Antigenic affinities of the root-nodule bacteria of legumes

Antisera prepared against 58 strains of root-nodule bacteria and against 16 strains belonging to the genusAgrobacterium were tested against 113 strains ofRhizobium, 20 strains ofAgrobacterium and 20 strains of other, possibly related, bacteria.Three serologically distinct groups of root-nodule bacteria were noted: (1)Rh. trifolii, Rh. leguminosarum andRh. phaseoli; (2)Rh. lupini, Rh. japonicum andRhizobium spp.; (3)Rh. meliloti. Strains ofRh. meliloti showed serological affinities withA. radiobacter andA. tumefaciens. All groups showed wider flagellar than somatic agglutination, and many different serotypes were apparent.The groupings obtained from this investigation are compared with those derived from other taxonomic studies, and the use of serological methods in rhizobial classification is discussed.     

DNA-DNA hybridization of some root-nodule bacteria indigenous in the salt-affected soils of egypt

The DNA-DNA hybridization was used to characterize thirty isolates of root-nodule bacteria indigenous to the salt-affected soils of Egypt. Total DNA from different bacterial isolates lacked homology with total DNA probes of the effective strains ofRhizobium leguminosarum andR. meliloti. It is suggested that the genomic structure of the root-nodule bacteria may be modified by salt stress and/or that the effective strains of these bacteria are to be eliminated from the salt-affected soil.     

Symbiosis between the root-nodule bacterium Sinorhizobium meliloti and alfalfa (Medicago sativa) under salinization conditions

Two hundred forty-three isolates of alfalfa root-nodule bacteria (Sinorhizobium meliloti) were obtained from nodules and soils sampled in the northern Aral region, experiencing secondary salinization. Isolates obtained from nodules (N isolates) were significantly more salt-tolerant than those from soils (T isolates) when grown in a liquid medium with 3.5% NaCl. It was found that wild species of alfalfa, melilot, and trigonella preferably formed symbioses with salt-tolerant root-nodule bacteria in both salinized and nonsalinized soils. Only two alfalfa species, Medicago falcata and M. trautvetteri, formed efficient symbioses in soils contrasting in salinity. The formation of efficient symbiosis with alfalfa in the presence of 0.6% NaCl was studied in 36 isolates (N and T) differing in salt tolerance and symbiotic efficiency. Fifteen isolates formed efficient symbioses in the presence of salt. The increase in the dry weight of the plants was 25–68% higher than in the control group. The efficiency of symbiotic interaction under salinization conditions depended on the symbiotic efficiency of the isolates under standard conditions but did not correlate with the source of root-nodule bacteria (soil or nodule) or their salt tolerance. The results indicate that the strains of root-nodule bacteria forming efficient symbioses under salinization conditions can be found.     

Accumulation of an Aromatic Amine, β-Phenethylamine, in Root Nodules of Adzuki Bean Vigna angularis

Neuroactive aromatic amines acting on the central nervous system are widespread in the plant kingdom. We have previously found β-phenethylamine (β-PHA), one of the aromatic alkaloids, in root nodules of various annual legume crops. The present study was undertaken to determine the site of β-PHA accumulation within root nodules of the adzuki bean Vigna angularis. High concentrations of β-PHA were always detected in the alkaloid fraction of adzuki bean root nodules. Related aromatic amines such as tyramine, dopamine, and other β-PHA derivatives, which are found in various medicinal plants, were not detected in adzuki bean root nodules. The amounts of β-PHA in root nodules varied not only with the growth stage of the host plant, but also with nodule age; β-PHA levels increased with nodule development, but declined with nodule senescence. Adzuki bean nodules, after crushing with a grinding medium, were separated into bacteroids and a nodule cytosol fraction. A large amount of β-PHA was detected in the bacteroids, while a very small amount was prsent in the nodule cytosol fraction derived from plant cells. The bacteroids in the mature nodules contained considerably higher amounts of β-PHA than did those in immature or senescent nodules. The formation of β-PHA in root-nodule bacteria was then tested using eight strains of Rhizobiaceae (Rhizobium, Bradyrhizobium and Sinorhizobium), including a strain isolated from root nodules of field-grown adzuki bean plant. None of the cultured cells produced β-PHA in liquid media in the presence or absence of phenylalanine, a putative precursor of β-PHA. Nitrogen-fixing bacteroids within nodules are the cells uniquely differentiated from root-nodule bacteria. The present results suggest that β-PHA is formed associated with the differentiation of vegetative bradyrhizobia into nitrogen-fixing bacteroids with the plant host cells.     

