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.     

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.     

Elucidating the interactions and phytotoxicity of zinc oxide nanoparticles with agriculturally beneficial bacteria and selected crop plants

There is a growing interest in the use of bioinoculants to assist mineral fertilizers in improving crop production and yield. Azotobacter and Pseudomonas are two agriculturally relevant strains of bacteria which have been established as efficient bioinoculants. An experiment involving addition of graded concentrations of zinc oxide (ZnO) nanoparticles was undertaken using log phase cultures of Azotobacter and Pseudomonas. Growth kinetics revealed a clear trend of gradual decrease with Pseudomonas; however, Azotobacter exhibited a twofold enhancement in growth with increase in the concentration of ZnO concentration. Scanning electron microscopy (SEM), supported by energy-dispersive X-ray (EDX) analyses, illustrated the significant effect of ZnO nanoparticles on Azotobacter by the enhancement in the abundance of globular biofilm-like structures and the intracellular presence of ZnO, with the increase in its concentration. It can be surmised that extracellular mucilage production in Azotobacter may be providing a barrier to the nanoparticles. Further experiments with Azotobacter by inoculation of wheat and tomato seeds with ZnO nanoparticles alone or bacteria grown on ZnO-infused growth medium revealed interesting results. Vigour index of wheat seeds reduced by 40–50% in the presence of different concentrations of ZnO nanoparticles alone, which was alleviated by 15–20%, when ZnO and Azotobacter were present together. However, a drastic 50–60% decrease in vigour indices of tomato seeds was recorded, irrespective of Azotobacter inoculation.     

Plant growth stimulation of wheat (Triticum aestivum L.) by inoculation of salinity tolerant Azotobacter strains

Five salinity tolerant Azotobacter strains i.e., ST3, ST6, ST9, ST17 and ST24 were obtained from saline soils. These Azotobacter strains were used as inoculant for wheat variety WH157 in earthen pots containing saline soil under pot house conditions, using three fertilizer treatment doses i.e., control (no fertilizer, no inoculation), 90 Kg N ha⁻¹ and 120 Kg N ha⁻¹. Inoculation with salinity tolerant Azotobacter strains caused significant increase in total nitrogen, biomass and grain yield of wheat. Maximum increase in plant growth parameters were obtained after inoculation with Azotobacter strain ST24 at fertilization dose of 120 kg N ha⁻¹ and its inoculation resulted in attaining 89.9 cms plant height, 6.1 g seed yield, 12.0 g shoot dry weight and 0.7 % total nitrogen. The survival of Azotobacter strain ST24 in the soil was also highest in all the treatments at 30, 60 and 90 days after sowing (DAS). However, the population of Azotobacter decreased on 90 DAS as compared to counts observed at 60 DAS at all the fertilization treatments.     

Characterization and diversity of native Azotobacter spp. isolated from semi-arid agroecosystems of Eastern Kenya

Declining food production in African agroecosystems is attributable to changes in weather patterns, soil infertility and limited farming inputs. The exploitation of plant growth-promoting soil microbes could remedy these problems. Such microbes include Azotobacter; free-living, nitrogen-fixing bacteria, which confer stress tolerance, avail phytohormones and aid in soil bioremediation. Here, we aimed to isolate, characterize and determine the biodiversity of native Azotobacter isolates from soils in semi-arid Eastern Kenya. Isolation was conducted on nitrogen-free Ashby''s agar and the morphological, biochemical and molecular attributes evaluated. The isolates were sequenced using DNA amplicons of 27F and 1492R primers of the 16S rRNA gene loci. The Basic Local Alignment Search Tool (BLASTn) analysis of their sequences revealed the presence of three main Azotobacter species viz., Azotobacter vinelandii, Azotobacter salinestris and Azotobacter tropicalis. Kitui County recorded the highest number of recovered Azotobacter isolates (45.4%) and lowest diversity index (0.8761). Tharaka Nithi County showed the lowest occurrence (26.36%) with a diversity index of (1.057). The diversity was influenced by the soil pH, texture and total organic content. This study reports for the first time a wide diversity of Azotobacter species from a semi-arid agroecosystem in Kenya with potential for utilization as low-cost, free-living nitrogen-fixing bioinoculant.     

Promising microbial consortia for producing biofertilizers for rice fields

Two cyanobacterial cultures from rice paddies of Kyzylorda Provence, Kazakhstan were isolated and characterized: Anabaena variabilis and Nostoc calsicola. Based on these cultures, new consortia of cyanobacteria, microalgae and Azotobacter were developed: ZOB-1 (Anabaena variabilis, Chlorella vulgaris, and Azotobacter sp.) and ZOB-2 (Nostoc calsicola, Chlorella vulgaris, and Azotobacter sp.). High growth rate and photosynthetic activity of microalgae were observed in these consortia. The active consortium ZOB-1 was selected, which improved germination and growth of rice plants. ZOB-1 was recommended as a biostimulator and biofertilizer for crops.     

