Lipopolysaccharide-binding protein of Bombyx mori participates in a hemocyte-mediated defense reaction against gram-negative bacteria

BmLBP is a lipopolysaccharide-binding protein in B. mori and participates in bacterial clearance in vivo. Here, we investigated the function of BmLBP more specifically. More than 90% of injected gram-negative rough strains to which BmLBP binds were removed from the plasma within 30 min post-injection, whereas it required 8h for the clearance of smooth strains to which BmLBP does not bind. Observation of the hemocoel after the injection of Escherichia coli rough strain showed that melanized nodules were formed at 30 min post-injection when the clearance of injected E. coli cells had occurred. Fluorescence microscope observation revealed that E. coli cells were actually trapped in the nodules formed in vivo. Furthermore, plasma pre-treated E. coli rough cells (BmLBP bound) added to hemocytes isolated in vitro caused vigorous hemocyte aggregations with the bacteria, while plasma pre-treated smooth cells did not. The formation of aggregates was inhibited by anti-BmLBP serum pre-treatment, suggesting that BmLBP causes the clearance of bacteria by promoting hemocyte nodule formation.     

Effect of the insect pathogenic bacterium Photorhabdus on insect phagocytes

Photorhabdus are insect pathogenic bacteria that replicate within the insect haemocoel following release from their entomopathogenic nematode symbionts. To investigate how they escape the cellular immune response we examined the effects of two strains of Photorhabdus, W14 and K122, on Manduca sexta phagocytes (haemocytes), in vitro and in vivo. Following injection of Esherichia coli into Manduca larvae, these non-pathogenic bacteria are rapidly cleared from the haemolymph and the number of free haemocytes transiently increases. In contrast, following injection of either strain of pathogenic Photorhabdus, the bacteria grow rapidly while the number of haemocytes decreases dramatically. In vitro incubation of haemocytes with either Photorhabdus supernatant reduced haemocyte viability, and the W14 supernatant caused distinct changes in the actin cytoskeleton morphology of different haemocyte cell types. In phagocytosis assays both Photorhabdus strains can inhibit their own phagocytosis whether the bacterial cells are alive or dead. Further, the supernatant of W14 also contains a factor capable of inhibiting the phagocytosis of labelled E. coli. Together these results suggest that Photorhabdus evades the cellular immune response by killing haemocytes and suppressing phagocytosis by mechanisms that differ between strains.     

柞蚕蛹抗菌肽D对大肠杆菌K_(12)D_(31)的作用

本文报道了不同浓度的柞蚕蛹抗菌肽D,对大肠杆菌K_(12)D_(31)的杀灭作用动力学。在LEG培养液中,抗菌从D的浓度在5微克/毫升时显效。浓度在10微克/毫升以上时,其杀菌速度大于细菌的增殖速度。固定抗菌肽浓度为10微克/毫升,细菌浓度在3×10~7个细菌/毫升,培养在磷酸钾盐缓冲液中,4小时后能全部杀灭。同样浓度细菌在LEG培养液中,4小时后细菌数下降到约为10~2个细菌/毫升,但不能全部杀灭。同时还提供了柞蚕蛹抗菌肽D和B对大肠杆菌K_(12)D_(31)作用不同时间的电镜照片。     

Characterization of the binding of diarrheagenic strains of E. coli to plant surfaces and the role of curli in the interaction of the bacteria with alfalfa sprouts

Diarrheagenic Escherichia coli were able to bind to plant surfaces, including alfalfa sprouts and open seed coats, and tomato and Arabidopsis thaliana seedlings incubated in water. The characteristics of the binding differed with the bacterial strain examined. Laboratory K12 strains of E. coli failed to show significant binding to any of the plant surfaces examined, suggesting that some of the genes present and expressed in pathogenic strains and absent or unexpressed in K12 strains may be required for binding to plants. When a plasmid carrying the mlrA gene (a positive regulator of curli biosynthesis) or a plasmid carrying the operons that encode the synthesis of curli (csgA-G) was introduced into K12 strains, the bacteria acquired the ability to bind to sprouts. CsgA mutants of an avian pathogenic E. coli and an O157:H7 strain showed no reduction in their ability to bind to sprouts. Thus, the production of curli appears to be sufficient to allow K12 strains to bind, but curli are not necessary for the binding of pathogenic strains, suggesting that pathogenic strains may have more than one mechanism for binding to plant surfaces.     

