Diversity of Bacillus thuringiensis strains in the maize and bean phylloplane and their respective soils in Colombia

AIMS: To evaluate the distribution of Bacillus thuringiensis strains from maize and bean phylloplane and their respective soils. METHODS AND RESULTS: B. thuringiensis was isolated from the phylloplane and soil of maize and bean from three municipalities in Antioquia, Colombia. Ninety six samples of phylloplane and 24 of soil were analyzed. A total of 214 isolates were obtained from 96 phylloplane samples while 59 isolates were recovered from 24 soil samples. Sixty five per cent and 12% of the phylloplane and soil isolates, respectively, showed activity against Spodoptera frugiperda. These isolates contained delta-endotoxin proteins of 57 and 130 kDa. The most toxic isolates against S. frugiperda had the genotype cry1Aa, cry1Ac, cry1B, and cry1D. In contrast, 27% of the phylloplane isolates and 88% of the soil isolates were active against Culex quinquefasciatus and had protein profiles similar to B. thuringiensis serovar. medellin and B. thuringiensis serovar. israelensis. The most active isolates contain cry4 and cry11 genes. CONCLUSIONS: The predominant population of B. thuringiensis on the phylloplane harbored the cry1 gene and was active against S. frugiperda, whereas in soil, isolates harboring cry11 gene and active against C. quinquefasciatus were the majority. SIGNIFICANCE AND IMPACT OF THE STUDY: The predominance of specific B. thuringiensis populations, both on the leaves and in the soil, suggests the presence of selection in B. thuringiensis populations on the studied environment.     

Bacillus thuringiensis populations naturally occurring on mulberry leaves: a possible source of the populations associated with silkworm-rearing insectaries

Mulberry leaves were examined for the occurrence of Bacillus thuringiensis. This organism was recovered from both abaxial and adaxial surfaces: a total of 186 B. thuringiensis colonies were isolated from 24 (96·0%) out of 25 mulberry trees, and from 112 (11·2%) out of 1004 leaves from 25 trees. The frequency of B. thuringiensis colonies was 3·2% among 5900 colonies belonging to the Bacillus cereus/B. thuringiensis group. Single colonies were associated with 75·9% of the B. thuringiensis -positive leaves and 2–16 colonies were occasionally found on a single phylloplane. Flagellar (H) serotypying of the isolates revealed that, among the 19 H serotypes (serovars) detected, the H serotype 13 (serovar pakistani ) was the predominant, followed by the H serotypes 3abc ( kurstaki ), 6ac ( oyamensis ), 16 ( indiana ), 24 ( neoleonesis ), 4ac ( kenyae ), 7 ( aizawai ) and 10 ( darmstadiensis ). Larvicidal activity, against the silkworm ( Bombyx mori ) and/or the mosquito ( Aedes aegypti ), was exhibited by 18 isolates (9·7%) belonging to H serovars kurstaki, kenyae, canadensis and aizawai , and an unidentified H serogroup.     

The Ecology of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> on the Phylloplane: Colonization from Soil,Plasmid Transfer,and Interaction with Larvae of <Emphasis Type="Italic">Pieris brassicae</Emphasis>

Seedlings of clover (Triflorium hybridum) were colonized by Bacillus thuringiensis when spores and seeds were co-inoculated into soil. Both a strain isolated in the vegetative form from the phylloplane of clover, 2810-S-4, and a laboratory strain, HD-1, were able to colonize clover to a density of about 1000 CFU/g leaf when seeds were sown in sterile soil and to a density of about 300 CFU/g leaf in nonsterile soil. A strain lacking the characteristic insecticidal crystal proteins produced a similar level of colonization over a 5-week period as the wild type strain, indicating that crystal production was not a mitigating factor during colonization. A small plasmid, pBC16, was transferred between strains of B. thuringiensis when donor and recipient strains were sprayed in vegetative form onto leaves of clover and pak choi (Brassica campestris var. chinensis). The rate of transfer was about 0.1 transconjugants/recipient and was dependent on the plant species. The levels of B. thuringiensis that naturally colonized leaves of pak choi produced negligible levels of mortality in third instar larvae of Pieris brassicae feeding on the plants. Considerable multiplication occurred in the excreted frass but not in the guts of living insects. Spores in the frass could be a source of recolonization from the soil and be transferred to other plants. These findings illustrate a possible cycle, not dependent on insect pathology, by which B. thuringiensis diversifies and maintains itself in nature.     

