Spirillum Lipoferum Research Articles

Determination of Increase in Yield of Tropical Grasses Inoculated with Spirillum lipoferum

Increase in yield of tropical grasses viz. digit grass (Digitaria decumbens), guinea grass (Panicum maximum) and pearl millet (Penisetum americanum) inoculated with tropical nitrogen-fixing bacterium, Spirillum lipoferum was investigated. The study was carried out for three consecutive years (2016-2018). Dry matter yields and protein content of the three tropical grasses were used for the assessment. In 2017, pearl millet (Penisetum americanum) and guinea grass (Panicum maximum) produced significantly higher protein content and dry matter yields. Projected yields using regression analysis of both pearl millet and guinea grass indicated that about 40kgN ha-1 yr-1 were replaced by inoculation. Although, protein production of guinea grass was lower during 2018, dry matter yield responses were similar to those of 2017. This research has shown that inoculation with Spirillum lipoferum, a tropical nitrogen-fixing bacterium reduced acetylene and increased yields or reduced nitrogen fertilizer requirement of the tropical grasses as well as replacement of up to 40KgN ha-1. This amount is agro-economically important and suggests the viability and potential for grass-bacteria systems.

THE EFFECT OF SALINITY TO ACTIVITY AND EFFECTIVITY PHOSPHATE SOLUBILIZING BACTERIA ON GROWTH AND PRODUCTION OF PADDY

<p>This study aimed to determine the extent of phosphate solubilizing bacteria resistant to salinity and still be able to provide P for paddy plant. Research using completely randomized design with fertilizer treatments: <br />(A) Bakteri Aerobacter aerogenes + Azotobacter indicus (B) Bakteri Bacillus thuringiensis + B. megaterium <br />+ Pseudomonas fluorescens, (C) Bakteri Nocardia mesentrica + Spirillum lipoferum, (D) Mix bakteri Pseudomonas fluorescens, Bacillus thuringiensis, B. megaterium, Nocardia mesentrica, Aerobacter aerogenes, Spirillum lipoferum, dan Azotobacter indicus, and (E) control (whitout inoculant), and salinity (NaCl): (1) 0% (0 grams / 7 kg soil), (2) 0.1% (7 grams / 7 kg soil), (3) 0.2% (14 g / 7 kg soil), (4) 0.3% (21 g / 7 kg soil), and (5) 0 , 4% (28 g / 7 kg soil). Thirty and one hundred days after transplanting (DAT), and then measured plant height, number of tillers, number and dry weight of whole grain paddy. The results showed that 0,1 % (7 gram/7 kg tanah) salinity is very good for the growth, activity and effectiveness of phosphate solubilizing bacteria and production of paddy, but 0.4% salinity (28 gr/7kg land) is still safe on the growth, activity and effectiveness of phosphate solubilizing bacteria (Pseudomonas fluorescens, Bacillus thuringiensis, <br />B. megaterium, Nocardia mesentrica, Aerobacter aerogenes, spirillum lipoferum and Azotobacter indicus) as biofertilizer or growth promoting rhizobacteria on growth and production of paddy.</p><p><br /><strong>Keywords</strong>: Salinity, Phosphate solubilizing bacteria, Paddy</p>

Nitrogen-fixing Bacteria in various Tissue Cells of Cyperus rotundus

Summary The bacterium, identified as Azotobacter chroococcum, was present within the cortical cells of the various organs of Cyperus rotundus, suggesting the possibility of symbiotic associations in grasses similar to legumes. Azotobacter was shown by Iswaran and Sen (1959) to occur in the rhizosphere of Cyperus rotundus. Dobereiner and Day (1976) have shown that Spirillum lipoferum occurs within the root cells of Digitaria decumbens. Bhide and Puran Dare (1979) reported the occurrence of Azotobacter within the root cells of the grass Cynodon dactylon. Since Cyperus rotundus is another widely distributed grass, it was thought worthwhile to find out whether a similar association occurs in this plant as well, and the method by Bhide and Puran Dare (1979) was followed.

