Gene manipulation in Bacillus thuringiensis : Biopesticide Development

Transcription

1 Gene manipulation in Bacillus thuringiensis : Biopesticide Development For safer Environment, Agriculture and Food! Samir JAOUA Univ. of Qatar, CAS, Dept of Biol. & Environ. Sc / Centre of Biotechno. Sfax, Tunisia samirjaoua@qu.edu.qa Dec 16th 09 - QU-CAS-DBES- PTS

2 Biopesticides Excellente alternative to substitute the chemical insecticides Bioinsecticides advantages -Biodegradable - Harmless to humans, birds and other mammals, -Respect the environment -Act specifically -Toxicity at low concentrations Biopesticides derive from natural materials as animals, plants, bacteria but essentially from micro-organisms (fungi, virus, bacteria)

3 BT : the star of biopesticides (90%) Bacillus thuringiensis (Bt) is a Gram + bacterium that kills insects larvae due to its parasporal crystals containing deltaendotoxins Spore crystal Crystal (δ-endotoxin)

4 *The Bt toxin is harmless to humans, birds and other mammals *Bts also are harmless to bees and other beneficial insect species (e.g., predators and parasites). >>>>>>>>>>>Specific

5 The delta-endotoxins are packaged in crystals that differ from one sub species to another by their number, composition, size and shape Bipyramidal Delta-endotoxins Carrier protein Passenger protein (Ricca and Cutting, 2003) Spherical apathetic ( LB pes., 2007, unpublished) Cubic They could be displayed at the B. thuringiensis spore surface, which improve its insecticidal activity. These spores could be used as recombinant protein display systems.

6 Bacillus thuringiensis is a source of different biological pesticides Bt Produces parasporal crystalline delta-endotoxin inclusions Cry proteins: insecticidal Cyt proteins Cytolytic activities B. thuringiensis Gram+ Bacteriocins Bactericidal activities Chitinases Enzymes having fungicide activities Two classes of Vegetative Insecticidal Proteins Vip1-Vip2 : binary toxins / Coleopteran Vip3 / Lepidopteran

7 Different classes of δ-endotoxins Genes MW (kda) Shape of de crystal Insectes cibles cry Bipyramidale cry2 cry3 70 Cubique 70 Rectangular cry4 cry1i Hétérogènes 80 Sphéric Rhomboïque -

8 Proposed mode of action of Cry toxins Ingestion & solubilisation of protoxin Proteolytic activation at N- and C- termini Interaction with cell surface binding protein Conformational change exposing a 4-5 helical hairpin Oligomerisation & insertion in membrane to form pores

9 BACILLUS THURINGIENSIS STRAINS COLLECTION Isolation of hundreds Bacillus thuringiensis strains from soil and environments:

10 MORPHOLOGICAL STUDY OF BT CRYSTALS OF NEWLY ISOLATED STRAINS cubical crystal & bipyramidal crystal Sp spherical crystal Sp Sp Amorphous crystal cubical crystal Sp Bipyramidal crystals

11 Identification of Bt Strains, using gyrb as a molecular marker La digestion par EcoRI produit : * 2 bandes de 37 et 315 pb pour HD1, 255 et BUPM 90 et DOT * 2 bandes de 134 et 218 pb pour 14T et BUPM40 PCR- DIGESTION by EcoRI MT ND HD H T DOT MT 352pb 315pb 218pb 134pb 37pb * 1 bande de 352 pb pour H3 Mireille Kallassy Awad, Imène Saadaoui, Souad Rouis, Slim Tounsi and Samir Jaoua (2007) Differentiation among strains of Bacillus thuringiensis by gyrb PCR-Sau3AI fingerprinting. Mol. Biotechnol, 35, 1-7

12 Insecticidal activitiesof delta-endotoxins Cry1, Cry2 and Cry5 of the strain BNS3 on Ephestia kuehniella larvae

13 Insecticidal activity on olive tree pathogenic insect Prays oleae

14 Comparaison of the insecticidal activities of different L.B.Pes Bacillus thuringiensis israelensis strains against C. pipiens