Studies on the cellular defense reactions of the madeira cockroach, Leucophaea maderae: in vitro phagocytosis of different strains of Bacillus cereus and their effect on hemocyte viability

Monolayers of Leucophaea maderae hemocytes, consisting of mainly plasmatocytes and coagulocytes, were incubated with three strains of Bacillus cereus of differing pathogenicities, and the levels of phagocytosis and hemocyte viability were determined. Incubation with viable B. cereus strains NCTC 2599, NCIB 3329, and B1 resulted in a significant drop in hemocyte viability after 60 min of incubation compared with the saline-only controls. The greatest effect, however, resulted from incubation with B. cereus B1 which is the most pathogenic of the three strains studied. The killing effect of the three B. cereus strains was abolished following their UV irradiation. Incubation of monolayers with viable B. cereus B1 resulted in a level of phagocytic activity at all time periods lower than that with the other two strains. The highest levels of phagocytosis were achieved with UV-killed B. cereus, although no significant differences were found in these values between the three strains at any of the incubation times. Phospholipase C, a lytic enzyme shown to be released by all three strains of B. cereus, although in varying amounts, also caused hemocyte death following its incubation with the monolayers. These data suggest that the hemocyte killing and resistance to phagocytosis by B. cereus are caused by the release of phospholipase C, or some other related toxin(s) by the bacteria.     

鸡肠源芽胞杆菌的分离、鉴定和抗菌活性

从人工饲养的健康三黄肉鸡的盲肠和大肠中分离出10株芽胞杆菌,对其进行菌落形态观察、革兰染色、芽胞染色和生理生化特性鉴定,确定为枯草芽胞杆菌、地衣芽胞杆菌、蜡状芽胞杆菌、巨大芽胞杆菌、球形芽胞杆菌、短小芽胞杆菌、凝结芽胞杆菌。同时测定了它们对动物病原性大肠埃希菌和链球菌的抑菌活性。其中有9株芽胞杆菌对大肠埃希菌有抑制作用,8株芽胞杆菌对链球菌有抑制作用。结果表明芽胞杆菌分离株Y2、Y3、Y4、Y5、Y6、Y7、Y8具有作为益生菌开发的潜力。     

Eicosanoids mediate nodulation reactions to bacterial Escherichia coli K 12 infections in larvae of the oriental blowfly, Chrysomya megacephala

Abstract  Nodulation is the predominant cellular defense reaction to bacterial challenges in insects. In this study, third instar larvae of Chrysomya megacephala were injected with bacteria, Escherichia coli K 12 (10⁶ CFU/mL, 2 μL), immediately prior to injection of inhibitors of eicosanoid biosynthesis, which sharply reduced nodulation response. Test larvae were treated with specific inhibitors of phospholipase A₂ (dexamethasone), cyclo-oxygenase (indomethacin, ibuprofen and piroxicam), dual cyclo-oxygenase/lipoxygenase (phenidone) and lipoxygenase (esculetin) and these reduced nodulation except esculetin. The influence of bacteria was obvious within 2 h of injection (5 nodules/larva), and increased to a maximum after 8 h (with 15 nodules/larva), and then significantly reduced over 24 h (9 nodules/larva). The inhibitory influence of dexamethasone was apparent within 2 h of injection (4 vs. 5 nodules/larva), and nodulation was significantly reduced, compared to control, over 24 h (5 vs. 8 nodules/larva). Increased dosages of ibuprofen, indomethacin, piroxicam and phenidone led to decreased numbers of nodules. Nodules continued to exist during the pupal stage. However, the effects of dexamethasone were reversed by treating bacteria-injected insects with an eicosanoid-precursor polyunsaturated fatty acid, arachidonic acid. These findings approved our view that eicosanoid can mediate cellular defense mechanisms in response to bacterial infections in another Dipteran insect C. megacephala .     