In vitro evaluation of bacteria for the biological control ofMacrophomina phaseolina

Sixty-four strains ofRhizobium and seven other rhizosphere bacteria were evaluated by streak-plate, double-layer, and spent-culture methods to determine their antibiotic activity againstMacrophomina phaseolina, causative agent of ashy stem blight of beans. Expression of inhibition varied among strains and depended on growth media and screening method. The streak-plate method with yeast extract/mannitol was the best bioassay. Results indicate that root-nodule bacteria have weak biofungicidal potential. A strain ofPseudomonas cepacia (UPR 5C) consistently restricted fungal growth.F. Perdomo, M. Alameda and E.C. Schröder are with the BNF Laboratory, Department of Agronomy and Soils, University of Puerto Rico, Mayagüez, PR 00681-5000, USA; R. Echávez-Badel is with the Department of Crop Protection, University of Puerto Rico, Mayagüez, PR 00681-5000, USA.     

Application of immunoprecipitation in agar gel for the serological typing of soybean root-nodules

The results of serological typing of root-nodule homogenates from soybean plants inoculated by mixed bacterial inocula under field and greenhouse conditions are submitted. The inocula were prepared from strains capable of serological differentiation on the basis of their somatic antigens. Individual root-nodule homogenates were typed serologically by immunoprecipitation in agar with antisera against the inoculum strains. This method gave accurate determination of the origin of 605 out of a total of 616 serologically typed root-nodules and of the individual bacterial strains which participated in their formation.     

4株羊瘤胃功能性细菌的筛选

通过DNS法测定羊瘤胃源功能性细菌产生的纤维素酶和淀粉酶的活力,福林酚法测定产生的蛋白酶的活力,检测细菌产生酶的特性。同时检测菌株的发酵液对大肠埃希菌(ATCC25922)、副溶血弧菌(ATCC17802)、藤黄八叠球菌(HY78)和产气杆菌(AS1489)等指示菌的抑制能力,分析它们的抑菌活性。结果表明,羊瘤胃源细菌C13产生的纤维素酶活力最高,产酶量也最高;而细菌C5产淀粉酶活力和蛋白酶活力最高,产生淀粉酶和蛋白酶的能力也最高。抑菌活性检测发现,细菌C9对副溶血弧菌(ATCC17802)有很高的抑制作用,而细菌C12对大肠埃希菌(ATCC25922)的抑制能力最明显。     

Oxalate utilisation is widespread in the actinobacterial genus Kribbella

The type strains of all 33 species in the genus Kribbella were tested for growth on oxalate (^?OOC-COO^?) as sole carbon source. Media were initially formulated to contain sodium oxalate, but even a concentration as low as 7.5 mM oxalate prevented growth. A modified medium based on calcium oxalate was very successful in characterising oxalate utilisation by Kribbella strains (metabolism of oxalate by oxalotrophic bacteria results in visible zones of clearing around the growth streaks on the opaque plates). To assess the variability of oxalate utilisation in Kribbella species, we also tested eight non-type strains for their ability to use oxalate. Thirty of 33 type strains (90.9%) and six of eight non-type strains (75%) were able to use oxalate as a sole carbon source. Based on these results, we propose that oxalate would be an excellent carbon source for the selective isolation of Kribbella strains. Based on the oxalate-utilisation phenotype and analyses of the 19 publicly available Kribbella type-strain genome sequences, we propose a pathway for oxalate metabolism in Kribbella. This pathway is significantly different from those previously proposed for oxalate metabolism in other bacteria, involving the indirect catabolism of oxalate to formate. Formate production is proposed to be involved in energy generation and to be crucial for oxalate import via an oxalate:formate antiporter. To our knowledge, this is the first report of an oxalate:formate antiporter in an aerobic, Gram-positive bacterium.     

The inhibitory activity of Lactobacillus spp. isolated from breast milk on gastrointestinal pathogenic bacteria of nosocomial origin

