Use of highly dispersed materials for culturing and isolation of granular Agrobacterium radiobacter preparations ]

The effects of synthetic and natural high-dispersion materials on the growth of Agrobacterium radiobacter were studied. Natural minerals montmorillonite and palygorskite (10 g/l nutrient medium) were more potent than high-dispersion silica and its modified forms in stimulating growth of Agrobacterium radiobacter. The interaction of Agrobacterium radiobacter with clay minerals increased the survival rate of bacteria at supraoptimal temperatures. We elaborated new granular bacterial preparation, which enhanced the productivity of cucumbers by 12-15%.     

Conditions for the production of Agrocin 84 byAgrobacterium radiobacter K84

Summary A chemically defined medium was developed that supported the growth ofAgrobacterium radiobacter K84 and the production of agrocin 84. Various supplements were investigated for their effect on growth rate and production of agrocin 84 using a well-diffusion assay method. Mannitol was found to be a better substrate for growth ofA. radiobacter K84 compared to the other sugar alcohols and sugars tested. By contrast,d-fructose was the better substrate for the production of agrocin 84. Biotin supplementation stimulated production of agrocin 84 but did not eliminate the diauxic lag seen with the basal medium. The opine, octopine, inhibited growth ofA. radiobacter K84 and production of agrocin 84 as did coenzyme B₁₂. By contrast, the cytokinin, isopentenyl adenosine, was marginally stimulatory to production, as was vitamin B₁₂. Acetosyringone supplementation had a negligible effect on growth rate and production of agrocin 84.     

Technical communication: A simple and economically viable medium for the growth of Agrobacterium radiobacter for the production of d-amino acid

A simple and cost effective medium for the growth and d-amino acid production by Agrobacterium radiobacter from hydantoin is reported. The effectiveness of this medium is compared with other synthetic media.     

Effect of vesicular-arbuscular mycorrhizal fungi and ofagrobacterium radiobacter on maize growth

An influence of dual inoculation with the rhizosphere bacteriumAgrobacterium radiobacter, and the VAM fungi,Glomus mosseae andGlomus sp., on maize growth and mycorrhizal infection was observed. Separate inoculations of bacteria or fungi showed significant positive effects on the shoot biomass production of pot-cultured plants only at the last of three consecutive harvests. Plant biomass production was enhanced substantially after a dual inoculation with bacteria and fungi. Synergistic interaction of fungal and bacterial inoculation and growth stimulation was evident at all three harvests compared to uninoculated plants and also compared to plants inoculated with fungi or bacteria only. The dual inoculation increased the shoot biomass of plants by approximately 30% as compared with control. No significant differences were found in mycorrhizal infection between plants uninoculated and inoculated with bacteria.Agrobacterium radiobacter seems to be compatible with mycorrhizal symbiosis and can act a synergistic partner of some VAM fungi.     

Isolation of acetonitrile-utilizing bacteria

Corynebacterium sp. 3 B and three unidentified bacterial strains, all utilizing acetonitrile as a sole carbon and nitrogen source, were isolated from soil by the enrichment culture technique.Agrobacterium radiobacter 8/4/1, also capable of growing on acetonitrile as a sole C and N source, was obtained from a bromoxynil-metabolizing strainA. radiobacter 8/4 using the same method. Resting cells of strains BSA 1 and 3 B produced acetamide and acetic acid during incubation with acetonitrile, whileA. radiobacter 8/4/1 produced acetic acid only.     

Wirkung des pflanzengesundheitsfördernden Rhizobakteriums Agrobacterium radiobacter Stamm G12 gegen den Kartoffelzystennematoden Globodera pallida bei unterschiedlicher Temperatur und Bodenfeuchte

Influence of temperature and soil moisture on the biological control of the potato-cyst nematode Globodera pallida using the plant-health-promoting rhizobacterium Agrobacterium radiobacter Treatment of potato tubers with the plant-health-promoting rhizobacterium A.radiobacter (G 12) resulted in significant (P 0.05) reductions in G. pallida penetration (25%) in green-house studies conducted in a non-sterilized sandy-loam soil. Significant reductions (P 0.05) in nematode infection were obtained when soil moisture was maintained between 60 and 90% of field capacity. When moisture levels were held at 30% of field capacity, nematode infection was also reduced, but not significantly. A. radiobacter repeatedly reduced nematode root-infection levels but did not affect the final population density. A. radiobacter reduced the hatch of G. pallida significantly (P 0.01) in vitro up to 70%. The bacterium effectively reduced nematode hatch at both 20 and 25°C.     

