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.

Isolation and identification by 16S rRNA sequence analysis of plant growth-promoting azospirilla from the rhizosphere of wheat

ABSTRACT The main objective of the present study was to isolate phytohormone-producing, phosphate-solubilizing strains of Azospirillum from wheat to be used as inoculants for plant growth promotion. Five Azospirillum strains were isolated from the rhizosphere of field-grown wheat (Triticum aestivum L.), and it was confirmed by BOX-polymerase chain reaction (PCR) that the isolates were different and not re-isolates of the same strain. Sequence analysis of the PCR-amplified 16S rRNA gene indicated that four isolates showed maximum similarity to Azospirillum brasilense and one isolate showed maximum similarity to Azospirillum zeae. This is the first report indicating the presence of an A. zeae like isolate in the wheat rhizosphere in Pakistan. The bacterial isolates were characterized for their plant growth-promoting traits, phosphate solubilization, and indole-3-acetic acid (IAA) production. None of the isolates showed phosphate solubilization activity in the commonly used Pikovskaya medium. However, all strains (except AzoK4) exhibited ability to solubilize tricalcium phosphate (TCP) in modified Pikovskaya medium in which sucrose was replaced by Na-malate, as well as in TCP-supplemented Luria-Bertani (LB) medium. Organic acids, such as acetic, citric, lactic, malic, and succinic acids, were detected in culture supernatants of the tested Azospirillum strains. All strains exhibited ability to produce IAA in the growth medium, except Azospirillum sp. AzoK1. Among the strains tested, the maximum IAA production (30.49 ± 1.04 mg L-1) and phosphate solubilization (105.50 ± 4.93 mg L-1) were shown by a pure culture of Azospirillum sp. AzoK2. In pot experiments, single-strain inocula of Azospirillum sp. AzoK1 and AzoK2 improved wheat plant growth.

Growth and yield responses of tomato [lycopersicon esculentum] grown under soilless cultivation to application of azospirillum, azotobacter and nitrogen

THIS STUDY was undertaken to investigate the possibility of using Azospirillum bras/lease and Azotobacter chroococcum applied with inorganic nitrogen to enhance tomato [Lycopersicon escnlentum, Mill] production in soilless systems. The effect of inoculation with A. brasilense and / or Azoto. chroococcum with the application of 75% [of the recommended dose] of N[2] on certain tomato growth parameters [shoot height, number of leaves, and fresh and dry weights of plants] after 30, 60 and 90 days of transplanting were determined. In addition, total yield, mean fruit weight and number of fruits per plant were evaluated in plants inoculated with the two bacteria [separately or in combination] with 75% N[2], and plants inoculated with 75% N[2] [control 1] and 100% N[2] [control 2], Plants inoculated with a mixed inoculum of A. bras/lease and Azoto. chroococcum with 75% N[2] gave the highest values of the growth parameters tested while plants inoculated with 75% N[2] [control 1] gave the lowest. The same treatment also resulted in the highest tomato yield, mean weight and number of fruits per plant followed by plants treated with A. brasilense and 75% N[2]. The responses to the tested biofertilizers on nitrogen, phosphorous and potassium [NPK] uptake, population of diazotrophic bacteria and nitrogenase and dehydrogenase activity of the tested bacteria on the rhizosphere of tomato showed that the mixed inoculum of A. brasilense and Azoto. chroococcum with 75% N[2] gave the highest NPK uptake levels after 30, 60 and 90 days of transplanting. The same treatment gave the highest population of diazotrophic bacteria and dehydrogenase and nitrogenase activity of the tested bacteria compared with the plants inoculated with a single organism or controls. In contrast, control 1 gave the least values among all treatments. This study shows that inoculation of tomato plants with Azospirillum brasilense and Azolobacter chroococcum have a significantly beneficial effect on tomato yield under soilless cultivation

Associative diazotrophic bacteria in grass roots and soils from heavy metal contaminated sites

