Survival of Rhizobiumin a Monoculture and Binary Population with Rhizosphere Bacteria

The survival of pure cultures of Rhizobium leguminosarumbv. pisumand Rhizobium trifoliiand their interaction with associative diazotrophic and phosphate-mobilizing bacteria after inoculation of sterile soil were studied. The viable heterotypic diazotrophic and rhizobial phosphate-mobilizing association was formed whose efficiency was 14% (clover) and 28% (pea) higher compared to monorhizobial inoculates.     

Possible Involvement of Phage-Like Structures in Antagonism of Cowpea Rhizobia by Rhizobium trifolii

A reduction in the viability of cowpea rhizobia was observed when Rhizobium trifolii IARI and cowpea Rhizobium strain 3824 were inoculated together in soil. The reduction in number of cowpea rhizobia in soil was found to be associated with the reduction in number of nodules per plant and retardation in plant growth. An antimicrobial substance was isolated from R. trifolii which, on electron microscopic investigation, demonstrated the presence of several phage-like structures.     

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.     

Interaction of yeasts and some nitrogen fixing bacteria on nodulation of legumes

Summary Combined inoculation ofRhizobium trifolii withSaccharomyces cerevisiae and other yeasts generally enhanced the number of nodules, length of plants and dry weight of Egyptian clover (Trifolium alexandrinum) seedlings grown on agar slopes. Similar effects were observed when seedlings were inoculated withR. trifolii in the presence of dialyzed culture filtrate ofS. cerevisiae.     

Effect of drought on three species of Rhizobium

Summary Three samples of soil were air dried and the reduction in population ofRhizobium meliloti, Rhizobium trifolii and a rhizobium of the ‘Lotus’ group was estimated by use of a plant-infection technique. The cells ofRhizobium trifolii proved to be more tolerant of the severe drought than did the cells of the other two species.     

Saprophytic competence of acid tolerant strains ofRhizobium trifolii in acid soil

Summary Laboratory prescreening ofRhizobium trifolii for acid tolerance, based upon the ability of rhizobia to grow in acid media (pH 4.2) containing Al (15 M), was successful for the selection of strains capable of survival in acid soil.Both sterile and non-sterile soils of varying acidity were inoculated with several strains ofR. trifolii.Acid tolerant strains generally had significantly higher populations at every sample period than an acid sensitive strain. Amelioration of soil acidity by liming improved persistence of all strains. Soil sterilization by autoclaving adversely affected survival of all strains at each soil acidity level.Paper Number 8766 of the Journal Series, North Carolina Agricultural Research Service, Raleigh, NC 27650, USA.     

Studies of natural populations and mutants of rhizobium in the improvement of legume inoculants

Summary A study has been made of the symbiotic effectiveness ofRhizobium trifolii in fields ofTrifolium subterraneum in south-eastern Australia, with the purpose of providing background information for a programme of inoculant strain improvement. The strains found varied widely in symbiotic effectiveness. The distribution patterns of effectiveness varied from year to year and from locality to locality. From 24 to 65% of strains ofR. trifolii from different localities exhibited antagonism to selectedR. trifolii indicator strains. These reactions were mainly due to mild antibiotic effects but bacteriocinogenic strains and strains producing specific, virulent or wide-range, temperate phages, were also common. Factors to be considered in the selection and evaluation of inoculant strains from among natural populations are discussed and the possible role of genetic manipulation is examined. It is concluded that the general application of DNA transformation and transduction is restricted because of the limited degree of DNA homology among strains, the limited host-range of transducing phages and the lack of suitablein vitro screening procedures for symbiotic characters. A mutational model is presented in which characters known to be associated with symbiotic effectiveness would be manipulated by mutation and back-mutation to effect quantitative increases in effectiveness.     

Competition between inoculum strains ofRhizobium japonicum in the process of soybean nodulation during three planting periods

Competition between inoculum strains ofRhizobium japonicum D 216 and 311 B applied to soybean seeds in mixed inocula depended, especially in the year of inoculation, directly on the ratio of the cell numbers of the two inoculum strains in mixtures. Uninoculated plants grown in original soil contaminated in August 1969 by inoculated seed and stored subsequently in original pots without watering for 8 months displayed, after the spring (May) and summer (August) sowing in 1970, a statistically significant nodulation shift in favour of the D 216 strain. The highest nodulatiou was achieved with all inoculation treatments during spring sowing in 1970.     

The effect of gum arabic on infection and nodulation of cluster clover by Rhizobium

Summary Gum arabic is an adhesive used in pelleting legume seeds with Rhizobium. The present study shows that the gum generally enhances the number of infected root hairs, promotes early nodulation and significantly increases the number of leaves during initial nodulation of cluster clover (Trifolium glomeratum) inoculated with Rhizobium trifolii under bacteriologically controlled conditions.     