15 I- Investigation and manipulation of delta-endotoxin coding genes from the best insecticidal strains

16 >>>>Evidence of interesting insecticidal strains against: 1- Lepidoptera: -BNS3 Novel genes encoding toxins active on Lepidoptera cry2 type Genes : TOUNSI S and JAOUA S (2003) Characterisation of a novel cry2aa-type gene from Bacillus thuringiensis subsp. kurstaki Biotechnol. Lett. 25, cry1a type Genes :.S. TOUNSI, M. DAMMAK, A. REBAI, AND S. JAOUA (2005) Response of larval Ephestia kuehniella (Lepidoptera: Pyralidae) to individual Bacillus thuringiensis kurstaki toxins and toxin mixtures Biological Control. 35, 27-31

17 cry1ia type Genes : 1*TOUNSI, S. and JAOUA, S. (2002) Identification of a promoter for the crystal protein-encoding gene cry1ia from Bacillus thuringiensis subsp. kurstaki. FEMS Microbiol. Lett. 208, *TOUNSI S, ZOUARI N and JAOUA S (2003) Cloning and study of the expression of a novel cry1ia-type gene from Bacillus thuringiensis subsp. kurstaki J. Appl. Microbiol. 94, 1-6

18 >>>>Evidence of interesting insecticidal strains against: 1- Lepidoptera: -BUPM5 Imen SAADAOUI, Souad ROUIS, and Samir JAOUA (2009) New Tunisian Bacillus thuringiensis kurstaki isolate having better toxicity and d- endotoxin yield Arch. Microbiol. 191,

19 Comparison of the amino acid sequence encoded by BLB1 Cry1Ac with those encoded by the other cry1ac genes published in the Nile-Crickmore data base. Only the regions containing differences are presented..

20 Comparaison of the insecticidal activities of different L.B.Pes Bacillus thuringiensis israelensis strains against C. pipiens LD50 (ng/ml ) after 10h H14 7,02 ± 1,8 BUPM97 - BUPM98 3,89 ± 0,28 BUPM100 3,36 ± 0,19 BLB124 14,6 ± 8,9 BUPM100 and BUPM98 are more toxic than the reference strain H14

21 Cloning and study of the organisation of genes encoding toxins active on diptera 1-Genes of cry4a type 2-Genes of cry4b types -R. ZRIBI ZGHAL, S.TOUNSI AND S. JAOUA (2006) Biotechnol. And Appl. Biochem. 44, R ZRIBI ZGHAL AND S JAOUA (2006) Mol. Biotechnol. 33, *very active strains against chemical insecticide resistant mosquitos 3- cyt 1A genes -R. ZRIBI ZGHAL, H. TRIGUI, M. BEN ALI AND S. JAOUA (2008) Mol. Biotechnol. 381,

22 (a) Evidence of plasmidic DNA Plasticity in Bti Evidence of DNA rearrangements in the 128- kilobase pbtoxis plasmid of Bacillus thuringiensis israelensis (b) EI EI EI H H H EV EV EV EV EV EV EV cry4b cry10a pbt048 EI 0.78 EI H H H EV EV 4.9 cry4b 5 EI Deletion of cry4a Deletion of cry10a Restriction Polymorphism downstream cry4b

23 STUDY OF THE SYNERGESTIC ACTIONS OF Bt DIFFERENTS DELTA- ENDOTOXINES AGAINST Ephestia kuehniella

24 Effect of crystal delta-endotoxins ratio on insecticidal activity *Slim TOUNSI, Mariam DAMMAK, Nabil ZOUARI, Ahmed REBAÎ and Samir JAOUA (2006) Evidence of the effect of deltaendotoxin ratio in Bacillus thuringiensis crystals on the toxicity against Ephestia kuehniella. Biological Control 37: * DAMMAK, MARIAM; TOUNSI, SLIM; ABDELKEFI MESRATI, LOBNA; SCHULTZ, PATRICK; JAOUA, SAMIR (2009) Restoration of the crystallisation of altered delta-endotoxins Cry1Ac, by the promotion of their in vivo integration into the Bacillus thuringiensis native crystal FEMS Microbiology Letters. 292, Strains LC 50 (ng toxins/mg flour) Improvement* BNS3 105 BNS3(pHTBlue) BNS3(pHTcry1Aa) BNS3(pHTcry1Ac) BNS3(pHTcry2Aa)

