Effect of strains of Rhizobium trifolii on the establishment of oversown white clover (Trifolium repens)

Strains of Rhizobium trifolii incorporated into commercial peat inoculants were compared for their effect on the establishment and growth of oversown white clover (Trifolium repens) on soils devoid of infective rhizobia.There were marked differences in numbers of seedlings establishing and clover dry matter production per hectare with the various strains. However, when adjusted to a constant number of established seedlings, dry matter production from all strains, apart from one strain at one site, were similar indicating that the strains did not appear to influence the growth of individual clover plants.The marked differences in establishment of clover inoculated with the various strains could not be accounted for by differences in the number of rhizobia in the peat inoculant.Selecting strains of rhizobia for ability to increase establishment is considered important where clover is oversown onto soils devoid of rhizobia.     

Nodulation of Trifolium subterraneum by introduced rhizobia in competition with naturalized strains

The ability of 4 strains of Rhizobium trifolii to compete with naturalized strains in nodulating Trifolium subterruneum cv. Mt Barker and cv. Woogenellup was assessed at 5 sites in New South Wales. The populations of naturalized rhizobia at these sites ranged from 4 × 10⁶ rhizobia/g to one where no rhizobia were detected. The introduced strains were inoculated singly or as mixed strain inocula onto seed of the host at 2 × 10⁶ rhizobia/seed. There were marked differences in competitive ability between the strains but these differences were modified by the host cultivar and the site.At the R. trifolii-free site the inoculum strain formed 100% of the nodules in the 1st yr; by the second year serologically unrelated strains had invaded the plots and these formed almost all of the nodules in the 3rd yr. At the site where competition was greatest (4 × 10⁶ naturalized rhizobia/g), there were no differences in the competitive abilities of the strains in the first year but at all other sites WU95 was superior whether used as a single strain or in a mixed strain inoculum. In these sites also the proportion of nodules formed by the inoculum strains declined markedly by the 2nd yr.     

Competitive ability of selected Cyclopia Vent. rhizobia under glasshouse and field conditions

This study tested the competitive ability of three locally isolated Cyclopia rhizobia and strain PPRICI3, the strain currently recommended for the cultivation of Cyclopia, a tea-producing legume. Under sterile glasshouse conditions, the three locally isolated strains were equally competitive with strain PPRICI3. In field soils, the inoculant strains were largely outcompeted by native rhizobia present in the soil, although nodule occupancy was higher in nodules growing close to the root crown (the original inoculation area). In glasshouse experiments using field soil, the test strains again performed poorly, gaining less than 6% nodule occupancy in the one soil type. The presence of Cyclopia-compatible rhizobia in field soils, together with the poor competitive ability of inoculant strains, resulted in inoculation having no effect on Cyclopia yield, nodule number or nodule mass. The native rhizobial population did not only effectively nodulate uninoculated control plants, they also out-competed introduced strains for nodule occupancy in inoculated plants. Nonetheless, the Cyclopia produced high crop yields, possibly due to an adequate supply of soil N.     

Inoculation responses of perennial forage legumes grown in fresh hill‐land ultisols as affected by soil acidity‐related factors

A growth chamber experiment was initiated with two field moist, marginal and acidic (pH 5.1–5.2) soils of the Lily series (Typic Hapludults) in order to determine the need for improved legume‐rhizobia symbioses for forage species of current, or potential, use in the renovation of Appalachian hill‐land pastures. One soil was from an abandoned pasture having broomsedge (Andropogon virginicus L.) as the predominant vegetation, whereas the other was from a minimally‐managed pasture dominated by orchardgrass (Dactylis glomerata L.). Treatments included inoculation (or no inoculation) and the addition of aluminum, nil, or lime to provide a range of soil acidities. Both soils contained effective populations of naturalized rhizobia for white clover (Trifolium repens L.) and red clover (Trifolium pratense L.), but low and/or ineffective naturalized populations of rhizobia for alfalfa (Medicago sativa L.), birdsfoot trefoil (Lotus corniculatus L.), bigflower vetch (Vicia grandiflora Scop.), and flatpea (Lathyrus sylvestris L.). Seed inoculation, by lime‐pelleting, was highly beneficial in establishing effective symbioses for all these latter species. The addition of low levels of aluminum or lime (1.5 and 2.0 cmol/kg soil, respectively) had little effect on any of the symbioses, with the exception of those for alfalfa. Thus, an improved legume rhizobia symbiosis would not seem to be a prerequisite for renovating pastures established on chemically similar ultisols with the forage legume species examined in this study, especially if the pasture has at least some history of management.     

