The classification of Bacillus thuringiensis strains has been revised and updated based on flagellar antigens which have been in use for many years. Sixty‐nine serotypes and 13 sub‐antigenic groups have now been identified, giving 82 serovars among the 3500 B. thuringiensis isolates of the IEBC Collection. The number of serovars has gradually increased with the total number of strains. The biochemical characters used have also been investigated and their value assessed for identification of B. thuringiensis at the subspecies level. A crystal analysis was carried out in terms of morphology, δ‐endotoxin profiles and larvicidal activity for the newly identified serovars. It was found that atypical crystals, some with novel components, are becoming more common. No insect susceptible to these serovars has been discovered among known target species. The number of cross‐reacting H‐antigens among B. cereus strains is increasing and may be of biological significance.

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The flagellar agglutination showed that both strains bore specific H antigens which allowed their classification as the new serotypes H57 and H59. The serovar names proposed for the type strains characterized in this work are B. thuringiensis ser. pirenaica, for the H serotype 57, and B. thuringiensis ser. iberica, for the H serotype 59. Further characterization of these strains, by means of SDS-PAGE, Western inmunodetection, plasmid profile and cry-gene identification by polymerase chain reaction, confirmed the originality of the two novel serotypes. Toxicity tests carried out against several insect species, belonging to the orders Lepidoptera, Diptera and Coleoptera, showed no detectable insecticidal activity for either of the B. thuringiensis strains.

MATERIALS AND METHODS Bacterial strains. B. thuringiensis H serotype reference strains were obtained from the collection of Dulmage (7) or from the Entomopathogenic Bacteria collection of the Institut Pasteur, Paris, France. Growth. For the production of crystal proteins, B. thuringiensis strains were grown as shake flask cultures in DSMG medium [0.4% (wt/vol) nutrient broth (Difco), 0.5% (wt/vol) glucose, 25 mM K2HPO4, 25 mM KH2PO4, 0.5 mM Ca(NO3)2, 0.5 mM MgSO4, 10 ,uM MnCl2, 10 ,uM FeSO4] at room temperature (21 to 24°C) for 4 to 5 days until sporulation and cell lysis had occurred. For the preparation of flagella, B. thuringiensis strains were grown as static liquid cultures in nutrient broth (Difco) plus 0.1% (wt/vol) glucose. Colony hybridization. A modified colony hybridization procedure of Grunstein and Hogness (12) was used. Environmental samples, consisting of soil, crop dust, and organic material, were suspended in spore buffer (0.06% [wt/vol] sodium phosphate [pH 7.0], 0.05% [wt/vol] potassium phosphate [pH 6.6], 0.005% [wt/vol] sodium dodecyl sulfate [SDS], 1 ,u1 of 1-octanol per ml; final pH of buffer approximately 7) to a concentration of 1 g of material per 10 ml of buffer, heated for 30 min at 60°C, diluted in spore buffer, and spread on NSM (19) agar medium. After overnight growth at 25°C, colonies were blotted from the agar medium to nitrocellulose filters and treated with 0.5 M NaOH-1.5 M NaCl for 30 min and then with 1 M ammonium acetate-0.02 M NaOH for 30 min. The filters were probed at 65°C in a solution containing 3x SSC (lx SSC is 0.15 M NaCl plus 0.015 M sodium citrate), lOx Denhardt's solution (lx Denhardt's solution is 0.02% [wt/vol] bovine serum albumin, 0.02% [vol/vol] Ficoll, 0.02% [wt/vol] polyvinylpyrrolidone), 200 ,ug of heparin per ml, and 0.1% SDS with the 2.0-kb HindIII-XbaI cryIlIA fragment of strain EG2158 (6) or with 3337