Competition studies withRhizobium trifolii in laboratory experiments

Summary Competition studies were carried out in soil cores by comparing the nodule forming ability of antibiotic resistance marked strains, inoculated at three inoculum levels onTrifolium repens versus an effective indigenous Rhizobium population of 1.5×10⁵/gm. It was seen that the indigenous marked isolate G1067 formed a high proportion of nodules sampled (>90%) at all three inoculum levels (10⁵, 10⁷ and 10⁹ cells/seed) wherein the introduced foreign strain G1032 formed >50% of the nodules at the highest inoculum level.In a test tube experiment, competition for nodule sites was examined by inoculating mixtures of twoR. trifolii strains at different input levels on two cultivars ofTrifolium repens var. Huia and Titan. It was seen that G1032 was less competitive than G1006 and G1067 on cv. Huia but was more competitive on cv. Titan than the other two strains.     

Selection of Bradyrhizobium strains and provenances of Acacia mangium and Faidherbia albida: Relationship with their tolerance to acidity and aluminium

This work was designed to determine the role of the acidity and aluminium stress in the selection of partners in the Acacia symbioses with relevance to the persistence of the microsymbiont Bradyrhizobium in the soil and the growth and nodulation of the host plant respectively. Fifteen strains of Bradyrhizobium from Acacia mangium and Faidherbia albida formed a very homogenous acid tolerant group as indicated by their ability to grow better in a medium at pH 4.5 than in a medium at pH 6.8. By contrast, a growth experiment using an acid liquid media (pH 4.5), containing different concentrations of aluminium successfully identified strains sensitive to aluminium toxicity and those able to grow even in the presence of 100 M AlCl₃.Our results suggest that high amounts of aluminium in the soil rather than acidity (pH 4.5) were a major soil factor for selection of Bradyrhizobium strains capable of establishing a permanently high population under natural conditions.Unlike the behaviour of the microsymbiont, growth and nodulation of Acacia mangium and Faidherbia albida were not affected by aluminium, even at 100 M, but they might be significantly affected by medium acidity (pH 4.5) depending on plant provenances. It is therefore suggested that ability of the host plant to tolerate acidity stress should be taken into account first when screening effective Acacia-Bradyrhizobium combinations for use in afforestation trials.     

Siderophore and organic acid production in root nodule bacteria

Nineteen strains of root nodule bacteria were grown under various iron regimes (0.1, 1.0 and 20 M added iron) and tested for catechol and hydroxamate siderophore production and the excretion of malate and citrate. The growth response of the strains to iron differed markedly. For 12 strains (Bradyrhizobium strains NC92B and 32H1, B. japonicum USDA110 and CB1809, B. lupini WU8, cowpea Rhizobium NGR234, Rhizobium meliloti strains U45 and CC169, Rhizobium leguminosarum bv viciae WU235 and Rhizobium leguminosarum bv trifolii strains TA1, T1 and WU95) the mean generation time showed no variation with the 200-fold increase in iron concentration. In contrast, in Bradyrhizobium strains NC921, CB756 and TAL1000, B. japonicum strain 61A76 and R. leguminosarum bv viciae MNF300 there was a 2–5 fold decrease in growth rate at low iron. R. meliloti strains WSM419 and WSM540 showed decreased growth at high iron.All strains of root nodule bacteria tested gave a positive CAS (chrome azurol S) assay for siderophore production. No catechol-type siderophores were found in any strain, and only R. leguminosarum bv trifolii T1 and bv viciae WU235 produced hydroxamate under low iron (0.1 and 1.0 M added iron).Malate was excreted by all strains grown under all iron regimes. Citrate was excreted by B. japonicum USDA110 and B. lupini WU8 in all iron concentrations, while Bradyrhizobium TAL1000, R. leguminosarum bv viciae MNF300 and B. japonicum 61A76 only produced citrate under low iron (0.1 and/or 1.0 M added iron) during the stationary phase of growth.Abbreviations CAS chrome azurol S - HDTMA hexadecyltrime-thylammonium bromide     

Siderophore production and utilization byRhizobium trifolii

Summary Several strains ofRhizobium trifolii were tested for their ability to synthesize and utilize phenolate or hydroxamate types of siderophores. None of the nodulating strains ofR. trifolii was able to produce detectable amounts of siderophores. Only the non-nodulating strainR. trifolii AR6 formed a phenolate siderophore, which stimulated the growth of the siderophore-negative mutant AR65. Other strains ofR. trifolii could not utilize iron from exogenously supplied Desferal, pseudobactin or citrate. The siderophore fromR. trifolii AR6 and 2,3-dihydroxybenzoic acid slightly stimulated the growth of someR. trifolii strains.     

