cells. Biotinylated-activated Cry1Da toxin bound protein of about 65 kDa on blots of S. littoralis brush border membrane preparations. This putative receptor differs in molecular size from those recognized by Cry1C and Vip3A which are active against this polyphagous insect. This difference in midgut receptors strongly supports the use of Cry1Da as insecticidal agent, particularly in case of Cry and/or Vip-resistance management.

Selection and characterization of thermotolerant Beauveria bassiana isolates and with insecticidal activity against the cotton-melon aphid Aphis gossypii (Glover) (Hemiptera: Aphididae).

BACKGROUND: Cotton-melon aphid Aphis gossypii (Glover) causes severe damage mainly to cucurbits. Twenty-two Beauveria sp. isolates were simultaneously assessed for their pathogenicity and heat tolerance. The selected isolates were identified molecularly and characterized in terms of conidial germination rate, mycelial growth, conidial yield and endophytic activity. RESULTS: Screening bioassays showed that the B. bassiana isolates B3, B7, B9 and B12 were the most toxic, inducing mortality equal to or slightly higher than the commercialized strain B. bassiana BNat (70.7%). Median lethal concentration (LC50 ) bioassays revealed that only isolate B12 had a significantly lower LC50 value (5.4 × 105 conidia ml-1 ) than strain BNat (5 × 106 conidia ml-1 ). The heat tolerance screening test (1 h of exposure to 45°C) allowed us to select isolates B3, B7, B9 and B12 with germination rates of 57.5% to 80.1% after 24 h incubation at 25°C, all significantly higher than strain BNat (22.1%). The germination rates of all isolates decreased significantly after 2 h of exposure to 45°C, with the exception of isolate B12 which displayed the highest thermotolerance (72% germination). The four selected isolates were able to endophytically colonize cucumber leaves when applied to the foliage. Inoculation of cucumber plants with isolate B12 did not affect cucumber plant growth. However, several plant growth parameters were improved 5 weeks after root inoculation. CONCLUSION: On the basis of its potent toxicity and thermotolerance, isolate B12 is a good candidate for further development as a biopesticide for use in integrated pest management strategies for aphid control. © 2022 Society of Chemical Industry.

Integration of a recombinant chitinase into Bacillus thuringiensis parasporal insecticidal crystal.

Chitinases have been successfully used in combination with Bacillus thuringiensis delta-endotoxins forming crystals in order to enhance their insecticidal activities. In this context, we opted for promoting the chitinase integration into these crystals. Thus, we engineered, for the first time, a fusion protein (CDF) consisting of the chitinase Chi255 and the carboxy-terminal half of Cry1Ac, both from B. thuringiensis subsp. kurstaki. The constructed transcriptional fusion (chi255Δsp-CTcry1Ac) was cloned into a shuttle vector (Escherichia coli/B. thuringiensis) downstream the sporulation-dependent promoters BtI-BtII and upstream the cry1Ac transcription terminator. The resulting plasmid, named pF, was transferred by electroporation to crystalliferous B. thuringiensis strain BNS3 and acrystalliferous strain BNS3Cry-. The functionality of the chimerical chitinase was demonstrated by an improvement of the relative chitinolytic activity of the recombinant strain BNS3/pF by 2.5 folds. Western blot analyses showed that, despite of the instability of CDF when expressed in the acrystalliferous strain, the C-terminal half of Cry1Ac succeeded to allow the integration of the chimerical chitinase into the crystal of BNS3. The recombinant strain BNS3/pF (LC(50) = 144.06 µg g(-1)) was 1.5 times more active against Ephestia kuehniella larvae than the wild strain (LC(50) = 212.10 µg g(-1)).

Investigation of the steps involved in the difference of susceptibility of Ephestia kuehniella and Spodoptera littoralis to the Bacillus thuringiensis Vip3Aa16 toxin.

BUPM95 is a Bacillus thuringiensis subsp. kurstaki strain producing the Vip3Aa16 toxin with an interesting insecticidal activity against the Lepidopteran larvae Ephestia kuehniella. Study of different steps in the mode of action of this Vegetative Insecticidal Protein on the Mediterranean flour moth (E. kuehniella) was carried out in the aim to investigate the origin of the higher susceptibility of this insect to Vip3Aa16 toxin compared to that of the Egyptian cotton leaf worm Spodoptera littoralis. Using E. kuehniella gut juice, protoxin proteolysis generated a major band corresponding to the active toxin and another band of about 22kDa, whereas the activation of Vip3Aa16 by S. littoralis gut juice proteases generated less amount of the 62kDa active form and three other proteolysis products. As demonstrated by zymogram analysis, the difference in proteolysis products was due to the variability of proteases in the two gut juices larvae. The study of the interaction of E. kuehniella BBMV with biotinylated Vip3Aa16 showed that this toxin bound to a putative receptor of 65kDa compared to the 55 and 100kDa receptors recognized in S. littoralis BBMV. The histopathological observations demonstrated similar damage caused by the toxin in the two larvae midguts. These results demonstrate that the step of activation, mainly, is at the origin of the difference of susceptibility of these two larvae towards B. thuringiensis Vip3Aa16 toxin.