Competitive abilities of common field isolates and a commercial strain of Rhizobium leguminosarum bv. trifolii for clover nodule occupancy

We previously reported that commercial Rhizobium leguminosarum bv. trifolii inoculants failed to outcompete naturalized strains for nodule occupation of clover sown into an alkaline soil [Aust. J. Agric. Res. 53 (2002) 1019]. Two field isolates that dominated nodule occupancy at the field site were labeled with a PnifH-gusA marker. Marked strains were chosen on the basis that they were equally competitive and fixed similar amounts of nitrogen in comparison to their parental strain. The minitransposon insertions were cloned and sequence analysis revealed that neither lesion disrupted the integrity of any known gene. The marked strains were then used to follow nodule occupancy of Trifolium alexandrinum in competition against the commercial inoculant TA1 under a range of experimental conditions. In co-inoculation experiments in sand-vermiculite, TA1 outcompeted each marked field isolate for nodule occupancy. However, using TA1-inoculated seed sown into alkaline soil containing a marked field strain, it was demonstrated that by increasing the cell number of marked rhizobia in the soil and reducing the cell number of the commercial inoculant, the proportion of nodules occupied by TA1 was reduced. These studies indicate that the ability of the field isolates to dominate nodule occupancy in the alkaline field soils was most likely caused by poor commercial inoculant survival providing the advantage for naturalized soil rhizobia to initiate nodulation.     

Growth of perennial forage legumes in acidic soils of the Appalachian Hill‐Lands after liming

A major constraint to the renovation of forage legume‐based pastures on acidic soils of the Appalachian hill‐lands is thought to be the absence of effective rhizobia. A growth chamber experiment was done with aluminum (Al) toxic, low pH (≥ 4.2) soils from four series (Berks, Lily, Tate, and Westmoreland) that were planted with alfalfa (Medicago sativa L.), red clover (Trifolium pratense L.), white clover (Trifolium repens L.), or birdsfoot trefoil (Lotus corniculatus L.). These soils, without lime addition, were previously shown not to contain effective, naturalized populations of rhizobia for these plant species. However, a non‐toxic, pH 6.8, Watauga soil was shown to have such rhizobia but only for alfalfa. In the present study, these five soils were reexamined after liming to pH ≥ 5.5 for effective, naturalized populations of rhizobia and the efficacy of soil inoculation with commercially available rhizobia. In addition to effective, naturalized R. meliloti for alfalfa in the Watauga soil, similar populations of R. trifolii for red clover, and R. lotus for birdsfoot trefoil, were now found. Such rhizobia were also found for alfalfa in the Lily soil and for red clover in the Lily and Tate soil. Thus, liming allowed the expression of effectiveness of natural rhizobia that otherwise would not have been detected in soil pot experiments without lime. Inoculation of the toxic soils after lime addition with commercial rhizobia was effective in about half of the soil‐plant combinations that did not contain populations of effective, naturalized rhizobia. Asymbiotic shoot growth of all the plant species was significantly (P ≤ 0.05) correlated with soil pH over a range of 5.5–6.6. These results indicate that, in the absence of effective, naturalized populations of rhizobia, improvement of rhizobial inocula could increase forage production by ～34% for some species on some of the toxic soils, even after the pH of the soils is increased to ≥ 5.5.     

Diversity of native rhizobia isolated in south Brazil and their growth promotion effect on white clover (Trifolium repens) and rice (Oryza sativa) plants

In this study, rhizobia strains isolated from white clover (Trifolium repens) root nodules were evaluated in an effort to identify an efficient nitrogen-fixing rhizobia strain that can also improve the growth of rice plants (Oryza sativa). White clover plants were collected from seven sites in south Brazil, and 78 native rhizobia isolates were obtained. The genetic diversity analysis of those isolates was carried out by BOX-polymerase chain reaction. Overall, the native rhizobia isolated showed a high genetic diversity, but when the bacterial isolates from the same site were compared, the diversity was lower. One native rhizobia, POA3 (isolated from the Porto Alegre locality), was able to promote the growth of both plants and is therefore a good candidate for new inoculant formulation. Finally, we can conclude that the community of native rhizobia symbiont of white clover plants in southern Brazil is highly diverse and the growth promotion effect of rhizobia inoculation on rice plants was more pronounced in a poor nutrient substrate condition than in a rich nutrient substrate condition.     

