Preliminary study of taxonomy ofAzotobacter andAzomonas by using rocket line immunoelectrophoresis

Rocket line immunoelectrophoresis was used to study the taxonomy ofAzotobacter andAzomonas assessed by reaction with antiserum to the AVO₂ strain ofAzotobacter. Forty-five cultures, comprising seventeen species in five genera, showed that antigen “β”, like high-titer somatic agglutination, was restricted to all 11 strains ofAzotobacter vinelandii and to one strain which has been namedAzotobacter macrocytogenes (10EM). A thermoresistant antigen (“γ”) was found to be shared by all strains and species ofAzotobacter andAzomonas investigated.     

Periodical variations in nitrogen fixation byAzotobacter

Summary Periodical nitrogen fixation experiments with two strains ofAzotobacter chroococcum for 16 months showed no definite correlation between gains of nitrogen and meteorological changes, as reported by Bortels. Variability in the gains of nitrogen seemed to be due largely to chance variation, together with certain abnormal depressions which did not clearly coincide with changes in atmospheric pressure. The conclusions of Bortels thus do not seem to be generally valid forAzotobacter. Certain long-term variations in nitrogen fixation seemed in one strain to be connected with the arising of a variant of lower nitrogen-fixing power.     

Performance and persistence of phosphate solubilizingAzotobacter chroococcum in wheat rhizosphere

Survival and establishment of inoculant strains ofAzotobacter chroococcum NV 11 and its mutant NV 43 were assessed in sterilized soil and rhizosphere soil of wheat plants at 7 and 15 d interval, respectively, after sowing,i.e., up to 45 d under pot-house conditions. There was an apparent decrease in population of both strains in bulk soil but a steady increase was observed in root zones of the wheat plant. 10³ to 10⁴ introduced bacteria per g root were found sticking to roots. Further studies indicated that inoculant strains ofAzotobacter could survive, proliferate and establish well in root zones.     

Characterization ofAzotobacter spp. and effect of Azotobacter and Azospirillum as inoculant on the yield and N-uptake of wheat crop

Out of ten isolates ofAzospirillum spp. isolated from the root and rhizosphere of wheat plant, seven belonged toA. brasilense and three belonged toA. lipoferum. All eight isolates ofAzotobacter spp. belonged toAzotobacter chroococcum. Two strains, one fromA. lipoferum and another fromA. chroococcum having high nitrogen fixing capacity with negative test for denitrification were used as inoculant to supplement the nitrogen need of wheat crop. Significant increases in the yield of wheat grain and uptake of nitrogen by the crop over the control were found in pot tests when the seeds were inoculated either withAzospirillum spp. orAzotobacter spp. or the combination of both the inoculants.     

Associative effect of inoculation of different strains of Azotobacter and homologous rhizobium on the yield of moong (Vigna radiata), soybean (Glycine max) and pea (Pisum sativum)

Summary It has been observed that in the case ofVigna radiata andGlycine max incorporation of suitable strain of Azotobacter gave higher yield than obtained by the use of Rhizobium as inoculant. In the case ofVigna radiata even a strain of Azotobacter isolated from the rhizosphere of berseem gave similar yields as Rhizobium. In the case ofPisum sativum association of Rhizobium with a strain ofAzotobacter chroococcum isolated from the rhizosphere of pea gave numerically higher yield than Rhizobium alone. It may be possible that statistically higher yield may be obtained when a suitable strain of Azotobacter is used after screening a large number of strains of Azotobacter from the rhizosphere of pea.     

Use of Bacteria of the Genus Azotobacter for Bioremediation of Oil-Contaminated Soils

The rate of self-purification of oil-contaminated soil increases after introduction of bacteria of the genus Azotobacter. The bacteria can assimilate oil hydrocarbons as the sole source of carbon and energy, both in the presence of fixed nitrogen and during nitrogen fixation. The species Azotobacter chroococcum activates growth of hydrocarbon-oxidizing bacteria present in Devoroil.     

