ofAzotobacter Chroococcum Research Articles

Description of Azotobacter chroococcum subsp. isscasi subsp. nov. isolated from paddy soil and establishment of Azotobacter chroococcum subsp. chroococcum subsp. nov.

Three aerobic, asymbiotic, N2-fixing bacterial strains, designated P205T, P204 and P207, were isolated from a paddy soil in Yanting County, China. Based on 16S rRNA gene sequences, the three strains were closely related to Azotobacter chroococcum IAM 12666T (=ATCC 9043T) (99.00-99.79 % similarities). Strain P205T formed an individual branch distinct from the other two newly isolated strains and other related type strains in phylogenetic analyses based on 16S rRNA gene and 92 core genes. The average nucleotide identity (ANI), average amino acid identity (AAI) and digital DNA-DNA hybridization (dDDH) values based on genome sequences of strain P205T and A. chroococcum ATCC 9043T, P204, P207 were near or slightly higher than the thresholds for species circumscription (95-96, 95-96 and 70 %, respectively), and the dDDH values were significantly lower than the threshold for delineating subspecies (79-80 %), which strongly supported that strain P205T belonged to A. chroococcum but was a novel subspecies distinct from the type strain of A. chroococcum. This finding was further corroborated by distinct phenotypic characteristics such as growth in Luria-Bertani (LB) medium, carbon source utilization and chemical sensitivity to vancomycin. Therefore, strain P205T represents a novel subspecies of Azotobacter chroococcum, for which the name Azotobacter chroococcum subsp. isscasi subsp. nov. is proposed with the type strain P205T (=KCTC 72233T=CGMCC 1.16846T=CCTCC AB 2019080T). The subspecies Azotobacter chroococcum subsp. chroococcum subsp. nov. is created automatically with the type strain ATCC 9043T (=DSM 2286T=JCM 20725T=JCM 21503T=LMG 8756T=NBRC 102613T=NCAIM B.01391T=NRRL B-14346T=VKM B-1616T).

The effect of inoculation with Azotobacter chroococcum on microorganisms in rhizosphere and sugar beet yield in organic farming

The effect on sugar beet yield parameters and microbiological soil status was studied using two techniques of sugar beet inoculation with strains of Azotobacter chroococcum. Cultivar ?Drena? was used in the study, and field trial was set under the conditions of organic farming system in Backi Petrovac. A mixture of three strains of Azotobacter chroococcum was used as microbial fertilizer. Inoculation was performed by: (A) incorporation of strains into soil before sowing; and (B) repeated incorporation of strains into soil two weeks after sowing. PGP characterization of the strains confirmed the ability of producing indole-3-acetic acid (IAA) from 12.63 ?g ml-1 to 14.95 ?g ml-1, nitrogen fixation, and P-solubilization. Positive effects on the number of azotobacter and free nitrogen fixers in rhizosphere were obtained by inoculation, as well as positive effects on the tested sugar beet yield parameters. The largest increase in root yield, yield of crystal sugar, and yield of polarised sugar compared with the control was obtained by repeated soil inoculation, ranging from 22 to 23%.

English

This study was designed to evaluate the effects of three pesticides (Imazetapir, Dimethoate and Bayleton 50) at the recommended concentration (in the field), on the growth of pure cultures ofAzotobacter chroococcum and Azotobacter vinelandii, on the amount of fixed nitrogen and nifgenes. Herbicide Imazetapir had no negative effect on nitrogen fixing bacteria, while Dimethoate and Bayleton 50 exhibited inhibitory effect on growth. Same effects were obtained on fixed nitrogen obtained when treated with studied pesticides. nifH1, nifH2, nifH3, nifU and nifV from A. chroococcum, and nifH, nifK, nifD, and nifM gene in A. Vinelandii were lost when pots were cultivated with wheat and treated with both Dimethoate and Bayleton 50, therefore, be deemed highly susceptible to them. While herbicide did not affect the nif genes, the bands on gel electrophoresis appeared as normal sample.   Key words: Azotobacter chroococcum, Azotobacter vinelandii, fungicide (Bayleton 50), herbicide (Imazetapir), insecticide (Dimethoate).

Granular preparations ofAzotobacter containing clay minerals

Interaction ofAzotobacter chroococcum 20 with clay minerals increased their cell viability at supraoptimal temperatures. Therefore, clay minerals were used to develop granular bacterial preparations with high viable cell counts and stable compositions during long-term storage. The titers of viable bacteria in the preparations remained 60–70% of the initial level after 12 months of storage.