Effect of bovicin HC5 on growth and spore germination of Bacillus cereus and Bacillus thuringiensis isolated from spoiled mango pulp

AIMS: To use bovicin HC5 to inhibit predominant bacteria isolated from spoiled mango pulp. METHODS AND RESULTS: Bovicin HC5 and nisin were added to brain heart infusion (BHI) medium (40-160 AU ml(-1)) or mango pulp (100 AU ml(-1)) and the growth of Bacillus cereus and Bacillus thuringiensis was monitored. Cultures treated with bovicin HC5 or nisin showed longer lag phases and grew slower in BHI medium. Bovicin HC5 and nisin were bactericidal and showed higher activity in mango pulp at acidic pH values. To determine the effect on spore germination and D values, mango pulp containing bovicin HC5 was inoculated with 10(6) and 10(9) spores per ml(-1), respectively, from each strain tested. Bovicin HC5 reduced the outgrowth of spores from B. cereus and B. thuringiensis, but thermal sensitivity was not affected. CONCLUSIONS: Bovicin HC5 was bactericidal against B. cereus and B. thuringiensis isolated from spoiled mango pulp. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacillus cereus and B. thuringiensis had not been previously isolated from spoiled mango pulp and bovicin HC5 has the potential to inhibit such bacteria in fruit pulps.     

Expression of a Bacillus thuringiensisdelta-endotoxin cry1Ab gene in Bacillus subtilis and Bacillus licheniformis strains that naturally colonize the phylloplane of tomato plants (Lycopersicon esculentum,Mills)

AIMS: To introduce a cry gene into microorganisms that naturally colonize the phylloplane of tomato plants to improve the persistence of the Cry proteins for controlling a South American tomato moth (Tuta absoluta, Meyrick, 1917). METHODS AND RESULTS: A cry1Ab gene isolated from a native Bacillus thuringiensis strain (LM-466), showing a relevant activity against T. absoluta larvae, was cloned into the shuttle vector pHT315 (Arantes and Lereclus 1991). The construct was introduced by electroporation into native Bacillus subtilis and Bacillus licheniformis strains, both natural inhabitants of the tomato phylloplane. Western analysis and toxicity assays against the target larvae proved that the successful expression of the gene was accomplished in host bacteria. Recombinant toxin displayed a similar LC50 value in comparison to native donor strain LM-466. Both transformed Bacillus survived for at least 45 days on the tomato leaf surface. CONCLUSIONS: Plant-associated microorganisms that naturally colonize the phylloplane could be useful as recombinant microbial delivery systems of toxin genes of B. thuringiensis. SIGNIFICANCE AND IMPACT OF THE STUDY: Modified microorganisms capable of surviving on leaf surfaces for several weeks with insecticidal activity should allow for a reduction in pesticide application.     

Selective Process for Efficient Isolation of Soil Bacillus spp

We were able to isolate Bacillus thuringiensis from environmental samples with a background of 10 bacteria per g of soil. Our selection process differed significantly from classical selection methods which permit only the desired organism to grow. In our process, germination of B. thuringiensis spores was selectively inhibited by sodium acetate, while most of the undesired sporeformers germinated. Next, all of the nonsporulated microbes were eliminated by heat treatment at 80 degrees C for 3 min. The surviving spores were then plated on a rich agar medium and allowed to grow until they sporulated. Of random colonies picked from agar, 20 to 96% were crystal-forming Bacillus species. B. thuringiensis and B. sphaericus were routinely selected by this method.     

Contribution of the Regulatory Gene lemA to Field Fitness of Pseudomonas syringae pv. syringae

