Novel cereal bran based low-cost liquid medium for enhanced growth, multifunctional traits and shelf life of consortium biofertilizer containing Azotobacter chroococcum, Bacillus subtilis and Pseudomonas sp.

The present study was carried out to valorise cereal (rice and wheat) bran for the development of low-cost liquid consortium bioformulation. Different concentrations of bran-based liquid media formulations were evaluated for the growth of consortium biofertilizer cultures (Azotobacter chroococcum, Bacillus subtilis and Pseudomonas sp.). Among the bran-based formulations, wheat bran-based formulation WB5, exhibited the highest viable cell of 10.68 ± 0.09 Log10 CFU/ml and 12.63 ± 0.04 Log10 CFU/ml for Azotobacter chroococcum and Bacillus subtilis whereas for Pseudomonas sp., rice bran based bioformulation RB5 recorded maximum viability (12.71 ± 0.05 Log10 CFU/ml) after 72 h of incubation. RB51 and WB52liquid formulations were further optimized for enhanced shelf life using 5, 10 and 15 mM of trehalose, 0.05 and 0.1% carboxymethyl cellulose, and 0.5 and 1.0% glycerol. Following the peak growth at 72 h of incubation, a gradual decrease in the viable population of consortium biofertilizer cultures was observed in all the liquid formulations. The WB5 and RB5 formulations with 15 mM trehalose and 0.1% CMC, not only recorded significantly highest cell count of consortium biofertilizer cultures, but also maximally supported multi-functional traits i.e., phosphate and zinc solubilization, ammonia and IAA production up to 150 days. Further evaluation of seedling emergence and growth of wheat (PBW 826) under axenic conditions recorded WB5 amended with 15 mM trehalose-based consortium bioformulation to exhibit maximum emergence and growth of wheat seedlings. This low-cost liquid formulation can be used for large-scale biofertilizer production as a cost-effective liquid biofertilizer production technology.

Bacterial inoculation positively affects the quality and quantity of flax under deficit irrigation regimes.

AIM: The present research was conducted to investigate the effect of plant growth-promoting rhizobacteria (PGPR) and deficit irrigation on quality and quantity of flax under field and pot conditions to determine bacterial efficiency and to decrease water deficit effects. METHODS AND RESULTS: Initially, in vitro experiments were performed to determine the growth-promoting characteristics of bacteria. Then in the field, the effects of bacterial inoculation (control, Azotobacter chroococcum, Azospirillum lipoferum, Bacillus amyloliquefaciens, Bacillus sp. strain1 and Pseudomonas putida) on flax traits were evaluated at different irrigation levels (100, 75 and 50% crop water requirement). Bacterial treatments in the pot experiment were selected based on the field experiment results. The irrigation regimes in the pot and field experiments were the same and bacterial treatments included single, doublet and triplet applications of the bacteria. All the bacterial strains could solubilize phosphate, produce ammonia (except for Bacillus sp. strain1), indole acetic acid and siderophore (except P. putida). Field results indicated that the bacteria significantly mitigated the effects of water deficit. Compared with control plants, bacterial treatments increased the oil, linolenic acid, protein and sulphur content; the number of shoots and capsules; and the harvest index in the flax plants. Pot experimental results revealed that the combined inoculations were more effective than single inoculum treatments. CONCLUSIONS: Bacterial inoculation alleviates deficit irrigation effects in flax plants. SIGNIFICANCE AND IMPACT OF THE STUDY: The effectiveness of applying A. chroococcum, B. amyloliquefaciens and Bacillus sp. strain1 was confirmed, especially as a combination to protect flax against water deficit and to improve its nutritional quality and growth.

Biocontrol of Rhizoctonia solani (Kühn) and Fusarium solani (Marti) causing damping-off disease in tomato with Azotobacter chroococcum and Pseudomonas fluorescens.