Classical and Bayesian predictions applied to Bacillus toxin production.

Bacillus thuringiensis is a bacterium with unusual properties that make it useful for pest control in ecoagriculture. It can form a parasporal crystal containing polypeptides (also called delta-endotoxins). These entomopathogenic toxins are made during the stationary phase of the bacterial growth cycle and were initially characterized as an insect pathogen. Nowadays, the use of saturated two-level designs is very popular. This method is especially used in industrial applications where the cost of experiments is expensive. Standard classical approaches are not appropriate to analyze data from saturated designs. It is due to the fact that they only allow to estimate the main factor effects and cannot assure enough freedom degrees to estimate the error variance. In this paper, we propose the use of empirical Bayesian procedures to get inferences for data obtained from saturated designs, inspired from Hadamard matrices. The proposed methodology is illustrated by assuming a dataset to prove the model robustness. The comparison between the two studied mathematical techniques, based on mean square error values (MSE), revealed that Bayesian method (BM) was more accurate than least square method (LSM): for example, the results showed that 2002 and 2000.7 mg/l for experimental and predicted (BM) data were obtained against 2002 and 1991 mg/l for experimental and predicted (LSM) data. This suggested method could be generalized in several application fields in biological sciences.

New Bacillus thuringiensis toxin combinations for biological control of lepidopteran larvae.

Cyt1Aa from Bacillus thuringiensis israelensis is known by its synergistical activity with B. thuringiensis and Bacillus sphaericus toxins. It is able to improve dipteran specific toxins activity and can prevent or overcome larval resistance to those proteins. The objective of the current study was to investigate the possible improvement of larvicidal activity of B. thuringiensis kurstaki expressing heterogeneous proteins Cyt1A and P20. cyt1A98 and p20 genes encoding the cytolytic protein (Cyt1A98) and the accessory protein (P20), respectively, were introduced individually and in combination into B. thuringiensis kurstaki strain BNS3. Immunoblot analysis evidenced the expression of these genes in the recombinant strains and hinted that P20 acts as molecular chaperone protecting Cyt1A98 from proteolytic attack in BNS3. The toxicities of recombinant strains were studied and revealed that BNS3pHTp20 exhibited higher activity than that of the negative control (BNS3pHTBlue) toward Ephestia kuehniella, but not toward Spodoptera littoralis. When expressed in combination with P20, Cyt1A98 enhanced BNS3 activity against E. kuehniella and S. littoralis. Thus, Cyt1Aa protein could enhance lepidopteran Cry insecticidal activity and would prevent larval resistance to the most commercialized B. thuringiensis kurstaki toxins.

Selection and characterisation of an HD1-like Bacillus thuringiensis isolate with a high insecticidal activity against Spodoptera littoralis (Lepidoptera: Noctuidae).

BACKGROUND: Spodoptera littoralis (Boisduval) larvae are known by their susceptibility to Bacillus thuringiensis subsp. aizawai strains. In order to prevent the appearance of B. thuringiensis (Bt) resistance and to develop economical Bt-based biopesticides, the selection and the characterisation of a B. thuringiensis isolate toxic against S. littoralis larvae and overproducing δ-endotoxins were investigated. RESULTS: Among 124 Tunisian B. thuringiensis isolates assessed against S. littoralis larvae, four isolates showed toxicity similar to and higher than the toxicity of the aizawai strain HD133 and the kurstaki strain HD1 respectively. The plasmid pattern of the selected isolates was similar to that of HD1. Polymerase chain reaction (PCR) analysis using specific primers revealed that these isolates present different gene contents. The only detected gene encoding Spodoptera-specific toxin was cry9. The selected isolates were found to produce bipyramidal and cubic crystals. The assessment of δ-endotoxin production by these isolates showed that BUPM28 produced 43.71 and 80.81% more δ-endotoxin than HD1 and HD133 respectively. The application of osmotic or heat shock stress on the BUPM28 isolate made it possible to enhance δ-endotoxin production by 22 and 23% respectively. CONCLUSION: On the basis of its potent insecticidal activity and its high level of δ-endotoxin production, the BUPM28 isolate can be considered to be an effective alternative for the control of S. littoralis.