In Pseudomonas syringae pv. syringae, lemA is required for brown spot lesion formation on snap bean and for production of syringomycin and extracellular proteases (E. M. Hrabak and D. K. Willis, J. Bacteriol. 174: 3011-3022, 1992; E. M. Hrabak and D. K. Willis, Mol. Plant-Microbe Interact. 6:368-375, 1993; D. K. Willis, E. M. Hrabak, J. J. Rich, T. M. Barta, S. E. Lindow, and N. J. Panopoulos, Mol. Plant-Microbe Interact. 3:149-156, 1990). The lemA mutant NPS3136 (lemA1::Tn5) was previously found to be indistinguishable from its pathogenic parent B728a in its ability to grow when infiltrated into bean leaves of plants maintained under controlled environmental conditions (Willis et al., Mol. Plant-Microbe Interact. 3:149-156, 1990). We compared population sizes of NPS3136 and B728aN (a Nal(supr) clone of wild-type B728a) in two field experiments to determine the effect of inactivation of lemA on the fitness of P. syringae pv. syringae. In one experiment, the bacterial strains were spray inoculated onto the foliage of 25-day-old bean plants. In the other, seeds were inoculated at the time of planting. In both experiments, the strains were inoculated individually and coinoculated in a 1:1 ratio. NPS3136 and B728aN achieved similar large population sizes on germinating seeds. However, in association with leaves, population sizes of NPS3136 were diminished relative to those of B728aN in both experiments. Thus, lemA contributed significantly to the fitness of P. syringae pv. syringae in association with bean leaves but not on germinating seeds under field conditions. When NPS3136 was coinoculated with B728aN, the mutant behaved as it did when inoculated alone. However, population sizes of B728aN in the coinoculation treatment were much lower than those when it was inoculated alone. Inactivation of the lemA gene appeared to have rendered the mutant suppressive to B728aN.     

Intestinal bacteria affect growth of Bacillus thuringiensis in larvae of the oriental tea tortrix, Homona magnanima diakonoff (Lepidoptera: tortricidae)

Spores and parasporal crystals of a Bacillus thuringiensis serovar aizawai were fed to fifth instar larvae of the oriental tea tortrix, Homona magnanima, that had been reared aseptically or that had been reared normally. Viable cell numbers of B. thuringiensis and other bacteria in H. magnanima larvae were estimated by homogenization of samples and dilution plating on peptone-polymyxin agar medium for B. thuringiensis cells and on nutrient agar medium for the other bacterial cells. B. thuringiensis did not grow in the larval cadavers of normally reared H. magnanima while bacteria other than B. thuringiensis grew rapidly. In contrast, B. thuringiensis within the larval cadavers of aseptically reared H. magnanima grew and increased 20 times. The bacteria other than B. thuringiensis from the sample homogenates of normally reared larvae that were fed on B. thuringiensis-treated diets had the same characteristics as the bacteria isolated from the guts of healthy H. magnanima larvae, which were putatively identified as Streptococcus spp. and Staphylococcus spp., typical intestinal bacteria of insects. The results strongly suggest that intestinal bacteria influence the growth of B. thuringiensis in the larvae.     

Recovery of Bacillus thuringiensis in vegetative form from the phylloplane of clover (Trifolium hybridum) during a growing season

Two media were developed which specifically allow the cultivation of Bacillus thuringiensis while it is in the vegetative as opposed to the spore form. Using these media B. thuringiensis was shown conclusively for the first time to exist in an active form on the phylloplane. The profile of its appearance in vegetative and spore form was followed over a growing season on clover (Trifolium hybridum) in the field. Three simultaneous and sudden rises and declines of both spore and vegetative cell densities were observed. The most common other spore-former on these leaves was Bacillus cereus but the fluctuations in appearance of these two very closely related species were not co-incident. Using specific PCR primers a considerable diversity of cry toxin gene types was found in isolates that had been recovered in vegetative form ('vegetative isolates') with the majority possessing multiple delta-endotoxin genes while some had only one of those tested. Bioassays against a lepidopteran insect of purified delta-endotoxins showed that they were no more potent than those from a laboratory-adapted strain. PCR primers for an internal region of the vip3A gene produced amplification in 70% of the vegetative isolates compared to 25% of the laboratory-adapted strains tested.     

Natural occurrence of Bacillus thuringiensis on cabbage foliage and in insects associated with cabbage crops

Bacillus thuringiensis was isolated from the phylloplane of organically grown cabbage in one field during two growth seasons (1992-93). The frequency of B. thuringiensis varied between 0.02 and 0.67 of the total B. cereus/B. thuringiensis population, with an average of 0.11. Characterization of the B. thuringiensis isolates from foliage showed that the majority (64% of 150 isolates) belonged to serovar kurstaki, had bipyramidal crystals and toxicity towards Pieris brassicae and/or Trichoplusia ni. Other serovars were also found on the foliage but occurred at very low frequencies (one to three isolates of each serovar). Bacillus thuringiensis was also isolated from insects associated with the cabbage crop (Pieris rapae (Lep.), Delia radicum (Dip.), Syrphidae ribesii (Dip.) and Aleochara bilineata (Col.)), which were collected alive at different developmental stages in the same field. Serologically these isolates were assigned to the serovars kurstaki, aizawai, tochigiensis, colmeri and indiana/colmeri.     

