Use of High-Dispersion Materials for Culturing and Obtaining Granular Agrobacterium radiobacterPreparations

The effects of synthetic and natural high-dispersion materials on the growth of Agrobacterium radiobacterwere 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 radiobacterwith clay minerals increased the survival rate of bacteria at supraoptimal temperatures. We elaborated a new granular bacterial preparation which enhanced the productivity of cucumbers by 12–15%.     

Phylogenetic assignment and mechanism of action of a crop growth promoting <Emphasis Type="Italic">Rhizobium radiobacter</Emphasis> strain used as a biofertiliser on graminaceous crops in Russia

The taxonomic position of “Agrobacterium radiobacter strain 204,” used in Russia as a cereal crop growth promoting inoculant, was derived by a polyphasic approach. The phenotypic analyses gave very similar biochemical profiles for strain 204, Rhizobium radiobacter NCIMB 9042 (formerly the A. radiobacter type strain) and R. radiobacter NCIMB 13307 (formerly the Agrobacterium␣tumefaciens type strain). High percentage similarities, above the species separation level, were observed between the 16S rRNA, fusA and rpoB housekeeping gene sequences of these three strains, and the genomic DNA–DNA hybridisation of strain 204 against the type strain of R. radiobacter NCIMB 9042 was over 70%. Strain 204 is not phytopathogenic and it does not fix atmospheric N₂ or form a physical association with the roots of barley. Strain 204 culture and culture supernatant stimulated the rate of mobilisation of seed reserves of barley in darkness and promoted its shoot growth in the light. Gibberellic acid (GA) concentration was 1.3 μM but indole acetic acid was undetectable (<50 nM) in cultures of strain 204. It is concluded that strain 204 is phenotypically and genotypically very similar to the current R. radiobacter type strain and that the mechanism of its effect on growth of cereals is via the production of plant growth promoting substances. GA is likely to play an important role in the strain 204 stimulation of early growth of barley.     

Effect on microorganisms of volatile compounds released from germinating seeds.

Volatile compounds evolved from germinating seeds of slash pine, bean, cabbage, corn, cucumber, and pea were evaluated for their ability to support growth of microorganisms in liquid mineral salts media lacking a carbon source. Growth of eight bacteria was measured turbidimetrically and of six fungi as dry weight of mycelium. Volatiles caused increased growth of Pseudomonas fluorescens, Bacillus cereus, Erwinia carotovora, Agrobacterium tumefaciens, A. radiobacter, Rhizobium japonicum, Mucor mucedo, Fusarium oxysporum f. conglutinans, Trichoderma viride, and Penicillium vermiculatum but not of Sarcina lutea, Serratia marcescens, Chaetomium globosum, or Schizophyllum commune. Spores of Trichoderma viride showed higher germination in the presence of volatiles. Effects on growth were apparent only during the first 3 or 4 days after planting the seeds. Killed or dried seeds had no effect. The volatiles did not support microbial growth in the absence of nitrogen nor did they supply growth factors. Passing volatiles through KMnO4 or hydrazone reduced growth of the bacteria, indicating that oxidizable organic compounds, primarily aldehydes, were the active components. The volatiles were not absorbed by sterile soil, clay minerals, or water, but they were absorbed by non-steril soil and activated charcoal.     

Toxicity and Mutagenesis of Chrysotile Asbestos toAgrobacterium radiobacter

The mutation of Agrobacterium radiobacter cells exposed to chrysotile asbestos was examined by the random amplified polymorphic DNA (RAPD) method. Approximately 1.4 kbp of DNA in A. radiobacter, which was not amplified strongly in the cells that were not exposed to asbestos, was amplified in the cells that were exposed to asbestos. Mutation in genomic DNA of A. radiobacter was found to be induced by asbestos. Specific DNA that was amplified by asbestos present in PCR products and that which exists latently in genomic DNA were cloned, and these sequences were then determined and compared. It was shown that one of the mutations by the asbestos in the A. radiobacter occurred only in the primer annealed region and was a point mutation or deletion.     