25 In vitro Study of the interaction of Bacillus thuringiensis toxins with Prays oleae and Ephestia kuehniella receptors Vesicle formation in the apical region of Prays cells. *S. ROUIS, M. CHAKROUN, I. SAADAOUI, AND S. JAOUA (2006) Proteolysis, histopathological effects and immunohistopathological localization of d-endotoxins of Bacillus thuringiensis subsp. kurstaki in the midgut of Lepidopteran olive tree pathogenic insect Prays oleae Mol. Biotechnol. 35,1-8 * S. ROUIS, M. CHAKROUN, AND S. JAOUA (2008) Comparative study of Bacillus thuringiensis -endo Cry1Aa and Cry1Ac toxin activation, inactivation and in situ Histopathological effect in Ephestia kuehniella (Lepidoptera: Pyralidae) Mol. Biotechnol. 38,

26 II- Investigation and manipulation of Vegetative insecticidal proteins

27 This class of vegetative insecticidal proteins (Vip) includes The binary toxin Vip1 and Vip2 with Coleopteran activity The toxin Vip3 with Lepidopteran activity

28 Vip3 is a secreted toxin Vip3 is active against the Lepidoperan pest Agrotis ipsilon that attacks more than 50 crops including cereal grains. This pest is not very sensitive to B. thuringiensis d- endotoxins In the midgut of susceptible larvae, Vip3 protein was activated and proteolysis generate 4 major products of about 62, 45, 33 and 22 kda Then, the active form of the toxin (62 kda) bounds to the midgut receptors causing larvae death

29 Screening of a collection of B. thuringiensis strains for the presence of vip3-type genes By PCR and Slot-blot hybridization, we detected the presence of vip3-type genes in 30% of the studied strains

30 Due to the interest provided by the insecticidal activity exhibited by the supernatant of the strain BUPM95 against the Lepidopteran larvae Ephestia kuehniella and Prays oleae, a vip gene named vip3lb was cloned from this strain

31 * ABDELKEFI MESRATI L, TOUNSI S, KAMOUN F AND JAOUA S (2005) Identification of a promoter for the vegetative insecticidal protein-encoding gene vip3lb from Bacillus thuringiensis. FEMS Microbiol. Lett. 247, * L ABDELKEFI MESRATI, S TOUNSI AND S JAOUA (2005) Characterization of a novel vip3-type gene from Bacillus thuringiensis and evidence of its presence on a large plasmid. FEMS Microbiol. Lett.. 244, Amino acid sequence similarity analysis revealed that Vip3LB is a new Vip3-type toxin, presenting several differences with other Vip3 proteins

32 Transcription start point of vip3 By primer extension analysis, we have identified the transcription start point of the vip3lb gene. Upstream from this tsp, was found a nucleotide sequence partially homologous to the consensus sequence for the E E holoenzyme of B. Subtilis

33 Gut juice ligand blotting *Lobna ABDELKEFI-MESRATI, Souad ROUIS, Sameh SELLAMI and Samir JAOUA (2009) Prays oleae midgut putative receptor of Bacillus thuringiensis Vegetative Insecticidal Protein Vip3LB differs from that of Cry1Ac toxin Mol. Biotechnol. 43: kda 65 kda Using the gut juice of Prays oleae, purified Vip3LB bounds to 65 kda protein whereas Cry1Ac bounds to 210 kda midgut receptor