Lime pelleting inoculated serradella (Ornithopus spp.) increases nodulation and yield

Lime pelleting of the inoculated seed is recommended for most pasture legume species to improve survival of the rhizobia on the seed and to counter deleterious effects of soil or fertiliser acidity on rhizobial numbers. Except for New South Wales, lime pelleting is specifically not recommended for serradella (Ornithopus spp.). Our objectives were to evaluate effects of lime pelleting on bradyrhizobial numbers on seed, and nodulation and growth of the serradella plants. Three experiments are reported at two acid-soil sites in northern New South Wales involving four cultivars of yellow serradella (Ornithopus compressus) and Bradyrhizobium sp. (Lupinus) strains WSM471 (current inoculant strain) and WU425 and WSM480. Lime pelleting increased bradyrhizobial numbers on seed, 24 h after inoculation, by an average of 90%. Similarly, lime pelleting increased nodulation and shoot dry matter of the inoculated plants by an average of 57 and 28%, respectively. The three strains were similar in effects on plant growth. Relative values for shoot dry weight, averaged over sites, were 100 for WSM471 and 98 for both WU425 and WSM480. Our results confirmed previous research that lime pelleting inoculated serradella seed was not deleterious to survival of the bradyrhizobial inoculum, and showed that it could result in enhanced symbiotic activity of the inoculum in some instances. We recommend lime pelleting of serradella and that WSM471 remain the inoculant strain.     

Ecological studies of root-nodule bacteria introduced into field environments—V.A critical examination of the stability of antigenic and streptomycin-resistance markers for identification of strains of Rhizobium tripolii

Marked strains of Rhizobium trifolii, distinguishable from other strains antigenically and by streptomycin resistance, were introduced by seed inoculation of subterranean clover (Trifolium subterraneum L.) into a field environment having a natural population of R. trifolii. Isolates from nodules obtained periodically during the following 41 months were classified using both methods of identification in parallel. This procedure made it possible to determine the reliability of each method independently.There was a gradual disappearance of the inoculum strains which occurred more rapidly in plots of cv. Woogenellup than in plots seeded with cv. Mount Barker. At five harvests, there was 95% (or greater) correspondence between inoculum survival using either method of identification. There was evidence that a small proportion of the progeny of the inocula sustained independent loss of antigenic character and/or streptomycin resistance in the field or, alternatively, that strains occurring naturally acquired these characteristics. A few nodules contained more than one strain of rhizobia. These exceptions occurred at low frequency and did not interfere substantially with identification results. It is concluded that gel immune diffusion serology and the use of streptomycin-resistant mutants are both reliable methods for identifying strains of rhizobia reisolated from field environments.     

Numbers of clover rhizobia needed for crown nodulation and early growth of clover in soil

Low soil populations of Rhizobium leguminosarum biovar trifolii indicate a need for inoculating clovers (Trifolium sp.) at planting. The number of rhizobia in soil varies considerably from field to field and the number needed for nodulation on the upper taproot and for vigorous seedling development is not known. Two experiments were undertaken using arrowleaf clover (T. vesiculosum Savi) and crimson clover (T. incarnatum L.) grown in pots filled with soil. Two soils were used; one contained 10 indigenous rhizobia g^-1 and the other contained fewer than three. The treatments consisted of amending each soil with two strains of inoculant rhizobia to contain from 10 to approximately 1×10⁶ rhizobia g^-1 followed by planting to clover. The number of nodules near the top of the root increased as the number of rhizobia in the soil increased to the highest inoculum level. A low number (approximately 1×10³ to 1×10⁴) of rhizobia was sufficient for maximal N content of seedlings. It seems that soil containing 100 or fewer rhizobia g^-1 may respond to inoculation with increased crown nodulation and seedling vigor.     

Survival and plasmid stability of rhizobia introduced into a contaminated soil

The behaviour of Rhizobium strains introduced separately into soil from a contaminated site with high concentrations of heavy metals (mainly Zn and Hg), and the role of plasmids in the ecology of these rhizobia strains were studied. Six Rhizobium leguminosarum biovar trifolii strains, from different sources and with different plasmid contents, were selected. Two of them were isolated from nodules of subterranean clover plants (Trifolium subterraneum) grown in the contaminated soil and four were from an uncontaminated soil. After inoculation with approximately 10⁷ cells g⁻¹ soil, of each strain, survival and plasmid stability were assessed over a period of 12-18 months. Differences in survival of Rhizobium strains were only detected more than 12 months after inoculation. After 18 months it was clear that survival in contaminated soil was greatest in the two strains originally isolated from that contaminated soil, and also by two of the strains originally isolated from uncontaminated soil. The latter two strains were also the only ones that showed changes in their plasmid profiles. The remaining isolates had the lowest populations, and their plasmid profiles were unchanged and similar to the parent strains.     

