Serotyping of Bacillus thuringiensis environmental isolates by extracellular heat-stable somatic antigens U E D A ,AND ~ KEIOA I Z A W A ~ MICHIOOHBA, KOUSUKE Institute of Biological Control, Faculty of Agriculture, Kyushu University, Fukuoka 812, Japan Received September 17, 1991 Revision received January 3, 1992
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by Depository Services Program on 11/12/14 For personal use only.
Accepted January 24, 1992 OHBA,M., UEDA,K., and AIZAWA, K. 1992. Serotyping of Bacillus thuringiensis environmental isolates by extracellular heat-stable somatic antigens. Can. J. Microbiol. 38: 694-695. A total of 525 Bacillus thuringiensis environmental isolates, belonging to the five flagellar (H) serovars (alesti, sotto, kenyae, aizawai, and morrisoni), were serotyped by extracellular heat-stable somatic antigens (HSSAs). The isolates belonging to a given H serovar were assigned to a single HSSA serogroup at a high frequency, 87-100%. This indicates that the extent of HSSA variation within a single H serovar is small in the field populations of these B. thuringiensis serovars. Key words: Bacillus thuringiensis, serotyping, extracellular heat-stable somatic antigen. OHBA,M., UEDA,K., et AIZAWA, K. 1992. Serotyping of Bacillus thuringiensis environmental isolates by extracellular heat-stable somatic antigens. Can. J. Microbiol. 38 : 694-695. Un total de 525 souches environnementales de Bacillus thuringiensis appartenant a cinq serovars (H) flagellaires (alesti, sotto, kenyae, aizawai et morrisoni) ont ete serotypees d'apres les antigenes somatiques extracellulaires thermostables (HSSAs). Les souches appartenant a un serovar H donne ont ete rattachees a un seul serogroupe HSSA selon une frequence elevee de 87 a 100%. Cela confirme que le degre de variation HSSA a l'interieur d'un serovar H precis est faible dans les populations sauvages de ces serovars de B. thuringiensis. Mots clks : Bacillus thuringiensis, serotypage, antigene somatique extracellulaire thermostable. [Traduit par la redaction]
Bacillus thuringiensis is among the most important bacterial agents for control of insect pests of agricultural and medical importance. It is generally accepted that this bacterium has two major antigens, the flagellar (H) antigen and the heat-stable somatic antigen (HSSA), on the surface of vegetative cells. The H serotyping of B. thuringiensis was established by de Barjac and Bonnefoi (1962) on the basis of the genetical stability and the group specificity of H antigens. This method has long been used successfully for classification and identification of the strains (de Barjac and Frachon 1990). However, earlier workers have suggested that the HSSA is also useful for intraspecific classification of B. thuringiensis (Sekijima and Ono 1982; Sekijima et al. 1987). In our preceding study (Mike et al. 1990), we demonstrated that B. thuringiensis produces the HSSA extracellularly during vegetative growth and this is detectable by the precipitin halo formation (PHF) test on antiserum-agar plates. More recently, Ueda et al. (1991) established, by using the P H F test, the 16 extracellular HSSA serogroups of B. thuringiensis. In the present study, we serotyped extracellular HSSAs of B. thuringiensis environmental isolates by P H F tests. The objective of our study was t o establish the degree of HSSA variation within a given H serovar of B. thuringiensis. All of the 525 B. thuringiensis isolates used were from the Bacillus thuringiensis Collection, Institute of Biological Control, Kyushu University. These isolates had been col-
lected from sericultural environments of the three main islands (Kyushu, Shikoku, and Honshu) of Japan over 30 years, and had been identified as H serovars alesti, sotto, kenyae, aizawai, and morrisoni in our earlier studies (Ohba and Aizawa 1978,1989; Ohba et al. 1979, 1981, 1984). These five H serovars are predominant in sericultural environments of Japan (Dulmage and Aizawa 1982). For identification of the extracellular HSSA serotype, the P H F tests were carried out following the method of Mike et al. (1990). Bacteria were precultured on nutrient agar (Difco) at 30°C overnight. The culture was then pin-spot inoculated on nutrient agar plates containing the reference HSSA antiserum at a concentration of 2070 (v/v). After incubation at 30°C for 18 h, the extracellular HSSA serotype of the isolate was determined according t o the HSSA serotyping scheme (Ueda et al. 1991) on the basis of the formation of immunoprecipitin halo surrounding the colonies. Reference HSSA antisera were raised in rabbits, as described previously (Ueda et al. 1991), against the strains of the 24 H serovars belonging to the 16 extracellular HSSA serogroups. The results are given in Table 1. Of 525 isolates tested, 500 were serotypable by P H F tests with 24 HSSA antiserumagar plates. Among the 165 isolates belonging t o H serovar alesti, 164 were assigned to HSSA serogroup 111. Similarly, .the HSSA of H serovar aizawai isolates was serologically highly homogenous; all 114 isolates from various geographical locations were referable t o HSSA serogroup IV. In the three other H serovars (kenyae, sotto, and morrisoni), 89-97% of the isolates fell into a single HSSA serogroup. Twenty-five isolates belonging t o H serovars alesti, sotto, kenyae, and morrisoni did not form visible precipitin halos 0" any of the refa-ence antiserum-agar plates. There are two likely explanations for this: (i) the amount of extracellular HSSA in these isolates is too small to be detected by the P H F tests and (ii) these isolates belong t o -
' ~ u t h o rto whom all correspondence should be addressed. address: National Federation of Waste Management Associations, Nagata-cho 1, Chiyoda-k~,Tokyo 100, Japan. 3~resentaddress: Department of Biosciences, Teikyo University, Utsunomiya 320, Japan. Printed in Canada / Imprime au Canada
-
NOTES
TABLE1. Serotyping of B. thuringiensis isolates by HSSAs
H serovar"
alesti (111) sotto (V)
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by Depository Services Program on 11/12/14 For personal use only.
kenyae (IV) aizawai (IV) morrisoni (VIII)
~ocalit~
No. of isolates tested
I11
IV
V
VIII
NSC
Honshu (1 1) Shikoku (1) Honshu (12) Shikoku (3) Kyushu (1) Honshu (3) Shikoku (3) Kyushu (4) Honshu (1 1) Shikoku (3) Kyushu (4) Honshu (10) Shikoku (4) Kyushu (3)
'The extracellular HSSA serotype to which the type strain of the indicated H serovar belongs is given in parentheses (Ueda et al. 1991). b ~ h number e of sericultural farms from which the indicated H serovar was detected is given in parentheses. CNS, not serotypable.
unknown HSSA serogroup(s). These possibilities should be clarified bv further investigation. " Previous investigators have demonstrated that variation is occasionally evident in the HSSA of several H serovars of B. thuringiensis (Padua et a / . 1980, 1984; Sekijima and these On0 l982; Krieg et al. 19g7; Mike et al. 1990). observations, it has often been assumed that there is a substantial HSSA heterogeneity within a single H serogroup of B. thuringiensis. However, the overall pattern of our results revealed that the degree of HSSA heterogeneity is rather small in the five H serovars tested. This suggests that the HSSA is a genetically stable character in these B. thuringiensis serovars. In another study (Mike et al. 1991), we have also shown that the HSSA is genetically stable within a B. thuringiensis strain, and the frequency of spontaneous HSSA variants is quite low. Interestingly, most isolates belonging to H serovars kenyae and aizawai were assigned to HSSA serogroup IV. This is in good agreement with our previous assignment of the type strains of these two H serovars to HSSA serogroup IV (Ueda et al. 1991). Our findings indicate that there is a discrepancy between the two serogrouping systems of B. thuringiensis. .-
No. of isolates assigned to extracellular HSSA serotype:
- -- - - - -
-
- 4
-
-
-
-
de Barjac, H., and Bonnefoi, A. 1962. Essai de classification biochimique et serologique de 24 souches de Bacillus du type B. thuringiensis. Entomophaga, 7: 5-3 1. de Barjac, H., and Frachon, E. 1990. Classification of Bacillus thuringiensis. Entomophaga, 35: 233-240. Dulmage, H.T., and Aizawa, K. 1982. Distribution of Bacillus thuringiensis in nature. In Microbial and viral pesticides. Edited by E. Kurstak. Marcel Dekkar, New York. pp 209-237. Krieg, A., Schnetter, W., Huger, A.M., and Langenbruch, G.A. 1987. Bacillus thuringiensis subsp. tenebrionis, strain BI256-82: a third pathotype within the H-serotype 8a8b. Syst. Appl. Microbiol. 9: 138-141.