Relationship ofAzotobacter chrooccum siderophores with nitrogen fixation

In iron-limited medium, a siderophore producing soil isolate ofAzotobacter chroococcum showed a high level of hydroxamate with relatively low level of nitrogen fixation. Inclusion of iron in the medium resulted in increased nitrogen fixation with decreased hydroxamate production. Under shake culture conditions, the level of both hydroxamate and catechol type of siderophores decreased after 2 d of incubation in iron-deficient medium. However, under iron-sufficient conditions, both siderophore production and nitrogen fixation increased with time although the level of siderophore was quite low. A number of soil isolates and mutants ofA. chrococcum were tested for nitrogen fixation, hydroxamate and catechol type of siderophore production. Wide variation was observed in the siderophore level and nitrogen fixation in the cultures tested. Nitrogen fixation was higher in the iron-sufficient medium than in iron-limited one while hydroxamate yield was higher in iron-limited medium than in the iron-sufficient one in all the cultures. Inclusion of ammonium acetate in the medium induced catechol synthesis in more than 60% of the cultures.

The role of cellulose-decomposing fungi in nitrogenase activity ofAzotobacter chroococcum

Azotobacter chroococcum was grown on cultures containing five carbon sources alone and also in co-cultures with three cellulolytic fungi (Aspergillus niger, Penicillium funiculosum andTrichoderma harzianum). In the absence of fungal species, nitrogenase activity was relatively low. The best nitrogenase activity was recorded in cultures containing faba bean straw followed by that in cultures having wheat straw, sugar cane leaves, carboxymethyl cellulose (CMC) or cellulose. In co-cultures with fungi,Azotobacter showed substantial nitrogenase activity on all tested substrates.Azotobacter —Trichoderma association showed the highest nitrogenase activity.

Effect of pesticides on growth, respiration and nitrogenase activity ofAzotobacter andAzospirillum

Pesticides (Brominal, Cuprosan and Fenvalerate) at 10 and 50 ppm suppressed growth, respiration and nitrogenase activity ofAzotobacter chroococcum, Azospirillum brasilense andAzospirillum lipoferum. The inhibitory effect on respiration ofAsm. lipoferum was most pronounced after 3 and 4 days.

Abscisic acid and its synthetic analog in relation to growth and nitrogenase activity ofAzotobacter chroococcum andNostoc muscorum

The plant hormone abscisic acid as well as its synthetic analog LAB 173711 significantly increased the nitrogenase activity of the bacterium Azotobacter chroococcum and the cyanobacterium Nostoc muscorum. The effect depended on the concentration of the substances (0.001-10 mg/L) and the age of the cultures. The biomass of the organisms was not significantly influenced.

Effect of nitrification inhibitors on N-fixing bacteriaRhizobium leguminosarum andAzotobacter chroococcum

The nitrification inhibitor dicyandiamide [DCD] did not inhibit growth and respiration of N-fixing bacteria (Rhizobium leguminosarum andAzotobacter chroococcum) in cell suspensions with concentrations of 400 ppm DCD. Growth ofRhizobium leguminosarum was inhibited by 17% with 100 ppm nitrapyrin (N-Serve), but respiration was not affected. Growth ofAzotobacter chroococcum was inhibited by 10 ppm (10%) and 100 ppm nitrapyrin (50%); in the latter case, respiration was also impaired (36%). Thiourea only caused a minor growth inhibition ofAzotobacter chroococcum with 100 ppm (8%) and had no effect onRhizobium leguminosarum.

Interaction between VA-mycorrhizal fungus and Azotobacter and their combined effects on growth of tall fescue

The effect ofAzotobacter chroococcum Beij. on tall fescue (Festuca arundinaceae Schreb.) isolated from spores ofGlomus mosseae (Nicol. and Gerd.) Gerd. and Trappe was investigated in a greenhouse experiment. Dry masses of leaves, culms, and roots of tall fescue were significantly higher when tall fescue was inoculated with both Azotobacter and Glomus than with Azotobacter alone. Molybdenum had no significant effect on fescue when added with Azotobacter alone. When molybdenum was added to Azotobacter plus Glomus, however, the dry mass of tall fescue increased significantly.

Root exudates ofZea mays and production of auxins, gibberellins and cytokinins byAzotobacter chroococcum

Growth ofAzotobacter chroococcum in N-free medium was stimulated in the presence of maize root exudates. Our results show that the production of auxins, gibberellins and cytokinins were significantly stimulated when maize root exudates from 7 to 30 d old plants were added to the culture media.

Role of ammonia assimilatory enzymes and extracellular ammonia accumulation in excretor and non-excretor strains ofAzotobacter chroococcum

Role of ammonia assimilatory enzymes and extracellular ammonia accumulation in excretor and non-excretor strains ofAzotobacter chroococcum

Nitrogen fixation in strains ofAzotobacter chroococcum bearing deletions of a cluster of genes coding for nitrogenase

Strains of the obligately aerobic nitrogen fixing organismAzotobacter chroococcum were constructed which contained defined chromosomal deletions in which the nitrogenase structural genenifHDK cluster (nifH for the polypeptide of the Fe-protein component of nitrogenase andnifD andnifK for the alpha and beta subunits respectively of the MoFe-protein component of the enzyme) was replaced by a kanamycin resistance gene. N2 fixation was nevertheless observed in deletion strains though only in a molybdenum-deficient medium or in spontaneously arising tungstate-resistant derivatives. In comparison with the parent strain growing in molybdenum-sufficient medium, diazotrophic growth was slow and the nitrogenase activity in vivo was characterised by disproportionately low rates of C2H2-reduction compared to H2-evolution and relative insensitivity of H2-evolution to inhibition by C2H2. The findings show reiteration of functional structural genes for nitrogenase inA. chroococcum consistent with our previous observation of twonifH genes in this organism and detection in this work of a secondnifK-like sequence in the genomes of both parent and deletion strains whenA. chroococcumnifK DNA was used as a probe.