Correlation of Ultrastructure in Azotobacter vinelandii with Nitrogen Source for Growth

Azotobacter synthesizes an extensive internal membranous nework when grown with air (N₂), i.e., under conditions when these bacteria fix nitrogen. Very slight quantities of internal membrane, concentrated mainly about the cell periphery, are formed when Azotobacter grows with fixed nitrogen, i.e., ammonia and amino acids. Compared to cells growing with ammonia, cells utilizing atmospheric nitrogen as the sole nitrogen source are smaller in size and volume, grow one-third slower, and lack detectable poly-β-hydroxybutyrate.     

Azotobacter: A potential bio-fertilizer for soil and plant health management

Stressor (biotic as well as abiotic) generally hijack the plant growth and yield characters in hostile environment leading to poor germination of the plants and yield. Among the plant growth promoting rhizobacteria, Azotobacter spp. (Gram-negative prokaryote) are considered to improve the plant health. Various mechanisms are implicated behind improved plant health in Azotobacter spp. inoculated plants. For example, acceleration of phytohormone like Indole-3-Acetic Acid production, obviation of various stressors, nitrogen fixation, pesticides and oil globules degradation, heavy metals metabolization, etc. are the key characteristics of Azotobacter spp. action. In addition, application of this bacteria has also become helpful in the reclamation of soil suggesting to be a putative agent which can be used in the transformation of virgin land to fertile one. Application of pesticides of chemical origin are being put on suspension mode as the related awareness program is still on. As far as the limitations of this microbe is concerned, commercial level formulations availability is still a great menace. Present review has been aimed to appraise the researchers pertaining to utility of Azotobacter spp. in the amelioration of plant health in sustainable agroecosystem. The article has been written with the target to gather maximum information into single pot so that it could reach to the dedicated researchers.     

Diversity of cultivable Azotobacter in the semi-arid alfisol receiving long-term organic and inorganic nutrient amendments

Monitoring the biological processes and microbial diversity is essential for sustaining the soil health for long-term productivity. In the present study, the impact of long-term nutrient management systems on changes in Azotobacter diversity of Indian semi-arid alfisol was assessed. Three soils, i.e., unfertilized control, soils amended with organic manures (OM), and with inorganic chemical fertilizers (IC) from century-old experimental fields were evaluated for Azotobacter diversity by Amplified Ribosomal DNA Restriction Analysis (ARDRA). Bray–Curtis’s similarity index of the ARDRA data of the isolates was analyzed by non-metric multi-dimensional scaling and hierarchical cluster analysis. The results revealed that the long-term organically managed soil recorded significantly higher soil organic carbon, microbial biomass carbon, and total culturable bacterial counts, whereas the chemical fertilized and control soils remained unaffected. Though the Azotobacter population was significantly higher in OM soil than IC and control soils, the genetic diversity was unaffected due to long-term addition of either organic manures or inorganic chemical fertilizers. This result implies the importance of continuous addition of organic manures and also the optimal use of inorganic chemical fertilizers without disturbing the biological properties of the soil.     

Biochemical and physiological changes produced by Azotobacter chroococcum (INIFAT5 strain) on pineapple in vitro-plantlets during acclimatization

This study highlights some of the effects of the application of Azotobacter chroococcum (INIFAT5 strain) on in vitro-pineapple plantlets during acclimatization. The bacteria were sprayed immediately after transplanting to the ex vitro environment; the plants were then sprayed every 4 week. Subsequently (4 months) the evaluated variables included plantlet fresh and dry weights, leaf and root lengths, and composition of minerals, amino-acids, carbohydrates and proteins. Photosynthesis indicators were also evaluated. Significant effects of the application of Azotobacter over pineapple plantlets during acclimatization were observed in the mineral, amino-acid, carbohydrate and protein levels, as well as, in the photosynthesis indicators. Contrastingly, plant growth parameters showed modest increases caused by the bacteria, although they were statistically significant. Looking into specific minerals, the following significant effects of Azotobacter should be highlighted: increased levels of nitrogen, phosphorous, potassium, magnesium, copper and zinc. Moreover, contents of all amino-acids recorded showed significant increases in their levels in sprayed plantlets. Carbohydrates were also increased in leaves of plantlets bio-fertilized with the bacteria, mainly sucrose and fructose. Chlorophyll b levels were also significantly increased by Azotobacter. The biofertilizer did not modify levels of calcium, iron or manganese.     