BACKGROUND AND OBJECTIVE: The Damping-off disease is one of the most reasons for low productively of tomato in the world, especially in Iraq. In the current study, two types of bacteria (Azotobacter chroococcum and Pseudomonas fluorescens) were used to evaluate their efficacy in inhibiting the growth of pathogenic fungi Rhizoctonia solani and Fusarium solani and protecting the seeds of tomato and increasing their germination percentage. MATERIALS AND METHODS: Dual culture technique and Food poisoning technique were used to study the effect of bacteria on the growth of fungi understudy, and study the effect of bacterial filtrates on germination of tomato seeds. RESULTS: A. chroococcum showed the strongest antagonistic activity followed by P. fluorescens with the percentage of inhibition ranging between 72.9-77.1 and 69.5-70.3% for R. solani and F. solani respectively after 7 days of incubation. The effect of A. chroococcum and P. fluorescens filtrates were increased and also increased the inhibition of growth of fungi understudy, A. chroococcum filtrate also showed the strongest inhibitory effect followed by P. fluorescens with the percentage of inhibition ranging between 86.0-87.0 and 83.0-83.5% for R. solani and F. solani respectively at 20% concentration of filtrate. The percentage of seeds germination reached 90% in the treatment of A. chroococcum filtrate and 80% in the treatment of P. fluorescens filtrate. CONCLUSION: It can be concluded that the filtrates of A. chroococcum and P. fluorescens have antifungal properties against R. solani and F. solani and provided a high protection and increasing tomato seeds germination percentage.

Bioinoculants play a significant role in shaping the rhizospheric microbial community: a field study with Cajanus cajan.

The present study is an attempt to understand the impact of bioinoculants, Azotobacter chroococcum (A), Bacillus megaterium (B), Pseudomonas fluorescens (P), on (a) soil and plant nutrient status, (b) total resident and active bacterial communities, and (c) genes and transcripts involved in nitrogen cycle, during cultivation of Cajanus cajan. In terms of available macro- and micro-nutrients, triple inoculation of the bioinoculants (ABP) competed well with chemical fertilizer (CF). Their 'non-target' effects were assessed in terms of the abundance and activity of the resident bacterial community by employing denaturing gradient gel electrophoresis (DGGE). The resident bacterial community (16S rRNA gene) was stable, while the active fraction (16S rRNA transcripts) was influenced (in terms of abundance) by the treatments. Quantification of the genes and transcripts involved in N cycle by qPCR revealed an increase in the transcripts of nifH in the soil treated with ABP over CF, with an enhancement of 3.36- and 1.57- fold at flowering and maturity stages of plant growth, respectively. The bioinoculants shaped the resident microflora towards a more beneficial community, which helped in increasing soil N turnover and hence, soil fertility as a whole.

Characterization of heavy metal toxicity in some plants and microorganisms-A preliminary approach for environmental bioremediation.

In situ bioremediation processes are important for control of pollution and clean-up of contaminated sites. The study and implementation of such processes can be designed through investigations on natural mechanisms of absorption, biotransformation, bioaccumulation and toxicity of pollutants in plants and microorganisms. Here, the phytotoxic effects of Cr(VI) and Cd(II) on seed germination and plant growth of Lepidium sativum have been examined at various concentrations (30-300 mg/L) in single ion solutions. The studies also addressed the ecotoxicity of metal ions on Azotobacter chroococcum and Pichia sp. isolated from soil. Microbial growth was estimated by weighing the dry biomass and determining the enzymatic activities of dehydrogenase and catalase. The results showed that Cr(VI) and Cd(II) can inhibit L. sativum seed germination and root development, depending on the metal ion and its concentration. The phytotoxic effect of heavy metals was also confirmed by the reduced amounts of dried biomass. Toxicity assays demonstrated the adverse effect of Cr(VI) and Cd(II) on growth of Azotobacter sp. and Pichia sp., manifested by a biomass decrease of more than 50 % at heavy metal concentrations of 150-300 mg/L. The results confirmed close links between phytotoxicity of metals and their bioavailability for phytoextraction. Studies on the bioremediation potential of soils contaminated with Cr(VI) and Cd(II) using microbial strains focusing on Azotobacter sp. and Pichia sp. showed that the microbes can only tolerate heavy metal stress at low concentrations. These investigations on plants and microorganisms revealed their ability to withstand metal toxicity and develop tolerance to heavy metals.

Response surface optimization of conditions for culturing Azotobacter chroococcum in Agaricus bisporus industrial wastewater.