Wirkung des pflanzengesundheitsfördernden Rhizobakteriums Agrobacterium radiobacter Stamm G12 gegen den Kartoffelzystennematoden Globodera pallida bei unterschiedlicher Temperatur und Bodenfeuchte

Influence of temperature and soil moisture on the biological control of the potato-cyst nematode Globodera pallida using the plant plant-health-promoting rhizobacterium Agrobacterium radiobacter Treatment of potato tubers with the plant-health-promoting rhizobacterium A. radiobacter (G 12) resulted in significant (P ≤ 0.05) reductions in G. pallida penetration (25%) in green-house studies conducted in a non-sterilized sandy-loam soil. Significant reductions (P ≤ 0.05) in nematode infection were obtained when soil moisture was maintained between 60 and 90% of field capacity. When moisture levels were held at 30% of field capacity, nematode infection was also reduced, but not significantly. A. radiobacter repeatedly reduced nematode root-infection levels but did not affect the final population density. A. radiobacter reduced the hatch of G. pallida significantly (P ≤ 0.01) in vitro up to 70%. The bacterium effectively reduced nematode hatch at both 20 and 25°C.     

Glyphosate degradation byAgrobacterium radiobacter isolated from activated sludge

Summary Two species of bacteria capable of growth onN-phosphonomethylglycine (glyphosate) were isolated from a bench scale sequencing batch reactor degrading a waste stream containing glyphosate. The enrichment and isolation medium contained defined salts and glyphosate as the sole carbon and energy source. Glyphosate was stoichiometrically degraded to aminomethylphosphonic acid (AMPA). The bacteria have been identified asAgrobacterium radiobacter andAchromobacter Group V D.     

Interaction between barley and mixed cultures of nitrogen fixing and phosphate-solubilizing bacteria

Pot experiments were carried out to investigate the effect of inoculation with pure and mixed cultures of nitrogen fixers Azospirillum lipoferum 137, Arthrobacter mysorens 7 and the phosphate-solubilizing strain Agrobacterium radiobacter 10 on growth and mineral nutrition of two barley cultivars. A significant positive effect on grain yield both of the studied barley cultivars was obtained after inoculation with mixtures of A. lipoferum 137 + A. radiobacter 10 and A. lipoferum 137 + A. mysorens 7 only. The acetylene reduction activity on roots or in batch culture was significantly higher when A. lipoferum 137 and A. radiobacter 10 were combined. Using ¹⁵N isotope dilution technique it was established that these mixed cultures significantly increased the accumulation of nitrogen fertilizer in the plants. The strain A. radiobacter 10 promoted a better accumulation of phosphorus fertilizer by plants and A. mysorens 7 increased the total phosphorus content in plant tissues. The maximum positive effect of joint inoculation on plant development was observed when the combined nitrogen in soil was in short supply. It was concluded that inoculation with bacterial mixtures provided a more balanced nutrition for the plants and the improvement in root uptake of nitrogen and phosphorus was the major mechanism of interaction between plants and bacteria. The introduced bacteria were able to colonize actively the rhizoplane of barley. No interspecific competition or antagonism were established between components of the bacterial mixtures in the rhizoplane. The strains A. mysorens 7 and A. radiobacter 10 improved viability of A. lipoferum 137 when the plants were grown in acid soil. Field experiments carried out on 3 barley cultivars confirmed the assertion that inoculation with mixed cultures significantly increases the grain yield and nitrogenous nutrition of plants as compared with single cultures.     

Effect ofAgrobacterium radiobacter and its polysaccharide on emergence and damping-off of tomato plants

Agrobacterium radiobacter B6 and agrobacteran (an exopolysaccharide of the succinoglycan group) stimulated seed germination and tomato plant emergence. The germination was most stimulated by dipping the seeds in 0.1 % agrobacteran for 30 min whereas plant emergence in garden soil was best with 0.4 % agrobacteran at 10–20°C. Treatment of the plants withA.radiobacter cells and agrobacteran solution at 30–35°C. had a lower effect.A.radiobacter cells applied on seed surface protected the plants against damping off in garden soil artificially inoculated with the fungiRhizoctonia solani andPythium ultimum; in soils contaminated withFusarium solani 0.1 to 0.2% agrobacteran had a higher protective effect than the bacterization. The difference can be attributed to the varying density ofA.radiobacter population in plant rhizosphere in the presence of different plantpathogenic fungi, different interactions of microorganisms in the rhizosphere and different mode of penetration of the pathogens into plant roots.     

Regulation of amino acid and glucose dissimilation in so-called ammonifiers and in other soil microorganisms

Pseudomonas acidovorans and P. putida, isolated from an enrichment culture with casein hydrolysate, and Agrobacterium radiobacter and Torulopsis sp., isolated from a glucose enrichment, were compared with respect to the physiology of ammonification. Decreasing ammonifying ability as well as increasing repression of the synthesis of amino acid degrading enzymes by glucose were found in the above order of organisms. In degradation sequences, observed with P. putida and A. radiobacter as test organisms, substances dissimilated prior to others had both, enhancing and repressing effects on the oxidation of the other compounds. This fact was parallelled by the observation, that in these two bacteria, glucose and single amino acids, when added to the same medium, exerted mutual repression of the synthesis of catabolic enzymes of their partners. The ecological significance of this type of regulation has been discussed.     