Production of Bacillus thuringiensis Berliner var. kurstaki Grown in Alternative Media

Agro - industrial residues and by - products available in southeastern Brazil were used as ingredients for low - cost culture media for liquid fermentation of Bacillus thuringiensis var. kurstaki. Highest spore yield was obtained with a medium containing cheese whey , soya bean milk and molasses (WSM) . Crystals and spores were produced in all media and potency of the final product was highest for nutrient broth + yeast extract medium (NBY) . There was no correlation between the number of spores in the fermented media and the potency of the preparations . Considering all three factors , the potencies , costs and yields of the final products , lowest relative cost was obtained with BMM medium ( Bombyx mori pupae + molasses) . NBY and WSM had intermediate relative cost approximately nine times higher than BMM . The cost analysis suggests that BMM medium should be preferred for local production of B. thuringiensis var . kurstaki in comparison to other media tested . The results also demonstrate the importance of considering yields , cost and potency of the B. thuringiensis preparations in selecting the production medium .     

A novel bacteriocin, thuricin 17, produced by plant growth promoting rhizobacteria strain Bacillus thuringiensis NEB17: isolation and classification

AIMS: The aim of this study was to identify and characterize a compound produced by the plant growth promoting bacterium, Bacillus thuringiensis non-Bradyrhizobium Endophytic Bacterium 17. METHODS AND RESULTS: The bacterial peptide was analysed and purified via HPLC. Using the disk diffusion assay this peptide inhibited the growth of 16/19 B. thuringiensis strains, 4/4 Bacillus cereus strains, among others, as well as a Gram-negative strain Escherichia coli MM294 (pBS42). Both bactericidal and bacteristatic effects were observed on B. cereus ATCC 14579 and bactericidal effects were observed on B. thuringiensis ssp. thuringiensis Bt1267. The molecular weight of the peptide was estimated via SDS-PAGE and confirmed with Matrix Assisted Laser Desorption Ionization Quadrapole Time of Flight mass spectrometry; its weight is 3162 Da. The peptide is biologically active after exposure to 100 degrees C for 15 min, and within the pH range 1.00-9.25. Its activity disappeared when treated with proteinase K and protease, but not with alpha-amylase or catalase. CONCLUSIONS: We conclude that this is the first report of a bacteriocin produced by a plant growth promoting rhizobacteria (B. thuringiensis) species and have named the bacteriocin thuricin 17. SIGNIFICANCE AND IMPACT OF THE STUDY: Our work has characterized a bacteriocin produced by a plant growth promoting bacterium. This strain is previously reported to increase soya bean nodulation.     

Inactivation of Escherichia coli O157:H7 and Salmonella on artificially or naturally contaminated mung beans (Vigna radiata L) using a stabilized oxychloro-based sanitizer

AIMS: To evaluate the efficacy of a stabilized oxychloro-based (SOC) sanitizer to decontaminate mung beans artificially or naturally contaminated with Escherichia coli O157:H7 or Salmonella. METHODS AND RESULTS: Naturally contaminated beans were produced by introducing a five-strain cocktail of E. coli O157:H7 or Salmonella onto the flowers of growing mung bean plants. Escherichia coli O157:H7 was only sporadically recovered from sprout lots (three testing positive from 10 tested) derived from harvested beans. In contrast, Salmonella was recovered from 18 of 20 lots screened. Pathogens present on naturally contaminated seed could be successfully inactivated with SOC applied at 200 ppm for 24 h at 28 degrees C. SOC treatment could also decontaminate artificially inoculated mung bean batches containing different levels of contaminated seed. SOC inactivated E. coli O157:H7, but not Salmonella introduced onto damaged (scarified) beans. CONCLUSIONS: SOC sanitizer could inactivate Salmonella or E. coli O157:H7 naturally or artificially introduced onto mung beans. However, the SOC treatment failed to inactivate Salmonella introduced onto damaged mung beans. SIGNIFICANCE AND IMPACT OF THE STUDY: SOC sanitizer represents an effective method for decontaminating undamaged mung beans.     