Binding-protein-dependent sugar transport by Agrobacterium radiobacter and A. tumefaciens grown in continuous culture

Binding-protein-dependent sugar transport has been investigated in Agrobacterium radiobacter and A. tumefaciens. A. radiobacter contained two high-affinity glucose-binding proteins (GBP1 and GBP2) that additionally bound D-galactose (KD 0.26 microM) and D-xylose (KD 0.04 microM) respectively and were involved in the transport of these sugars. Partial sequencing of GBP1 and GBP2 showed that GBP2 exhibited significant homology with both the arabinose-binding protein (ABP) and the galactose-binding protein (GalBP) from Escherichia coli, whereas GBP1 exhibited significant homology only with ABP. Antiserum raised against GBP1 cross-reacted with GBP1 but not with GBP2, and vice versa. Anti-GBP1 and anti-GBP2 also cross-reacted with proteins corresponding to GBP1 and GBP2 respectively in A. tumefaciens, but little or no cross-reaction was observed with selected members of the Enterobacteriaceae, Rhizobiaceae and Pseudomonadaceae families grown under glucose limitation. GBP1 was less strongly repressed than GBP2 following batch growth of A. radiobacter on various carbon sources. The growth of A. radiobacter for more than approximately 10 generations in continuous culture under galactose or xylose limitation (D 0.045 h-1) led to the emergence of new strains which exhibited increased rates of glucose/galactose or glucose/xylose uptake, and which respectively hyperproduced GBP1 (strain AR18a) or GBP2 (strain AR9a). Similarly, growth of A. tumefaciens for more than approximately 15 generations under glucose or galactose limitation produced new strains which exhibited increased rates of glucose/xylose or glucose/galactose uptake and which respectively hyperproduced proteins analogous to GBP2 (strain AT9) or GBP1 (strain AT18a). It is concluded that growth of Agrobacterium species under carbon-limited conditions leads to the predictable emergence of new strains which specifically hyperproduce the transport system for the limiting nutrient. The GBP1-dependent system of A. radiobacter is unique amongst these transport systems in that the mutations that lead to its hyperproduction under carbon limitation render it least susceptible to repression by excess glucose during ammonia limitation, with the result that succinoglucan exopolysaccharide is produced from glucose at an enhanced rate.     

Plasmids in avirulent strains of Agrobacterium.

Twelve strains of Agrobacterium radiobacter isolated from naturally occurring crown galls or soil were found to be avirulent on sunflower, tomato, Kalanchoe, and carrot. Eleven strains contained plasmids of molecular weights 77 X 10(6) to 182 X 10(6) as determined by electron microscopy. One strain contained only a smaller plasmid (50 X 10(6) daltons). Several strains had both large and small (ca. 11 X 10(6) daltons) plasmids; one strain contained two large plasmids (112 X 10(6) and 136 X 10(6) daltons). Hybridization reactions of virulence plasmids from Agrobacterium tumefaciens strains C58 and A6 with plasmids from each of the A. radiobacter strains revealed that some A. radiobacter plasmids had less than 10% homology to either the C58 or A6 plasmids. Plasmids from some strains had approximately 50% homology with the C58 plasmid, but only one A. radiobacter plasmid contained more than 10% homology to the A6 plasmid. The presence of large plasmids in A. radiobacter strains did not correlate with sensitivity to agrocin 84; however, the utilization of the amino acid derivatives octopine and nopaline was generally correlated to partial base sequence homology to the C58 plasmid. We conclude that all large plasmids found in Agrobacterium strains are not virulence associated, although they may share base sequence homology with a virulence-associated plasmid. Further, plasmids from tumorigenic strains may be more closely related by base sequence homology to plasmids from nonpathogenic strains than to plasmids from other pathogenic strains.     

Effects of the humic substances of de-inking paper sludge on the antagonism between two compost bacteria and Pythium ultimum

We investigated the in vitro influence of humic substances (HS) extracted from de-inking paper sludge compost on the inhibition of Pythium ultimum by two compost bacteria, Rhizobium radiobacter (Agrobacterium radiobacter) and Pseudomonas aeruginosa. When low concentrations (5 or 50 mg l(-1)) of HS were added to the culture medium, fungal inhibition by R. radiobacter significantly increased (P<0.01) by 2-3%. In contrast, these low levels of HS had no effect on P. ultimum inhibition by P. aeruginosa. The Fe, chelated by HS, was in part responsible for the decrease of P. ultimum inhibition by the bacteria when increasing amounts of HS were added in the culture medium. The addition of 500 mg l(-1) of humic acids isolated from de-inking paper sludge compost or from fossil origin completely eliminated the inhibition of P. ultimum by R. radiobacter. This Fe effect also stimulated growth of R. radiobacter and reduced its siderophore production in a minimal medium supplemented with HS as sole source of Fe. The results showed that HS influence microbial antagonism when added to a culture medium. However, this effect varies with different factors such as the type of bacteria, concentration of HS, molecular weight and Fe content.     