In the present study, the conditions for Azotobacter chroococcum fermentation using Agaricus bisporus wastewater as the culture medium were optimized. We analyzed the total number of living A. chroococcum in the fermentation broth, using multispectral imaging flow cytometry. Single-factor experiments were carried out, where a Plackett-Burman design was used to screen out three factors from the original six processing factors wastewater solubility, initial pH, inoculum size, liquid volume, culture temperature, and rotation speed that affected the total number of viable A. chroococcum. The Box-Behnken response surface method was used to optimize the interactions between the three main factors and to predict the optimal fermentation conditions. Factors significantly affecting the total number of viable A. chroococcum, including rotation speed, wastewater solubility, and culture temperature, were investigated. The optimum conditions for A. chroococcum fermentation in A. bisporus wastewater were a rotation speed of 200 rpm, a solubility of 0.25%, a culture temperature of 26°C, an initial pH of 6.8, a 5% inoculation volume, a culture time of 48 h, and a liquid volume of 120 mL in a 250 mL flask. Under these conditions, the concentration of total viable bacteria reached 4.29 ± 0.02 ✕ 107 Obj/mL A. bisporus wastewater can be used for the cultivation of A. chroococcum.

Root inoculation with Azotobacter chroococcum 76A enhances tomato plants adaptation to salt stress under low N conditions.

BACKGROUND: The emerging roles of rhizobacteria in improving plant nutrition and stress protection have great potential for sustainable use in saline soils. We evaluated the function of the salt-tolerant strain Azotobacter chroococcum 76A as stress protectant in an important horticultural crop, tomato. Specifically we hypothesized that treatment of tomato plants with A. chroococcum 76A could improve plant performance under salinity stress and sub-optimal nutrient regimen. RESULTS: Inoculation of Micro Tom tomato plants with A. chroococcum 76A increased numerous growth parameters and also conferred protective effects under both moderate (50 mM NaCl) and severe (100 mM NaCl) salt stresses. These benefits were mostly observed under reduced nutrient regimen and were less appreciable in optimal nitrogen conditions. Therefore, the efficiency of A. chroococcum 76A was found to be dependent on the nutrient status of the rhizosphere. The expression profiles of LEA genes indicated that A. chroococcum 76A treated plants were more responsive to stress stimuli when compared to untreated controls. However, transcript levels of key nitrogen assimilation genes revealed that the optimal nitrogen regimen, in combination with the strain A. chroococcum 76A, may have saturated plant's ability to assimilate nitrogen. CONCLUSIONS: Roots inoculation with A. chroococcum 76A tomato promoted tomato plant growth, stress tolerance and nutrient assimilation efficiency under moderate and severe salinity. Inoculation with beneficial bacteria such as A. chroococcum 76A may be an ideal solution for low-input systems, where environmental constraints and limited chemical fertilization may affect the potential yield.

Plant Growth-Promoting Rhizobacteria Improved Salinity Tolerance of Lactuca sativa and Raphanus sativus.

Salinity stress is an important environmental problem that adversely affects crop production by reducing plant growth. The impacts of rhizobacterial strains to alleviate salinity stress on the germination of Lactuca sativa and Raphanus sativus seeds were assessed using different concentrations of NaCl. Plant growth-promoting rhizobacteria (PGPR) strains were also examined to improve the early germination of Chinese cabbage seeds under normal conditions. Lactobacillus sp. and P. putida inoculation showed higher radicle lengths compared with non-inoculated radish (Raphanus sativus) seeds. LAP mix inoculation increased the radicle length of lettuce (Lactuca sativa) seedlings by 2.0 and 0.5 cm at salinity stress of 50 and 100 mM NaCl concentration, respectively. Inoculation by Azotobacter chroococcum significantly increased the plumule and radicle lengths of germinated seeds compared with non-inoculated control. A. chroococcum increased the radicle length relative to the uninoculated seeds by 4.0, 1.0, and 1.5 cm at 50, 100, and 150 mM NaCl concentration, respectively. LAP mix inoculation significantly improved the radicle length in germinated radish seeds by 7.5, 1.3, 1.2, and 0.6 cm under salinity stress of 50, 100, 150, and 200 mM NaCl concentration, respectively. These results of this study showed that PGPR could be helpful to mitigate the salinity stress of different plants at the time of germination.