This work aimed to evaluate density of associative diazotrophic bacteria populations in soil and grass root samples from heavy metal contaminated sites, and to characterize isolates from these populations, both, phenotypically (Zinc, Cadmium and NaCl tolerance in vitro, and protein profiles) and genotypically (16S rDNA sequencing), as compared to type strains of known diazotrophic species. Densities were evaluated by using NFb, Fam and JNFb media, commonly used for enrichment cultures of diazotrophic bacteria. Bacterial densities found in soil and grass root samples from contaminated sites were similar to those reported for agricultural soils. Azospirillum spp. isolates from contaminated sites and type strains from non-contaminated sites varied substantially in their in vitro tolerance to Zn+2 and Cd+2, being Cd+2 more toxic than Zn+2. Among the most tolerant isolates (UFLA 1S, 1R, S181, S34 and S22), some (1R, S34 and S22) were more tolerant to heavy metals than rhizobia from tropical and temperate soils. The majority of the isolates tolerant to heavy metals were also tolerant to salt stress as indicated by their ability to grow in solid medium supplemented with 30 g L-1 NaCl. Five isolates exhibited high dissimilarity in protein profiles, and the 16S rDNA sequence analysis of two of them revealed new sequences for Azospirillum.

Objetivou-se avaliar a densidade de populações de bactérias diazotróficas associativas em amostras de solos e de raízes de gramíneas oriundas de sítios contaminados com metais pesados, e caracterizar isolados destas populações através da análise fenotípica (tolerância aos metais pesados zinco e cádmio e à NaCl in vitro, perfis protéicos), e genotípica (seqüenciamento de 16S rDNA), comparados às estirpes tipo das mesmas espécies. As densidades foram avaliadas nos meios NFb, Fam e LGI, comumente utilizados para culturas de enriquecimento de populações de bactérias diazotróficas associativas. As densidades encontradas em amostras de solo e raiz de sítios contaminados foram semelhantes àquelas relatadas na literatura para solos agrícolas. Isolados de Azospirillum spp. de solos contaminados e estirpes tipo oriundas de solos não contaminados variaram substancialmente com relação à tolerância a Zn+2 e Cd+2, sendo que Cd+2 mais tóxico que Zn+2. Dentre os isolados mais tolerantes (UFLA 1S, 1R, S181, S34, e S22), alguns(1R, S34 e S22) foram mais tolerantes a metais pesados que rizóbios isolados de solos de áreas tropicais e temperadas. A maioria dos isolados mais tolerantes a metais pesados também foi tolerante ao estresse salino, o que foi indicado por seu crescimento em meio sólido suplementado com 30 g L-1 de NaCl in vitro. Cinco isolados apresentaram alta dissimilaridade em perfis protéicos e o seqüenciamento do gene 16S rDNA em dois deles revelou que apresentam novas seqüências de Azospirillum.

Efecto de la inoculación de Azospirillum brasilense y Glomus sp: en Gmelina arborea durante su germinación y manejo en vivero / Response of gmelina arborea to glomus sp. and azospirillum brasilense Inoculation in greenhouse conditions

Se realizó la inoculación de Azospirillum brasilense inmovilizado en microperlas de alginato y de los hongos Glomus manihotis y Glomus occultum en semillas de Gmelina arborea en tres grados de madurez. Las semillas inoculadas se sembraron en suelo y en turba compactada. Cuarenta y un días después de la siembra se determinó el efecto de los sistemas de siembra y de los microorganismos sobre la germinación. Cuarenta y siete días después del transplante a bolsa se determinaron las variables de micorrización y altura de las plantas. El sustrato de siembra (p<0.01) y la inoculación de A. brasilense (p<0.01) influyeron en la germinación de las semillas de G. arborea. Se presentó correlación positiva entre micorrización y la altura de las plantas durante el establecimiento en vivero (0.61 p=0.03). Además se presentó un efecto sinérgico de los microorganismos sobre la micorrización.

Seeds of Gmelina arborea at three different maturity degrees were inoculated with Glomus manihotis,Glomus occultum and Azospirillum brasilense immobilized in alginate microbeads. Inoculated seeds were sown in two different growing systems: soil and compacted- peat-Jiffy®. Forty-one days after sowing (das), the effects of growing system and microorganism application on seed germination were determined. Forty-seven das, mycorrhization percentages and plant height were evaluated. Results showed that the growing system and the inoculation of A. brasilense have a significant effect (p<0.01) on the germination of G. arborea seeds. A positive correlation between mycorrhization and plantheight was found during the initial stage of establishment in greenhouse conditions (0.61 p=0.03). In addition, there is a synergic effect of both types of microorganisms on mycorrhization.

