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Recombinant Escherichia coli strains harboring pAG1, pAG2, pKBB100, and pKBB101 were cloned by using antiserum constructed against 130-kDa crystal protein antigen of Bacillus thuringiensis serovar japonensis strain Buibui. DNAs in the recombinant strains hybridized to the 26-base oligonucleotide probe corresponding to N-terminal amino acids of the 130-kDa crystal protein of strain Buibui. Cultures of the recombinant strains were toxic to larvae of the cupreous chafer, Anomala cuprea. Furthermore, the production of the 130-kDa polypeptide was demonstrated in the cells harboring pAG1 and pAG2 by immunoblot analysis with antiserum against the 130-kDa crystal protein. Southern hybridization analysis showed that the 130-kDa crystal protein gene is located on the chromosomal DNA of strain Buibui. On the other hand, DNA probes derived from cryIA(a) and cryIIIA genes did not hybridize to the DNA of strain Buibui.

A new isolate of Bacillus thuringiensis serovar japonensis strain Buibui, which was specific to scarab beetles (M. Ohba et al., Lett. Appl. Microbiol. 14:54, 1992), was shown to have a 130-kDa insecticidal crystal protein (ICP) (H. Hori et al., J. Appl. Bacteriol. 76:307, 1994). ClaI restriction enzyme fragments of total cell DNA of the isolate were cloned into E. coli (Sato et al., Curr. Microbiol. 28:15, 1994). Whole 3480-bp nucleotide sequence of the gene encoding 130-kDa ICP was determined, and the molecular weight of the ICP was estimated to be 130,424. The strongly conserved five blocks that occur in almost all ICP genes of B. thuringiensis were detected in the ORF with the same order and almost the same intervals as elsewhere. The amino acid sequence homologies of the whole ICP or N-terminus half portion to that of the CryIIIA, B, C, D, and CryV were about 35%.

Vertical dispersal, host-finding through movement in sand and infectivity to Galleria mellonella and Popillia japonica in three strains of Steinernema glaseri, S. anomali and S. kushidai were evaluated to determine which nematode species/strain showed the best potential for control of soil-inhabiting insects such as white grubs in turf. The dispersal of S. glaseri # 328, S. kushidai and S. carpocapsae was significantly different from S. glaseri # 326 and # 330 and S. anomali in sand columns without host. Steinernema glaseri # 326 and # 330 migrated over 10 cm. Over 58 % nematodes of S. anomali were recovered from 0-10 cm sections, but the few dispersed deeper. A small portion of S. glaseri # 328 and S. kushidai dispersed in the sand column but the majority remained near the top. Three strains of S. glaseri and S. anomali caused higher mortality against G. mellonella than S. kushidai and S. carpocapsae in host-finding through movement in a sand column. Even with the application of 120 infective juveniles of S. kushidai, the mortality of G. mellonela by this nematode was lower than that by 10 infective juveniles of S. glaseri # 326 and # 330. In one-on-one assays , G. mellonella mortalities by S. glaseri # 328, S. anomali and S. carpocapsae were higher compared with those by S. glaseri # 326 and # 330, and S. kushidai. The three strains of S. glaseri, S. anomali and S. kushidai were effective against P .japonica at 1,000 infective juveniles/larva. Infectivities to G. mellonella and P. japonica also differed among the test nematode species. Jpn. J. nematol. 25 (1) 24-32 (1995 (8 , 9, 22). In addition, S. anomali (KozoDol) and S. kushidai MAMIYA were isolated from larvae in the genus Anomala (11,14), suggesting that they may be effective against white grubs. Thus, S. kushidai was highly pathogenic to some scarabaeid larvae (Allomyrina dichotoma (L)., Anomala cuprea HOPE, Heptophylla picea MOTSCHULSKY, Maladera japonica (MOTSCHULSKY) , and Protaetia orientalis (GORY et PERCHELON)), but not to lepidopteran larvae (Acanthoplusia agnata (STAUDIN- * RESULTSVertical dispersal: Table 1 shows the vertical distribution of six nematode species/strains in 30-cm sand-column and the depth (cm) of their dispersal. Steinernema glaseri # 326 was found in all sections, but the majority occurred in the upper 5-10 and 10-15 cm sections. Those of # 328 were recovered primarily from the first two sections (0-5, 5-10 cm) , whereas # 330 was equally distributed in the deeper three sections (5-10, 10-15 and 15-20 cm) . For S. anomali, most juveniles were recovered from the 0-10 cm sections, but 6.3-14.0% of the juveniles were detected in four sections below 10 cm. However, 64.1% of S. kushidai were detected from the first section (0-5 cm) , with the remaining found primarily at 5-10 cm. Most S . carpocapsae were recovered from the first section.The dispersal depth of S. glaseri # 326 averaged 10.2 cm, which was not significantly different from S. glaseri # 330 (12.1 cm) and S. anornali (10.6 cm) (F =31.63; df = Table 1. Vertical distrib...

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