Effect of dimethylsulfoxide on derepression of nitrogenase in Spirillum lipoferum

FEBS LettersVolume 126, Issue 1 p. 77-80 Full-length articleFree Access Effect of dimethylsulfoxide on derepression of nitrogenase in Spirillum lipoferum Sikha Rauth, Sikha Rauth Department of Biochemistry, Bose Institute, Calcutta-700009, IndiaSearch for more papers by this authorSudhamoy Ghosh, Sudhamoy Ghosh Department of Biochemistry, Bose Institute, Calcutta-700009, IndiaSearch for more papers by this author Sikha Rauth, Sikha Rauth Department of Biochemistry, Bose Institute, Calcutta-700009, IndiaSearch for more papers by this authorSudhamoy Ghosh, Sudhamoy Ghosh Department of Biochemistry, Bose Institute, Calcutta-700009, IndiaSearch for more papers by this author First published: April 06, 1981 https://doi.org/10.1016/0014-5793(81)81037-XCitations: 4AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat References 1 J.W.F. Von Bülow, J. Döbereiner, Proc. Natl. Acad. Sci. USA, 72, (1975), 2389– 2393. 2 R.A. Parejko, P.N. Wilson, Can. J. Microbiol., 16, (1970), 681– 685. 3 K.T. Shanmugam, F. O'Gara, K. Anderson, R.C. Valentine, Annu. Rev. Plant. Physiol., 29, (1978), 263– 276. 4 S.L. Streicher, K.T. Shanmugam, F. Ausubel, C. Morandi, R.B. Goldberg, J. Bacteriol., 120, (1974), 815– 821. 5 M.J. Leonardo, R.B. Goldberg, J. Bacteriol., 142, (1980), 99– 110. 6 J.K. Gordon, W.J. Brill, Biochem. Biophys. Res. Commun., 59, (1974), 967– 971. 7 Y. Okon, S.L. Albrecht, R.H. Burris, J. Bacteriol., 128, (1976), 592– 597. 8 J.E. Brenchley, J. Bacteriol., 114, (1973), 666– 673. 9 F.M. Ausubel, S.C. Bird, K.J. Durbin, K.A. Janssen, R.F. Margolskee, A.P. Peskin, J. Bacteriol., 140, (1979), 597– 606. 10 S. Nakanishi, S. Adhya, M. Gottesman, I. Pastan, Cell, 3, (1974), 39– 46. 11 Y. Okon, S.L. Albrecht, R.H. Burris, J. Bacteriol., 127, (1976), 1248– 1254. 12 Y. Okon, S.L. Albrecht, R.H. Burris, Appl. Environ. Microbiol., 33, (1977), 85– 88. 13 R.A. Bender, K.A. Janssen, A.D. Resnick, M. Blumberg, F. Foor, B. Magasanik, J. Bacteriol., 129, (1976), 1001– 1009. 14 B.M. Shapiro, E.R. Stadtman, Methods Enzymol., 17A, (1970), 910– 922. 15 A.H. Neilson, S. Norlund, J. Gen. Microbiol., 91, (1975), 53– 62. 16 R.S. Tubb, Nature, 251, (1974), 481– 485. 17 P.W. Ludden, Y. Okon, R.H. Burris, Biochem. J., 173, (1978), 1001– 1003. 18 D. Herbert, P.J. Phipps, R.E. Strange, Methods Microbiol., 5B, (1971), 249– 252. Citing Literature Volume126, Issue1April 06, 1981Pages 77-80 ReferencesRelatedInformation

Isolation of Azospirullum from diverse geographic regions.

We have isolated Azospirillum (Spirullum lipoferum) from roots of grasses of several genera collected from a number of tropical and subtropical-temperate locations. Pure cultures were obtained from a small percentage of samples; no higher percentage was secured from tropical than from other grasses. Acetylene reduction and distinctive growth in N-free soft agar deeps were inadequate to identify this genus, although helpful in initial screening. Fluorescent antibody tests with antiserum against characterized strains were helpful. There is some evidence that this genus of bacteria may be favored in the rhizoplane.