Factors effecting chitinase activity of <Emphasis Type="Italic">Rhizobium</Emphasis> sp. from <Emphasis Type="Italic">Sesbania sesban</Emphasis>

Twenty six Rhizobium strains isolated from root nodules of Sesbania sesban were studied for chitinase activity on chitin agar plates. Among them, only 12 strains showed chitinase activity. The strain showing the highest chitinase activity was selected based on maximum clear zone/colony size ratio on chitin agar plates and chitinase activity in culture filtrate. The strain was identified as Rhizobium sp. which showed a high degree of similarity with Rhizobium radiobacter (= Agrobacterium radiobacter). The cultural and nutritional conditions were optimized for maximum chitinase activity. The Rhizobium sp. exhibited maximum chitinase activity after 36 h of incubation, at neutral pH. Among the different nutritional sources, arabinose and yeast extract were found to be good inducers for chitinase activity. Rhizobium sp. could degrade and utilize dead mycelia of Aspergillus flavus, Aspergillus niger, Curvularia lunata, Fusarium oxysporum and Fusarium udum.     

Assessment of the Integral Toxicity of Aquatic Medium Contamination with Oil and Oil Products Using Bacterial Tests

A biotest kit was used to assess the integral toxicity level of aquatic environment contamination with oil and oil products. The integral toxicity dynamics were also monitored during biodegradation of oil and oil products by an association of oil-degrading strains, including Acinetobacter sp., Mycobacterium flavescens, and Rhodococcussp. The following bacterial tests were used: the bioluminescence (BL) test based on Photobacterium leiognathi; electroorientation (EO), optoosmotic (OO), and growth tests; and the reducing activity (RA) test based on an Agrobacterium radiobacter culture. No significant increase in the integral toxicity level of the aquatic medium was observed when diesel fuel and kerosene contamination had been subjected to biodegradation. Although rapid biotests (EO, OO, RA, and BL) detected a pronounced increase in the integral toxicity of the aquatic environment, long-term growth biotests revealed no statistically significant increase in the toxicity level.     

Elicitor activity ofAgrobacterium radiobacter in plants

Agrobacterium radiobacter was tested for the ability to induce the accumulation of phytoalexins and hypersensitive necrotic reaction in pea, bean and potato. A live bacterial suspension with a cell concentration of 1/pL and a solution of a crude polysaccharide produced by the bacteria caused the hypersensitive reaction in potato and bean and the production of phytoalexins in all three species of plants. The results obtained are discussed in connection with the previously found protective effect of the studied strain ofA. radiobacter against soil phytopathogenic fungi. A contribution of defense reactions to the determination of host specificity of the pathogenic strains of theAgrobacterium genus has been proposed.     

Cloning and Identification of anAgrobacterium radiobacter5D-1 Chromosome Fragment Involved in Controlling Nitrogen Metabolism, Biosynthesis of Indolylacetic Acid, and Replication of ColE1 Plasmids

Pleiotropic chromosomal mutations were earlier identified in saprophytic associative bacterium Agrobacterium radiobacter5D-1. The mutations changed nitrogen metabolism, disturbed synthesis of indolylacetic acid (IAA), and conferred the ability to sustain replication of ColE1 plasmid derivatives, which are not normally maintained in bacteria other than Escherichia.The mutations were designated Nr(Nitrogen metabolism) and assigned to a single cluster on an A. radiobactergenetic map. A 420-bp fragment AGH23.1.1 was cloned from an agrobacterial genomic library. Introduced in the Nr mutants as a part of a pUC18-based recombinant plasmid, the AGH23.1.1 fragment complemented the Nrmutations with respect to nitrogen metabolism and IAA biosynthesis, but transformants still sustained replication of ColE1 plasmids. Transformation with the linear AGH23.1.1 fragment was due to substitution of a mutant allele of the nrgene with its wild-type counterpart as a result of recombination and completely restored the wild phenotype in the Nr mutants, including the inability to maintain ColE1 plasmids. The AGH23.1.1 fragment and its flanking regions were sequenced. The established sequence was shown to contain two open reading frames (ORFs) coding for proteins with unknown functions. Thus, the cloned fragment contained a gene(s) that controls nitrogen metabolism and IAA synthesis and replication of ColE1 plasmids inA. radiobactercells. Possible variants of the genetic control of these processes are considered.     