Stimulation of Nodulation in the Clover-Rhizobium trifolii 0403 System by Penicillin and Mecillinam

The number of nodules produced per clover seedling inoculated with Rhizobium trifolii 0403 can be increased almost 2-fold by the addition of penicillin or mecillinam. Two-day-old dutch white clover seedlings grown in 250 milliliter boston round jars containing agar-solidified plant growth medium were inoculated with exponentially growing Rhizobium trifolii 0403 cells. Penicillin or mecillinam (100 micrograms per milliliter) were added immediately or after 24 hours. Following 42 days growth, 10 replicate sets of 5 plants for each treatment were assayed for nodule number, plant dry weight, and Kjeldahl nitrogen. Both antibiotics increased nodule number, plant dry weight, and Kjeldahl nitrogen. Increases in nodule number and dry weight were statistically significant. The range of values in Kjeldahl nitrogen was so extensive as to make the data insignificant at the P < 0.05 level, however nodule number, plant dry weight, and Kjeldahl nitrogen displayed a significant correlation with each other. There were no significant differences in treatment with either antibiotic or with time of treatment. Nodule number increased by about 85%, and plant dry weight and nitrogen increased by about 30%.     

Isolation and characterization ofSporomusa acidovorans sp. nov., a methylotrophic homoacetogenic bacterium

Exopolysaccharides (EPS) of nodulating strains of Rhizobium trifolii and Rhizobium leguminosarum added to red clover seedlings before inoculation reduced the number of nodules. The inhibition of the nodulation was correlated with the amount of EPS. The preparations of EPS from mutants defective in early stages of nodulation (Roa^- or Hac^-) did not affect the nodulation, whereas EPS from mutants deficient in late stages (post Hac^-) exerted an inhibitory effect.Inactive preparation of EPS contained less O-acetyl groups and pyruvic acid residues. Deacetylation and depyruvylation of EPS from R. trifolii Nod⁺ abolished it inhibitory effect. It was concluded that noncarbohydrate substitutions (acetate, pyruvate) are involved in EPS effect.Abbreviations CPS capsular polysaccharide - EPS exopolysaccharide - LPS lipopolysaccharide - Nod nodulation - Fix nitrogen fixation - Hac root hairs curling - Roa root adhesion     

Effect of lime-pelleting on the nodulation of lucerne (Medicago sativa L.) in an acid soil: A comparative study carried out in the field,in pots and in rhizotrons

The nodulation of lucerne was studied in soil (pH-H₂O 5.2) with seeds either inoculated with Rhizobium meliloti (R), or inoculated and pelleted with lime (RP). For comparison, experiments were done in the field and in two types of micro-cosmos: pots and rhizotrons. In the field experiments, lime-pelleting improved the establishment of seedlings and augmented the nitrogen yield of the first harvest. These positive responses in plant growth were the consequence of a better nodulation on the upper 10 mm of the seedling tap root. The number of seedlings carrying crown nodules increased from 18% (R) to 56% (RP) at 26 days after sowing.In both, pots and rhizotrons, lime-pelleting also increased crown nodulation: in pots from 32% (R) to 60% (RP), and in rhizotrons from 5% (R) to 90% (RP). Rhizotrons, made of plastic petri dishes, allowed for continuously following of early root developments and nodule formation. Crown nodulation could already be measured after 14 days. Based on these experiments, it was concluded (i) that crown nodulation is an adequate parameter to quantify the benefit of lime-pelleting, and (ii) that rhizotrons, because of the more pronounced effects and shorter incubation time, are more suitable to study the nodulation responses in the soil caused by the addition of rhizobia and lime.     

Short-term survival of rhizobia on arrowleaf clover seed sown at different depths

Seed of arrowleaf clover (Trifolium vesiculosum Savi) were inoculated with a streptomycin resistant mutant ofRhizobium leguminosarum biovartrifolii and planted on the surface of a Norwood fine sandy loam and at 10 and 25 mm depths. Populations of rhizobia declined from an excess of 10,000 seed⁻¹ immediately after inoculation to less than 100 within three to four days after sowing on the soil surface when water was the peat inoculant adhesive. Gum arabic as the adhesive promoted the survival of rhizobia. Populations of rhizobia on surface sown seed declined much more rapidly than on seed buried in soil. Although, the soil was nearly air dry, rhizobia on buried seed survived at populations exceeding 1,000 seed⁻¹. The maximum soil temperatures ranged between 21 and 36°C over the sampling time and did not seem to have a major influence on short term survival of rhizobia. Delayed germination of seed due to the higher temperature would indirectly influence the number of viable rhizobia present at germination.     

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.     

Conjugational transfer of the nodulation-conferring plasmid pWZ2 in Rhizobium trifolii

Summary When the nodulating Rhizobium trifolii strain 24Vio^r containing plasmid RP4 was conjugated with the non-nodulating R. trifolii mutant strain 24Str^rNod^-35, plasmid RP4 was transferred at a frequency 10^-3–10^-4. Two out of nearly three thousand tested transconjugants which contained plasmid RP4 had acquired the ability to form nodules on clovers. Molecular studies of the DNA of both these nodulating transconjugants showed the presence of plasmid RP4 and another plasmid which was not found in the original recipient strain. The size of this second plasmid corresponded to that of the plasmid pWZ2, the elimination of which was correlated with irreversible loss of the nodulating ability of R. trifolii strain 24 (Zurkowski and Lorkiewicz 1979). Plasmid RP4 was eliminated from cells by ethidium bromide, without the loss of nodulating properties. The nodulation capacity, however, was eliminated from transconjugants after incubation of bacteria at elevated temperature. Non-nodulating clones obtained after such incubation did not contain the plasmid pWZ2. The results indicate that the plasmid pWZ2 is a necessary element for induction of nodules by R. trifolii, and that it can be mobilized by plasmid RP4.     