Cellulase production by Rhizobium

Summary The production of cellulase byRhizobium species was studied.Rhizobium trifolii cellulase was induced by a variety of polysaccharides, including celluloses and hemicelluloses. Cellobiose and myo-inositol also allowed enzyme expression but mannitol prevented it at concentrations higher than 0.25%. Both soluble and insoluble plant root substances moderately stimulated cellulase production byRhizobium trifolii.Most substances tested did not induce the production of cellulases by the slow-growing, cowpea type rhizobia strain CIAT 79. Effective inducers were carboxymethylcellulose, gluconate and myo-inositol.Cellulase production was very low under all conditions tested. In most cases the enzyme activity was loosely bound to the capsular material. The enzyme in fast-growers is an 1,4--D-glucan-4-glucanohydrolase (endo-glucanase EC 3.2.1.4) with specificity for high molecular weight polysaccharides.There was no correlation between infectiveness ofRhizobium trifolii strains and cellulase production. One strain, which lacks the nodulation plasmid, produced cellulase at the same rate as its parental infective strain.     

Effects of pH,Ca and Al on the exudation from clover seedlings of compounds that induce the expression of nodulation genes inRhizobium trifolii

The expression of nodulation genes inR. trifolii is induced by flavone compounds present in clover root exudates. In the present experiments a bioassay with an indicator strain ofR. trifolii, which contained thelacZ gene fromEscherichia coli fused to theR. trifolii nodA gene, was used to measure the level ofnod gene expression inR. trifolii. Compounds that stimulatednodA gene expression were shown to be present in exudates of white clover (Trifolium repens L.) and nine cultivars of subterranan clover (T. subterraneum L.) seedling sgrown at a range of pH between pH 3.0 and pH 8.0. Thenod gene-induction activity of exudates was, however, reduced when seedlings of all clover species were grown at pH>7.0 and at pH<4.0 and pH<5.0 for white clover and subterranean clover respectively. No major differences were apparent in the activity of exudates from seedlings of the various cultivars of subterranean clover.Nod gene-induction activity of exudates was shown to increase markedly with seedling age. The presence of Ca at concentrations up to 10 mM in seedling culture solutions also resulted in marked increases in thenod gene-induction activity of seedling exudates. Increases in activity due to the presence of Ca were most apparent at low pH where between 5 and 10-fold increases were observed for white clover and subterranean clover respectively. Conversely, the presence of Al at concentrations up to 60 M in seedling culture solutions had no effect on thenod gene-induction activity of seedling exudates.The observations that both low pH and Ca concentrations affected thenod gene-induction activity of seedling exudates suggested that the net presence of stimulatory flavones in root exudates was an important contributing factor to the acid-sensitive step in nodule formation.     

Survival and multiplication ofRhizobium japonicum strains in silt loam

Summary Antibiotic resistant mutants 8-0 Str^R, 110 Tet^R and 138 Kan^R derived from wild typeRhizobium japonicum strains were inoculated into silt loam soil to cell concentrations greater than 2×10⁸/g of soil. Population changes were monitored using antibiotic media and strain identification was done using immunodiffusion assay on microcores of soil. Immunodiffusion bands formed by the mutant strains with homologous antisera essentially duplicated bands formed by the parent strain. Strains 110 Tet^R and 8-0 Str^R had cross reacting antigens whereas antigens of strain 138 Kan^R reacted only with the homologous antiserum. Populations ofR. japonicum strains introduced into sterile soil increased over a period of four weeks under both single and mixed culture inoculations. All populations decreased by the end of six weeks and thereafter remained constant. When theseR. japonicum strains were introduced into non-sterile soil, the population did not increase over the initial population added. Population decreased gradually for two weeks and then maintained thereafter. It was possible to recover very low populations of antibiotic resistantR. japonicum strains from both sterile and unsterile soils using media containing specific antibiotics. Detection ofR. japonicum strains by immunodiffusion was accomplished only when the population was 10⁹ cells/g of soil. The method using antibiotic resistant mutants permitted an evaluation of the interactions of variousR. japonicum strains in soil with respect to their survival and multiplication.     