Breaking continuous potato cropping with legumes improves soil microbial communities, enzyme activities and tuber yield.

This study was conducted to explore the changes in soil microbial populations, enzyme activity, and tuber yield under the rotation sequences of Potato-Common vetch (P-C), Potato-Black medic (P-B) and Potato-Longdong alfalfa (P-L) in a semi-arid area of China. The study also determined the effects of continuous potato cropping (without legumes) on the above mentioned soil properties and yield. The number of bacteria increased significantly (p < 0.05) under P-B rotation by 78%, 85% and 83% in the 2, 4 and 7-year continuous cropping soils, respectively compared to P-C rotation. The highest fungi/bacteria ratio was found in P-C (0.218), followed by P-L (0.184) and then P-B (0.137) rotation over the different cropping years. In the continuous potato cropping soils, the greatest fungi/bacteria ratio was recorded in the 4-year (0.4067) and 7-year (0.4238) cropping soils and these were significantly higher than 1-year (0.3041), 2-year (0.2545) and 3-year (0.3030) cropping soils. Generally, actinomycetes numbers followed the trend P-L>P-C>P-B. The P-L rotation increased aerobic azotobacters in 2-year (by 26% and 18%) and 4-year (40% and 21%) continuous cropping soils compared to P-C and P-B rotation, respectively. Generally, the highest urease and alkaline phosphate activity, respectively, were observed in P-C (55.77 mg g-1) and (27.71 mg g-1), followed by P-B (50.72 mg mg-1) and (25.64 mg g-1) and then P-L (41.61 mg g-1) and (23.26 mg g-1) rotation. Soil urease, alkaline phosphatase and hydrogen peroxidase activities decreased with increasing years of continuous potato cropping. On average, the P-B rotation significantly increased (p <0.05) tuber yield by 19% and 18%, compared to P-C and P-L rotation respectively. P-L rotation also increased potato tuber yield compared to P-C, but the effect was lesser relative to P-B rotation. These results suggest that adopting potato-legume rotation system has the potential to improve soil biology environment, alleviate continuous cropping obstacle and increase potato tuber yield in semi-arid region.

Sequence of inoculation influences the nature of extracellular polymeric substances and biofilm formation in Azotobacter chroococcum and Trichoderma viride.

Extracellular polymeric substances (EPS) are important structural components of biofilms. In the present study, the EPS in biofilms developed using two agriculturally beneficial organisms-Azotobacter chroococcum (Az) and Trichoderma viride (Tv) were quantified and characterised. Time course experiments were undertaken to optimise the EPS yield of biofilm samples resulting from coculture and staggered inoculation. The EPS produced during biofilm formation was found to differ quantitatively and qualitatively in individual cultures (Az alone, Tv alone), and in treatments differing in the sequence of inoculation of bacterium and fungus (Az + Tv coculture, staggered inoculation of Az followed by Tv i.e. Az - Tv, or Tv followed by Az i.e. Tv - Az). Significant enhancement in terms of growth and biofilm formation, as compared to individual inoculation was recorded, with Tv - Az exhibiting higher values of these attributes. The EPS from biofilms showed significantly higher concentrations of protein, acetyl, and uronic acids, while planktonic EPS recorded higher total carbohydrates. Fourier transform infrared spectroscopy analyses illustrated the significant influence on chemical and structural aspects of EPS (planktonic and biofilm). This represents a first report correlating EPS production, cell aggregation and biofilm formation during bacterial-fungal biofilm development, which can have implications in the colonisation of soil and plants.

Uso de Leersia hexandra (Poaceae) en la fitorremediación de suelos contaminados con petróleo fresco e intemperizado / Use of Leersia hexandra (Poaceae) for soil phytoremediation in soils contaminated with fresh and weathered oil