Effect of cadmium on fungi and on interactions between fungi and bacteria in soil: influence of clay minerals and pH.

Fungi (Rhizopus stolonifer, Trichoderma viride, Fusarium oxysporum f. sp. conglutinans, Cunninghamella echinulata, and several species of Aspergillus and Penicillium) tolerated higher concentrations of cadmium (Cd) when grown in soil than when grown on laboratory media, indicating that soil mitigated the toxic effects of Cd. In soil amended with clay minerals, montmorillonite provided partial or total protection against fungistatic effects of Cd, whereas additions of kaolinite provided little or no protection. Growth rates of Aspergillus niger were inhibited to a greater extent by 100 or 250 mug of Cd per g in soil adjusted to pH 7.2 than in the same soil at its natural pH of 5.1. However, there were no differences in the growth rates of Aspergillus fischeri with 100 or 250 mug of Cd per g in the same soil, whether at pH 5.1 or adjusted to pH 7.2. Growth of A. niger and A. fischeri in a soil contaminated with a low concentration of Cd (i.e., 28 mug/g), obtained from a site near a Japanese smelter, did not differ significantly from growth in a soil collected some distance away and containing 4 mug of Cd per g. Growth of A. niger in sterile soil amended with 100 mug of Cd per g and inoculated with Bacillus cereus or Agrobacterium tumefaciens was reduced to a greater extent than in the same soil containing 100 mug of Cd per g but no bacteria. The inhibitory effects of Agrobacterium radiobacter to A. niger were slightly reduced in the presence of 100 mug of Cd per g, whereas the inhibitory effects of Serratia marcescens were enhanced.     

Utilization of octopine and nopaline by Agrobacterium

Tests for utilization of d-octopine and nopaline in defined media containing a carbon and nitrogen source were made on 60 strains of Agrobacterium representing four species and on a representative of each of five species of Rhizobium. Among 46 virulent strains of Agrobacterium, only two strains were found which utilized neither compound, while three strains were found which could utilize both. Of the remaining virulent strains, 27 utilized octopine and 14 utilized nopaline. Each of six strains of A. rhizogenes tested utilized only octopine but at a slower rate relative to growth than most A. tumefaciens. All eight of the A. radiobacter strains failed to utilize either compound, as did four of six nonvirulent strains of A. tumefaciens. The rhizobia did not utilize octopine or, with the possible exception of R. japonicum, nopaline. Virulence in the genus Agrobacterium is concluded to be highly correlated with the ability to utilize one or both of these compounds.     

Biodegradation of Glycerol Trinitrate and Pentaerythritol Tetranitrate by Agrobacterium radiobacter

Bacteria capable of metabolizing highly explosive and vasodilatory glycerol trinitrate (GTN) were isolated under aerobic and nitrogen-limiting conditions from soil, river water, and activated sewage sludge. One of these strains (from sewage sludge) chosen for further study was identified as Agrobacterium radiobacter subgroup B. A combination of high-pressure liquid chromatography and nuclear magnetic resonance analyses of the culture medium during the growth of A. radiobacter on basal salts-glycerol-GTN medium showed the sequential conversion of GTN to glycerol dinitrates and glycerol mononitrates. Isomeric glycerol 1,2-dinitrate and glycerol 1,3-dinitrate were produced simultaneously and concomitantly with the disappearance of GTN, with significant regioselectivity for the production of the 1,3-dinitrate. Dinitrates were further degraded to glycerol 1- and 2-mononitrates, but mononitrates were not biodegraded. Cells were also capable of metabolizing pentaerythritol tetranitrate, probably to its trinitrate and dinitrate analogs. Extracts of broth-grown cells contained an enzyme which in the presence of added NADH converted GTN stoichiometrically to nitrite and the mixture of glycerol dinitrates. The specific activity of this enzyme was increased 160-fold by growth on GTN as the sole source of nitrogen.     