Effect ofAzotobacter chroococcum on the hatching of egg masses and eggs ofMeloidogyne incognita

Investigations were carried out to study the effect ofAzotobacter chroococcum VP-5 on the hatching of egg masses and eggs ofMeloidogyne incognita in vitro. Eggs were more susceptible and required lesser time for antagonistic action of Azotobacter than egg masses.

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

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.

Biological control of Rhizoctonia solani on potatoes by antagonists. 4. Inoculation of seed tubers with Verticillium biguttatum and other antagonists in field experiments

Inoculation of seed potatoes with the mycoparasiteVerticillium biguttatum, isolate M73 (combined withGliocladium roseum in 1981, either alone or mixed with isolate M180 plus antibiotics-producing isolates ofAzotobacter chroococcum in 1982) repeatedly proved successful in reducingRhizoctonia solani on stolons and stems. In field experiments, this ultimately led to a reduced formation of sclerotia on new tubers, particularly in neutral sandy loam and clay loam soils. In 1981 inoculation with antagonists led, when compared with no inoculation, to average reductions of 22 and 42% for the harvest from clean, and 15 and 26% for the harvest from infected seed tubers grown on slightly acid sandy soils and on neutral loam soils, respectively. The harvest from clean, inoculated seed tubers had the lowest sclerotium index. In 1982 inoculation of seed tubers planted in slightly acid sandy soils gave reductions of the sclerotium index of up to 22%. In the neutral marine loam soils considerable reductions were often achieved, viz., in slightly infected loams 51–68% and in rather heavily infected ones 4–43%. Chemical disinfection of seed tubers proved effective only in loam soils that were slightly infested withR. solani. In both years inoculation of seed tubers with antagonists led to significantly lower sclerotium indices of the harvest (p=0.1% in 1981; p=5% in 1982).

Enumeration and characterization of nitrogen-fixing bacteria in a stripmine site

Nitrogen-fixing heterotrophic bacteria were isolated from a stripmine reclamation site in western North Dakota. Of 232 isolates, 164 wereBacillus spp., 38 were members of the Enterobacteriaceae, and 21 wereClostridium spp. Single isolates ofAzotobacter chroococcum andAzospirillum lipoferum were identified, as were several isolates considered to beXanthobacter autotrophicum. Fewer nitrogen-fixing bacteria adhered to the root system of crested wheatgrass (Agropyrum desertorum) in sites with replaced topsoil than in sites with no topsoil. Several media and techniques were compared in enumeration of nitrogen fixers by most probable numbers.

Suppressive effect of Azotobacter chroococcum on Rhizoctonia solani infestation of potatoes

Isolates ofAzotobacter chroococcum were found to be promising for the control of infestation of potato plants withRhizoctonia solani. Inoculation with an isolate ofVerticillium biguttatum in combination with isolates ofA. chroococcum effectively protected sprouts, stems and stolons against infestation withR. solani. The effect of inoculation varied with soil temperature.

Effect ofAzotobacter chroococcum andAzospirillum brasilense inoculation under graded levels of nitrogen on growth and yield of wheat

A field experiment was conducted to study the effect ofAzotobacter chroococcum andAzospirillum brasilense inoculation under graded levels of fertilizeer nitrogen (0, 30, 60, 90, 120 kg/ha) on wheat (Triticum durum Desf. var. HD2189). The results revealed that both the inolculants sigificnatly increased the growth parametersviz. tilles, dry-matter accumulation, grain yield and protein content of wheat gains. Fertilizer—N @ 90 kg/ha with both inoculations was found to be the most responsive, however 120 kg/ha with Azospirillum inoculation recorded maximum number of tillers (3.5/plant), dry-matter accumulation (13.5 g/plant), grain yield (41.9 g/ha) and protein content in grains (13.3%). Maximum percent increase in grain yeidl with Azospirillum (25.8%) and Azotobacteer (19.2%) over uninoculated control was observed at 90 kg N/ha level. Azospirillum inoculation, Azotobacter inoculation and uninoculated control significnatly differed between each other.

Antagonism of Azotobacter chroococcum isolates to Rhizoctonia solani

Antagonism of isolates ofAzotobacter chroococcum toRhizoctonia solani on agar plates was studied, and isolates were tested for their ability to controlR. solani infection of potato sprouts in sterilized and unsterilized soil. The degree of antagonism exhibited varied strongly among the isolates and was also found to be temperature-dependent.