ResumenLa industria petrolera ha generado derrames crónicos de petróleo y su acumulación en Gleysoles en zonas anegadas en el estado de Tabasco, en el sureste de México. El anegamiento es un factor que limita el uso de tecnologías de remediación por el alto costo y los bajos niveles de degradación del petróleo, sin embargo, Leersia hexandra Sw. es un pasto que crece en estas zonas contaminadas con petróleo intemperizado. El objetivo del estudio fue evaluar la densidad de bacterias, producción de biomasa vegetal y fitorremediación de L.hexandra en suelo contaminado con petróleos fresco e intemperizado, bajo condiciones experimentales de anegamiento. Se realizaron dos experimentos (E1 y E2) en un túnel de plástico. El E1 se basó en ocho dosis: 6 000, 10 000, 30 000, 60 000, 90 000, 120 000, 150 000 y 180 000 mg kg-1 base seca (b.s.) de hidrocarburos totales de petróleo fresco (HTPF), y en el E2 se evaluaron cinco dosis: 14 173, 28 400, 50 598, 75 492 y 112 142 mg kg-1 b. s. de hidrocarburos totales de petróleo intemperizado (HTPI), con ocho repeticiones en cada experimento, además se utilizó un testigo con 2 607 mg kg-1 b. s. de HTP de origen biogénico. Las variables evaluadas a los tres y seis meses fueron 1) densidad microbiana de las bacterias fijadoras de nitrógeno de vida libre totales (BFN), del grupo Azospirillum (AZP) y Azotobacter (AZT), por cuenta viable en placa seriada; 2) producción de materia seca total (MS), se cuantificó por el peso seco por gravimetría, y 3) el porcentaje de descontaminación de los hidrocarburos (DSC) por extracción en equipo soxhlet. En suelos con HTPF, la población de BFN, AZP y AZT se estimuló hasta cinco veces más que el tratamiento testigo a los tres y seis meses; sin embargo, concentraciones de 150 000 y 180 000 mg kg-1 b. s. inhibieron entre un 70 y 89 % la densidad bacteriana. A su vez, en suelos con PI, la inhibición se registró hasta en un 90 %, a excepción del tratamiento con 14 173 mg kg-1 b. s., el cual estimuló las BNF y AZT en 2 y 0.10 veces más que testigo, respectivamente. La producción de MS fue continua en los experimentos hasta los seis meses, con valores de 63 y 89 g en PF y PI, respectivamente; sin diferencias significativas con el testigo (p ≤ 0.05). El DSC alcanzó valores del 66 % al 87 % en HTPF como HTPI a los seis meses, respectivamente. Estos resultados demuestran la habilidad del L. hexandra para desarrollar una rizósfera con alta densidad de BFN, producir biomasa vegetal y fitorremediar Gleysoles con petróleo fresco e intemperizado en ambientes tropicales inundados.

Abstract:The oil industry has generated chronic oil spills and their accumulation in wetlands of the state of Tabasco, in Southeastern Mexico. Waterlogging is a factor that limits the use of remediation technologies because of its high cost and low levels of oil degradation. However, Leersia hexandra is a grass that grows in these contaminated areas with weathered oil. The aim of the study was to evaluate the bacteria density, plant biomass production and phytoremediation of L. hexandra in contaminated soil. For this, two experiments in plastic tunnel were performed with fresh (E1) and weathered petroleum (E2) under waterlogging experimental conditions. The E1 was based on eight doses: 6 000, 10 000, 30 000, 60 000, 90 000, 120 000, 150 000 and 180 000 mg.kg-1 dry basis (d. b.) of total petroleum hydrocarbons fresh (TPH-F), and the E2, that evaluated five doses: 14 173, 28 400, 50 598, 75 492 and 112 142 mg. kg-1 d. b. of total petroleum hydrocarbons weathered (TPH-W); a control treatment with 2 607 mg.kg-1 d. b. was used. Each experiment, with eight replicates per treatment, evaluated after three and six months: a) microbial density of total free-living nitrogen-fixing bacteria (NFB) of Azospirillum (AZP) and Azotobacter group (AZT), for viable count in serial plate; b) dry matter production (DMP), quantified gravimetrically as dry weight of L. hexandra; and c) the decontamination percentage of hydrocarbons (PDH) by Soxhlet extraction. In soil with TPH-F, the NFB, AZP y AZT populations were stimulated five times more than the control both at the three and six months; however, concentrations of 150 000 and 180 000 mg.kg-1 d. b. inhibited the bacterial density between 70 and 89 %. Likewise, in soil with TPH-W, the FNB, AZP and AZT inhibitions were 90 %, with the exception of the 14 173 mg.kg-1 d. b. treatment, which stimulated the NFB and AZT in 2 and 0.10 times more than the control, respectively. The DMP was continued at the six months in the experiments, with values of 63 and 89 g in fresh and weathered petroleum, respectively; had no significant differences with the control (p≤0.05). The PDH reached values of 66 to 87 % both TPH-F and TPH-W at six months, respectively. These results demonstrated the ability the L. hexandra rhizosphere to stimulate the high NFB density, vegetal biomass production and phytoremediation of contaminated soils (with fresh and weathered petroleum), in a tropical waterlogging environment. Rev. Biol. Trop. 65 (1): 21-30. Epub 2017 March 01.