Screening clover and Lotus rhizobia for tolerance of acidity and aluminium

Clover rhizobia (55 strains) were screened for tolerance of acidity and Al, using the technique of Keyser and Munns (1979). Assessment of visible turbidity after 14 days indicated three strains tolerant of pH 4.5 (although growth rate was reduced), 25 strains tolerant of 5μm Al and no strains tolerant of 50 μ m Al at pH 5.5.50 μmAl caused a decrease in the numbers of acid-tolerant strains at pH 4.5. Tolerance of acidity or Al was not associated with the pH or Al status of the soil from which a strain was isolated.Screening of eight strains of clover rhizobia and nine strains of Lotus rhizobia using turbidity assessment and viable counts indicated seven strains of clover rhizobia with different degrees of tolerance of 20 μm Al but none tolerant of 50 μm Al at pH 5.5. All Lotus rhizobia (both slow- and fast-growers) were tolerant of 20 and 50 μm Al at pH 5.5, with 50 μm Al causing a reduction in growth rate.Subculturing of strains in non-stressed and stressed media had no effect on the response to 50 μmAl at pH 5.5.     

Population densities of clover rhizobia in Texas pastures and response to liming

Summary Clovers are widely used forage legumes on acidic soils in Texas and need inoculation with appropriate rhizobia when first introduced. Acidic soils are not conducive to survival of clover rhizobia. A survey of pastures was undertaken to determine the number of rhizobia present. The effect of liming acidic soils on the survival of clover rhizobia was also evaluated in the laboratory. The number of clover rhizobia was more than 100 cells g^-1 soil in 70% of the pastures surveyed but populations within pastures varied by more than two orders of magnitude. The number of years of clover production beyond 1 year did not affect the rhizobial population density. The soil pH of twelve samples was below 5.0 and six samples had populations of rhizobial lower than 100 g^-1 soil. Eleven out of sixteen samples from fields that had grown clover and had pH values above 6.0 had populations exceeding 1000 g^-1 soil and only three samples had populations lower than 100 g^-1 soil. Incubating indigenous or inoculated rhizobia in well-mixed soils having pH values of 5.1 or below resulted in populations declining to below 10 g^-1 soil in 6 weeks. Mixing of soils with pH values of up to 5.4 induced reduction of rhizobial numbers, possibly by destroying microsites. Liming of soils to increase pH values above 5.5 improved survival of native or inoculated rhizobia in most cases.     

Sampling effects on the assessment of genetic diversity of rhizobia associated with soybean and common bean

Biological nitrogen fixation plays a key role in agriculture sustainability, and assessment of rhizobial diversity contributes to worldwide knowledge of biodiversity of soil microorganisms, to the usefulness of rhizobial collections and to the establishment of long-term strategies aimed at increasing contributions of legume-fixed N to agriculture. Although in recent decades the use of molecular techniques has contributed greatly to enhancing knowledge of rhizobial diversity, concerns remain over simple issues such as the effects of sampling on estimates of diversity. In this study, rhizobia were isolated from nodules of plants grown under field conditions, in pots containing soil, or in Leonard jars receiving a 10⁻² or a 10⁻⁴ serially-diluted soil inoculum, using one exotic (soybean, Glycine max) and one indigenous (common bean, Phaseolus vulgaris) legume species. The experiments were performed using an oxisol with a high population (10⁵ cells g⁻¹ soil) of both soybean rhizobia, composed of naturalized strains introduced in inoculants and of indigenous common-bean rhizobia. BOX-PCR was used to evaluate strain diversity, while RFLP-PCR of the ITS (internally transcribed spacer) region with five restriction enzymes aimed at discriminating rhizobial species. In both analyses the genetic diversity of common-bean rhizobia was greater than that of soybean. For the common bean, diversity was greatly enhanced at the 10⁻⁴ dilution, while for the soybean dilution decreased diversity. Qualitative differences were also observed, as the DNA profiles differed for each treatment in both host plants. Differences obtained can be attributed to dissimilarity in the history of the introduction of both the host plant and the rhizobia (exotic vs. indigenous), to host-plant specificity, rhizobial competitiveness, and population structure, including ease with which some types are released from microcolonies in soil. Therefore, sampling method should be considered both in the interpretation and comparison of the results obtained in different studies, and in the setting of the goals of any study, e.g. selection of competitive strains, or collection of a larger spectrum of rhizobia. Furthermore, effects of sampling should be investigated for each symbiosis.     