Enumeration and Relative Importance of Acetylene-Reducing (Nitrogen-Fixing) Bacteria in a Delaware Salt Marsh

Three groups of N₂-fixing bacteria were enumerated from the top 1 cm of the surface in four vegetational areas in a Delaware salt marsh. The results over the 9-month sampling period showed that there were no discernible seasonal patterns for any of the groups enumerated (Azotobacter sp., Clostridium sp., and Desulfovibrio sp.). Azotobacter sp. was present in numbers of 10⁷ per g of dry mud, whereas the two anaerobic fixers were present in much lower numbers (10³ to 10⁴ per g of dry mud). There were no differences in the numbers of each group among the different vegetational areas, indicating that there was a heterogeneous population of N₂ fixers present. Additional studies indicate that the activity of sulfate reducers (Desulfovibrio sp.) may account for as much as 50% of the total observed acetylene reduction activity. Oxygen was found to exert little effect on the observed acetylene reduction activity, indicating that stable aerobic and anaerobic microenvironments exist in the surface layer of marsh sediments.     

Agro-industrial wastes as carriers for bacterial inoculants

Peat is an ideal carrier for Rhizobium and Azotobacter, but high-quality peat is not abundantly and readily available in India. The experiments have shown the usefulness of indigenously available agro-industrial wastes such as dehydrated powdered bagasse with traces of mahua (Madhuca latifolia) cake and silk cocoon waste as substrates for the production of bioinoculants.     

Effect of Inoculation with N2-Fixing Spirilla and Azotobacter on Nitrogenase Activity on Roots of Maize Grown Under Subtropical Conditions

Inoculated and non-inoculated seedlings of maize were grown in fertile clayloam soils of Egypt and Belgium under subtropical conditions provided in a greenhouse. Acetylene-reducing activity and microbial counts were determined during a period ranging from 6 to 12 weeks after sowing. Irrespective of soil origin, N₂-fixing spirilla and Azotobacter were common under maize cultivation. Inoculation resulted in a transitional increase in their numbers at early stages of growth. Nitrogenase activity was not detected in the rhizosphere of young plants. The maximum activities measured (81 to 1,436 nmol of C₂H₄ g⁻¹ h⁻¹) occurred close to the 50 to 70% silking stage. Inoculation with N₂-fixing spirilla, particularly in Nile Delta soil, doubled the amount of N₂ fixed in a late period of growth (12 weeks), whereas inoculation with Azotobacter had no noticeable effect.     

Non-pyridine nucleotide dependent l-(+)-glutamate oxidoreductase in Azotobacter vinelandii

l-(+)-Glutamate oxidation that is non-pyridine nucleotide dependent is readily carried out by a membrane-bound enzyme in Azotobacter vinelandii strain O. Enzyme activity concentrates in a membranous fraction that is associated with the Azotobacter electron transport system. This l-glutamate oxidation is not dependent on externally added NAD⁺, NADP⁺, FAD, or FMN for activity. O₂, phenazine methosulfate and ferricyanide all served as relatively good electron acceptors for this reaction; while cytochrome c and nitrotetrazolium blue function poorly in this capacity. Paper chromatographic analyses revealed that the 2,4-dinitrophenylhydrazine derivative formed from the enzymatic oxidation of l-glutamate was α-ketoglutarate, while microdiffusion studies indicated that ammonia was also a key end product. These findings suggest that the overall reaction is an oxidative deamination. Ammonia formation was found to be stoichiometric with the amount of oxygen consumed (2 : 1 respectively, on a molar basis). The oxidation of glutamate was limited to the l-(+)-enantiomer indicating that this reaction is not the generalized type carried out by the l-amino acid oxidase. This oxidoreductase is functionally related to the Azotobacter electron transport system: (a) the activity concentrates almost exclusively in the electron transport fraction; (b) the l-glutamate oxidase activity is markedly sensitive to electron transport inhibitors, i.e. 2-n-heptyl-4-hydroxyquinoline-N-oxide, cyanide, and 4,4,4-trifluoro-1-(2-thienyl)-1,3-butanedione; and (c) spectral studies on the Azotobacter R₃ fraction revealed that a substantial amount of the flavoprotein (non-heme iron) and cytochrome (a₂, a₁, b₁, c₄ and c₅) are reduced by the addition of l-glutamate.     

Biofilm of <Emphasis Type="Italic">Aureobasidium pullulans</Emphasis> var. <Emphasis Type="Italic">pullulans</Emphasis> on winter wheat kernels and its effect on other microorganisms

Winter wheat, grown under greenhouse conditions, was protected four times with a cell suspension of Aureobasidium pullulans var. pullulans during the growing season. After harvest, the distribution and survival rates of the studied biocontrol agent were analyzed under a scanning electron microscope. The abundance of filamentous fungi, yeasts, pseudomonads and Azotobacter bacteria was determined by inoculation onto selective agar media. A. pullulans produced mostly unicellular chlamydospores on the surface and in the brush of kernels. Multicellular blastospore conglomerates secreted extracellular polymeric substances (EPS), and their biofilms were found in the brush and crease of kernels. The application of a cell suspension of A. pullulans with the density of 10⁴ CFU to winter wheat spikes, repeated four times, inhibited the growth of pseudomonads, Azotobacter bacteria and filamentous fungi.     