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.

[Use of Leersia hexandra (Poaceae) for soil phytoremediation in soils contaminated with fresh and weathered oil]. / Uso de Leersia hecandra (Poaceae) en la fitorremediación de suelos contaminados con petróleo fresco e intemperizado.

The oil industry has generated chronic oil spills and their accumulation in wetlands of the state of Tabasco, in Southeastern Mexico. Waterlogging is a factor that limits the use of remediation technologies because of its high cost and low levels of oil degradation. However, Leersia hexandra is a grass that grows in these contaminated areas with weathered oil. The aim of the study was to evaluate the bacteria density, plant biomass production and phytoremediation of L. hexandra in contaminated soil. For this, two experiments in plastic tunnel were performed with fresh (E1) and weathered petroleum (E2) under waterlogging experimental conditions. The E1 was based on eight doses: 6 000, 10 000, 30 000, 60 000, 90 000, 120 000, 150 000 and 180 000 mg.kg-1 dry basis (d. b.) of total petroleum hydrocarbons fresh (TPH-F), and the E2, that evaluated five doses: 14 173, 28 400, 50 598, 75 492 and 112 142 mg. kg-1 d. b. of total petroleum hydrocarbons weathered (TPH-W); a control treatment with 2 607 mg.kg-1 d. b. was used. Each experiment, with eight replicates per treatment, evaluated after three and six months: a) microbial density of total free-living nitrogen-fixing bacteria (NFB) of Azospirillum (AZP) and Azotobacter group (AZT), for viable count in serial plate; b) dry matter production (DMP), quantified gravimetrically as dry weight of L. hexandra; and c) the decontamination percentage of hydrocarbons (PDH) by Soxhlet extraction. In soil with TPH-F, the NFB, AZP y AZT populations were stimulated five times more than the control both at the three and six months; however, concentrations of 150 000 and 180 000 mg.kg-1 d. b. inhibited the bacterial density between 70 and 89 %. Likewise, in soil with TPH-W, the FNB, AZP and AZT inhibitions were 90 %, with the exception of the 14 173 mg.kg-1 d. b. treatment, which stimulated the NFB and AZT in 2 and 0.10 times more than the control, respectively. The DMP was continued at the six months in the experiments, with values of 63 and 89 g in fresh and weathered petroleum, respectively; had no significant differences with the control (p≤0.05). The PDH reached values of 66 to 87 % both TPH-F and TPH-W at six months, respectively. These results demonstrated the ability the L. hexandra rhizosphere to stimulate the high NFB density, vegetal biomass production and phytoremediation of contaminated soils (with fresh and weathered petroleum), in a tropical waterlogging environment.

[THE COMPLEX ESTIMATE OF THE RHIZOBIUM NODULATION ABILITY AND THE FEATURES OF SOYBEAN SYMBIOTIC SYSTEMS FORMATION AT THE MICROBIAL COMPOSITIONS SEED INOCULATION].

The features of the soybean symbiotic systems formation and carry out the complex es- timate of the rhizobium nodulation ability at the seed inoculation of the microbial composi- tions on the bases of nodule bacteria, azotobacter and phytolectins (soybean seeds lectin, wheat germ agglutinin) were studied in the green-house experiments with a soil cultures. It was shown, that complex inoculants accelerate the process of becoming infected of plants by rhizobia in the early stages of soybean development; contribute to the expansion of the spectrum of genetically determined ability of nodule bacteria in the formation of a certain number of nodules on the host plant during the growing season as well as the formation of more root nodules with more of their weight during the second half of the growing season of soybean and significant increase mass of the one nodule and also slow the root nodules aging process at the end of the growing season compared with a rhizobial monoinoculant. It was proposed to use a complex of criteria in the estimating of the rhizobia nodulation ability in the microbial compositions: "nodulation activity", "nodulation range", "the num- ber of nodules on the plant", "mass of nodules per plant", "mass of one nodule", which are indicative for different stages of the symbiosis formation.