34 Heterologous expression of the Bacillus thuringiensis vegetative insecticidal Vip3LB in Photorhabdus temperata strain K122 and oral toxicity against insect *Kaïs Jamoussi, Sameh Sellami, Lobna Abdelkefi-Mesrati, Alain Givaudan and Samir Jaoua (2009) Heterologous Expression of Bacillus thuringiensis Vegetative Insecticidal Protein-Encoding Gene vip3lb in Photorhabdus temperata Strain K122 and Oral Toxicity against the Lepidoptera Ephestia kuehniella and Spodoptera littoralis Mol. Biotechnol. 43: * W JALLOULI, W HAMMAMI, NL ZOUARI, S JAOUA (2008) Medium optimization for biomass production and morphology variance overcome of Photorhabdus temperata ssp. temperata strain K1 Process Biochem.43,

35 Express the vip3lb (vip3aa16) gene in Photorhabdus temperata strain K122 and to determine Vip3LB sub-cellular location. Test the oral toxicity of the recombinant bacterium against larvae of Ephestia kuehniella and Spodoptera littoralis species.

36 Oral toxicity of recombinant P. temperata K122 against Spodoptera littoralis L2 growing larvae (whole cultures) Photo : Sabine Tiessen Average Mass of 10 larvae (mg) K122 (pbad) 75 µl/gr ( Bact.) Cutworm Highly polyphagous specie (Solanaceae, Cruciferaceae, Maize, cotton, tomato, fodder crops ) Time (hours) Control K122 (pbad-vip3lb) 75 µl/gr ( Bact.) - Photorhabdus temperata does not cause growth inhibition of Spodoptera littoralis larvae - But, expressed Vip3LB causes growth Inhibition of Spodoptera littoralis larvae

37 III-Enhancement of the insecticidal activity of Bacillus thuringiensis subsp. kurstaki strain via the production of chitinasecontaining crystals Chitinases are enzymes that degrade the chitin of insect and fungi. se of chitinases in cultures protection: As an anti-fungal enzyme Synergistic action with bioinsecticides

38 Screening of Bt strains collection for anti-fungal and chitinase activities Best chitinolytic activity (a) Screening Selection of strain BUPM cm Test de l activité fongicide sur Aspergillus niger (b) Best antifungal activity cm