Introduction and recovery of Clavibacter michiganensis subsp. michiganensis from agricultural soil

Twelve phytopathogenic Clavibacter michiganensis subsp. michiganensis strains were introduced into non-sterile agricultural loam soil at an inoculum density of about log. 6.0 cfu g^–1 dry weight soil. The soil samples were incubated at 22°C under a 12h light, 12h dark cycle and the population densities followed over a 30-day period by plating subsamples of serial dilutions of soil on Brain Heart Infusion agar amended with 0.5% (w/v) yeast extract and 30 g mL^–1 nalidixic acid. In 5 soil samples C. michiganensis cfu were not detected after 30 days incubation. Initially, C. michiganensis cfu accounted for about 90% of the cfu recovered but decreased to less than 10% after 30 days. These results suggested that some C. michiganensis strains survive in this particular soil, while other strains exhibit poor survival and/or may be difficult to detect when present in low numbers.     

Utilization of the tricarboxylic acid cycle intermediates and symbiotic effectiveness inRhizobium meliloti

Summary The utilization of the tricarboxylic acid cycle intermediates and related compounds was studied in strains ofRhizobium meliloti having different symbiotic effectiveness. In general, the very effective (VE) strains used these compounds as sole carbon source better than the ineffective (I) strains. However, a significant different was observed between VE and I strains in their ability to use acetate or oxaloacetate for growth. In fact, at a concentration of 2 mM, 80% of the VE strains used acetate or oxaloacetate white 50% of the I strains used acetate and none was able to grow on oxaloacetate. No correlation was found between the symbiotic effectiveness of the strains and their ATP content, when grown on mannitol. The highest ATP content (9.21 nM×g protein^–1) was found in the I strain S₂₀ and the lowest (0.69 nM×g protein^–1 was found in the effective strain S₈. Numerical analysis of the patterns of utilization of the TCA cycle intermediates and related compounds indicated that the 49 strains tested formed 11 distinct groups at 86% similarity, according to Jaccard's coefficient. Several strains showed unique patterns of utilization and can be clearly identified under laboratory conditions.Contribution no.225 Station de Recherches, Agriculture Canada.     

Survival and symbiotic characteristics of rhizobium in saline-alkali soils

Summary Survival, growth and symbiotic performance of rhizobia isolated from normal, saline-sodic and mildly acidic soils were studied in original and amended saline-alkali soils. Rhizobia of 4 out of 9 legumes studied for nodulation were found to be present in a highly saline-sodic soil. Majority of the strains of these bacteria did not survive in the original saline-sodic soil of pH 10.5 but as the pH was amended to lower than 10.0, all the strains survived in the soil. Virtually no differences were noticed in the survival and symbiotic characteristics of native and exotic strains ofRhizobium leguminosarum andRhizobium trifolii in the saline-sodic soil, though wide variations were observed among individual strains irrespective of their ecological origin. Rhizobia were found to possess greater tolerance for alkalinity than their host legumes. However, delayed nodulation in lentil (Lens esculenta) and berseem (Trifolium alexandrinum) resulting in decreased yield of the plants at pH values higher than 9.0 was observed.     

Studies on the respiration of efficient and inefficient strains of Rhizobium

Summary In a pot culture experiment using sterilised soil, growth and nitrogen content of Berseem (Trifolium alexandrinum) inoculated with 15 strains of Rhizobium trifolii and of pea (Pisum sativum) inoculated with 10 strains of Rhizobium leguminosarum were found to vary considerably depending on the strains of the respective Rhizobium used. Out of the 15 strains of Rhizobium trifolii, 6 strains were found to be highly efficient (increasing the nitrogen content by more than 70 per cent over the control-uninoculated) and the rest as either moderately efficient (increase in N by 30–50 per cent) or inefficient (increase in N by 4 to 20 per cent) strains. Similarly, out of the 10 strains of Rhizobium leguminosarum 5 strains were found to be highly efficient, 1 moderately efficient and the rest were more or less inefficient strains.The respiration rate (l O₂ consumed per hour per mg dry cells) of efficient and inefficient strains of Rhizobium trifolii and Rhizobium leguminosarum in glucose, maltose and mannitol substrate did not bear any relation to their efficiency. However, the stimulation of the respiratory rate of the Rhizobium strains due to the addition of glycine to the glucose substrate was found to be significantly more in case of efficient strains of Rhizobium trifolii and Rhizobium leguminosarum than those of the inefficient ones.The data presented in the paper is taken from the thesis submitted by the Senior author, to the P. G. School, IARI, N. Delhi, in 1968, for Ph. D. degree.     