Solubilization of tricalcium phosphate by P(3HB) accumulating Azotobacter chroococcum MAL-201.

Cells of Azotobacter chroococcum MAL-201 (MTCC 3853) are capable of accumulating the intracellular poly(3-hydroxybutyric acid) [P(3HB)], accounting for 65-71 % of its cell dry weight and also capable of synthesizing the enzyme alkaline phosphatase (APase), when grown in glucose and tricalcium phosphate containing nitrogen-free modified Stockdale medium. The concentration of insoluble phosphate in broth medium was optimized as 0.25 % (w/v) for growth and biosynthesis of APase. However, the suboptimal concentration of phosphate (0.1 %, w/v) appeared as the best suited for accumulation of P(3HB) by the strain. The significant differences were observed in biosynthesis of polymer and APase enzyme under variable phosphate concentrations. Glucose, 3.0 % (w/v) was recorded as the optimum concentration for all of the three parameters. The continuation of APase biosynthesis was observed during the period of significant decline in the cellular content of the polymer in the late phase of growth. In order to study the role of P(3HB), the rate of autodigestion of biopolymer and phosphate solubilization rate (k, mineralization constant) were determined in carbon-free medium under batch cultivation process and the parameters were found to be positively correlated. The maximum phosphate solubilization rate (k = 0.0154) by the strain MAL-201 timed at the 10th hour of incubation when the rate of polymer degradation concomitantly attained its peak corresponding to 87 mg/l/h and then declined gradually. Only a negligible amount of residual polymer remained undigested. These data strongly support the functional role of P(3HB) in response to multinutritional stress condition.

Phenotypic and molecular characterisation of efficient nitrogen-fixing Azotobacter strains from rice fields for crop improvement.

Biological nitrogen fixation (BNF) is highly effective in the field and potentially useful to reduce adverse effects chemical fertilisers. Here, Azotobacter species were selected via phenotypic, biochemical and molecular characterisations from different rice fields. Acetylene reduction assay of Azotobacter spp. showed that Azotobacter vinelandii (Az3) fixed higher amount of nitrogen (121.09 nmol C2H4 mg(-1) bacteria h(-1)). Likewise, its plant growth functions, viz. siderophore, hydrogen cyanide, salicylic acid, IAA, GA3, zeatin, NH3, phosphorus solubilisation, ACC deaminase and iron tolerance, were also higher. The profile of gDNA, plasmid DNA and cellular protein profile depicted inter-generic and inter-specific diversity among the isolates of A. vinelandii. The PCR-amplified genes nifH, nifD and nifK of 0.87, 1.4 and 1.5 kb , respectively, were ascertained by Southern blot hybridisation in isolates of A. vinelandii. The 16S rRNA sequence from A. vinelandii (Az3) was novel, and its accession number (JQ796077) was received from NCBI data base. Biofertiliser formulation of novel A. vinelandii isolates along with commercial one was evaluated in rice (Oriza sativa L. var. Khandagiri) fields. The present finding revealed that treatment T4 (Az3) (A. vinelandii) are highly efficient to improved growth and yield of rice crop.

Novel route of tannic acid biotransformation and their effect on major biopolymer synthesis in Azotobacter sp. SSB81.