Dinitrogen fixation (C2H2 reduction) by bacterial strains at various temperatures

Acetylene-reducing activities (ARA) of strains ofEnterobacter agglomerans, Azospirillum brasilense, Azotobacter chroococcum, and Bacillus, isolated from temperate or tropical soils, were compared at different temperatures to study temperature adaptability. All Enterobacter strains and Bacillus strain C-11-25 reduced C₂H₂ at temperatures as low as 5°C. ARA by Enterobacter strains declined sharply above 30°C but ARA by Bacillus strain C-11-25 continued to increase with an increase in temperature.A. brasilense strain sp 245, isolated from wheat roots in Brazil, reduced more C₂H₂ at lower temperatures than strain Cd, isolated from a Californian soil. Similarly, the temperate strain ofA. chroococcum was a better N₂ fixer than the tropicalA. chroococcum strain at lower temperatures. Tropical strains ofA. brasilense andA. chroococcum reduced more C₂H₂ than temperate strains at higher temperatures. Therefore, it appears that temperate and tropical N₂-fixing organisms adapt themselves to their particular environment and should have more potential to benefit crops grown at the particular temperatures favorable to them. Only Bacillus strain C-11-25 has potential to benefit both temperate and tropical crops because it reduced significant acetylene over a wide temperature range.     

Diversity and characterization of <Emphasis Type="Italic">Azotobacter</Emphasis> isolates obtained from rice rhizosphere soils in Taiwan

Azotobacter species, free-living nitrogen-fixing bacteria, have been used as biofertilizers to improve the productivity of non-leguminous crops, including rice, due to their various plant growth-promoting traits. The purposes of this study were to characterize Azotobacter species isolated from rice rhizospheres in Taiwan and to determine the relationship between the species diversity of Azotobacter and soil properties. A total of 98 Azotobacter isolates were isolated from 27 paddy fields, and 16S rRNA gene sequences were used to identify Azotobacter species. The characteristics of these Azotobacter strains were analyzed including carbon source utilization and plant growth-promoting traits such as nitrogen fixation activity, indole acetic acid production, phosphate-solubilizing ability, and siderophore secretion. Of the 98 strains isolated in this study, 12 were selected to evaluate their effects on rice growth. Four species of Azotobacter were identified within these 98 strains, including A. beijerinckii, A. chroococcum, A. tropicalis, and A. vinelandii. Of these four species, A. chroococcum was predominant (51.0%) but A. beijerinckii had the highest level of nucleotide diversity. Strains within individual Azotobacter species showed diverse profiles in carbon source utilization. In addition, the species diversity of Azotobacter was significantly related to soil pH, Mn, and Zn. Members of the same Azotobacter species showed diverse plant growth-promoting traits, suggesting that the 98 strains isolated in this study may not equally effective in promoting rice growth. Of the 12 strains evaluated, A. beijerinckii CHB 461, A. chroococcum CHB 846, and A. chroococcum CHB 869 may be used to develop biofertilizers for rice cultivation because they significantly promoted rice growth. This study contributes to the selection of suitable Azotobacter strains for developing biofertilizer formulations and soil management strategies of Azotobacter for paddy fields.     

Ammonia excretion byAzotobacter chroococcum in liquid culture and soil in the presence of manganese and clay minerals

Summary Ammonia excretion was studied in the presence of Mn⁺⁺ in liquid culture and soil with amendments of the minerals using two ammonia excreting strains ofAzotobacter chroococcum (AC2 and AC50) and a non-excreting strain (AC45) under sterilized as well as unsterilized conditions. Ammonia excretion increased in the presence of 1×10⁻⁷M Mn⁺⁺ in liquid culture as well as in soil. But there was a decrease in ammonia excretion with increasing clay content.     

Influence of analogue-resistant mutants ofAzotobacter chroococcum solubilizing phosphate on yield and quality of sunflower (Helianthus annuus)

Two phosphate-solubilizing analogue-resistant mutants ofAzotobacter chroococcum showed a positive interactive effect on sunflower (Helianthus annuus). Plant height, leaf area, seed mass, seed yield, protein and oil content increased significantly by inoculation withA. chroococcum mutants. Both levels of fertilizers applied (80N+30P₂O₅ and 80N+60P₂O₅ kg/hm² showed increased seed yield. Likewise, inoculation resulted in a significantly higher yield. Application ofP-solubilizingA. chroococcum strains with the lower fertilizer level showed no significant difference on plant growth parameters when compared with the higher fertilizer level, indicating that inoculation of P-solubilizingAzotobacter strains can reduce the necessary levels of fertilization from 60P₂O₅ to 30P₂O₅ kg/hm².     