39 Cloning and sequencing of chi255 genegttttcatatatagtt * F. DRISS, M. KALLASSY-AWAD, N. ZOUARI AND S. JAOUA (2005) Molecular characterization of a novel chitinase from Bacillus thuringiensis subsp. kurstaki J. Appl. Microbiol. 99, F. DRISS, A. BAANANNOU, S. ROUIS, I. MASMOUDI, N. ZOUARI AND S. JAOUA (2007) Effect of the chitin binding domain deletion from Bacillus thuringiensis subsp. kurstaki chitinase Chi255 on its stability in Escherichia coli Mol. Biotechnol. 36, EMBL accession number AJ TGTATTCAAGCCTTTTTGTATTGAGAAAGTCTTTTTCAACTTAATAAAGCGTTTACACTAAATCTTACATTTGTTACGATTTAATCACCCCCAGCTCCCTTGTAT AGACTTCGTGATGTCTGATCATTTTATCTAGACGTTTCACACGTCTAGATTCATTTTGATTTATTGGCGTATGCCTTTAAATATATCTTTTATTTTGAAAGGAGAA ATG GCT ATG AGG TCT CAA AAA TTC ACA CTG CTA TTA CTA TCC CTA CTA CTT TTC TTA CCT CTT TTT CTC ACA AAT 75 M A M R S Q K F T L L L L S L L L F L P L F L T N 25 TTT ATT ACT CCA AAT CTC GCA TTA GCA GAT TCA CCA AAG CAA AGT CAA AAA ATT GTT GGG TAC TTT CCT TCG TGG 150 F I T P N L A L A D S P K Q S Q K I V G Y F P S W 50 GGC GTT TAC GGA CGT AAT TAT CAA GTT GCT GAC ATT GAT GCA TCA AAA CTT ACT CAC CTT AAC TAT GCT TTC GCG 225 G V Y G R N Y Q V A D I D A S K L T H L N Y A F A 75 GAT ATT TGT TGG AAT GGA AAA CAT GGA AAC CCT TCT ACT CAT CCT GAT AAT CCA AAT AAA CAA ACG TGG AAC TGT 300 D I C W N G K H G N P S T H P D N P N K Q T W N C 100 AAA GAA TCT GGT GTA CCA TTG CAA AAT AAA GAG GTT CCT AAT GGT ACT CTC GTA CTC GGG GAC CAT GGG CTG ATG 375 K E S G V P L Q N K E V P N G T L V L G D H G L M 125 TTA CCA AAT CGG TAT CCT GGC TCA GGG ACA ACT TGG GAA GAT TGC GAT AAA TAT GCC CGT TGC GGA AAT TTC GGG 450 L P N R Y P G S G T T W E D C D K Y A R C G N F G 150 GAA CTA AAA CGA TTA AAA GCT AAA TAT CCT CAC TTA AAA ACA ATT ATT TCC GTT GGT GGC TGG ACT TGG TCT AAC 525 E L K R L K A K Y P H L K T I I S V G G W T W S N 175 CGC TTT TCT GAT ATG GCC GCT GAT GAA AAA ACA AGA AAA GTA TTT GCT GAA TCT ACA GTA GCT TTT CTT CGC GCA 600 R F S D M A A D E K T R K V F A E S T V A F L R A 200 TAT GGG TTT GAT GGC GTA GAT TTA GAC TGG GAA TAT CCG GGC GTT GAA ACG ATT CCT GGT GGT AGT TAT CGT CCT 675 Y G F D G V D L D W E Y P G V E T I P G G S Y R P 225 GAA GAT AAA CAA AAT TTC ACT CTC CTT CTT CAA GAC GTC CGA AAT GCT TTG AAT AAA GCA GGT GCT GAA GAT GGC 750 E D K Q N F T L L L Q D V R N A L N K A G A E D G 250 AAA CAA TAT TTA CTA ACA GTC GCT TCA GGT GCA AGC CAA CGC TAC GCT GAT CAT ACA GAA CTA AAG AAA ATT TCT 825 K Q Y L L T V A S G A S Q R Y A D H T E L K K I S 275 CAA ATA CTC GAT TGG ATT AAT ATT ATG ACA TAT GAT TTC CAC GGC GGA TGG GAA GCT ACT TCT AAT CAT AAT GCA 900 Q I L D W I N I M T Y D F H G G W E A T S N H N A 300 GCT CTA TAT AAG GAT CCA AAT GAT CCA GCA GCA AAT ACG AAT TTT TAC GTA GAT GGT GCT ATA AAT GTT TAT ACA 975 A L Y K D P N D P A A N T N F Y V D G A I N V Y T 325 AAT GAA GGT GTT CCA GTC GAT AAA CTA GTA TTA GGC GTA CCC TTT