Enantioselective hydrolysis of O-acetylmandelonitrile to O-acetylmandelic acid by bacterial nitrilases

Bacteria were enriched from soil samples, using benzylcyanide, -methyl-, -ethyl- or -methoxybenzyl-cyanide as the sole source of nitrogen. All isolated strains belonged to the genus Pseudomonas. Resting cells of the isolates hydrolysed O-acetylmandelonitrile to O-acetylmandelic acid, O-acetylmandelic acid amide and mandelic acid. From racemic O-acetylmandelonitrile all isolates preferentially formed R(–)-acetylmandelic acid ( = d-acetylmandelic acid). The enantioselective hydrolysis of O-acetylmandelonitrile could also be demonstrated in vitro. Crude extracts did not hydrolyse O-acetylmandelic acid amide indicating an enantioselective nitrilase rather than a nitrile hydratase/amidase system.     

SelectingRhizobium phaseoli strains for use with beans (Phaseolus vulgaris L.) in Kenya: Infectiveness and tolerance of acidity and aluminium

Forty strains ofRhizobium phaseoli, isolated from Kenyan soils, were tested for infectiveness on common bean (Phaseolus vulgaris L.). 28 strains were infective and a cultivar × Rhizobium interaction was observed. 48 strains were screened for tolerance of acidity and Al in liquid culture. Assessment of visible turbidity after 14 days indicated 34 strains tolerant of pH 4.5 but none tolerant of pH 3.5. No strain was tolerant of 50 M Al at pH 5.5. Three strains were tolerant of 20 M Al at pH 5.5 and 10 M Al at pH 4.5. Screening on a solid medium identified strains tolerant of 20 and 50 M Al at pH 5.5 and 4.5 which were sensitive to these treatments in liquid culture. Those strains tolerant to 20 M Al at pH 4.5 and 5.5 in liquid culture were correctly identified on the solid medium.     

Action ofN-methyl-N′-nitro-N-nitrosoguanidine inRhizobium trifolii

Rhizobium trifolii was highly resistant to the lethal effect ofN-methyl-N-nitro-N-nitrosoguanidine (MNNG), but it was sensitive to the mutagenic action of this chemical. A concentration of 500g/ml yields a survival of between 1% and 10%, which allows us to obtain a higher number of mutants than lower concentrations that yield higher survival rates. Lethal damage produced by nitrosoguanidine was repaired, and repair is inhibited by acriflavine.     

Genetic organization ofAcetobacter for acetic acid fermentation

Plasmid vectors for the acetic acid-producing strains ofAcetobacter andGluconobacter were constructed from their cryptic plasmids and the efficient transformation conditions were established. The systems allowed to reveal the genetic background of the strains used in the acetic acid fermentation. Genes encoding indispensable components in the acetic acid fermentation, such as alcohol dehydrogenase, aldehyde dehydrogenase and terminal oxidase, were cloned and characterized. Spontaneous mutations at high frequencies in the acetic acid bacteria to cause the deficiency in ethanol oxidation were analyzed. A new insertion sequence element, IS1380, was identified as a major factor of the genetic instability, which causes insertional inactivation of the gene encoding cytochromec, an essential component of the functional alcohol dehydrogenase complex. Several genes including the citrate synthase gene ofA. aceti were identified to confer acetic acid resistance, and the histidinolphosphate aminotransferase gene was cloned as a multicopy suppressor of an ethanol sensitive mutant. Improvement of the acetic acid productivity of anA. aceti strain was achieved through amplification of the aldehyde dehydrogenase gene with a multicopy vector. In addition, spheroplast fusion of theAcetobacter strains was developed and applied to improve their properties. ADH membrane-bound alcohol dehydrogenase - ALDH membrane-bound aldehyde dehydrogenase - IS insertion sequence - NTG N-methyl-N-nitro-N-nitrosoguanidine - PQQ pyrroloquinoline quinone     

Use of an organic buffer for the selection of acid tolerantRhizobium meliloti strains

Summary Nine media used to grow rhizobia were examined for their ability to maintain a stable low pH during the growth ofR. meliloti Large fluctuations in the pH of all media were recorded within 72 h, indicating their unsuitability for use in the selection of acid tolerant rhizobia. Morpholino-ethanesulphonic acid (MES) was assessed for its ability to buffer the pH of the media whilst still permitting rapid growth ofR. meliloti, R. trifolii, andBr. lupini. With 30.7 mM MES, the pH of a defined medium containing galactose, arabinose, and glutamate did not change from the initial value of 5.5 even though rhizobial numbers increased from 10⁴ to 10⁹ cells.ml^–1. Even at a buffer concentration of 15.3 mM, pH only increased from 5.5 to 5.6. There was no effect of the buffer on rhizobial growth.     