Growth enhancement of Sesamum indicum L. by rhizosphere-competent Azotobacter chroococcum AZO2 and its antagonistic activity against Macrophomina phaseolina

Indigenous strains isolated from rhizosphere may contain highly competent genotypes to enhance the plant growth and often perform better than the introduced isolates. The present study deals with the characterisation of plant growth-promoting (PGP) attributes and antagonistic activity of Azotobacter chroococcum AZO2 against Macrophomina phaseolina causing charcoal rot disease and their effect on the growth of sesame (Sesamum indicum L.). Eight strains of Azotobacter were isolated from sesame rhizosphere on nitrogen-free medium, which exhibited significant PGP parameters such as phosphate solubilisation, indole acetic acid and siderophore production. The strain A. chroococcum AZO2 (EU274299) was characterised by 16S rDNA gene sequencing. Amplification of 781 bp nif H gene confirms nitrogenase activity of all the strains. A. chroococcum AZO2 exhibited strong antagonistic activities against M. phaseolina causing 81% colony growth inhibition and resulted in hyphal perforations, empty cell (halo) formation, hyphal twisting, shrinking and lysis of fungal mycelia along with degeneration of sclerotia. A. chroococcum AZO2 produced chitinase that caused degradation and digestion of the cell wall component of M. phaseolina. Different vegetative and reproductive parameters of sesame were found to be enhanced significantly upon application of A. chroococcum AZO2 + half doses of chemical fertilisers. A. chroococcum AZO2 was also found to be an effective root coloniser, plant growth promoter and potential antagonistic bacterium. It can be concluded that A. chroococcum AZO2 strain bears the characteristics of technological applications for inoculant preparation and growth enhancement of sesame besides being utilised as a better PGP bacterium as well as an effective agent for biocontrol of M. phaseolina.     

Intergeneric transformation betweenRhizobium andAzotobacter

Attempts were made to transform two strains ofAzotobacter, viz.A. chroococcum andA. vinelandii with the DNA ofRhizobium leguminosarum, R. japonicum, R. meliloti andR. trifoli. Resistance to streptomycin or crystal violet was used as marker for screening purposes. The frequency of transformation varied between 0.05 to 0.95 percent. The transformants also acquired a few other characters of the donor strains.     

Specific substrates for isolation and differentiation of Azotobacter vinelandii

Summary Resorcinol, ethylene glycol and glutaric acid have been found to be specific substrates for the enrichment of Azotobacter vinelandii in the presence of other Azotobacter species. The three compounds may also be used for species differentiation in the genus Azotobacter. In contrast to other reports strains of A. chroococcum and A. beijerinckii have been isolated which are able to use L-rhamnose for growth.Conditions for the production of a yellow pigment and green fluorescence by A. vinelandii from non-aromatic substrates on agar-plates have been tested.The production of water soluble yellow pigments in benzoate containing media cannot be used as a criterium for the presence of A. vinelandii. The pigment seems to be -hydroxy-muconic semialdehyde, the splitting product resulting from metacleavage of catechol. It may be formed by all Azotobacter species that can metabolize aromatic substrates.     

Effect of the composition of the growth medium on morphology and reproduction ofAzotobacter chroococcum

Summary The growth of threeAzotobacter chroococcum strains was investigated in a number of media. The cultures were incubated at 29°C. and studied by means of a Wild phase contrast microscope. The experiments have clearly demonstrated that the morphology and reproduction ofA. chroococcum are influenced greatly on the one hand by conditions of nutrition and on the other hand also by the properties of the strains. Although all strains used in this work were able to produce gonidia and regenerative rods, these were not found in all media. The formation of filamentous cells, and vegetative cells inside such cells, depended also on the medium and, in some cases, on the strain. Cysts could sometimes resist a temperature of 50°C. for 5.5 minutes, but not a single strain with cysts survived temperatures of 60°C. or higher. Spores could not be detected by the usual staining methods.     