Choice of liquid, semisolid, or soil suspension media: An important factor modifying the effect of pesticides on the nitrogenase (C 2H 2) activity of Clostridium pasteurianum, Azotobacter chroococcum, and Spirillum lipoferum beijerinck

Experiments with 10, 20, and 100 μg ml −1 of active ingredients in commercial formulations of dinoseb-acetate, ioxynil, paraquat, atrazine, and dodine in liquid, soil suspension, and semisolid agar cultures of Clostridium pasteurianum, Azotobacter chroococcum, and Spirillum lipoferum revealed various types of interactions as indicated by the nitrogenase (C 2H 2) activity. Strong inhibitions of paraquat and dodine could be reversed by their adsorption to soil. Other observations indicated utilization of test compounds, subtoxic stimulation, and delayed toxicity. The organisms differed also in susceptibility against different compounds. It is stressed that tests for side effects against functional groups of microorganisms should be sufficiently diversified to cover all possible harmful interactions.

Nitrogen‐fixing Bacteria Associated with Graminaceous Roots with Special Reference to Spirillum lipoferum Beijerinck

AbstractMethods and results for field estimation of nitrogen fixation in the rhizosphere of grasses and cereals and the problem of choosing a reliable method for estimates based on acetylene reduction tests are reviewed. Growth and nitrogen fixation of Azotobacter spp. and of Spirillum lipoferum in plant rhizospheres are discussed on the basis of their physiology. Special consideration is given to the oxygen sensitivity of the nitrogenase reaction and adaptation to microaerophilic conditions, plant root exudates, plant specificity and genotype, mineral nitrogen and to microbial synergism, antagonism and competition. A better understanding of factors essential for a successful saprophytic competition of S. lipoferum in the root environment could allow utilization of its nitrogen fixing potential in crop production. Examples of growth promoting and ineffective associations of this organism with various graminaceous host plants are given.

Fluorescent antibody technique to identify Azospirillum brasilense associated with roots of grasses

Bacteria associated with roots of grasses from Florida, Ecuador and Venezuela were isolated and their N 2-fixing ability was demonstrated by C 2H 2 reduction assay. The bacterial isolates have been classified as Azospirillum brasilense (formerly Spirillum lipoferum). These N 2-fixing isolates have been compared with several Brazilian strains. Fluorescent antibody (FA) techniques were used to assist identifying isolates of N 2-fixing bacteria from grass roots. Tests with antisera prepared against four strains of Azospirillum were used to define serological groups. Antigen-antibody specificity was demonstrated using both Azotobacter and Azospirillum antisera against known species of other soil microorganisms and numerous unidentified soil bacteria. Several applications of the FA technique are suggested to identify N 2-fixing bacteria associated with grass roots.

Response of Pearl Millet Inbreds and Hybrids to Inoculation with Azospirillum brasilense1

Plant yield and acetylene reduction were measured on six hybrids and 15 inbreds (including the hybrid parents) of pearl millet, Pennisetum americanum (L.) K. Shum., after field inoculation with Azospirillum brasilense, Sp 13t (reclassified from Spirillum lipoferum Beijerinck). During the first year of testing, inoculation responses were investigated among all plant genotypes by measuring plant dry weight, % N, total N, and acetylene reduction activity. A smaller population of genotypes were tested a second year for repeatability of inoculum response. The first year, one hybrid, Tift 23DA ✕ Tift 186 (‘Gahi 3’), gave significantly higher plant dry weight and total N in response to inoculation. Inoculated Gahi 3 produced 31.7% more dry weight and 37.4% more total plant N when compared to autoclaved inoculum controls. No inbred was found to respond. Acetylene reduction values were low among all genotypes (range 0.54 nmole/[g dry root ✕ hour] and did not support yield effects or confirm inoculum treatments. Inoculation responses were found to be repeatable for all genotypes tested during the second year (positive or negative) but not of a statistically significant magnitude. Of the genotypes tested both years, a combined analysis of the 2‐year yields revealed significant dry weight increases after inoculation of 19.2 and 14.0% with Gahi 3 and the inbred, Bil 3B, respectively. Again, acetylene reduction values did not explain any data. A N balance study was conducted in greenhouse containers on inoculated Gahi 3 plants in an attempt to repeat the yield differences observed in the field and to monitor inputs of N into the soil‐plant system. No significant yield increase of N into the soil‐plant system was found due to inoculation. Sampling error possibly negated an accurate measurement of all N.