Growth and development kinetics of Bacillus thuringiensis in batch culture

The kinetics of Bacillus thuringiensis growth and its assimilation of nutrient substances were studied under the conditions of batch cultivation in a complex medium containing yeast extract and in a chemically defined medium with amino acids. The growth of B. thuringiensis can be divided into five phases: exponential growth; decelerated growth; stationary phase when protein crystals are formed; stationary phase when spores are formed; lysis of sporangia releasing spores. The first phase may in turn be subdivided into three stages according to changes in the specific growth rate and substrate assimilation: a high specific growth rate and no glucose assimilation; an abrupt drop in mu and the beginning of intensive glucose assimilation from the medium; a new rise in the specific growth rate. As follows from the results of studying the kinetics of B. thuringiensis growth in a chemically defined medium, the above changes in the exponential growth phase are due to the fact that the culture assimilates yeast extract components in the complex medium or amino acids in the chemically defined medium during this phase, and then starts to assimilate glucose and ammonium in the following phases of growth.     

Degradation of African locust bean oil by Bacillus subtilis and Bacillus pumilus isolated from soumbala,a fermented African locust bean condiment

AIMS: To investigate predominant isolates of Bacillus subtilis and B. pumilus in soumbala, a fermented African locust bean condiment, for their ability to degrade African locust bean oil (ALBO). METHODS AND RESULTS: Agar diffusion test in tributyrin and ALBO agar was used for screening of the isolates for esterase and lipase activity, respectively. The quantity and the profile of free fatty acids (FFA) during 72 h of degradation of ALBO by the Bacillus isolates were studied by titration and gas chromatography. The degradation of tributyrin and ALBO was variable among the isolates. Two strains of B. subtilis and two strains of B. pumilus showed significantly higher esterase and lipolytic activities than the others. The degradation ALBO was most pronounced in enriched nutrient agar except for one isolate of B. pumilus degrading ALBO to the same extent regardless of the enrichment. The quantity of FFA released from ALBO by the most lipolytic strains of Bacillus increased mainly between 0 and 24 h and differed among the isolates. The profile of FFA was similar for the Bacillus isolates with oleic acid (C18:2) occurring as the major FFA in all the samples except in samples incubated with B. subtilis B9 where stearic acid (C18) was dominant. CONCLUSION: Bacillus isolates from soumbala showed high strain dependent lipolytic activity against ALBO. SIGNIFICANCE AND IMPACT OF THE STUDY: This study contributes to the selection of Bacillus strains to be used as starter cultures for controlled production of soumbala.     

Diversity of Colombian strains of Bacillus thuringiensis with insecticidal activity against dipteran and lepidopteran insects

AIM: To evaluate the genetic and molecular diversity and insecticidal activity of Bacillus thuringiensis isolates from all the natural regions of Colombia. METHODS AND RESULTS: A total of 445 isolates from a collection of B. thuringiensis were characterized. The parasporal crystal morphology that was most abundant was bipyramidal (60%). Almost 10% of the isolates were toxic to Spodoptera frugiperda and 5.6% against Culex quinquefasciatus larvae. cry gene content determined by PCR indicated that 10.6% of the isolates contained cry1 genes and 1.1% contained cry2, cry4 or cry11 genes. Protein content of the parasporal crystal was determined by SDS-PAGE; 25 and 18 different protein profiles were found in isolates active against S. frugiperda and C. quinquefasciatus, respectively. CONCLUSIONS: Bacillus thuringiensis presents great genetic and molecular diversity even in isolates from the same soil sample. Moreover, the diversity and activity of the isolates might have a relationship with the geographical origin of the samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The results obtained here indicate that some of the B. thuringiensis isolates characterized in this study are potential control agents that could be used in programmes against mosquitoes and S. frugiperda.     