Mike, A., Ohba, M., and Aizawa, K. 1990. Extracellular production of a heat-stable somatic antigen by Bacillus thuringiensis. LettMicrObiO1247-250. Mike, A*, Ohba, M.9 and Aizawa, K- 19g1. Spontaneous occurrence of heat-stable somatic antigen variants in Bacillus thuringiensis. J. Appl. Bacterial. 70: 408-413. Ohba, M., and Aizawa, K. 1978. Serological identification of Bacillus thuringiensis and related bacteria isolated in Japan. J . Invertebr. Pathol. 32: 303-309. Ohba, M., and Aizawa, K. 1989. Distribution of the four flagellar (H) antigenic subserotypes of Bacillus thuringiensis H serotype 3 in Japan. J . Appl. Bacteriol. 67: 505-509. Ohba, M., Aizawa, K., and Furusawa, T. 1979. Distribution of Bacillus thuringiensis serotypes in Ehime Prefecture, Japan. Appl. Entomol. Zool. 14: 340-345. Ohba, M., Aizawa, K., and Shimizu, S. 1981. A new subspecies of Bacillus thuringiensis isolated in Japan: Bacillus thuringiensis subsp. tohokuensis (serotype 17). J. Invertebr. Pathol. 38: 307-309. Ohba, M., Aizawa, K., and Sudo, S. 1984. Distribution of Bacillus thuringiensis in sericultural farms of Fukuoka Prefecture, Japan. Proc. Assoc. Plant Prot. Kyushu, 30: 152-155. Padua, L.E., Ohba, M., and Aizawa, K. 1980. The isolates of Bacillus thuringiensis serotype 10 with a highly preferential toxicity to mosquito larvae. J. Invertebr. Pathol. 36: 180-186. Padua, L.E., Ohba, M., and Aizawa, K. 1984. Isolation of a Bacillus thuringiensis strain (serotype 8a:8b) highly and selectively toxic against mosquito larvae. J. Invertebr. Pathol. 44: 12-17. Sekijima, Y., and Ono, K. 1982. Grouping of Bacillus thuringiensis by heat-stable somatic antigens. Appl. Entomol. Zool. 17: 393-397. Sekijima, Y., Fujikura, Y., and Ono, K. 1987. Presence of specificity of heat-stable somatic antigens among Bacillus thuringiensis 17 subspecies. Appl. Entomol. Zool. 22: 403-409. Ueda, K., Ohba, M., and Aizawa, K. 1991. Serogrouping of Bacillus thuringiensis by extracellular heat-stable somatic antigens. Syst. Appl. Microbiol. 14: 291-294.
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by Depository Services Program on 11/12/14 For personal use only.
Accepted January 24, 1992 OHBA,M., UEDA,K., and AIZAWA, K. 1992. Serotyping of Bacillus thuringiensis environmental isolates by extracellular heat-stable somatic antigens. Can. J. Microbiol. 38: 694-695. A total of 525 Bacillus thuringiensis environmental isolates, belonging to the five flagellar (H) serovars (alesti, sotto, kenyae, aizawai, and morrisoni), were serotyped by extracellular heat-stable somatic antigens (HSSAs). The isolates belonging to a given H serovar were assigned to a single HSSA serogroup at a high frequency, 87-100%. This indicates that the extent of HSSA variation within a single H serovar is small in the field populations of these B. thuringiensis serovars. Key words: Bacillus thuringiensis, serotyping, extracellular heat-stable somatic antigen. OHBA,M., UEDA,K., et AIZAWA, K. 1992. Serotyping of Bacillus thuringiensis environmental isolates by extracellular heat-stable somatic antigens. Can. J. Microbiol. 38 : 694-695. Un total de 525 souches environnementales de Bacillus thuringiensis appartenant a cinq serovars (H) flagellaires (alesti, sotto, kenyae, aizawai et morrisoni) ont ete serotypees d'apres les antigenes somatiques extracellulaires thermostables (HSSAs). Les souches appartenant a un serovar H donne ont ete rattachees a un seul serogroupe HSSA selon une frequence elevee de 87 a 100%. Cela confirme que le degre de variation HSSA a l'interieur d'un serovar H precis est faible dans les populations sauvages de ces serovars de B. thuringiensis. Mots clks : Bacillus thuringiensis, serotypage, antigene somatique extracellulaire thermostable. [Traduit par la redaction]