Tolerance ofRhizobium leguminosarum bv.phaseoli to acidity and drought

Ten strains ofRhizobium leguminosarum bv.phaseoli isolated from soils of Morocco were more tolerant than three culture collection strains to acid conditions in culture media or in sterile soil. The survival rate of a tolerant strain in a sandy acid soil was greater than a sensitive strain at different humidity levels. These properties should give locally selected strains an advantage in nodulatingPhaseolus vulgaris roots in soils similar to those used here.     

Ethanol production from sulphuric acid wood hydrolysate ofPinus radiata using free and immobilized cells ofPichia stipitis

Summary Ethanol was produced by a strain ofPichia stipitis adapted to an inhibitory acid wood hydrolysate ofPinus radiata. The best ethanol productivity for batch cultures was 0.21 g/l h at 0.7% ethanol. Varying culture conditions increased ethanol concentration to 0.76%, however the productivity decreased to 0.18 g/l h. A decrease in ethanol concentration in the culture fluid was noted late in the batch which suggested ethanol catabolism. Values of kinetic parameters (K _m,K _s, _max, andV _max) were evaluated for this system. The use of calcium alginate immobilized cells in a continuous-flow stirred tank reactor lead to enhanced fermentative performance, namely a maximum productivity of 0.27 g/l h and 1.13% ethanol yield. The immobilized cells in continuous flow reactors represent an attractive option for fermenting sugars released by sulphuric acid hydrolysis ofP. radiata wood.     

Competitive growth of slow growingRhizobium japonicum against fast growingEnterobacter andPseudomonas species at low concentrations of succinate and other substrates in dialysis culture

A cultivation system with simultaneous growth of six bacterial cultures in separate bags in dialysis culture was developed. In a medium with no added carbon source (one half concentrated Hoagland solution, water deionized and distilled), cell number ofRhizobium japonicum increased during a 7 day period by a factor of 35, whereas the number ofEnterobacter aerogenes cells decreased to one half. With a concentration of 100 nM succinate as an additional carbon source in the inflow,Rhizobium japonicum 61-A-101 cell number increased by a factor of 50 during an 8 day period, whereas cell number ofEnterobacter cloacae NCTC 10005 only doubled and ofEnterobacter aerogenes NCTC 10006 decreased. At 10 mM concentration of succinate in the inflow, doubling time the twoEnterobacter strains was about 12 h, compared to about 24 h for theRhizobium japonicum strain. Varying the succinate concentration from 10 mM to 100 nM in the inflow,Rhizobium japonicum 61-A-101 surpassed theEnterobacter aerogenes strains in the growth rate between 1 mM and 100 M succinate in the inflowing medium. Three otherRhizobium japonicum strains (fix⁺ and fix^-) did grow with a similar rate as strain 61-A-101 at very low concentrations of substrate. Growth rates for the strains were confirmed by protein data per culture. Growing in competition with twoPseudomonas strains,Rhizobium japonicum RH 31 Marburg (fix^-) did overgrow alsoPseudomonas fluorescens, was however outgrown byPseudomonas putida. In utilizing low concentrations of a¹⁴C labelled organic acid (malonate), three strains ofRhizobium japonicum left 2–4 times smaller amounts of¹⁴C in the medium than two species ofPseudomonas and two species ofArthrobacter.On sabbatical leave at ANU     

Production and properties of xylanases from thermophilic actinomycetes

30 strains of xylanolytic thermophilic actinomycetes were isolated from composted grass and cattle manure and identified as members of the generaThermomonospora, Saccharomonospora, Microbispora, Streptomyces andActinomadura. Screening of these strains for extracellular xylanase indicated that strains ofSaccharomonospora andMicrobispora generally were poor xylanase producers (0.5–1.5 U/ml) whereas relatively high activities were observed in cultures ofStreptomyces andActionomadura (4–12 U/ml).A preliminary characterization of the enzymes of strains of the latter genera suggested that xylanases of all the strains ofActinomadura exhibited higher thermostabilities than those ofStreptomyces. To evaluate the potential of thermophilicActinomadura for industrial applications, xylanases of three strains were studied in more detail. The highest activity levels for xylanases were observed in cultures grown on xylan and wheat bran. The optimal pH and temperature for xylanase activities ranged from 6.0 to 7.0 and 70 to 80°C. The enzymes exhibited considerable thermostability at their optimum temperature. The half-lives at 75°C were in the range from 6.5 to 17h. Hydrolysis of xylan by extracellular xylanases yielded xylobiose, xylose and arabinose as principal products. Estimated by the amount of reducing sugars liberated the degree of hydrolysis was 55 to 65%. Complete utilization of xylan is presumably achieved by -xylosidase activities which could be shown to be largely cell-associated in the 3Actinomadura strains.