AIMS: To examine tannic acid (TA) utilization capacity by nitrogen-fixing bacteria, Azotobacter sp. SSB81, and identify the intermediate products during biotransformation. Another aim of this work is to investigate the effects of TA on major biopolymers like extracellular polysaccharide (EPS) and polyhydroxybutyrate (PHB) synthesis. METHODS AND RESULTS: Tannic acid utilization and tolerance capacity of the strain was determined according to CLSI method. Intermediate products were identified using high-performance liquid chromatography, LC-MS/MS and (1) H NMR analysis. Intermediates were quantified by multiple reactions monitoring using LC-MS/MS. The strain was able to tolerate a high level of TA and utilized through enzymatic system. Growth of Azotobacter in TA-supplemented medium was characterized by an extended lag phase and decreased growth rate. Presence of TA catalytic enzymes as tannase, polyphenol oxidase (PPO) and phenol decarboxylase was confirmed in cell lysate using their specific substrates. PPO activity was more prominent in TA-supplemented mineral medium after 48 h of growth when gallic to ellagic acid (EA) reversible reaction was remarkable. Phase contrast and scanning electron microscopic analysis revealed elongated and irregular size of Azotobacter cells in response to TA. (1) H NMR analysis indicated that TA was transformed into gallic acid (GA), EA and pyrogallol. Biopolymer (EPS and PHB) production was decreased several folds in the presence of TA compared with cells grown in only glucose medium. CONCLUSIONS: This is the first evidence on the biotransformation of TA by Azotobacter and also elevated level of EA production from gallotannins. Azotobacter has developed the mechanism to utilize TA for their carbon and energy source. SIGNIFICANCE AND IMPACT OF THE STUDY: The widespread occurrence and exploitation of Azotobacter sp. strain SSB81 in agricultural and forest soil have an additional advantage to utilize the soil-accumulated TA and detoxifies the allelopathic effect of constant accumulated TA in soil.

Effects of lindane on lindane-degrading Azotobacter chroococcum; evaluation of toxicity of possible degradation product(s) on plant and insect.

The effects of lindane on growth and plant growth-promoting traits of two lindane-degrading Azotobacter chroococcum strains (JL 15 and JL 104) were determined. The potential of both A. chroococcum strains to degrade lindane was also determined. Lower concentrations of lindane had a stimulatory effect, and higher concentrations generally had an inhibitory effect on growth and plant growth-promoting activities. A high percentage (>90%) of lindane was degraded by both strains at a lindane concentration of 10 ppm. Lindane at 1,000 ppm decreased seed germination and reduced seedling fresh weight. However, the possible degradation products for a starting lindane concentration of 10 ppm was found to be non-phytotoxic. Toxicity studies with larvae of Spilarctia obliqua resulted in an LC50 estimate of 3.41 ppm for lindane solutions into which leaf discs were dipped. No toxicity was observed for possible degradation products.

[Dynamics of diazotrophic bacteria number in the root zone of wheat Vrn lines isogenic by genes].

The number of diazotrophic bacteria and nitrogenase activity in the root zone of isogenic monogene-dominant Vrn lines were measured in the field experiments throughout their vegetation from tillering to heading. The total number of diazotrophic bacteria and nitrogenase activity in the root zone of these lines during this period were increased irrespective of their genotypes. The above indices of the winter cultivar (Vrn loci bottom recessive) were lower than those of the spring lines--Vrn-A1, Vrn-B1 and Vrn-D1. Plants of Vrn-B1 line have the lowest indices among the spring lines with the exception of some indices. This line plants flowered later than those of Vrn-A1 and Vrn-D1 lines. We hypothesized the differences between plants of these lines as to nitrogen fixation activity and the number of diazotrophic bacteria are mediately determined by Vrn loci through their effects on metabolism intensity and assimilate reflux in the form of root exudates, therefore the total number of diazotrophic bacteria and nitrogenase activity increases.

Biotransformation of p-coumaric acid and 2,4-dichlorophenoxy acetic acid by Azotobacter sp. strain SSB81.

A comprehensive study was made on biotransformation of p-coumaric acid and 2,4-dichlorophenoxyacetic acid by an Azotobacter sp. strain SSB81. The strain was able to tolerate a high amount of both the phenolic acids and p-coumaric acid degraded maximum (50%) than 2,4-D (29%) after five days of incubation. The intermediate products during transformation have been identified and quantified using UV-Vis and LC-MS/MS analysis. Para-coumaric acid was degraded via p-hydroxybenzoic acid and protocatechuic acid, a non-oxidative pathway whereas 2,4-D via 4-chlorophenoxyacetic acid, 4-chlorophenol and 4-chlorocatechol, an oxidative pathway. The results suggest that SSB81 developed both the oxidative and non-oxidative pathway to degrade the soil accumulated phenolic acids. Thus, Azotobacter provides an advantage to reduce the toxic level of soil accumulated phenolic acids in addition to increase the soil fertility.