Bacillus thuringiensis is among the most important bacterial agents for control of insect pests of agricultural and medical importance. It is generally accepted that this bacterium has two major antigens, the flagellar (H) antigen and the heat-stable somatic antigen (HSSA), on the surface of vegetative cells. The H serotyping of B. thuringiensis was established by de Barjac and Bonnefoi (1962) on the basis of the genetical stability and the group specificity of H antigens. This method has long been used successfully for classification and identification of the strains (de Barjac and Frachon 1990). However, earlier workers have suggested that the HSSA is also useful for intraspecific classification of B. thuringiensis (Sekijima and Ono 1982; Sekijima et al. 1987). In our preceding study (Mike et al. 1990), we demonstrated that B. thuringiensis produces the HSSA extracellularly during vegetative growth and this is detectable by the precipitin halo formation (PHF) test on antiserum-agar plates. More recently, Ueda et al. (1991) established, by using the P H F test, the 16 extracellular HSSA serogroups of B. thuringiensis. In the present study, we serotyped extracellular HSSAs of B. thuringiensis environmental isolates by P H F tests. The objective of our study was t o establish the degree of HSSA variation within a given H serovar of B. thuringiensis. All of the 525 B. thuringiensis isolates used were from the Bacillus thuringiensis Collection, Institute of Biological Control, Kyushu University. These isolates had been col-
lected from sericultural environments of the three main islands (Kyushu, Shikoku, and Honshu) of Japan over 30 years, and had been identified as H serovars alesti, sotto, kenyae, aizawai, and morrisoni in our earlier studies (Ohba and Aizawa 1978,1989; Ohba et al. 1979, 1981, 1984). These five H serovars are predominant in sericultural environments of Japan (Dulmage and Aizawa 1982). For identification of the extracellular HSSA serotype, the P H F tests were carried out following the method of Mike et al. (1990). Bacteria were precultured on nutrient agar (Difco) at 30°C overnight. The culture was then pin-spot inoculated on nutrient agar plates containing the reference HSSA antiserum at a concentration of 2070 (v/v). After incubation at 30°C for 18 h, the extracellular HSSA serotype of the isolate was determined according t o the HSSA serotyping scheme (Ueda et al. 1991) on the basis of the formation of immunoprecipitin halo surrounding the colonies. Reference HSSA antisera were raised in rabbits, as described previously (Ueda et al. 1991), against the strains of the 24 H serovars belonging to the 16 extracellular HSSA serogroups. The results are given in Table 1. Of 525 isolates tested, 500 were serotypable by P H F tests with 24 HSSA antiserumagar plates. Among the 165 isolates belonging t o H serovar alesti, 164 were assigned to HSSA serogroup 111. Similarly, .the HSSA of H serovar aizawai isolates was serologically highly homogenous; all 114 isolates from various geographical locations were referable t o HSSA serogroup IV. In the three other H serovars (kenyae, sotto, and morrisoni), 89-97% of the isolates fell into a single HSSA serogroup. Twenty-five isolates belonging t o H serovars alesti, sotto, kenyae, and morrisoni did not form visible precipitin halos 0" any of the refa-ence antiserum-agar plates. There are two likely explanations for this: (i) the amount of extracellular HSSA in these isolates is too small to be detected by the P H F tests and (ii) these isolates belong t o -
' ~ u t h o rto whom all correspondence should be addressed. address: National Federation of Waste Management Associations, Nagata-cho 1, Chiyoda-k~,Tokyo 100, Japan. 3~resentaddress: Department of Biosciences, Teikyo University, Utsunomiya 320, Japan. Printed in Canada / Imprime au Canada
-
NOTES
TABLE1. Serotyping of B. thuringiensis isolates by HSSAs
H serovar"
alesti (111) sotto (V)
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by Depository Services Program on 11/12/14 For personal use only.
kenyae (IV) aizawai (IV) morrisoni (VIII)
~ocalit~
No. of isolates tested
I11
IV
V
VIII
NSC
Honshu (1 1) Shikoku (1) Honshu (12) Shikoku (3) Kyushu (1) Honshu (3) Shikoku (3) Kyushu (4) Honshu (1 1) Shikoku (3) Kyushu (4) Honshu (10) Shikoku (4) Kyushu (3)
'The extracellular HSSA serotype to which the type strain of the indicated H serovar belongs is given in parentheses (Ueda et al. 1991). b ~ h number e of sericultural farms from which the indicated H serovar was detected is given in parentheses. CNS, not serotypable.