Bacterias fijadoras asimbióticas de nitrógeno de la zona agrícola de San Carlos. Córdoba, Colombia / Non-symbiotic bacterial diazotrophs from of agricultural crops of San Carlos. Córdoba, Colombia

Se aislaron bacterias diazotróficas de los géneros Azotobacter sp y Azospirrillum sp a partir de la rizosfera de cultivos de plátano, pastos, maíz y de rastrojos (zonas sin cultivar) en el municipio de San Carlos (Valle del Sinú medio en el departamento de Córdoba, Colombia). Las poblaciones microbianas se identificaron mediante pruebas bioquímicas y observación macroscópica y microscópica con tinción de Gram en diferentes medios de cultivo: a) Burk´s, Asbhy y Jensen´s, (género Azotobacter sp), y b) Burk´s, nfb y rojo congo (género Azospirillum sp). El objetivo del presente trabajo fue determinar la producción del ión amonio a partir de los géneros bacterianos aislados;la cuantificación del ión amonio fue llevada a cabo por el método colorimétrico de Berthelot (fenol-hipoclorito) empleando un espectrofotómetro Perkin-Elmer Lamba 11 uv-Vis; la técnica fue modificada y estandarizada de acuerdo con las condiciones del equipo. Como resultado seobtuvieron 14 aislados que produjeron concentraciones de 0,9 hasta 5,2 mg/l, siendo los más destacados para el género Azotobacter sp, A16PG y A26M1P (5,1545 y 5,1743 mg/l de amonio, respectivamente), y para el género Azospirrillum spp, A5M1G (4,6741 mg/l de amonio). La fijación biológica del nitrógeno (fbn) por bacterias diazotróficas ha contribuido a incrementar el rendimiento en las cosechas, reduciendo la necesidad de fertilizantes nitrogenados y la emisión de gases tóxicos como el N2O, obteniendo beneficios económicos y ambientales en las granjas.

Molecular and functional characteristics, growth promoting effect and persistence of selected parent isolates and streptomycin resistant derivatives of rice rhizobacteria.

Molecular and functional characteristics of seven azospirilla and five phosphorus solubilizing bacteria (PSB) isolates of rice rhizosphere, growth promotion ability of two efficient strains, Azospirillum amazonense A10 (MTCC4716) and Bacillus megaterium P5 (MTCC4714) and their persistence based on streptomycin resistant derivatives (SRD), were determined. SDS-PAGE and isozyme banding patterns of the isolates were used to arbitrarily group the azospirilla into 4 and PSB into 3 clusters and as markers to ascertain their identity. The azospirilla produced 2.0 to 10.5 ppm of IAA like substances and showed nitrogenase activity of 0.02 to 3.55 nmole C2H4/hr/ml of pure culture. PSB isolates produced 7.8 to 15.0 ppm IAA like substances and 20 to 128 ppm soluble P. Induction of resistance to streptomycin resulted in changes of these properties. Co-inoculation of rice with SRD A10 and SRD P5 and their parental strains in separate treatments enhanced grain yield over control by 31 and 12.4%, respectively. Nitrogenase activity of rice roots under SRD co-inoculated treatment was higher (4.16 nmole C2H4/hr/hill) than that-under parental strains co-inoculated treatment (3.76 nmole C2H4/hr/hill). SDS-PAGE profile and population count of the strains confirmed their establishment in rice rhizosphere and persistence over a year after inoculation.

Survival of endophytic diazotrophic bacteria in soil under different moisture levels

Neste trabalho foi avaliado o efeito da umidade do solo na sobrevivência de três espécies de bactérias diazotróficas (Azospirillum amazonense, Gluconacetobacter diazotrophicus e Azospirillum brasilense). O teor de umidade apresentou pouca influência na sobrevivência de A. brasilense, considerada uma espécie cosmopolita, enquanto A. amazonense e G. diazotrophicus, consideradas endofíticas, aumentaram o período de culturabilidade na presença de umidade no solo. Os resultados demonstram que o teor de umidade do solo possui maior influência nas espécies endofíticas, em comparação às espécies associativas.