Expression ofRhodopseudomonas sphaeroides carotenoid photopigment genes in phylogenetically related nonphotosynthetic bacteria

The carotenoid photopigment genes of the purple nonsulfur photosynthetic bacteriumRhodospeudomonas sphaeroides have been cloned into the kanamycin resistance transposon Tn5 to create a carotenoid transposon-Tn5-Crt⁺. Transposition of Tn5-Crt⁺ onto the broad host range plasmid pR751 produced a broad host range carotenoid plasmid-pJP115. Transfer of pJP115 to the phylogenetically related nonphotosynthetic bacteriaParacoccus denitrificans, Agrobacterium tumefaciens, Agrobacterium radiobacter, andAzotomonas insolita resulted in carotenoid biosynthesis by these strains. Expression of carotenoid photopigment genes in nonphotosynthetic bacteria lends credence to the evolutionary relationships between photosynthetic and non-photosynthetic bacteria.     

Purification and characterization of a novel type of protocatechuate 3,4-dioxygenase with the ability to oxidize 4-sulfocatechol

4-Aminobenzenesulfonate is degraded via 4-sulfocatechol by a mixed bacterial culture that consists of Hydrogenophaga palleronii strain S1 and Agrobacterium radiobacter strain S2. From the 4-sulfocatechol-degrading organism A. radiobacter strain S2, a dioxygenase that converted 4-sulfocatechol to 3-sulfomuconate was purified to homogeneity. The purified enzyme also converted protocatechuate with a similar catalytic activity to 3-carboxy-cis,cis-muconate. Furthermore, the purified enzyme oxidized 3,4-dihydroxyphenylacetate, 3,4-dihydroxycinnamate, catechol, and 3- and 4-methylcatechol. The enzyme had a mol. wt. of about 97,400 as determined by gel filtration and consisted of two different types of subunits with mol. wt. of about 23,000 and 28,500. The NH₂-terminal amino acid sequences of the two subunits were determined. An isofunctional dioxygenase was partially purified from H. palleronii strain S1. A. radiobacter strain S2 also induced, after growth with 4-sulfocatechol, an „ordinary“ protocatechuate 3,4-dioxygenase that did not oxidize 4-sulfocatechol. This enzyme was also purified to homogeneity, and its catalytic and structural characteristics were compared to the „4-sulfocatechol-dioxygenase“ from the same strain. Received: 5 February 1996 / Accepted: 18 April 1996     

Employment of Rhizobacteria for the Inoculation of Barley Plants Cultivated in Soil Contaminated with Lead and Cadmium

In laboratory experiments, the rhizobacteria Azospirillum lipoferum 137, Arthrobacter mysorens7, Agrobacterium radiobacter 10, and Flavobacterium sp. L30 were found to have a relatively high resistance to the toxic heavy metals lead and cadmium (except that strain L30 was found to be sensitive to Cd). When introduced by means of seed bacterization, the heavy metal–resistant strains actively colonized the rhizosphere of barley plants cultivated in uncontaminated and contaminated soils. In both pot and field experiments, seed bacterization improved the growth of barley plants and the uptake of nutrient elements from soil contaminated with Pb and Cd. The bacterization also prevented the accumulation of Pb and Cd in barley plants, thereby mitigating the toxic effect of these heavy metals on the plants.     

Characterization and identification of bacteria isolated from micropropagated mint plants

Summary Bacterial isolates from contaminated mint shoot cultures were characterized and identified as a preliminary step in determining an elimination treatment. The 22 bacteria were characterized using biochemical and morphological tests and subjected to sensitivity tests with four antibiotics. The isolates were compared with known organisms and assigned to genera according to similarities in characteristics. Seven isolates were analyzed by fatty acid analysis carried out by a commercial laboratory. Six were classified asAgrobacterium radiobacter; eight asXanthomonas; one each asPseudomonas fluorescens, Micrococcus spp.,Corynebacterium spp., andCurtobacterium spp.; four could not be assigned to genera. Inhibition of growth of the bacteria by most antibiotics was best at pH 7.5. Minimal inhibitory concentration and minimal bactericidal concentrations of gentamicin, rifampicin, streptomycin sulfate, and Timentin varied with genotype.     

Production of exopolysaccharide from ethanol inAgrobacterium radiobacter

Agrobacterium radiobacter produces an extracellular polysaccharide from various carbon sources. The exopolysaccharide is produced from ethanol also in a minimal medium containing nitrate as the sole nitrogen source. On cultivation in a medium without CaCO₃ only a minute amount of ethanol is converted to the exopolysaccharide. Both ethanol and nitrate in higher concentrations inhibit the growth rate. In a medium with CaCO₃ the proportion of ethanol converted to the polysaccharide is about ten-fold higher and the inhibitory effect of ethanol and nitrate on the growth rate is analogous to that found in the medium without CaCO₃-Comparison of results of mass and electron balance with a kinetic model shows that the parameters obtained in cultivations with CaCO₃ are less reliable. The balances point to the possibility of formation of other extracellular products.