Milk acts as a mean for transporting many essential substances from the mother to the child. In human beings, milk includes several predominant bacteria, such as staphylococci, streptococci, micrococci, lactobacilli, enterococci, lactococci and bifidobacteria. Besides, its intake favors the predominance of bifidobacteria and lactobacilli in the child’s intestinal microbiota. The present work explores the isolation and selection of lactobacilli strains with probiotic potential, focusing in their degree of hydrophobicity and antagonism against important gastrointestinal nosocomial pathogens. 98 lactobacilli were isolated from 48 breast milk samples, with most strains belonging to the obligately homofermentative group (36.7%). 63% of the isolated strains showed a high degree of hydrophobicity when tested on three solvents and were selected for detecting antimicrobial activity against gastrointestinal pathogens, including Escherichia coli, Shigella spp, Pseudomonas spp and Salmonella spp strains. When applying the agar diffusion test, many isolated strains presented inhibitory activity against pathogenic strains. We observed that: Salmonella enteriditis was the most inhibited pathogen, and the strains with the most inhibitory power were AR2 and O1 (both highly hydrophobic lactic acid bacteria), which showed an opposing effect against all nosocomial pathogens tested. Although more in vitro, in vivo or clinical data would be needed before any conclusion on the probiotic properties of the strains can be drawn, our results demonstrate that some of the tested strains may have good probiotic potential for their inclusion in products targeting infants.     

Adaptation of chickpea to desiccation stress is enhanced by symbiotic rhizobia

This study examined the influence of three inoculant strains of Bradyrhizobium japonicum (Thal-8, Tal 620, Dulawala) on the ability of chickpea (Cicer arietinum (L.) to adapt to drought-stress. Strain Thal-8 was most effective in the root-nodule symbiosis and also partially alleviated decreased growth and yield imposed by drought stress. Strain Thal-8, in pure culture, also produced higher amounts of gibberellic acid (GA) and indole-3-acetic acid (IAA) and lower amounts of abscisic acid (ABA) than the other two test strains. Thal-8 increased the root biomass, GA and IAA contents of leaves of chickpea plants, including ICC 4948NN, a non-nodulating line. These results are consistent with the hypothesis that GA and IAA is produced by the Thal-8 strain and/or elevates levels of these phytohormones in chickpeas. This contributes to its high performance as a nitrogen-fixing microsymbiont. The growth-promoting response evoked by different strains of Bradyrhizobium correlated with higher ratios of GA and IAA relative to ABA phytohormones in the plants.     

The association of algicidal bacteria and raphidophyte blooms in South Carolina brackish detention ponds

Over the past 5 years, raphidophyte blooms have been frequently observed along the South Carolina coastal zone. During the 2002, 2003, and 2004 sampling seasons, we investigated temporal fluctuations of algicidal bacteria abundance against raphidophycean flagellates (Heterosigma akashiwo, Chattonella subsalsa, and Fibrocapsa japonica) using the microplate most probable number (MPN) method in three Kiawah Island brackish stormwater detention ponds (K1, K2, and K75). Local axenic isolates of H. akashiwo, C. subsalsa, and F. japonica were obtained and their susceptibility to algicidal bacteria tested. A total of 195 algicidal bacterial strains were isolated from raphidophyte blooms in the study ponds, and 6 of them were identified at the genus level, and the taxonomic specificity of their algicidal activity was tested against local (pond) and nonlocal isolates of raphidophytes (3 species, 10 total strains). In the ponds, a consistent association was found between raphidophyte bloom development and an increase in bacteria algicidal to the bloom species. In 12 of 15 cases, bloom decline followed the increase in algicidal bacteria to maximum abundances. Although variability was found in the taxonomic specificity of the algicidal bacteria effect (i.e. the number of raphidophyte species affected by a particular bacteria strain) and raphidophyte susceptibility (i.e. the number bacteria strains affecting a particular raphidophyte species), a toxic effect was always found when strains of a raphidophyte species were exposed to algicidal bacteria isolated from a bloom caused by that same species. The results suggest that algicidal bacteria may be an important limiting factor in raphidophyte bloom sustenance and can promote bloom decline in brackish lagoonal eutrophic estuaries.     

Behavior of Variable V3 Region from 16S rDNA of Lactic Acid Bacteria in Denaturing Gradient Gel Electrophoresis

Separation of amplified V3 region from 16S rDNA by denaturing gradient gel electrophoresis (DGGE) was tested as a tool for differentiation of lactic acid bacteria commonly isolated from food. Variable V3 regions of 21 reference strains and 34 wild strains referred to species belonging to the genera Pediococcus, Enterococcus, Lactococcus, Lactobacillus, Leuconostoc, Weissella, and Streptococcus were analyzed. DGGE profiles obtained were species-specific for most of the cultures tested. Moreover, it was possible to group the remaining LAB reference strains according to the migration of their 16S V3 region in the denaturing gel. The results are discussed with reference to their potential in the analysis of LAB communities in food, besides shedding light on taxonomic aspects. Received: 2 August 2000 / Accepted: 5 September 2000     