Role of some soil bacteria and acyionomycetes in controlling cucumber root-rot disease

Twenty five isolates of N2-fixing bacteria and eleven actinomycetes were chosen from 117 microorganisms isolated from desert soils and different plant roots to study their effect on Rhizoctonia solani, the causative of the root-rot disease. The data revealed that Azotobacter Rf and Azospirillium Kc were the most active for nitrogen fixation [210 and 133 ppm], root colonization [61.3% and 65%] and antagonistic effect on the pathogenic fungus R. solani [15 and 17 mm]. The two strains were identified as Azotobacter chroococcum and Azospirillium lipoferum. The actinomycetes isolate, Streptomyces Ncu was the most active strain for root colonization ability [41.4%] and antagonistic effect against R. solani [22 mm]. This strain was completely identified as Streptomyces lydicus

Partial characterization of nif genes from the bacterium Azospirillum amazonense

Azospirillum amazonense revealed genomic organization patterns of the nitrogen fixation genes similar to those of the distantly related species A. brasilense. Our work suggests that A. brasilense nifHDK, nifENX, fixABC operons and nifA and glnB genes may be structurally homologous to the counterpart genes of A. amazonense. This is the first analysis revealing homology between A. brasilense nif genes and the A. amazonense genome. Sequence analysis of PCR amplification products revealed similarities between the amino acid sequences of the highly conserved nifD and glnB genes of A. amazonense and related genes of A. brasilense and other bacteria. However, the A. amazonense non-coding regions (the upstream activator sequence region and the region between the nifH and nifD genes) differed from related regions of A. brasilense even in nitrogenase structural genes which are highly conserved among diazotrophic bacteria. The feasibility of the 16S ribosomal RNA gene-based PCR system for specific detection of A. amazonense was shown. Our results indicate that the PCR primers for 16S rDNA defined in this article are highly specific to A. amazonense and can distinguish this species from A. brasilense

Azospirillum amazonense: su presencia en el área cañera de la provincia de Tucumßn / Azospirillum amazonense: its presence in the sugar cane area of the province of Tucumßn

We confirm the presence of Azospirillum amazonense in the sugar cane area of the Province of Tucumßn. Several strains were isolated from sugar-cane roots in different places. Phenotypic and genotypic characteristics coincided with those described for this species. This new one is being added to the other two species, A. brasilense and A. lipoferum, already reported.

Enhancement of polygalacturonase activity during auxin induced para nodulation and endorhizosphere colonization of Azospirillum in rice roots.

Effect of different auxins, namely, 2,4-dichlorophenoxyacetic acid (2,4-D), naphthalene acetic acid (NAA) and indole acetic acid (IAA) and Azospirillum brasilense bioinoculation on the enhancement of polygalacturonase (PG) activity in rice roots during para nodulation and endorhizosphere colonization of Azospirillum was studied under in vitro condition. It was observed that Azospirillum bioinoculation could augment PG activity of rice roots to a lesser extent without any root morphogenesis whereas auxin application together with Azospirillum bioinoculation enhanced PG activity of rice roots to a higher level which resulted in better root morphogenesis (para nodule) and endorhizosphere colonisation of A. brasilense. Among the three auxins tested, 2,4-D, even at lower concentration (0.5 ppm) enhanced the rice root PG activity, root morphogenesis and endorhizosphere colonization of Azospirillum while it was 2.0 ppm with NAA and variable with IAA. It is concluded that there is a positive correlation existing among PG activity, degree of root morphogenesis and endorhizosphere colonization of Azospirillum brasilense in rice roots and the degree of correlation is determined by the chemical composition, concentration and mode of action of the auxin utilised.

Azospirillum inoculation of cereal crops in upper Egypt

This investigation aimed to select efficient Azospirillum strains for inoculation of maize and sorghum at Assiut and Sohag Governorates and reach the best method[s] for inocula application under field condition. Different irrigation regimes and different N-fertilization levels were tested with inoculation to secure the highest yields with least input. The results showed that seed inoculation with peat inocula was better than seed soaking in broth cultures. The double inoculation of maize cultivars [seed treatment + soil spray] gave better growth and yield than the single inoculation as seed treatment. Maize and sorghum cultivars showed variations in response to inoculation with azospirilla. The two-week irrigation intervals were the best regime for maize cultivars, whereas the four-week intervals were the optimum for sorghum cultivars. Significant positive interactions found between inoculation and the two-week irrigation regime in case of maize and between the three-week regime and inoculation in case of sorghum reflected a significant increase in grain yields

Differences in the levels of aminoacylation and contents of modified nucleotides between total tRNAs from N2- and NH4(+)-grown Azospirillum lipoferum cells.