The nitrogenase system of Spirillum lipoferum.

The nitrogenase system of Spirillum lipoferum was separated into three components, the normal Mo-Fe and Fe proteins as well as an activating factor for the Fe protein. The rate of activation is increased by Mn2+ or by an excess of Mg2+, and the process requires ATP.

A taxonomic study of the Spirillum lipoferum group, with descriptions of a new genus, Azospirillum gen. nov. and two species, Azospirillum lipoferum (Beijerinck) comb. nov. and Azospirillum brasilense sp. nov.

Sixty-one strains of the root-associated nitrogen fixer Spirillum lipoferum exhibited a similar morphology in peptone--succinate salts medium: vibrioid cells having a diameter of 1.0 micrometer. When grown in broth the cells had a single polar flagellum, but when grown on agar at 30 degrees C lateral flagella of shorter wavelength were also formed. The DNA base composition was 69--71 mol% guanine + cytosine when determined by thermal denaturation. DNA homology experiments indicated the occurrence of two distinct but related homology groups: 46 strains were in group I and 15 strains were in group II. Group II strains were distinguished by their ability to use glucose as a sole carbon source for growth in nitrogen-free medium, by their production of an acidic reaction in a peptone-based glucose medium, by their requirement for biotin, and by their formation of wider, longer, S-shaped or helical cells in semisolid nitrogen-free malate medium. The results indicate that two species exist, and on the basis of their characteristics it is proposed that they be assigned to a new genus, Azospirillum. Strians belonging to group II are named A. lipoferum (Beijerinck) comb. nov., while those belonging to group I are named A. brasilense sp. nov. Strain Sp 59b (ATCC29707) is proposed as the neotype strain for A. lipoferum, and strain Sp 7 (ATCC 29145) is proposed as the type strain for A. brasilense.

Nitrate and nitrite reductase negative mutants of N2-fixing Azospirillum spp.

Chlorate resistant spontaneous mutants of Azospirillum spp. (syn. Spirillum lipoferum) were selected in oxygen limited, deep agar tubes with chlorate. Among 20 mutants from A. brasilense and 13 from A. lipoferum all retained their functional nitrogenase and 11 from each species were nitrate reductase negative (nr-). Most of the mutants were also nitrite reductase negative (nir-), only 3 remaining nir+. Two mutants from nr+ nir+ parent strains lost only nir and became like the nr+ nir- parent strain of A. brasilense. No parent strain or nr+ mutant showed any nitrogenase activity with 10 mM NO3-. In all nr- mutants, nitrogenase was unaffected by 10 mM NO3-. Nitrite inhibited nitrogenase activity of all parent strains and mutants including those which were nir-. It seems therefore, that inhibition of nitrogenase by nitrate is dependent on nitrate reduction. Under aerobic conditions, where nitrogenase activity is inhibited by oxygen, nitrate could be used as sole nitrogen source for growth of the parent strains and one mutant (nr- nir-) and nitritite of the parent strains and 10 mutants (all types). This indicates the loss of both assimilatory and dissimilatory nitrate reduction but only dissimilatory nitrite reduction in the mutants selected with chlorate.

Light microscopy observations of tetrazolium-reducing bacteria in the endorhizosphere of maize and other grasses in Brazil.