Scanning Electron Microscopy of Rhizobium trifolii Infection Sites on Root Hairs of White Clover

White clover root hairs which were inoculated with Rhizobium trifolii 4S (infectious strain) contained infection threads which were observed by light microscopy and scanning electron microscopy. Three morphological types of root hairs retaining infection threads were recognized. The bacteria were strongly attached between the surfaces of two plant cell walls as follows: between surfaces of a root hair tip curled back on itself, between a protuberance from a root hair and its cell surface, or between two root hair tips clinging together. An anatomical analysis documented the attachment site of the infection thread sheath from the inside of the root hair cell.     

Interaction of Azospirillum and Rhizobium Strains Leading to Inhibition of Nodulation

Rhizobium-Azospirillum interactions during establishment of Rhizobium-clover symbiosis were studied. When mixed cultures of Azospirillum and Rhizobium trifolii strains were simultaneously inoculated onto clover plants, no nodulation by R. trifolii was observed. R. trifolii ANU1030, which nodulated clover plants without attacking root hairs, i.e., does not cause root hair curling (Hac⁻), did not show inhibition of nodulation when inoculated together with Azospirillum strains. Isolation of bacteria from surface-sterilized roots showed that azospirilla could be isolated both from within root segments and from nodules. Inhibition of nodulation could be mimicked by the addition of auxins to the plant growth medium.     

Microscopic analysis of the effect ofRhizobium leguminosarum biovartrifolii host specific nodulation genes in the infection of white clovers

Summary A microscopic assessment is presented of the comparative infection capacity of wild-type and hybrid strains ofRhizobium leguminosarum bv.viciae withR. l. bv.trifolii strain ANU 843 on white clover seedlings. TheR. l. bv.viciae hybrid strains contained defined DNA segments coding for different combinations ofR. l. bv.trifolii host-specific nodulation genes. White clover plants were examined over a 72 h period to assessRhizobium infectivity, the morphological changes in root hair growth; colonisation ability of rhizobia; infection thread initiation and the ability to induce cortical cell division.R. l. bv.viciae strain 300 induced root hair curling more slowly than strain ANU 843 or any of the hybrid strain 300 bacteria, and when curling had taken place, there was poorer colonization by strain 300 within the folded hair cell, no evidence of infection thread formation and only limited cortical cell division 72 h after inoculation. The addition of the host-specific nodulation genes ofR. l. bv.trifolii to strain 300 was necessary to induce infection threads and establish a normal pattern of nodulation of the roots of white clovers.     

Effects of some organophosphate and carbamate insecticides on nitrification and legume growth

Summary Nine insecticides (six organophosphates and three carbamates) were tested for their effects on soil nitrification, growth of legume seedlings, and growth of four species of rhizobia bacteria. No inhibition of nitrification was found at normal field rates (5 ppm) of application. Some instances of inhibition were observed at 50 ppm and at 500 ppm. Similarly, 5 ppm applications did not inhibit growth of alfalfa or sweetclover seedlings ... with one exception. Disc inhibition tests of the rhizobia bacteria showed thatRhizobium leguminosarum andRhizobium trifolii were most sensitive to the pesticides.Rhizobium meliloti, and particularlyRhizobium japonicum, were resistant to the insecticides. No consistent correlation was observed between tests on the nodulating bacteria and the tests on legume growth.Published with the approval of the Director of the North Dakota Agricultural Experiment Station as Journal Article No.309. Portion of a thesis presented by the senior author in partial fulfillment of the requirements for the M.S. degree in bacteriology at North Dakota State University.     

Effect of soil Pseudomonads on colonization of hazel roots by the ecto-mycorrhizal species Tuber melanosporum and its competitors

Hazel (Corylus avellana L.) seedlings, inoculated with the ecto-mycorrhizal fungus Tuber melanosporum (Vitt.) were grown in pots with soil previously inoculated with fluorescent Pseudomonads. These bacteria have been previously isolated from a typical truffle orchard soil. The effect of Pseudomonads on T. melanosporum synthesis proceeded by a two-stage process. Tuber melanosporum infection first declined and then resumed to almost the original level. Infection by competing mycorrhizal fungi declined after 6 months and was very low after 12 months, at the end of the experiment. The same dynamic process occurred with all bacterial strains tested, regardless of their taxonomical group or iron-chelating power. However, some isolates of Pseudomonads seemed to be more efficient than others. A protective effect of Pseudomonads against soil-borne competitors of mycorrhizae was postulated to occur in orchards, consequently favouring the continuation of the T. melanosporum symbiosis. Prospects for use of Pseudomonads in nurseries are discussed.