Total tRNAs isolated from N2- and NH4(+)-grown Azospirillum lipoferum cells were compared with respect to amino acid acceptance, isoacceptor tRNA species levels and extent of nucleotide modifications. Amino-acylation of these two tRNA preparations with ten different amino acids indicated differences in the relative acceptor activities. Comparison of aminoacyl-tRNA patterns by RPC-5 column chromatography revealed no qualitative differences in the elution profiles. However, quantitative differences in the relative amounts of some isoacceptors were observed. These results indicate that alterations of relative amounts of functional tRNA species occur to match cellular requirements of the bacterial cells using N2 or NH4+ as nitrogen source. In addition, the content of modified nucleotides in total tRNAs of N2- and NH4(+)-grown cells was determined. In the NH4(+)-grown cells, content of most of the modified nucleotides decreased significantly. Based upon these results, the relationship of chargeability of tRNAs to base modifications is discussed.

Chemical nature of antibiotic produced by pseudomonas fluorescens strain a inhibiting azospirillum spp growth

The antagonistic activity of three Pseudomonas fluorescens strains against Azospirillum spp. was investigated. Data revealed that Pseudomonas fluorescens strain A was the most potent against Azospirillum lipoferum when grown on 523 agar medium, with initial pH of 7.0 and incubation for one day at 30C. Extraction of Pseudomonas fluorescens strain A active ingredient was achieved by using acetone followed by diethyl ether. Using paper chromatography technique, an antibiotic was obtained as purified yellowish brown amorphous powder. It is soluble in methanol, acetone, ethyl acetate, n-butanol, diethyl ether and chloroform. The physicochemical characteristics, i.e., melting point, ultraviolet spectra [UV], infrared spectra [IR], color reactions, the 1H nuclear magnetic resonance [1H-NMR], mass spectra and elemental analysis are given. The purified antibiotic exerted a sharp melting point at 126C, the UV absorption spectra was maximum at 245 [sh] nm in ethylacetate, the molecular weight was found to be 279. Relative abundance [RA], 9.9% and elemental analysis of the antibiotic revealed that the antibiotic substance contains carbon 81.7%, hydrogen 7.5%, oxygen 5.7% and nitrogen 5.0%. The molecular formula of the antibiotic was proposed to C19H21NO based on the MS spectra and elemental analysis

Construction of azospirillum lipoferum gene Library and cloning of Trans-zeatin secretion [TZS] gene

A gene library of total DNA of A. lipoferum ATCC 29708 was constructed using the cosmid vector pV 34. More than two thousand transductants were isolated with inserted fragments of 36.45 kb in average. Trans- zeatin secretion [tzs] gene was isolated by screening the gene library by DNA hybridization using the tzs gene of Agrobacterium tumefaciens as a probe. Three clones were found, p MAZ 1, 2 and 3. The clone p MAZ 1 was 43.5 kb and the tzs gene was localized on the eight kb-Sal I-fragment or on the 5.2 kb fragment of the double digestion Eco RI/Sal I of this plasmid. The successful cloning of tzs gene of A. lipoferum is an evidence for the involvement of DMA transferase enzyme in trans-zeatin [tz] production in Azospirillum

Relation between cellulolytic bacteria and Azospirillum brasilense

The association of different strains of azospirilla and cellulose decomposing bacteria was studied in sterile soil. Azospirilla alone failed to use cellulose as a sole carbon source, while inoculation of a cellulose decomposer markedly increased azospirilla densities. Inoculation of active cellulose-solubilizing bacteria in combination with azospirilla in soil containing cellulose increased the acetylene reducing activity over that due to inoculation with azospirilla alone. Nitrogen gains and efficiency of fixation were markedly increased as a result of the association between Azospirillum and cellulose - solubilizing bacteria. Between 286-402 ug/g N were gained at the end of the incubation period which corresponded to 68.9-80.1 mg N fixed/g carbon oxidized