TAC GGA CGT GGC TGG AAA AGT TGT GGC AAA 1050 N E G V P V D K L V L G V P F Y G R G W K S C G K 350 GAA AAT AAC GGA CAA TAT CAA CCT TGC AAA CCA GGT AGT GAT GGG AAA CTT GCT TCT AAA GGT ACT TGG GAT GAT 1125 E N N G Q Y Q P C K P G S D G K L A S K G T W D D 375 TAC TCT ACC GGT GAC ACA GGT GTC TAT GAT TAC GGT GAT TTA GCA GCC AAT TAC GTT AAT AAA AAT GGT TTT GTA 1200 Y S T G D T G V Y D Y G D L A A N Y V N K N G F V 400 CGC TAC TGG AAT GAC ACA GCT AAA GTA CCT TAC TTA TAT AAT GCA ACT ACA GGC ACA TTT ATT AGC TAC GAT GAC 1275 R Y W N D T A K V P Y L Y N A T T G T F I S Y D D 425 AAT GAA TCT ATG AAA TAC AAA ACA GAC TAT ATA AAG ACG AAA GGT TTA AGT GGA GCA ATG TTT TGG GAA CTA AGC 1350 N E S M K Y K T D Y I K T K G L S G A M F W E L S 450 GGA GAT TGC CGT ACA AGT CCA AAA TAT AGT TGC AGT GGT CCA AAA TTA CTT GAT ACG CTA GTA AAA GAA TTA CTT 1425 G D C R T S P K Y S C S G P K L L D T L V K E L L 475 GGT GGA CCT ATT AAT CAA AAA GAT ACT GAG CCA CCA ACG AAT GTT AAA AAC ATT GTA GTT ACG AAT AAA AAT TCA 1500 G G P I N Q K D T E P P T N V K N I V V T N K N S 500 AAC TCA GAA CAA TTA AAC TGG ACT GCA TCT ACT GAT AAC GTA GGA GTT ACC GAA TAT GAA ATT ACT GCT GGA GAA 1575 N S E Q L N W T A S T D N V G V T E Y E I T A G E 525 GAG AAA TGG AGT ACA ACA ACA AAT AGC ATT ACA ATT AAA AAC TTA AAA CCT AAT ACG GAA TAC AAA TTT TCG GTA 1650 E K W S T T T N S I T I K N L K P N T E Y K F S V 550 ATT GCC AAA GAT GCT GCT GGA AAT AAA TCA CAA CCC ACC GCT CTT ACT GTC AAA ACG GAT GAA GCT AAT ACG ACA 1725 I A K D A A G N K S Q P T A L T V K T D E A N T T 575 CCT CCT GAT GGA AAT GGT ACT GCT ACA TTT TCA GTC ACT TCG AAT TGG GGC AGC GGT TAT AAC TTC TCG ATT ATA 1800 P P D G N G T A T F S V T S N W G S G Y N F S I I 600 ATC AAA AAT AAT GGA ACG AAC CCT ATT AAA AAT TGG AAA TTA GAA TTT GAT TAT AGC GGC AAT TTA ACA CAA GTT 1875 I K N N G T N P I K N W K L E F D Y S G N L T Q V 625 TGG GAT TCT AAA ATT AGT AGT AAA ACA AAT AAT CAT TAT GTA ATT ACG AAC GCA GGA TGG AAT GGT GAA ATT CCT 1950 W D S K I S S K T N N H Y V I T N A G W N G E I P 650 CCT GGT GGA TCT ATT ACA ATT GGC GGT GCA GGA ACA GGT AAT CCT GCC GAA CTT TTA AAA GCC GTC ATT AGC GAA 2025 P G G S I T I G G A G T G N P A E L L K A V I S E 675 AAC TAG 2031 N * 677 ACGTAATATCCATTAATTACTTCACTGAAGTTTGAATTTAGGTTTGAGCAATACCTCCTAAATTCAAACTTTTAATTTTATGAAAAATCAAAAACATCTCAATAAC TTGTTATAATTATATTTAGTTTCTAATATGAAGTTTATATGAAGTTCATTTGAATTTTACATGGATTTCCTTTAATTTCTATTGTCTTCCTATAATATCCATATTA TCCTTTACATAAAGGAATTATTTTCATTCCATTTTGAACTATCTATATATATAAGAAGATAGTCACCTGTTAGGAAAGCTTTCGTATTATCATAAATATAACAAAAG AAAAAAGCACGCTTATAAGCGCACCATAAGAGTGCTTCTTCATACAATTCTCATGTAAGAGTTCTCGAAAATCATGTTAATCACAAAGAAAAGGCATTAGCTCATAA AAGCTAATGCCTTTTCTTTATTCATATTGTTCAGCATTCCAATCTTCTATATTCCATACTTTTGTTATCCACGATTCATAGAAAGAAGGCTCATGTGAGACAATTAA AACCGTTCCTTTGTATTGTTGTAATGCTTCCTATAAAGCCATCTTAGTTTCTACATCCAAATGATTAGTCCGTTCGTCTAAAATTAAAACAATC