Symbiotic effectiveness of Bradyrhizobium japonicum in acid soils can be predicted from their sensitivity to acid soil stress factors in acidic agar media

In acid soil, low pH, reduced availability of nutrients, and toxicity of Al and Mn limit plant growth and the survival and effectiveness of rhizobia. The symbiosis between legumes and rhizobia is particularly sensitive to acid soil stress. A pot experiment evaluated whether Bradyrhizobium japonicum strain growth on acidic agar media would predict ability to colonize the rhizosphere and form effective nodules in acidic soils. Three Indonesian strains of B. japonicum with similar effectiveness at neutral pH in sand culture but with different tolerance of acid soil stress factors in agar media, and an acid-tolerant commercial strain (CB1809) of comparable effectiveness, were tested in three acid soils using the Al tolerant soybean (Glycine max cv PI 416937). At 7 days after inoculation all strains had achieved large rhizosphere populations, but by day 14 the rhizosphere population of the acid-sensitive strain had decreased, while the more acid-tolerant strains increased. The acid-tolerant strains had significantly greater nodulation and symbiotic effectiveness than plants inoculated with the acid-sensitive strain. Laboratory prescreening of B. japonicum for acid, Al and Mn tolerance in acid media successfully identified strains which were symbiotically competent in low pH soils.     

Colonization of rhizospheres and nodulation of two Vigna species by rhizobia inoculated onto seed: Influence of soil

Changes in the populations of Rhizobium strains CB756str, CB985 and CB1024strspc in the rhizospheres of cowpea (Vigna unguiculata) and black gram (V. mungo) grown at three sites were evaluated. The population dynamics of the three rhizobia varied with soil type but the strain responses on the two legumes were generally similar. Most noticeable was the ability of CB756str to grow in the sandy soil (Beerwah) but not in the heavy clays (Narayen and Emerald). In contrast, the levels of CB1024strspc and CB985 generally increased in the clay soils.Nodulation (% due to the inoculum strain) did not always reflect events within the rhizosphere. Although not suited to Narayen, CB756str formed a similar proportion of the nodule population of black gram as CB1024strspc but this may have been due to higher seed inoculum levels of CB756str. At Emerald nodulation by all three strains of rhizobia was poor regardless of the success in colonization of the rhizosphere. Successful competition for nodule sites by native rhizobia may contribute to this discrepancy between Narayen and Emerald although lower seed inoculum levels at Emerald may also have been important.Nodule decay was consistently associated with a large increase in the number of rhizobia per root system. This is likely to be important in the survival of strains into the following season.Comparisons of nodulation by parent and mutant rhizobia suggested that resistance to antibotics may have slightly reduced nodule forming ability for CB1024strspc on black gram at Emerald.     

Displacement of native rhizobia nodulating chickpea (Cicer arietinum L.) by an inoculant strain through soil solarization

Summary Soil solarization greatly reduced the native chickpea Rhizobium population. With inoculation, it was possible to increase the population of the Rhizobium in solarized plots. In the 1st year, 47% nodulation was obtained with chickpea inoculant strain IC 59 when introduced with a cereal crop 2 weeks after the soil solarization and having a native Rhizobium count of <10 g^-1 soil, and only 13% when introduced 16 weeks after solarization at the time the chickpeas were sown, with 2.0×10² native rhizobia g^-1 soil. In the non-solarized plots inoculated with 5.6×10³ native rhizobia g^-1 soil, only 6% nodulation was obtained with the inoculant. In the succeeding year, non-inoculated chickpea was grown on the same plots without any solarization or Rhizobium inoculation. The treatment that showed good establishment of the inoculant strain in year 1 formed 68% inoculant nodules. Other treatments indicated a further reduction in inoculant success, from 1%–13% to 1%–9%. Soil solarization thus allowed an inoculant strain to successfully displace the high native population in the field and can serve as a research tool to compare strains in the field, irrespective of competitive ability. In year 1, Rhizobium inoculation of chickpea gave increased nodulation and increased plant growth 20 and 51 days after sowing, and increased dry matter, grain yield, and grain protein yield at maturity. These beneficial effects of inoculation on plant growth and yield were not measured in the 2nd year.Submitted as Journal Article No. JA 945 by the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru, Andhra Pradesh 502 324, India     