Investigation of ampicillin effect on morphological and mechanical properties of Escherichia coli and Bacillus cereus cells with atomic force microscopy

The effect of subbacteriostatic concentrations of ampicillin on morphological and mechanical properties of gramnegative and grampositive cells of Escherichia coli K12 TG1 and Bacillus cereus IP 5832 respectively was studied with atomic force microscopy. Significant heterogeneity of the bacterial populations was shown by the character of the response to the antibiotic effect. The common feature was increase of the cell size likely due to the effect of the inner osmotic pressure on the lowered cell wall strength. In the E. coli population there were besides observed anomalous elongated cells with signs of septation disorder, as well as their structurs, lacking the cytoplasmic liquid fraction. In the B. cereus the inner osmotic pressure mainly enlarged the cell cross section, changing the cell shape from rod to sphere, that was accompanied by significant impairment of the surface structure with liberation of the peptidoglycane fragments to the medium. The particular features of the E. coli K12 TG1 and B. cereus IP 5832 respond to the ampicillin effect were attributed to the differences in the structure of their cell wall, also due to specific properties of the peptidoglycane synthesis and three-dimensional organization.     

抗动物病原菌芽孢杆菌的筛选、初步鉴定和抗菌活性

从鸡肠道分离、挑选的18株芽孢杆菌经培养特征、形态观察、生理生化实验,初步被鉴定为枯草芽孢杆菌(Bacillus subtilus)、地衣芽孢杆菌(Bacillus licheniformis)、蜡状芽孢杆菌(Bacillus cereus)、巨大芽孢杆菌(Bacillus megaterium)和凝结芽孢杆菌(Bacillus coagulans)。同时测定了它们对5种常见动物病原菌大肠杆菌(E.coli)、金黄色葡萄球菌(Staphylococcus aureus)、肠炎沙门氏菌(Salmonella typhimurium)、猪链球菌(Streptococcus suis)、奇异变形杆菌(Proteus mirabilis)的抑菌活性,其中有4株芽孢杆菌对5种病原菌都有抑制作用。还分别测定了它们产木聚糖酶(从0.895 U1到3.239 U1)和纤维素酶活性(分别为0.391 U2和0.465 U2)。结果表明,芽孢杆菌分离株BC17、BC32、BC106、BC228、BC247和BC261具有作为益生菌的开发潜力。     

Effects of Rosa rugosa petals on intestinal bacteria

The effects of pulverized petal of Rosa rugosa on the growth of 10 species of intestinal and pathogenic bacteria were investigated. Growth of bifidobacteria and lactobacilli was not affected by the addition of the petal in plate cultivation. However, the growth of Bacteroides vulgatus, Escherichia coli, Staphylococcus aureus, and Bacillus cereus was completely inhibited by the addition of 0.1, 0.5, 0.1, and 0.05% (w/v) of the petal respectively. In liquid cultivation, the addition of the petal (0.5%) stimulated the growth of Bifidobacterium breve and slightly inhibited the growth of Lactobacillus salivarius. But the growth of E. coli, S. aureus, B. cereus, and Salmonella sp. was inhibited by nearly 50%. Hydrolyzable tannins isolated from R. rugosa, rugosin D, and tellimagradin II showed antibacterial activities against E. coli, S. aureus, B. cereus, and Salmonella sp., but little or no effect against Bif. breve and L. salivarius. R. rugosa petal showed selective antibacterial activities against intestinal and pathogenic bacteria, and the selectivity resembled that of prebiotics such as oligosaccharides and dietary fiber. Hydrolyzable tannins in R. rugosa, such as rugosin D and tellimagradin II, must be active constituents.     

Eicosanoids rescue Spodoptera exigua infected with Xenorhabdus nematophilus, the symbiotic bacteria to the entomopathogenic nematode Steinernema carpocapsae

Xenorhabdus nematophilus is a pathogenic bacterium causing insect haemolymph septicemia, which leads to host insect death. To address the fundamental mechanisms underlying this haemolymph septicemia, or the immunodepressive response of the host insects following bacterial infection, we tested a hypothesis that the insect immune-mediating eicosanoid pathway is blocked by inhibitory action of the bacterium. Haemocoelic injection of the bacteria into the fifth instar larvae of Spodoptera exigua reduced the total number of living haemocytes with postinjection time and resulted in host death in 16 h at 25 degrees C. The lethal efficacy, described by the median lethal bacterial dose (LD(50)), was estimated as 33 colony-forming units per fifth instar larva of S. exigua. The lethal effect of the bacteria on the infected larvae decreased significantly with the addition of exogenous arachidonic acid (10 μg), a precursor of eicosanoids. In comparison, injections of dexamethasone (10 μg), a specific inhibitor of phospholipase A(2), and other eicosanoid biosynthesis inhibitors elevated significantly the bacterial pathogenicity. Live X. nematophilus induced the infected larvae to form less nodules than did the heat-killed bacteria, but the addition of arachidonic acid increased the number of nodules formed significantly in response to live bacterial injection. The treatment with dexamethasone and other inhibitors, however, decreased the nodule formation after injection of heat-killed bacteria. These results indicate that eicosanoids play a role in the immune response of S. exigua, and suggest strongly that X. nematophilus inhibits its eicosanoid pathway, which then results in immunodepressive haemolymph septicemia.     