On the sensitivity of isolates of Rhizobium spp. and Azotobacter chroococcum to TMTD and its degradation product NaDDC

Summary The in-vitro effect of the seed dressing fungicide tetramethyl thiuram disulphide (TMTD, Thiram) and its degradation product sodium dimethyl dithiocarbamate (NaDDC) on the sensitivity of Rhizobium spp. and Azotobacter chroococcum isolates was studied at different pH values. The Azotobacter chroococcum isolates were more sensitive than Rhizobium spp. isolates at all pH values. The sensitivity of these isolates to TMTD and NaDDC had a maximum at pH 7, and it decreased on either side of this value. The Rhizobium isolates from urid (Phaseolus radiatus) were resistant to TMTD at pH 7 but proved sensitive to its degradation product. A difference between the power of the sensitive and resistant isolates to reduce TMTD was revealed.     

Pesticide tolerant Azotobacter isolates from paddy growing areas of northern Karnataka, India

A total of 14 Azotobacter strains were isolated from different paddy cultivating soils with pH ranging from 6.5 to 9.5 by using serial dilution agar plate method. The strains were Gram negative, rod shaped, cyst forming and developed brown to black colored colonies, which were glistening, smooth, slimy on Ashby’s agar plates. Biochemically they were positive for biochemical tests namely, indole production, citrate, catalase, carbohydrate fermentation and Voges–Proskauer test. Further, sequence analysis of PCR amplicons obtained from these cultures revealed the presence of five different Azotobacter species viz., Azotobacter vinelandii, Azotobacter salinestris, Azotobacter sp., Azotobacter nigricans subsp. nigricans and Azotobacter tropicalis. Phylogenetically these strains were grouped into two distinct clusters. These strains were tested for their ability to grow on a media containing four different pesticides such as pendimethalin, glyphosate, chloropyrifos and phorate, which are commonly used for the paddy. Out of 14 strains tested, 13 strains were able to grow on a media containing herbicides such as pendimethalin, glyphosate and insecticides like chloropyrifos and phorate. However, five Azotobacter strains were able to grow at higher concentration of 5 % pesticides, without affecting their growth rate. Further, the effect of pesticides on the indole acetic acid (IAA) production by Azotobacter strains was also estimated. Azotobacter-16 strain was found to produce 34.4 μg ml^?l of IAA in a media supplemented with 1,000 mg of tryptophan and 5 % of pendimethalin. Present study reveals that species of Azotobacter are able to grow and survive in the presence of pesticides and no significant effects were observed on the metabolic activities of Azotobacter species.     

Bdellovibrio possesses a prey-derived OmpF protein in its outer membrane

Bdellovibrio bacteriovorus 109D andBdellovibrio stolpii derive one of their major outer membrane proteins from the outer membrane of their prey. This prey-derived protein corresponds to the OmpF protein ofEscherichia coli. Bdellovibrios cultivated onSalmonella typhimurium prey acquire theSalmonella OmpF protein; this protein is distinguishable electrophoretically from the OmpF protein ofE. coli. Bdellovibrios containing the prey-derived OmpF protein are sensitive to killing by colicin A but not colicin E1, whereas bdellovibrios without this protein are completely resistant to colicin killing.     

A new phenol oxidase produced during melanogenesis and encystment stage in the nitrogen-fixing soil bacterium Azotobacter chroococcum

Laccases are copper-containing phenol oxidases that are commonly found in many types of plant, insect, fungi and bacteria. Whilst phenol oxidases have been well characterized in fungal species, laccase-type enzymes originating from bacteria have been much less well defined. Bacteria belonging to the family Azotobacteraceae share many morphological characteristics with strains already known to exhibit polyphenol and phenol oxidase activity; and hence the aim of this work was to identify and characterize a novel laccase from the isolated strain Azotobacter chroococcum SBUG 1484 in an attempt to provide further understanding of the roles such enzymes play in physiological development. Laccase activity was clearly observed through oxidation of 2,6-dimethoxyphenol, other typical substrates including: methoxy-monophenols, ortho- and para-diphenols, 4-hydroxyindole, and the non-phenolic compound para-phenylenediamine. A. chroococcum SBUG 1484 showed production of a cell-associated phenol oxidase when grown under nitrogen-fixing conditions, and was also observed when cells enter the melanogenic and encystment stages of growth. Catechol which is structurally related to melanin compounds was also released from Azotobacter cells into the surrounding culture medium during nitrogen-fixing growth. From our results we propose that a membrane-bound laccase plays an important role in the formation of melanin, which was monitored to correlate with progression of A. chroococcum SBUG 1484 cells into the encystment stage of growth.     