Responses to rhizobial inoculation by two promiscuous soybean cultivars in soils of the Southern Guinea savanna zone of Nigeria

TGX soybean lines were bred at IITA Ibadan for promiscuity with indigenous rhizobia in Nigerian soils. Two cultivars, TGX1456-2E and TGX1660-19F, were tested in a 2-year trial for their response to rhizobial inoculation in five farmers' fields within a 60-km radius of Minna town, in the Southern Guinea savanna zone of Nigeria. Using the ELISA method, the competitiveness and persistence of the two elite strains of rhizobia contained in the inoculant mixture were also studied. There was a close relationship between nodulation and the size of resident rhizobial populations, with wide variation in nodulation across the various sites irrespective of the treatments. Cultivar effect on height and nodule number was significant only in the first cropping season of the trial. The inoculant strains appeared to be less competitive, but more effective, than the indigenous populations. The proportions of the nodules occupied by the inoculant strains were 17% in the first cropping season, and 24% in the second. Inoculation with rhizobia increased the percent arbuscular mycorrhizal infection by an average of 50%. Although grain yield varied between sites, no significant cultivar effect was observed. However, inoculation increased grain yield by 40% in the first cropping season, while no such yield differences occurred in the second season. The proportion of nitrogen derived from N₂ fixation ranged from 27% to 50% in both cropping seasons, and this was dependent on crop management on farmers' fields, rather than any cultivar or inoculation effect.     

Response of field-grown bean (Phaseolus vulgaris L.) to Rhizobium inoculation and nitrogen fertilization in two Cerrados soils

 Most soils sown with field beans (Phaseolus vulgaris L.) contain indigenous rhizobia which might interfere with the establishment of inoculated strains. As a consequence, the benefits of bean inoculation are usually questioned, and the use of N fertilizer is gradually becoming a common practice. The present study had the objective of evaluating the effectiveness of inoculation and N fertilization in field soil with (site 1) and without (site 2) a previous bean-cropping history. At site 1, which had a rhizobial population of 7×10² cells g^–1 soil, inoculation had no effect on nodulation or yield, whereas at site 2 (<10 cells g^–1 soil) inoculation increased nodulation, nodule occupancy by the inoculated strain and grain yield. N fertilizer decreased nodulation at both sites, but increased grain yield at site 1 but not at site 2, indicating that the response to inoculation and N fertilization depends on the cropping history. When bean was cultivated for the first time, indigenous populations of rhizobia were low and high yields were accomplished solely with seed inoculation, with no further response to N fertilizer. In contrast, previous cultivation of bean increases soil rhizobia, preventing nodule formation by inoculated strains, and N fertilizer may be necessary for maximum yields. A significant interaction effect between N fertilizer and inoculation was detected for serogroup distribution only at site 2, with N fertilizer decreasing nodule occupancy by the inoculated strain and increasing the occurrence of indigenous strains. Consequently, although no benefits were obtained by the combination of inoculation and N fertilizer, this practice may be feasible with the selection of appropriate N-tolerant strains from the indigenous rhizobial population. Received: 26 May 1999     

Rhizobia in tropical legumes—XI. survival in the seed environment

Factors affecting rhizobia in the seed environment of the legumes Cajanus cajan, Centrosema pubescens, Glycine max, and Psophocarpus tetragonolobus were investigated. These included the effects of fungicides, insecticides, and herbicides on the survival and growth of rhizobia on agar and in liquid cultures. The pesticides used varied in their toxicity towards rhizobia but many fungicides and insecticides could be used in the vicinity of inoculated seeds. Certain fungicides and insecticides were shown to have little or no effect on the Rhizobium-legume symbiosis of C. cajan, C. pubescens, G. max, and P. tetragonolobus when added directly to inoculated seed. Various protective pelleting procedures were investigated including preparation of coating materials containing rock phosphates from different localities, talc, and bonemeal. These were applied to the seed in mixtures suspended in aqueous solutions of substituted celluloses, or gum arabic. Talc and Christmas Island rock phosphate both proved satisfactory, while “Cellofas” is recommended as the adhesive in this formulation. Soaking seeds before pelleting in an effort to remove anti-rhizobial substances from G. max seeds was beneficial in the case of soyabean cultivar KS437, but harmful when applied to Palmetto seeds.