Roots of field-grown tropical maize, Panicum maximum Jacq. and Digitaria decumbens Stent., and of sorghum and wheat grown in monoxenic culture with the diazotroph Spirillum lipoferum (syn. Azospirillum spp.) were examined for tetrazolium-reducing bacteria following incubation of roots in a malate-phosphate buffer-2,3,5-triphenyltetrazolium chloride medium. Bacteria were observed between and in cells of the cortex, in intercellular spaces between the cortex and endodermis, in xylem cells, and in and between pith cells. In maize, colonization of the inner cortex and stele appears to occur in the absence of significant bacterial colonization or collapse of outerlying tissues. Bacteria in the stele remained viable after a 6-h treatment of roots with chloramine-t, indicating that the endodermis was intact. Infection of the inner cortex and stele appears to occur initially in branches, and then to spread longitudinally into main roots. Inter- and intra-cellular infections of the cortex were observed in monoxenic systems. Tetrazolium reduction and prominent crystal formation was not specific for diazotrophic bacteria, but S. lipoferum was isolated from surface-sterilized roots, and S. lipoferum-like organisms were observed in the endorhizosphere. A correlation of inner cortex and stele infections with the presence of branches appears to explain previous observations that excised roots of grasses exhibiting high nitrogenase activity are characteristically branched roots with an intact cortex.

Ferredoxins from Nitrogen‐Fixing Bacteria

Two ferredoxins have been purified from N2‐fixing Mycobacterium flavum. Ferredoxin I had a minimum molecular weight (by amino acid analysis) of 11230 and contained 7.4 mol Fe and 7.0 mol S2− per mol. The ratio of the absorbance at 400 nm to that at 275 nm was 0.7. Ferredoxin II had a minimum molecular weight of 13478 and contained 3.4 mol Fe and 3.2 mol S2− per mol. The ratio of the absorbance at 400 nm to 268 nm was 0.42.Both ferredoxins were paramagnetic when oxidised by potassium ferricyanide and diamagnetic when reduced by sodium dithionite. This suggests that, like Azotobacter ferredoxin, they operate solely between the – 2 and – 1 oxidation states. A ferredoxin fraction of similar properties was isolated from Spirillum lipoferum. The electron paramagnetic resonance (EPR) spectra of both ferredoxins had features at g= 2.023, 2.017 and 2.00. The signals were affected by pH: below pH 6.0 that at g= 2.023 predominated, whereas above pH 6.0 the g= 2.017 features was clearly present. The g= 2.00 features was broader between pH 6.0 and 9.0 than outside this pH range. The change to the low‐pH form was not reversible.Ferredoxin I had redox centres with mid‐point potentials at – 420 mV and + 230 mV; ferredoxin II had one redox centre of mid‐point potential at approximately – 420 mV. Above + 300 mV ferredoxin I suffered irreversible oxidative damage and lost the g= 2.023 feature of the EPR spectrum. Both ferredoxins were damaged by prolonged exposure to potassium ferricyanide. Ferredoxin I took up 1.8 electrons per molecule when reduced by sodium dithionite but the EPR signal of the oxidised form integrated to only 0.35 electron per molecule. Ferredoxin II took up 0.9 electron per molecule on reduction and the EPR signal of the oxidised form integrated to 0.66 electron per molecule.It was concluded that M. flavum ferredoxin I resembles Azotobacter ferredoxin and is thus the second ferredoxin isolated that has two redox centres with mid‐point potentials approximately 700 mV apart. Ferredoxin II has no exact counterpart in other systems. Aerobic nitrogen‐fixing bacteria apparently contain ferredoxins which operate between the – 2 and – 1 oxidation states.

Inducing effect of plant cells on nitrogenase activity by Spirillum and Rhizobium in vitro.

Eleven different plant cell tissue cultures of both legume and non-legume origin have been grown in direct association, and in separate but close proximal association with both Spirillum lipoferum and Rhizobium sp. 32H1. Basic similarities were found in the nutritional requirement for the induction of nitrogenase activity (C2H2) in both organisms. In the absence of plant cell cultures both organisms need to be provided with a pentose sugar and a tricarboxylic acid to induce high levels of nitrogen-fixing activity. Plant cell callus tissue appears only capable of supplying the tricarboxylic acid to induce high levels of nitrogen-fixing activity. Plant cell callus tissue appears only capable of supplying the tricarboxylic acids needed but not the sugar component. The plant tissue, however, seems able to activate certain carbohydrates, which in themselves are incapable of substituting for the pentose additive.