Rhizobium meliloti nodulation genes allow Agrobacterium tumefaciens and Escherichia coli to form pseudonodules on alfalfa.

Regions of the Rhizobium meliloti symbiotic plasmid (20 to 40 kilobase pairs long) containing nodulation (nod) genes were transferred to Agrobacterium tumefaciens or Escherichia coli by conjugation. The A. tumefaciens and E. coli transconjugants elicited root hair curling and the formation of ineffective pseudonodules on inoculated alfalfa plants. A tumefaciens elicited pseudonodules formed at a variable frequency, ranging from 15 to 45%, irrespective of the presence of the Ti plasmid. These pseudonodules developed characteristic nodule meristems, and in some nodules, infection threads were found within the interior of nodules. Infrequently, infection threads penetrated deformed root hairs, but these threads were found only in a minority of nodules. There was no evidence of bacterial release from the infection threads. In addition to being found within threads, agrobacteria were also found in intercellular spaces and within nodule cells that had senesced . In the latter case, the bacteria appeared to invade the nodule cells independently of infection threads and degenerated at the same time as the senescing host cells. No peribacteroid membranes enclosed any agrobacteria , and no bacteroid differentiation was observed. In contrast to the A. tumefaciens-induced pseudonodules , the E. coli-induced pseudonodules were completely devoid of bacteria; infection threads were not found to penetrate root hairs or within nodules. Our results suggest that relatively few Rhizobium genes are involved in the earliest stages of nodulation, and that curling of root hairs and penetration of bacteria via root hair infection threads are not prerequisites for nodule meristem formation in alfalfa.     

Interaction of Escherichia coli K1 and K5 with Acanthamoeba castellanii trophozoites and cysts

The existence of symbiotic relationships between Acanthamoeba and a variety of bacteria is well-documented. However, the ability of Acanthamoeba interacting with host bacterial pathogens has gained particular attention. Here, to understand the interactions of Escherichia coli K1 and E. coli K5 strains with Acanthamoeba castellanii trophozoites and cysts, association assay, invasion assay, survival assay, and the measurement of bacterial numbers from cysts were performed, and nonpathogenic E. coli K12 was also applied. The association ratio of E. coli K1 with A. castellanii was 4.3 cfu per amoeba for 1 hr but E. coli K5 with A. castellanii was 1 cfu per amoeba for 1 hr. By invasion and survival assays, E. coli K5 was recovered less than E. coli K1 but still alive inside A. castellanii. E. coli K1 and K5 survived and multiplied intracellularly in A. castellanii. The survival assay was performed under a favourable condition for 22 hr and 43 hr with the encystment of A. castellanii. Under the favourable condition for the transformation of trophozoites into cysts, E. coli K5 multiplied significantly. Moreover, the pathogenic potential of E. coli K1 from A. castellanii cysts exhibited no changes as compared with E. coli K1 from A. castellanii trophozoites. E. coli K5 was multiplied in A. castellanii trophozoites and survived in A. castellanii cysts. Therefore, this study suggests that E. coli K5 can use A. castellanii as a reservoir host or a vector for the bacterial transmission.     

Antibacterial spectrum of lisosubtilin G10X

Data on the antibacterial spectrum of lysosubtilin G10X, a preparation of lytic enzymes from Bacillus subtilis SK-52 are presented. Lysosubtilin was active against grampositive and gramnegative pathogenic bacteria. When it was used as a substrate of live lyophilized microbial cells the highest lysis levels were observed in B. brevis, B. cereus, B. pumilis, B. subtilis and S. faecalis. Preincubation of the substrate in acid media mainly increased the levels of the lysis by enzyme preparation. Sometimes the increase was very high (B. sphaericus, B. subtilis 720, E. coli K12 and MRE-600). Such a preincubation provided cell lysis in some strains not liable to the effect of lysosubtilin (B. cereus 1312, C. renale, M. luteus, S. aureus KP, 800, 805 and 126001, S. pyogenes 291). An increase in the lysosubtilin concentration in the reaction mixture in the majority of the cases did not provide favourable results. However, some strains resistant to the preparation at a concentration of 1000 units/ml were lysed with its 10 times higher doses. An increase in the lysis level was also achieved with increasing the time of the incubation with the enzyme preparation. Proceeding from the preparation antibacterial spectrum it is possible to recommend it for treatment of diseases in agricultural animals. Its use in veterinary was a success.     