Binding Characteristics of N2-Fixing Bacteria to Cereal Roots

The attachment of Rhizobium japonicum 61A89 and Rhizobium spp. 32H1 to the roots of wheat and rice seedlings is analyzed in terms of an equilibrium model. A Langmuir adsorption isotherm describes the binding. Strain 61A89 binds to a greater extent than does strain 32H1, and the equilibrium constants for each strain binding to wheat are strongly temperature dependent. Both time-dependent dissociation and association, predicted by an equilibrium model, have been found. The dissociation rate constant for 32H1 is approximately twice that of 61A89, and each is weakly temperature dependent. The rate equation for the binding of exponentially growing 61A89 to wheat roots has been solved as a function of time. Theory and experiment both indicate that the binding at very short times is much less than the equilibrium values. The binding of Azotobacter vinelandii 12837 to wheat roots has also been measured. Root-associated Azotobacter fixes nitrogen, whereas under aerobic growth conditions, root-associated 61A89 and 32H1 do not. The effect of metabolic inhibitors and antibiotics on the binding of Rhizobia and Azotobacter was examined.     

Improved biological treatment of nitrogen-deficient wastewater by incorporation of N2-fixing bacteria

The N₂-fixing bacterium, Azotobacter vinelandii, was used both in single culture and in combination with activated sludge culture for the treatment of nitrogen-deficient wastewaters as an alternative to external nitrogen supplementation. Azotobacter-supplemented activated sludge culture removed more total organic carbon (TOC), especially at low initial TN/COD (total nitrogen/chemical oxygen demand) ratios, than the Azotobacter-free culture. Up to 95% TOC removal efficiencies were obtained with synthetic media of TN/COD<4 when Azotobacter was used singly or with activated sludge. The results indicated clear advantage of using Azotobacter in the activated sludge to improve TOC removal from nitrogen-deficient wastewaters.     

Impact of bio-fertilizers and different levels of cadmium on the growth,biochemical contents and lipid peroxidation of Plantago ovata Forsk

Plantago ovata Forsk. (isabgol) is a valuable medicinal plant; its seeds and shell have a significant role in pharmacy as a laxative compound. Increasing soil contamination with cadmium (Cd) is one of the major concerns and is responsible for toxic effects in plants. This investigation was aimed to analyze the role of biofertilizers in alleviation of cadmium stress, given at the rate of 0, 50, and 100 mg kg⁻¹ of soil. The plants of isabgol, were grown in pots with and without application of AM fungi and Azotobacter (alone and combination). Cadmium showed negative effect on growth and biochemical component whereas proline and MDA content increase with increasing cadmium concentration. Addition of bio-fertilizer showed better growth and higher pigment concentration under cadmium stress as compared to the control. The dual inoculation of AM fungi and Azotobacter was found to be the best in reduction of cadmium stress and promotion of growth parameters.     

Beobachtungen über das Verhalten von Azotobacter im Wurzelbereich höherer Pflanzen

Zusammenfassung Es wurde der Azotobacter-Gehalt innerhalb der Rhizosphäre zahlreicher landwirtschaftlicher Nutzpflanzen während ihrer frühsommerlichen Wachstumsphase untersucht. Die Zunahmen gegenüber dem wurzelfreien Boden bewegen sich im allgemeinen in sehr engen Grenzen, können jedoch erheblich sein, falls der Boden arm an organischen Stoffen ist. Eine Verdrängung von Azotobacter durch Wurzelausscheidungen ließ sich bei den Versuchspflanzen nicht feststellen. N-Gewinne ergeben sich nicht so sehr aus der Azotobacter-Dichte als vielmehr aus dem laufenden Umsatz an Azotobacter-Zellen; an diesem Geschehen dürften Antagonisten, insbesondere Fluoreszenten, die sich im Wurzelbereich stark anreichern können, wesentlich beteiligt sein. In Sterilsynthesen (Weizen und Azotobacter) ließ sich bei nachträglicher Beimpfung mit Pseudomonas fluorescens ein spontaner Abbau von Azotobacter-Zellen und deren Verwertung als Nahrungsquelle nachweisen.Meinem hochverehrten Lehrer, Herrn Professor Dr. Dr. h. c. Rippel-Baldes, zum 70. Geburtstage.