Biological Control of Crown Gall: Seed and Root Inoculation

S ummary . Experiments on the control of crown gall by inoculating susceptible plants with a non-pathogenic strain of Agrobacterium radiobacter have continued. In all experiments, highly significant disease control was achieved. In one experiment, 42% of untreated plants growing in soil heavily infested with A. radiobacter var. tumefaciens died; inoculation of seed with the non-pathogenic strain reduced this to nil. Combined seed and root inoculation was more efficient than seed inoculation alone. In naturally infested soil, combined seed and root inoculation at transplanting gave 99% control of gall formation (as dry weight). A significant increase in plant growth resulted from combined seed and root inoculation. At transplanting, roots should probably be inoculated within 2 h of lifting. This method of biological control is now widely practised by commercial growers in South Australia.     

Attachment of Agrobacterium tumefaciens B6 and A. radiobacter K84 to Tomato Root Tips

Agrobacterium tumefaciens B6 and the avirulent Agrobacterium radiobacter strain K84 attached to in vitro-cultured tomato root tips, but the binding of strain B6 to root tips was greater than the binding of strain K84. Strain K84 was not able to block the attachment of A. tumefaciens B6 to in vitro-cultured tomato root tips.     

Urea and Biuret as Nitrogen Sources for Rhizobium Spp.

Ability to utilize urea as nitrogen source proved to be universal in 80 fast and 40 slow growing strains of Rhizobium spp. from 23 genera of host plants, and also in 10 strains of Agrobacterium radiobacter. Rhizobium meliloti (32 strains) as well as A. radiobacter constantly failed to utilize biuret. Most rhizobia from other host plants (with 8 exceptions among 88) were able to use biuret as a somewhat suboptimal source of nitrogen, which was generally assimilated at a slower rate than urea and rarely resulted in the same amount of growth, particularly in the fast growing strains; in exceptional cases and mostly among the slow growing strains biuret appeared slightly superior to urea. Adaptation experiments showed that urease occurred as a constitutive enzyme in Rh. leguminosarum , while the biuret decomposing enzyme appeared to be inducible.     

Modeling growth and succinoglucan production by Agrobacterium radiobacter NCIB 9042 in batch cultures

Wild-type Agrobacterium radiobacter NCIB 9042 has been cultivated in batch cultures on a synthetic medium which was adapted for growth and succinoglucan production. Experiments were carried out in a 4-L stirred-tank aerated reactor. Glucose, biomass, polysaccharide, protein, and inorganic- and organic-nitrogen concentrations were measured, and oxygen consumption and CO(2) production rates were obtained by a gas-balance technique. Nitrogen balance shows that inorganic nitrogen is entirely recovered into proteins. The carbon balance is satisfied with in +/-5%. Stoichiometric equations for biomass growth and succinoglucan synthesis were established. The biosyntheticpolymer pathways including ATP and cofactor consumption were investigated. From previous studies, a (P/O) value of 1.66 is selected for oxygen sufficient cultures. The actual ATP requirements of 25.4 mmol ATP/g succinoglucan (38.5 mol ATP/mol succinoglucan), determined by a metabolic analysis, is 2.39 times the stoichiometric value. Experimental results were modeled by a system of differential equations. The exponential growth phase was described by a nitrogen-limited Monod equation. Subsequent succinoglucan synthesis followed a slightly modified Luedeking-Piret relation partitioning internal and external polysaccharide. Experimentally determined coefficients are compared with published results for continuous culture of A. radiobacter NCIB 11883.     

The relationship between glucose transport and the production of succinoglucan exopolysaccharide by Agrobacterium radiobacter

Agrobacterium radiobacter NCIB 11883 was grown in ammonia-limited continuous culture at low dilution rate with glucose as the carbon source. Under these conditions the organism produced an extracellular succinoglucan polysaccharide and transported glucose using the same periplasmic glucose-binding proteins (GBP1 and GBP2) as during glucose-limited growth. Transition from glucose- to ammonia-limited growth was accompanied by a very rapid decrease in glucose uptake capacity, whereas the glucose-binding proteins were diluted out much more slowly (t1/2 approximately 1 h and 14 h respectively). Although the rate of glucose uptake and the concentrations of GBP1 and GBP2 were much lower during ammonia limitation, the activities of enzymes involved in the early stages of glucose metabolism and in the production of succinoglucan precursors were essentially unchanged. Glucose transport was also investigated in two new strains of A. radiobacter which had been isolated following prolonged growth under glucose limitation. Glucose uptake by strain AR18 was significantly less repressed during ammonia limitation compared with either the original parent strain or strain AR9, and this was reflected both in its relatively high concentration of GBP1 and in its significantly higher rate of succinoglucan synthesis. Flux control analysis using 6-chloro-6-deoxy-D-glucose as an inhibitor of glucose transport showed that the latter was a major kinetic control point for succinoglucan production. It is concluded that glucose uptake by A. radiobacter, particularly via the GBP1-dependent system, is only moderately repressed during ammonia-limited growth and that the organism avoids the potentially deleterious effects of accumulating excess glucose by converting the surplus into succinoglucan.     