Methods of isolation and cultivation of Frankia species from actinorhizas

A critical review is given about the isolation and cultivation methods of Frankia species fromAlnus glutinosa root-nodules. The best results so far are obtained with a combination of sucrose (60% w/v)-sedimentation of root-nodule homogenate and subsequent suspension in the top-layer of a doubleagar layer system. The top-layer needs to contain a suitable C-source, in this study often a lipid factor from an alcoholic root-extract and an organic N-source.The isolation and cultivation of Sp(–) and Sp(+) strains fromAlnus glutinosa root nodules and a Frankia from the root-nodules ofMyrica gale is reported. The regular observation of growing colonies appears to be very important for the interpretation of results. The latter was illustrated by the remarkable diauxic growth of the strains isolated fromAlnus glutinosa Sp(+) root nodules.     

Antibacterial activity of the leaf essential oil of <Emphasis Type="Italic">Blumea mollis</Emphasis> (D. Don) Merr.

The antibacterial activity of the leaf essential oil of Blumea mollis was assayed against 14 clinically isolated bacterial strains on Muller–Hinton Agar medium and Muller–Hinton Agar medium with 5% sheep blood. The essential oil had promising antibacterial activity against all the bacterial strains tested. The highest mean zone of inhibition and lowest values of minimum inhibitory concentration were recorded against methicillin-resistant Staphylococcus aureus followed by beta hemolytic Streptococcus pyogenes. The Gram-positive bacteria were more sensitive than Gram-negative bacteria. Among the bacterial strains tested, Psudomonas aeruginosa was resistant to the essential oil. The results of the present study suggest that the essential oil of B. mollis is one of the new medicinal resources as an antibacterial agent against the bacterial strains tested.     

Influence of rhizobia,azotobacter and blue-green algae on N content and yield of rice

Summary Inoculation with root-nodule bacteria had favourable influence on N-uptake and yield of wheat. Since waterlogged root region of rice permits higher nitrogenase activity a pot culture experiment was conducted using same nine strains of rhizobia,Azotobacter chroococcum and bluegreen algae as inoculants.R. leguminosarum in combination with 50 kg N ha⁻¹;R. japonicum and a strain of rhizobium isolated from moong bean increased the yield of paddy cv. Pusa-33. On the other hand an adverse effect of bacterial inoculation and of applied N was observed in case of Azotobacter, and rhizobia isolated from green gram, cicer, soyabean and clover. The importance of plant type, growth conditions and application of inorganic N in determining the success of plant-rhizobial associations is emphasised.     

Formation of poly(3-hydroxyalkanoates) by phototrophic and chemolithotrophic bacteria

The formation of poly(3-hydroxyalkanoic acid), PHA, by various strains of chemolithotrophic and phototrophic bacteria has been examined. Chemolithotrophic bacteria were grown aerobically under nitrogen-limiting conditions on various aliphatic organic acids. Phototrophic bacteria were grown anaerobically in the light on a nitrogen-rich medium and were subsequently transferred to a nitrogen-free medium containing acetate, propionate, valerate, heptanoate or octanoate as carbon source. All 41 strains investigated in this study were able to synthesize and accumulate PHA. All 11 strains of chemolithotrophic bacteria and all 15 strains belonging to the non-sulfur purple bacteria synthesized a polymer, which contained 3-hydroxy-valerate (3HV) beside 3-hydroxybutyrate (3HB), if the cells were cultivated in the presence of propionate, valerate or heptanoate. Many non-sulfur purple bacteria synthesized copolyesters of 3HB and 3HV even with acetate as carbon source. In contrast, most sulfur purple bacteria did not incorporate 3HV at all. Among 15 strains tested, only Chromatium vinosum strain 1611, C. purpuratum strain BN5500 and Lamprocystis roseopersicina strain 3112 were able to synthesize polyesters containing 3HV with propionate, valerate or heptanoate as carbon source.     