Rapid and Sensitive Detection of Bacteria by Gas Chromatography

A gas chromatograph fitted with electron capture and flame ionization detectors was employed for the rapid detection of bacteria by analysis for their metabolic products. The presence of Proteus vulgaris, Streptococcus faecalis, S. liquefaciens, Escherichia coli B, Bacillus cereus, and B. popilliae was detected in 2 to 4 hr in media inoculated with less than 10(4) cells per ml, whereas a 7- to 12-hr growth period was required for the detection of products formed in cultures of Serratia marcescens, Aerobacter aerogenes, E. coli K-12, Staphylococcus aureus, and Salmonella typhimurium. Metabolites elaborated by the equivalent of less than a single cell of B. cereus, S. faecalis, P. vulgaris, or E. coli B were sensed by the electron capture detector. The flame ionization detector was generally not as sensitive. Volatile metabolites were identified, and their concentrations were determined.     

Transepithelial activation of human leukocytes by probiotics and commensal bacteria: Role of Enterobacteriaceae-type endotoxin

The goal of the current study was to clarify whether commercially available probiotics induce greater trans-epithelial activation of human leukocytes than do commensal, food-derived and pathogenic bacteria and to identify the compounds responsible for this activation. Eleven different bacterial strains, and some of their pathogen-associated molecular patterns, were incubated apically on a confluent layer of intestinal epithelial cells (Caco-2), which were basolaterally co-cultured with human mononuclear leukocytes. Only Gram-negative bacteria having Enterobacteriaceae -type endotoxin (commensal Escherichia coli K12, probiotic E. coli Nissle, EPEC) induced basolateral production of TNF-α, IFN-γ, IL 6, 8, and 10. Gram-positive probiotics ( Lactobacillus spp. and Bifidobacterium spp. ) had virtually no effect. In addition, commensals ( Enterococcus faecalis , Bacteroides vulgatus ) and food fermenters ( Lactobacillus spp. ) did not stimulate leukocytes transepithelially. Endotoxin from E. coli and Salmonella enterica Typhimurium induced comparable transepithelial stimulation of leukocytes, but not endotoxin from B. vulgatus or lipoteichoic acid from E. faecalis . Endotoxin-binding agents (polymyxin, colistin) completely abrogated transepithelial activation of leukocytes. Enterobacteriaceae -type endotoxin is a crucial factor in transepithelial stimulation of leukocytes, regardless of whether it is produced by probiotics or other bacteria. Hence, transepithelial stimulation of leukocytes' innate immune response seems to not be linked to the health-promoting effects of probiotics.     

Comparison of in vitro models to study bacterial adhesion to the intestinal epithelium

Aims:  To evaluate the adhesion ability of intestinal bacteria to different in vitro models of intestinal epithelia, and to estimate the suitability of these models and the type of interactions involved.
Methods and results:  The adhesion of probiotic ( Lactobacillus rhamnosus GG and Bifidobacterium animalis subsp . lactis Bb12), commensal ( B. animalis IATA-A2 and B. bifidum IATA-ES2) and potentially pathogenic bacteria ( E. coli and L. monocytogenes ) was determined. The adhesion models used were polycarbonate-well plates, with or without mucin, and different configurations of Caco-2 and/or HT29-MTX cell cultures. All bacteria adhered to wells without mucin (2·6–27·3%), the values being highly variable depending on the bacterial strain. Adhesion percentages of potentially probiotic bacteria to Caco-2 cultures were remarkably lower ( P  <   0·05) than those to mucin, and more similar to those of pathogenic strains. The lowest adhesion of different bacterial strains was detected on HT29-MTX (0·5–2·3%) cultures and Caco-2/HT29-MTX (0·6–3·2%) cocultures, while these values were increased in Caco-2 cultures plus mucin.
Conclusions:  The results suggested that bacterial strains exhibit different capacities to adhere to cellular components and several types of mucin present in different models, showing preferences for intestinal MUC2.
Significance and impact of the study:  The use of Caco-2 cells monolayer plus mucin (type II) better approaches the physiological characteristics of in vivo situation, providing a reliable and suitable in vitro model to evaluate bacterial adhesion.     