Starch Gel Electrophoresis of Catalase and Esterase Isoenzymes from Some Rhizobium and Agrobacterium spp.

S ummary : The esterase and catalase isoenzymes of Agrobacterium tumefaciens and A. radiobacter have been compared to those of the fast growing rhizobia using starch gel electrophoresis. An overall similarity was found for the agrobacteria-rhizobia group although the majority of the agrobacteria possessed only one catalase. Those agro-bacteria with 2 catalases gave patterns similar to Rhizobium meliloti and were 3-ketolactose negative. Agrobacterium pseudotsugi isoenzymes, although fitting the general agrobacteria-rhizobia pattern, were not identical to any specific isogram. The taxonomic value of these findings is discussed.     

Dissimilatory ammonia production vs. denitrification in vitro and in inoculated agricultural soil samples

A dissimilatory ammonia-producing isolate identified as Enterobacter amnigenus and a denitrifier identified as Agrobacterium radiobacter isolated from the same soil were studied. The products of nitrate reduction in a minimal medium, enriched with glucose and containing nitrate N as the sole nitrogen source, were quantified when each of these isolates was cultured anaerobically, alone or mixed together in the presence or absence of C(2)H(2). When they were cultured together, ammonia was the principal product of nitrate reduction. The distribution between denitrification and dissimilatory ammonia production (DAP) for nonsterilised soil samples inoculated with E. amnigenus or A. radiobacter, or a mixture of these two isolates, was also investigated. Production of NH(4)(+) was increased under these conditions (strict anaerobiosis and much available fermentable carbon), but the inoculation of soil samples with 1.2?×?10(7) cells of E. amnigenus·g dried soil(-1) was not sufficient to shift nitrate reduction from nitrous oxide (denitrification) to ammonia production, suggesting that inoculation with a greater number of DAP bacteria than introduced would probably be required to enable ammonia production to exceed nitrous oxide release. Key words: dissimilatory ammonia production, denitrification, Enterobacter amnigenus, Agrobacterium radiobacter.     

Carbohydrate Catabolism of Selected Strains in the Genus Agrobacterium

Radiorespirometric and enzyme analyses were used to reveal the glucose-catabolizing mechanisms functioning in single strains of seven presumed Agrobacterium species. The Entner-Doudoroff and pentose cycle pathways functioned in A. radiobacter, A. tumefaciens, A. rubi, and A. rhizogenes. Whereas both catabolic pathways were utilized to an almost equal degree in the A. radiobacter and A. tumefaciens strains, use of the Entner-Doudoroff pathway predominated in the A. rubi and A. rhizogenes strains. A stellulatum catabolized glucose almost solely through the Entner-Doudoroff pathway. In A. pseudotsugae and A. gypsophilae, glucose was metabolized mainly through the Emden-Meyerhof-Parnas pathway; the pentose phosphate pathway was also utilized.     

Biological Control of Crown Gall: Seed Inoculation

S ummary : Peach seeds were inoculated with the nonpathogenic isolate 84 of Agrobacterium radiobacter var. radiobacter biotype 2 before sowing in natural soil heavily inoculated with the tumour inducing biotype 2 isolate 27 ( A. radiobacter var. tumefaciens ). Nonpathogens (presumably isolate 84) became predominant in the biotype 2 population on roots, on underground stems and in the soil round plant crowns. Significant ( P < 0·001) biological control of crown gall was achieved. Total gall incidence on plants grown from inoculated seed was 31% and from uninoculated seed 79%; the corresponding gall incidence on plant crowns was 12% and 76%. Dusting seed with Thiram (3·1 g/kg seed) did not significantly reduce disease incidence. Infection appeared to occur through undamaged lenticels indicating that wounding is not a necessary prerequisite for crown gall induction in peach.