红海榄内生细菌多样性及其抑制鱼类致病菌活性研究

红海榄为红树科红海属植物,广泛分布于热带海岸的红树林中,为真红树植物的典型代表。该研究以广西山口红树林国家级自然保护区红海榄为对象,应用稀释涂布法和三线法从红海榄各组织中分离出17株内生细菌,通过各菌株形态特征观察及利用16S rRNA序列基因和韦恩图分析其内生细菌多样性。结果表明:17株内生细菌分属3个门5科7属8种,Micromonospora和Mangrovibacter属为其优势类群。进一步分析发现红海榄根茎叶等组织的内生细菌类群差别较大,仅有1个相同菌属,17株菌中有3株菌与已有细菌物种典型菌株的全长16S rRNA基因相似性低于97%,代表着潜在的新属或新种。运用纸片法研究8株内生细菌发酵液的乙酸乙酯提取物对鱼类致病菌副溶血弧菌活性抑制效果,发现3株内生细菌(H003、H013、H009,浓度5 mg·m L-1)的代谢产物对副溶血弧菌具有较强的抑菌活性,其抑菌圈直径分别达到(8.4±0.07)、(8.2±0.07)、(8.3±0.14)mm。该研究结果表明红海榄中具有较好的内生细菌多样性和抑菌活性,为今后研究其内生细菌的化学多样性及其应用提供了重要的物质基础。     

Fimbriation ofEscherichia coli in urinary tract infections. Comparisons between bacteria in the urine and subcultured bacterial isolates

Midstream urine samples from 37 patients with urinary tract infections were studied by electron microscopy, hemagglutination, and the salt aggregation test (SAT) to measure the hydrophobicity of the bacterial surface.Escherichia coli subcultured from these urine samples were tested in the same way. Fimbriae were visualized onE. coli in the urine of 31 specimens, and all these urines containedE. coli that expressed pronounced surface hydrophobicity and aggregated in ammonium sulfate of 0.1–1.6 M final concentration. Hemagglutination of human and/or guinea pig erythrocytes was expressed by 21E. coli in the urine. TheE. coli strains subcultured from these 31 urine samples were also fimbriated, but the number of fimbriae per bacterium as well as the percentage of fimbriated bacteria varied compared with the directly collected strains. The surface hydrophobicity and hemagglutination were similar to the results with the directly collected bacteria. However, after serial transfer in CFA-broth under static conditions, all non-hemagglutinating strains expressed mannose-sensitive hemagglutination of guinea pig erythrocytes, and three strains also expressed weak mannose-resistant hemagglutination of human erythrocytes. Following serial transfer, fimbriae were also visualized on the sixE. coli strains that appeared non-fimbriate in the urine. It is thus concluded thatE. coli causing urinary tract infection are often fimbriated and express surface hydrophobicity in the urine. Based on these findings, a rapid method to isolate hydrophobic, possibly fimbriated bacteria was tried in which the urine was mixed with a hydrophobic gel. Hydrophobic bacteria bound to the gel and could be eluted from the sedimented gel.     

川蔓藻内生及根际细菌多样性与抑菌活性研究

该研究采用稀释涂布法结合形态观察、16S rRNA基因序列分析,对广西北海川蔓藻(Ruppia maritima)内生及根际细菌的物种多样性进行了研究,并采用琼脂扩散法和光度计法分析了其粗提物抑制马尔尼菲青霉菌活性。结果表明:从川蔓藻中分离到可培养内生细菌26株,根际可培养细菌31株。分别将内生细菌归属为10科12属13种,根际分离出细菌归属为9科14属19种,其中5株根际细菌可能为潜在新种。获得8株细菌对马尔尼菲青霉菌有抑制活性,总阳性率为25.0%。其中,菌株BGMRC 2015、BGMRC 2059、BGMRC 2043的粗提物表现出较强的抑制马尔尼菲青霉菌效果,其MIC分别为(1.800±0.045)、(1.881±0.061)、(1.604±0.021)mg·m L~(-1)。川蔓藻中可培养细菌具有较高的物种多样性,蕴藏着丰富的新物种资源,且富含抑菌活性良好的菌株。     

海南西海岸真红树内生细菌多样性及其延缓衰老活性研究

该文设计9种分离培养基,采用稀释涂布法从14份真红树植物的46份组织样品中分离纯化内生细菌。并基于菌株形态学特征和16S rRNA基因序列确定分离菌株的种属及分析其物种多样性,采用秀丽隐杆线虫模型筛选菌株延缓衰老活性。结果表明:(1)通过基因序列去重复后从46份真红树植物组织样品中获得32株海洋细菌,基于菌株16S rRNA基因序列信息分析,覆盖12科17属,其中芽孢杆菌属(Bacillus)为优势菌属,并获得1株疑似橙单胞菌属(Aurantimonas)新种,16S rRNA基因序列相似性低于97%;(2)经过秀丽隐杆线虫粗筛发现3株海洋细菌具有显著延缓线虫衰老的活性(P0.05)。以上结果表明海南西海岸真红树内生细菌具有物种多样性,部分菌株具有延缓线虫衰老活性。