INFLUENCE OF NITROGEN ON GROWTH, FREE AMINO ACIDS AND NITROGENASE ACTIVITY IN SPIRILLUM LIPOFERUM

Growth and nitrogenase activity were studied in cultures of Spirillum lipoferum growing with dinitrogen and combined nitrogen. Not much difference is observed in the growth rates in culture growing with and without dinitrogen except that the lag phase is shortened in culture growing with combined nitrogen. The nitrogenase activity was repressed by ammonium chloride but, after exhaustion of ammonia, the activity was derepressed. With respect to PO2, maximum nitrogenase activity in this bacterium is observed at PO2=0.004atm, indicating that nitrogen is fixed microaerophilically by Spirillum. Free amino acid pools extracted from growing cultures of Spirillum lipoferum is also reported here, suggesting that the free amino acid pool contains a variety of amino acids with glutamic acid and amide glutamine being present in much higher concentrations than others.

Nitrogen fixation, denitrification, and pleomorphic growth in a highly pigmented Spirillum lipoferum.

A strain of Spirillum lipoferum with intense red pigmentation was isolated from the roots of Cynodon dactylon "Coastal." This isolate vigorously reduced acetylene when grown in N-free, Na-malate, semisolid agar, and it was identical to S. lipoferum strain SP7 by standard taxonomic tests. Both S. lipoferum SP7 and the C. dactylon root isolate displayed the unique features of being denitrifiers as well as N2 fixers. The N2-dependent growth curve was biphasic: cells in younger cultures showed the characteristic spiral shape and motility, but those in older cultures developed larger, nonmotile, cystlike forms. Nitrogenase activity seemed associated only with younger spiral forms. The red pigment may be a b- or c-type cytochrome. The strong red color, which this strain develops, could be used as a marker in evaluating soil inoculation experiments.

Effects of Light and Temperature on the Association between Zea mays and Spirillum lipoferum

Zea mays was grown on a low N nutrient solution under 16 conditions of light and temperature in a crossed-gradient room in an attempt to determine whether or not variation in climatic conditions influences N(2) fixation by the association between maize and Spirillum lipoferum. Temperatures were 28, 32, 36, and 40 C and 10 C lower at night; light intensities were 500, 1,250, 2,400, and 3,000 ft-c. Plants harvested after 94 days showed no significant benefit from association with S. lipoferum either in dry weight production or in total N content; variations in temperature and light had only a small influence on N(2) fixation under the conditions tested. Measurements of total N, together with designated assumptions, indicated that less than the equivalent of 0.5 kilogram of N was fixed/hectare during the entire growing period by the maize-S. lipoferum association. Rates of C(2)H(2) reduction by replicate root samples generally were low and variable and did not correlate with the measurements of total N.

Relationship between glutamine synthetase and nitrogenase in Spirillum lipoferum

Relationship between glutamine synthetase and nitrogenase in Spirillum lipoferum

Nitrate reduction nitrogenase activity in Spirillum lipoferum1.

Nitrate and nitrite reduction under aerobic, microaerophillic, and anaerobic conditions was demonstrated in Spirillum lipoferum (ATCC 29145). Nitrite did not accumulated during assimilatory nitrate reduction in air. The nitrite produced during dissimilatory nitrate reduction accumulated in the medium but not in the cells. On exposure of the bacteria to nitrate and anaerobiosis, a low initial rate (lag) was followed by accelerated rates of nitrite accumulation. A 3-h anaerobic pretreatment, in the absence of nitrate, did not a void the lag phase. No nitrate reductase activity (NRA) developed in the presence of chloramphenicol. The data suggest that induction of anaerobic NRA in S. lipoferum required nitrate and protein synthesis. Anaerobic N2-ase by S. lipoferum was greatly stimulated in the presence of nitrate. The time course of nitrate reduction was coincidental with the pattern of nitrate-stimulated N2-ase activity inidcating that a relationship exists between these two processes.