Characterization of a NewBacillus thuringiensisSubsp.higoStrain Isolated from Rice Bran in Korea

JOURNAL OF INVERTEBRATE PATHOLOGY ARTICLE NO.

71, 95–96 (1998)

IN974697

NOTE Characterization of a New Bacillus thuringiensis Subsp. higo Strain Isolated from Rice Bran in Korea A new Bacillus thuringiensis strain was isolated from grain dusts of rice brans collected from a rice mill located in Changwon, Kyungnam province in South Korea and was named BT205. Serotyping revealed that this isolate belongs to serotype H44 and accordingly was classified as B. thuringiensis subsp. higo. B. thuringiensis subsp. higo was reported as a new subspecies isolated from the silkworm-rearing litter, in Kumamoto, Japan and having an insecticidal activity against the anopheline mosquito (Ohba et al., 1995). BT205 is only the second known isolate of serotype H44 and there has been no report on the detailed characterization of B. thuringiensis subsp. higo. Here we report the characterization of B. thuringiensis subsp. higo BT205. This isolate was a Gram-positive and motile rod with dimensions of 1.0–1.1 3 2.8–4.8 µm. There were no significant differences in the shape and size of the vegetative cells between BT205, B. thuringiensis subsp. higo and the known B. thuringiensis serotypes. The parasporal inclusion bodies (crystals) produced by BT205 was spherical in shape (Fig. 1). BT205 and the original B. thuringiensis subsp. higo were both examined for their biochemical characteristics. They both showed positive reactions in VogesProskauer, anerobic growth; production of catalase, arginine dihydrolase, oxidase, urease, gelatinase; and utilization of casein, esculine, glucose, maltose, salicine, starch, glycerol, ribose, fructose, N-acetylglucosamine, amygdaline, arbutine, cellobiose, trehalose, glycogene. However, both were negative in nitrate reduction; production of indole, H2S, b-galactosidase, phenylalanine deaminase, tryptophane deaminase, lysine decarboxylase, ornithine decarboxylase; and utilization of adonitol, arabinose, citrate, dulcitol, inositol, lactose, mannitol, raffinose, rhamnose, sorbitol, sucrose, xylose, mannose. The minimum inhibitory concentration (µg/ml) of various antibiotics against BT205 were as follows: amikacin, 6.25; ampicillin, .100; bacitracin, .100; cephalothin, 100; chloramphenicol, 12.5; chlortetracycline, 3.125; colistin, .100; erythromycin, ,1.56; gentamycin, 12.5; kanamycin, 12.5; methicillin, .100; neomycin, 6.25; novobiocin, 3.125; penicillin G, .100; polymyxin B sulfate, .100; rifampicin, 6.25; streptomycin, 12.5; tetracycline, 6.25; and tobramycin, 3.125.

Insecticidal activity of the isolate BT205 was examined on the second to third instar of Bombyx mori (Lep.: Bombycidae), Hyphantria cunea (Lep.: Arctiidae), and Culex tritaeniorhynchux (Dip.: Culicidae). BT205 did not show any insecticidal activity against these insects. BT205 and B. thuringiensis subsp. higo were assayed on the third instar of Leptinotarsa decemlineata (Col.: Chrysomelidae). Neither showed any insecticidal activity. It has been reported that B. thuringiensis subsp. higo showed low to moderate insecticidal toxicities against two mosquito species, Anopheles stephensi and C. pipiens molestus while displaying no activity against B. mori and H. cunea (Ohba et al., 1995). We have shown through PCR using specific primers and Southern blotting hybridization with specific probes that B. thuringiensis subsp. higo BT205 had neither cryIVA nor -IVBgene encoding crystal protein toxic to dipteran larvae (Jung et al., 1997). It is considered that although the reported B. thuringiensis subsp. higo has a larvicidal activity for two mosquito species, the isolate

FIG. 1. Transmission electron micrograph of B. thuringiensis subspecies higo BT205 after incubation for 48 hr at 30°C. The isolate BT205 has a spherical inclusion. Arrowheads indicate parasporal inclusions. 37,900. 95

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BT205 may have novel cry-type genes other than cryIV-type genes. SDS–PAGE analysis of sucrose gradient-purified parasporal bodies showed that crystal proteins from the isolate BT205 consist of two main polypeptides of approximately 95 and 52 kDa (data not shown). The result indicates that the isolate BT205 produces parasporal inclusions (crystals) of unique molecular weight different from those of known B. thuringiensis subspecies (Ho¨fte and Whitely, 1989; Stahly et al., 1992). So far, only two B. thuringiensis subsp. higo strains are known. Both were isolated in North-East Asia. It would be interesting to know whether both strains are geographically restricted to this part of the world or are more widely distributed. If the former holds true than both strains might be pathogens of invertebrates also restricted to North-East Asia. KEY WORD: Bacillus thuringiensis subsp. higo. REFERENCES Ho¨fte, H., and Whitely, H. R. 1989. Microbiol. Rev. 53, 242–255. Jung, Y. C., Kim, S. U., Bok, S. H., Park, H. Y., Coˆte´, J.-C., and Chung, Y. S. 1997. Can. J. Microbiol. 43, 403–410. Ohba, M., Saitoh, H., Miyamoto, K., Higuchi, K., and Mizuki, E. 1995. Lett. Appl. Microbiol. 21, 316–318. Stahly, D. P., Andrews, R. E., and Yousten, A. A. 1992. In ‘‘The Prokaryotes’’ (A. Balows, H. G. Tru¨per, M. Dworkin, W. Harder, and

K.-H. Schleifer, Eds.), Vol. 2, pp. 1697–1745. Springer-Verlag, New York.

Yong Chul Jung* Sung Uk Kim† Jean-Charles Coˆte´ ‡,1 Marguerite-M. Lecadet§ Young Sup Chung* Song Hae Bok† *De´partement de Sciences Biologiques Universite´ de Montre´al Que´bec, Canada, H3C 3J7 †Bioproducts Research Group Korea Research Institute of Bioscience & Biotechnology, K.I.S.T. P.O. Box 115, Yu-Sung Taejon, 305-600, Korea ‡Agriculture and Agri-Food Canada Research Centre 430 Boul. Gouin, St-Jean-sur-Richelieu Que´bec, Canada, J3B 3E6 §Unite´ des Bacte´ries Entomopathoge`nes Institut Pasteur, Paris, France Received March 11, 1997, accepted June 26, 1997 1 To whom correspondence should be addressed. Fax: 1-514-3467740. E-mail: [email protected].