40 Construction of a recombinant chitinase able to integrate B. thuringiensis crystals by its fusion with the C-terminal part of a delta-endotoxin Fusion of the chitinase Chi255 of B. thuringiensis with the C- terminal domain of the δ-endotoxin Cry1Ac responsible for its crystallization.

41 BIOINSECTICIDES BASED ON DELTA- ENDOTOXINS OF BACILLUS THURINGIENSIS- PROCESS OF PRODUCTION

42 -BIOINSECTICIDES PRODUCTION BY FERMENTATION Stress conditions Adaptation Classical Mutagenesis B. thuringiensis Control of metabolism oxidative metabolism and Catabolic Repression Combination of different improvement factors

43 *ZOUARI N, BEN SIK ALI S and JAOUA S (2002) Production of delta-endotoxins by several Bacillus thuringiensis strains exhibiting various entomocidal activities towards lepidoptera and diptera in gruel and fish-meal media, Enz. Microb. Technol. 31, *ZOUARI N, Achour O and JAOUA S (2002) Production of delta-endotoxin by Bacillus thuringiensis subsp. kurstaki and overcome of catabolite repression, by using highly concentrated gruel and fish meal media in 2 and 20 dm3 fermenters J. Chem. Technol. Biotechnol. 77, *GHRIBI D, ZOUARI N, and JAOUA S (2004) Improvement of Bioinsecticide Production through mutagenesis of Bacillus thuringiensis subsp. kurstaki by UV and Nitrous acid affecting metabolic pathways and/or delta-endotoxins synthesis J. Appl. Microbiol. 97, *D. GHRIBI, N. ZOUARI AND S. JAOUA (2005) Improvement of bioinsecticides production through Adaptation of Bacillus thuringiensis to heat treatment and NaCl addition J. Appl. Microbiol. 98, D. GHRIBI, N. ZOUARI AND S. JAOUA (2007) Use of sea water as salts source in starch and soya bean based media, for the production of Bacillus thuringiensis bioinsecticides Process Biochem. 42, Dhouha Ghribi, Nabil Zouari*, Hassen Trabelsi and Samir Jaoua (2006) Improvement of Bacillus thuringiensis delta-endotoxin production by overcome of carbon catabolite repression through adequate control of aeration Enz. Microbiol Technol. 40,

44 Scale up of the Bioinsecticides production 300 litres fermenter used for Bt Bioinsecticides production Recovery by Ultrafiltration Formulation (liquid form)

45 Result: 100 % insects mortality after 2 days post treatment of 39 olive trees Application at small field scale, for the control of olive tree entomopathogenic insect: Prays olea

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48 LABORATORY OF BIOPESTICIDES (L.B.Pes.) AOUA Samir, Professor, Director of the Laboratory (previously), now in QU OUARI Nabil, Professor OUNSI Slim, Associate-Professor AMMOUSSI Kais, Assistant-Professor OUIS Souad, Assistant-Professor RIGUI Mohamed, Assistant-Professor ZZOUZ Hichem, Assistant-Professor BDELKAFI Lobna, Assistant Professor RIBI Raïda, Assistant Professor li Hacina, Engineer ELGHITH Najeh, Technician ALGHOUTHI Aïcha, Technician AMOUN Fakher, Post-Doc AMMAK Mariam, Engineer, PhD ALLOULI Wafa, Engineer, PhD EN KHEDHER Saoussan, PhD RISS Fatma, Post Doc AADAOUI Imène, PhD ILANI Olfa, PhD EN HSOUNA Anis, PhD OUHKRIS Mariam, PhD ELLAMI Sameh, PhD EN FGUIRA Ines, PhD NNOURI Karim, PhD Master degree students L.B.Pes. Centre of Biotechnology of Sfax TUNISIA

49 In the CAS, Dept Biol &Env Sc, we are developing an integrated approach that will explore activities of new Bt isolates from the Qatar and Gulf region, unexplored area, by performing a big programme of Bt strain isolation from different places in this region, screening using modern molecular techniques, gene investigation, bioassays. The novel strains with high insecticidal activity will be used for the optimization of bioinsecticide production at large scale fermentation. Success in any of these actions will have great impact on Qatar and Gulf agriculture and health in respect to respectively food safety and control of disease vector insects and possibly also socio-economic impact Thanks, With my best regards