Bdellovibrio bacteriovorus gen. et sp. n., a predatory,ectoparasitic, and bacteriolytic microorganism

Bdellovibrio bacteriovorus, gen. et sp. n., a predatory and ectoparasitic microorganism with lytic activity against susceptible bacteria, is described, as are techniques for isolation and cultivation. These unusual bacteria cause reactions that are similar in their outward manifestations to bacteriophage-induced lysis. Upon plating a mixture of host bacteria and parasites, confluent lysis or single plaque formation occurs, just as in titration experiments with bacteriophage. However, the parasite plaques develop more slowly than phage plaques. Lysis of host bacteria in liquid culture is accompanied by a decrease in optical density; actually, a population of infected host bacteria is replaced by a population of the tiny parasite.Individual cells of the presently known strains ofBdellovibrio bacteriovorus are typically about 0.3 µ in width and, thus, are considerably narrower than ordinary bacteria. Therefore, they can pass Millipore filters of 0.45 µ pore size diameter. Their shape is often vibrio-like. They possess one unusually thick polar flagellum of about 50 mµ diameter, and they show a distinctive type of motility.The interaction betweenBdellovibrio and the attacked host bacterium can be followed in the phase-contrast microscope; it is characterized by a physical attack of the highly motile parasite, attachment to the bacterial cell surface, and lysis of the host cell.It has not yet been possible to cultivateBdellovibrio in its parasitic form on any artificial substrate. All parasitic strains require living host cells for their propagation. However, saprophytic mutants can be selected from a population of the parasite. These saprophytic derivatives are unable to lyse living bacteria as does the wild-type parasite. On the basis of morphological and physiological properties, a saprophyte strain which has been examined in some detail shows no close relationship to any of the already known categories of bacteria.A study of the kinetics of growth ofBdellovibrio in mixed culture with a susceptible host has disclosed that the number of parasites produced is not proportional to the number of host bacteria killed during the same period. After the majority of the host cells has been destroyed, there is still a considerable increase in parasites, indicating that they grow at the expense of material released from the lysed bacteria. Under the conditions of this trial, the generation time is about 100 minutes.All presently known isolates ofBdellovibrio possess lytic activity only against gram-negative bacteria. The individual strains, however, show certain differences in their host activity spectra; some have a restricted host range, while others are able to attack a broad spectrum of host bacteria.     

基于基因组的一株土壤固氮菌分离菌株鉴定及其促生作用

[目的] 为获得高效固氮菌株,充分研究利用土壤固氮菌资源。[方法] 选取固氮能力较高的紫色土发育水稻土,采用富集纯化法分离固氮微生物菌株。通过16S rRNA基因系统发育分析和全基因组相关指数比较对新分离菌株进行物种鉴定。采用乙炔还原法和¹⁵N₂示踪法定量测定新分离菌株的固氮能力,通过培养特性和接种效果初步研究固氮菌株的促生作用。[结果] 从紫色土发育水稻土中分离得到1株可在无氮培养基上快速生长的菌株P208。基于16S rRNA基因和基因组92个核心基因的系统发育分析结果表明,新分离菌株P208与Azotobacter chroococcum IAM 12666^T（=ATCC 9043^T）系统发育距离最近（16S rRNA基因相似度为99.79%）。菌株P208与A.chroococcum ATCC 9043^T的基因组平均核苷酸一致性（ANI）、平均氨基酸一致性（AAI）和数字DNA-DNA杂交值（dDDH）高于物种分类阈值（ANI>95%-96%,AAI>95%-96%,dDDH>70%）,最大唯一匹配指数（MUMi）低于物种分类阈值（<0.33）,得出新分离菌株P208为褐球固氮菌（A.chroococcum）。A.chroococcum P208固氮活性为模式菌株A.chroococcum ATCC 9043^T的2.61倍。除固氮能力外,A.chroococcum P208具有IAA生成、溶磷活性和铁载体生成等促进植物生长潜力的培养特性,室内培养条件下接种A.chroococcum P208能够促进水稻、小麦幼苗根系的生长。[结论] 从固氮能力较强的水稻土中分离纯化得到1株具有较强固氮、促生潜力的固氮菌,具有潜在的开发应用价值,可为研究利用生物固氮提供微生物资源。