Conditions are described that led to the isolation of NRRL B-2309M, a strain of Bacillus popilliae which sporulates regularly in laboratory culture. Colonies grown on a medium formulated with yeast extract and the ingredients of Mueller-Hinton with phosphate, trehalose, and agar, produced 20% spores in 10 to 12 days. The quantity and kind of yeast extract determine the extent of sporulation, although there are other requirements for optimal growth and sporulation. Spore inocula free of viable vegetative cells are necessary to maintain sporogenicity since asporogenic substrains arise spontaneously on solid and in liquid media. One such substrain, NRRL B-2309N, is also asporogenic in larvae, but lethal, owing to vigorous vegetative growth. Strain B-2309M is infective when vegetative cells or spores are injected into Japanese beetle larvae but fewer spores are formed in vivo than when infections are caused by NRRL B-2309. The characteristics of four related strains of B. popilliae are tabulated.

A survey of 272 free-living rhizobia strains showed that a majority of the Rhizobium japonicum and unclassified Rhizobium isolates are capable of both growth and acetylene reduction on a Casamino Acids-minimal medium. Two other taxonomic categories of Rhizobium were discernible: R. meliloti and R. trifolii, which grow but do not reduce acetylene; and R. leguminosarum, which fails to grow on the minimal medium. Sixty-nine strains were acetylene reducers obtained from root nodules of the plant hosts: Albizzia, Cassia (partridge pea), Baptisia (wild indigo), Crotalaria, Ulex (thornbroom), Indigofera, Lespedeza, Desmodium (Florida beggarweed), Arachis (peanut), Phaseolus, Strophostyles, Stizolobium (velvet bean), Pueraria (kudzu), Vigna (cowpea), Erythrina, and Glycine (soybean). The extent of acetylene reduction varied depending on both the Rhizobium strain and 0 2 tension, suggesting that reduction activity differs according to the cellular organization of individual strains.Taxonomic approaches to separate rhizobia have included: nutritional and antigenic specificities (20); deoxyribonucleic acid base composition (4); deoxyribonucleic acid homology (9); and glutamate and phosphate uptakes by cells (21). Based on such studies, rhizobia that have been classified fall generally into three groups: (i) Rhizo bium leguminosarum-R. phaseoli-R. trifolii, (ii) R. meliloti, and (iii) R. japonicum-R. lupini. Many other fast-and slow-growing rhizobia, however, await specific designation. The introduction of acetylene reduction as an assay for nitrogen fmation capability (3,5,8) and the production of acetylene-reducing enzyme by Rhizobium strains under nonsymbiotic conditions (2, 11, 13, 15, 17, 19) have made possible a rapid survey in an attempt to categorize 272 strains of rhizobia from the NRRL collection.To acknowledge the years Lewis T. Leonard MATERIALS AND METHODS Cultivation of strains.The lyophilized Rhizobium strains and corresponding record of the L-collection were obtained from L. K. Nakamura (Agricultural Research Service Culture Collection, Peoria, Ill.). The complete list (NRRL numbers L-1 through L-344) includes 29 strains that failed to grow and consequently were discarded over the years. Of the remaining 315 lyophilized strains in the present L-collection, 32 failed to grow on conventional yeast extract-mannitol-soil extract agar (YMS medium 111; 18) and were omitted from this survey. No further attempt was made to ensure purity of each culture, but a small number of cultures were discarded since they yielded more than one distinct type of colony or growth on the various media and were not consistent with the recorded growth rate before lyophilization. A cowpeatype strain, Rhizobiurn sp. 32H1, was generously provided by J. C. Burton (Nitragin Co., Milwaukee, Wis.).The lyophilized strains were suspended in a small volume of yeast extract-malt extract broth before transfer to YMS slants. After 5 to 20 days of incubation at 25"C, a loopful of culture was transferred to a "preconditioning" liquid medium, adj...

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Conditions are described that led to the isolation of NRRL B-2309M, a strain of Bacillus popilliae which sporulates regularly in laboratory culture. Colonies grown on a medium formulated with yeast extract and the ingredients of Mueller-Hinton with phosphate, trehalose, and agar, produced 20% spores in 10 to 12 days. The quantity and kind of yeast extract determine the extent of sporulation, although there are other requirements for optimal growth and sporulation. Spore inocula free of viable vegetative cells are necessary to maintain sporogenicity since asporogenic substrains arise spontaneously on solid and in liquid media. One such substrain, NRRL B-2309N, is also asporogenic in larvae, but lethal, owing to vigorous vegetative growth. Strain B-2309M is infective when vegetative cells or spores are injected into Japanese beetle larvae but fewer spores are formed in vivo than when infections are caused by NRRL B-2309. The characteristics of four related strains of B. popilliae are tabulated.