Properties of Bacillus thuringiensis isolated from bank voles

AIMS: To assess the properties of B. thuringiensis naturally occurring in the intestines of bank voles. METHODS AND RESULTS: Seventeen Bacillus thuringiensis strains, exhibiting typical growth on selective medium for the B. cereus group and characterized by the ability to produce parasporal crystals, were isolated from bank voles trapped in the ?omza Landscape Park of the Narew River Valley (north-east Poland). All isolates were characterized by pulsed field gel electrophoresis (PFGE) of chromosomal DNA and SDS polyacrylamide gel electrophoresis (SDS-PAGE) of whole-cell proteins. Six pulsotypes were found with PFGE typing, using SmaI or NotI as restriction enzymes. Significant differences in chromosome size, ranging from 2.4 to 4.2 Mb for the B. thuringiensis strains studied, were noted. Strain heterogeneity in pulsotypes was also reflected by the similarity of whole-cell protein profiles of the strains. Environmental isolates and reference strains grouped at 71% similarity according to SDS-PAGE data and at 84% on the basis of biochemical tests. CONCLUSIONS: B. thuringiensis from intestines of bank voles demonstrated an important level of heterogeneity. The comparison of PFGE profiles and SDS-PAGE of whole-cell protein patterns may be useful to evaluate the relationship between B. thuringiensis isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: The results presented in this paper may help to explain the diversity of B. thuringiensis.     

Inorganic phosphate has a crucial effect on Cry3Aa delta-endotoxin production

AIMS: The study aimed at increasing Cry3Aa delta-endotoxin production by a local isolate of Bacillus thuringiensis (B.t. strain Mm2). To this end, different nutritional conditions were tested for their effects on Cry3Aa yields. METHODS AND RESULTS: Bacillus thuringiensis Mm2 was grown by shaking at 30 degrees C in different media. Samples were taken from the cultures at intervals and used for protein extraction. SDS-PAGE was performed for toxin analysis. Inclusion of inorganic phosphate (Pi) into the Difco's sporulation medium at an increased level of 200 mmol l-1 caused a fivefold increase (from 3 to 15.6 microg ml-1) in toxin production. Omission of FeSO4 from the medium decreased this yield by half. Resuspension experiments suggested catabolite repression of toxin biosynthesis by glucose. The inclusion of high Pi invariably increased toxin synthesis, even in the absence of sugars. CONCLUSIONS: Inorganic phosphate had the most striking effect on toxin biosynthesis. Iron effect was found to be unique to our isolate whereas Pi effect seemed to be common to the biosynthesis of Cry3Aa-type toxins. Stimulation of toxin synthesis by Pi did not seem to represent a relief from glucose repression. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacillus thuringiensis is the most versatile biopesticide for use in pest management. Regarding cost-effectiveness of related fermentations, high Pi supplement drastically increases Coleoptera-specific toxin synthesis.     

Thuricin 7: a novel bacteriocin produced by Bacillus thuringiensis BMG1.7, a new strain isolated from soil

AIMS: Detection and identification of new antagonistic activities towards Bacillus cereus and relatives. METHODS AND RESULTS: Twenty Bacillus thuringiensis strains were screened for their capacity to express bacteriocin-like agents. Strain BMG1.7, isolated from soil, showed an antagonistic activity called thuricin 7. Thuricin 7 was active against several species of the genus Bacillus, including three of the four known B. thuringiensis/B. cereus bacteriocin producers, as well as against Streptococcus pyogenes and Listeria monocytogenes strains. Antimicrobial activity was lost after treatment with proteinase K. The active protein had an apparent molecular weight of 11.6 kDa, and was secreted at the end of the exponential growth phase. Thuricin 7 retained 55% of the activity after incubation at 98 degrees C for 30 min. The mode of action of thuricin 7 was shown to be bactericidal and bacteriolytic. CONCLUSION: Thuricin 7 is a novel bacteriocin produced by a newly isolated Bacillus thuringiensis strain BMG1.7. SIGNIFICANCE AND IMPACT OF THE STUDY: The characteristics of thuricin 7 indicate that it is a new bacteriocin which may have interesting biotechnological applications due to its relatively large activity spectrum.     

Co-inoculation with Bacillus sp. CECT 450 improves nodulation in Phaseolus vulgaris L.

The strain Bacillus sp. CECT 450 increased nodulation on bean (Phaseolus vulgaris L.) when co-inoculated with Rhizobium tropici CIAT 899. This positive effect occured under controlled conditions on perlite-vermiculite, sand, or in a mixture of soil and sand. This increase was also observed in a field assay. Nodulation kinetic studies suggested that the synergistic effect is pronounced during the latter stages of cultivation. In contrast, the same bacteria co-inoculated with Bradyrhizobium japonicum USDA 110 reduced nodulation on soybean (Glycine max (L.) Merr.). Inoculation with Bacillus sp. CECT 450 alone had no effect on bean plants, but reduced root growth in soybean. The survival of Bacillus sp. CECT 450 on inoculated seeds was high, even when inoculated seeds were maintained for several months at room temperature.