Effect of dark repair on ultraviolet sensitivity of bacteriophage-infected bacteria

Feiner, R. R. (Columbia University, New York, N.Y.), and R. F. Hill. Effect of dark repair on ultraviolet sensitivity of bacteriophage-infected bacteria. J. Bacteriol. 91:1239-1247. 1966.-Changes in ultraviolet (UV) sensitivity of phage-host complexes during phage development have been studied for the following systems: T1 and Escherichia coli B, T1 and E. coli K-12S, lambda and E. coli K-12S. Complexes were formed with bacterial strains differing in ability to dark-repair UV damage to deoxyribonucleic acid and, after irradiation, were plated on bacteria differing similarly. In the first half of the latent period, the resistance of complexes formed with nonrepairing bacteria increased considerably; with T1 and E. coli B hcr(-), in 4 min the resistance became the same as that of complexes formed with repairing bacteria. The repair ability of plating bacteria affected survival curves only upon irradiation in the second half of the latent period after mature phages were present in the initial complex. Use of nonrepairing bacteria both for initial infection and for plating of late complexes resulted in a series of survival curves showing for all three systems the same pattern of change originally reported for T2-E. coli B complexes. Thus, a hitherto unexplained difference between radiation survival curves for T-even and T-odd phages seems due to repair of T-odd phages by the host.     

Studies on the in vivo cellular reactions of insects: Clearance of pathogenic and non-pathogenic bacteria in Galleria mellonella larvae

The fate of various doses of bacteria of different pathogenicities injected into Galleria mellonella larvae was monitored over time, from haemocyte and bacterial counts, phagocytic responses and the speed and extent of formation of melanized cell aggregates (nodules). An initial haemocytopenia was recorded in all larvae, probably as a result of wound healing, an increased stickiness of the haemocytes for host tissues and/or cell clump or nodule formation. The results also showed that phagocytosis is the primary cellular defence reaction of this insect for doses of bacteria below ca. 10³ μl^?1 haemolymph while above this level phagocytosis and bacterial clearance are usually rapidly augmented by nodule formation. The extent to which these processes are elicited depends greatly upon the nature of the bacteria injected. In general, the more pathogenic strains produced greater responses than the relatively non-pathogenic forms. This enhanced cellular reactivity was, however, soon overcome by the pathogens which rapidly induced a secondary bacteraemia, a huge drop in haemocyte numbers and death of the larvae. The relative importance of phagocytosis and nodule formation in dealing with various doses of bacteria of differing pathogenicities is discussed.     

Nodulation of Glycine max by Six Bradyrhizobium japonicum Strains with Different Competitive Abilities

The root nodule locations of six Bradyrhizobium japonicum strains were examined to determine if there were any differences which might explain their varying competitiveness for nodule occupancy on Glycine max. When five strains were added to soybeans in plastic growth pouches in equal proportions with a reference strain (U.S. Department of Agriculture, strain 110), North Carolina strain 1028 and strain 110 were the most competitive for nodule occupancy, followed by U.S. Department of Agriculture strains 122, 76, and 31 and Brazil strain 587. Among all strains, nodule double occupancy was 17% at a high inoculum level (10⁷ CFU pouch⁻¹) and 2% at a low inoculum level (10⁴ CFU pouch⁻¹). The less competitive strains increased their nodule representation by an increase in the doubly occupied nodules at the high inoculum level. Among all strains, the number of taproot and lateral root nodules was inversely related at both the high and low inoculum levels (r = −0.62 and −0.69, respectively; P = 0.0001). This inverse relationship appeared to be a result of the plant host control of bacterial infection. Among each of the six strains, greater than 95% of the taproot nodules formed at the high inoculum density were located on 25% of the taproot length, the nodules centering on the position of the root tip at the time of inoculation. No differences among the six strains were observed in nodule initiation rates as measured by taproot nodule position. Taproot nodules were formed in the symbiosis before lateral root nodules. One of the poorly competitive strains (strain 76) occupied three times as many taproot nodules as lateral root nodules when competing with strain 110 (nodules were harvested from 4-week-old plants). Among these six wild-type strains of B. japonicum, competitive ability evidently is not related to nodule initiation rates.