unknown HSSA serogroup(s). These possibilities should be clarified bv further investigation. " Previous investigators have demonstrated that variation is occasionally evident in the HSSA of several H serovars of B. thuringiensis (Padua et a / . 1980, 1984; Sekijima and these On0 l982; Krieg et al. 19g7; Mike et al. 1990). observations, it has often been assumed that there is a substantial HSSA heterogeneity within a single H serogroup of B. thuringiensis. However, the overall pattern of our results revealed that the degree of HSSA heterogeneity is rather small in the five H serovars tested. This suggests that the HSSA is a genetically stable character in these B. thuringiensis serovars. In another study (Mike et al. 1991), we have also shown that the HSSA is genetically stable within a B. thuringiensis strain, and the frequency of spontaneous HSSA variants is quite low. Interestingly, most isolates belonging to H serovars kenyae and aizawai were assigned to HSSA serogroup IV. This is in good agreement with our previous assignment of the type strains of these two H serovars to HSSA serogroup IV (Ueda et al. 1991). Our findings indicate that there is a discrepancy between the two serogrouping systems of B. thuringiensis. .-
No. of isolates assigned to extracellular HSSA serotype:
- -- - - - -
-
- 4
-
-
-
-
de Barjac, H., and Bonnefoi, A. 1962. Essai de classification biochimique et serologique de 24 souches de Bacillus du type B. thuringiensis. Entomophaga, 7: 5-3 1. de Barjac, H., and Frachon, E. 1990. Classification of Bacillus thuringiensis. Entomophaga, 35: 233-240. Dulmage, H.T., and Aizawa, K. 1982. Distribution of Bacillus thuringiensis in nature. In Microbial and viral pesticides. Edited by E. Kurstak. Marcel Dekkar, New York. pp 209-237. Krieg, A., Schnetter, W., Huger, A.M., and Langenbruch, G.A. 1987. Bacillus thuringiensis subsp. tenebrionis, strain BI256-82: a third pathotype within the H-serotype 8a8b. Syst. Appl. Microbiol. 9: 138-141.
Mike, A., Ohba, M., and Aizawa, K. 1990. Extracellular production of a heat-stable somatic antigen by Bacillus thuringiensis. LettMicrObiO1247-250. Mike, A*, Ohba, M.9 and Aizawa, K- 19g1. Spontaneous occurrence of heat-stable somatic antigen variants in Bacillus thuringiensis. J. Appl. Bacterial. 70: 408-413. Ohba, M., and Aizawa, K. 1978. Serological identification of Bacillus thuringiensis and related bacteria isolated in Japan. J . Invertebr. Pathol. 32: 303-309. Ohba, M., and Aizawa, K. 1989. Distribution of the four flagellar (H) antigenic subserotypes of Bacillus thuringiensis H serotype 3 in Japan. J . Appl. Bacteriol. 67: 505-509. Ohba, M., Aizawa, K., and Furusawa, T. 1979. Distribution of Bacillus thuringiensis serotypes in Ehime Prefecture, Japan. Appl. Entomol. Zool. 14: 340-345. Ohba, M., Aizawa, K., and Shimizu, S. 1981. A new subspecies of Bacillus thuringiensis isolated in Japan: Bacillus thuringiensis subsp. tohokuensis (serotype 17). J. Invertebr. Pathol. 38: 307-309. Ohba, M., Aizawa, K., and Sudo, S. 1984. Distribution of Bacillus thuringiensis in sericultural farms of Fukuoka Prefecture, Japan. Proc. Assoc. Plant Prot. Kyushu, 30: 152-155. Padua, L.E., Ohba, M., and Aizawa, K. 1980. The isolates of Bacillus thuringiensis serotype 10 with a highly preferential toxicity to mosquito larvae. J. Invertebr. Pathol. 36: 180-186. Padua, L.E., Ohba, M., and Aizawa, K. 1984. Isolation of a Bacillus thuringiensis strain (serotype 8a:8b) highly and selectively toxic against mosquito larvae. J. Invertebr. Pathol. 44: 12-17. Sekijima, Y., and Ono, K. 1982. Grouping of Bacillus thuringiensis by heat-stable somatic antigens. Appl. Entomol. Zool. 17: 393-397. Sekijima, Y., Fujikura, Y., and Ono, K. 1987. Presence of specificity of heat-stable somatic antigens among Bacillus thuringiensis 17 subspecies. Appl. Entomol. Zool. 22: 403-409. Ueda, K., Ohba, M., and Aizawa, K. 1991. Serogrouping of Bacillus thuringiensis by extracellular heat-stable somatic antigens. Syst. Appl. Microbiol. 14: 291-294.
