Persistence of Rhizobium japonicum on the Soybean Seed Coat Under Controlled Temperature and Humidity

When Rhizobium japonicum strain 61A68 was added to surface-sterilized soybean (Glycine max) seed along with 12 different coating materials, a definite effect of temperature upon survival was observed both with and without coating materials. At a storage temperature of 15°C and 50 ± 5% relative humidity, from 0.9 to 14.1% of the original inoculum survived for 3 weeks. At 22.5°C, from 0.5 to 7.2% of the original inoculum survived. At 30°C, from 0.1 to 1.6% of the original inoculum survived. The data indicated that extremely large numbers of R. japonicum would have to be added to the seed to have numbers adequate for nodulation survive for 3 weeks of storage at ordinary temperatures.     

Dilution of Liquid Rhizobium Cultures To Increase Production Capacity of Inoculant Plants

Experiments were undertaken to test whether peat-based legume seed inoculants, which are prepared with liquid cultures that have been deliberately diluted, can attain and sustain acceptable numbers of viable rhizobia. Liquid cultures of Rhizobium japonicum and Rhizobium phaseoli were diluted to give 10⁸, 10⁷, or 10⁶ cells per ml, using either deionized water, quarter-strength yeast-mannitol broth, yeast-sucrose broth, or yeast-water. The variously diluted cultures were incorporated into gamma-irradiated peat, and the numbers of viable rhizobia were determined at intervals. In all of the inoculant formulations, the numbers of rhizobia reached similarly high ceiling values by 1 week after incorporation, irrespective not only of the number of cells added initially but also of the nature of the diluent. During week 1 of growth, similar multiplication patterns of the diluted liquid cultures were observed in two different peats. Numbers of rhizobia surviving in the various inoculant formulations were not markedly different after 6 months of storage at 28°C. The method of inoculant preparation did not affect the nitrogen fixation effectiveness of the Rhizobium strains.     

Bacteriocin-Like Substances Produced by Rhizobium japonicum and Other Slow-Growing Rhizobia

Bacteriocin-like substances were commonly produced by slow-growing Rhizobium japonicum and cowpea rhizobia on an L-arabinose medium. Antagonism between strains of R. japonicum was not detected in vitro; however, such strains were often sensitive to some bacteriocins produced by cowpea rhizobia. Inhibitory zones (2 to 8 mm from colony margins), produced by 58 of 66 R. japonicum test strains, were reproducibly detected with Corynebacterium nebraskense as an indicator. Quantitative production was not related to symbiotic properties of effective strains, since nine noninfective strains and one ineffective strain produced bacteriocin. Eight R. japonicum strains that did not produce bacteriocin nevertheless formed effective nodules on soybeans. R. japonicum strains that produced bacteriocin in vitro had no antagonistic effect on nonproducer strains during soybean nodulation. Under controlled conditions, a nonproducer (3I1b135) predominated over a bacteriocin producer (3I1b6) when inoculated at 1:1 and 1:9 ratios. Depending on the particular ratio, up to 38% of the total nodules formed were infected with mixed combinations. The bacteriocin(s) had a restricted host range and antibiotic-like properties which included the ability to be dialyzed and resistance to heat (75 to 80°C, 30 min), Pronase, proteinase K, trypsin, ribonuclease, and deoxyribonuclease. R. japonicum strains representing genetic, serological, cultural, and geographic diversity were differentiated into three groups on the basis of bacteriocin production.     

Adsorption of slow- and fast-growing rhizobia to soybean and cowpea roots

Roots of soybean (Glycine max [L.] Merr. cv Hardee) and cowpea (Vigna unguiculata [L.] Walp. cv Pink Eye Purple Hull) were immersed in suspensions containing 10⁴Rhizobium cells per milliliter of a nitrogen-free solution. After 30 to 120 minutes the roots were rinsed, and the distal 2-centimeter segments excised and homogenized. Portions of the homogenates then were plated on a yeast-extract mannitol medium for bacterial cell counts. The adsorption capacities of four slow-growing rhizobia and a fast-growing R. meliloti strain varied considerably. Adsorption was independent of plant species and of the abilities of the Rhizobium strains to infect and nodulate. R. lupini 96B9 had the greatest adsorption capacity, and Rhizobium sp. 3G4b16 the least. Rhizobium sp. 229, R. japonicum 138, and R. meliloti 102F51 were intermediate, except on cowpea, where the adsorption of strain 102F51 was similar to that of strain 3G4b16. The initial adsorption rates of bacteria cultured in synthetic media and in the presence of soybean roots were about the same. Addition of soybean lectin to the bacterial inoculum failed to influence initial adsorption rates. Both treatments, however, reduced the numbers of bacteria that bound after incubation with roots for 120 minutes. The relationship between the logarithm of the number of strain 138 cells bound per soybean root segment and the logarithm of the density of bacteria in the inoculum was linear over five orders of magnitude. Binding of strain 138 to soybean roots was greatest at room temperature (27°C) and substantially attenuated at both 4 and 37°C. Although R. lupini 96B9 strongly rejected a model hydrophobic plastic surface, there were no simple correlations between bacterial binding to model hydrophobic and hydrophilic plastic surfaces and bacterial adsorption to roots.     

Interactions between Rhizobia and Lectins of Lentil, Pea, Broad Bean, and Jackbean

A quantitative method was developed to measure the binding of fluorescent-labeled lentil (Lens esculenta Moench), pea (Pisum sativum L.), broad bean (Vicia faba L.), and jackbean (Canavalia ensiformis L., DC.) lectins to various Rhizobium strains. Lentil lectin bound to three of the five Rhizobium leguminosarum strains tested. The number of lentil lectin molecules bound per R. leguminosarum 128C53 cell was 2.1 × 10⁴. Lentil lectin also bound to R. japonicum 61A133. Pea and broad bean lectins bound to only two of the five strains of R. leguminosarum, whereas concanavalin A (jackbean lectin) bound to all strains of R. leguminosarum, R. phaseoli, R. japonicum, and R. sp. tested. Since these four lectins have similar sugarbinding properties but different physical properties, the variation in bindings of these lectins to various Rhizobium strains indicates that binding of lectin to Rhizobium is determined not only by the sugar specificity of the lectin but also by its physical characteristics.     

Inoculant Production with Diluted Liquid Cultures of Rhizobium spp. and Autoclaved Peat: Evaluation of Diluents, Rhizobium spp., Peats, Sterility Requirements, Storage, and Plant Effectiveness

Fully grown broth cultures of various fast- and slow-growing rhizobia were deliberately diluted with various diluents before their aseptic incorporation into autoclaved peat in polypropylene bags (aseptic method) or mixed with the peat autoclaved in trays (tray method). In a factorial experiment with the aseptic method, autoclaved and irradiated peat samples from five countries were used to prepare inoculants with water-diluted cultures of three Rhizobium spp. When distilled water was used as the diluent, the multiplication and survival of rhizobia in the peat was similar to that with diluents having a high nutrient status when the aseptic method was used. In the factorial experiment, the mean viable counts per gram of inoculant were log 9.23 (strain TAL 102) > log 8.92 (strain TAL 82) > log 7.89 (strain TAL 182) after 24 weeks of storage at 28°C. The peat from Argentina was the most superior for the three Rhizobium spp., with a mean viable count of log 9.0 per g at the end of the storage period. The quality of inoculants produced with diluted cultures was significantly (P = 0.05) better with irradiated than with autoclaved peat, as shown from the factorial experiment. With the tray method, rhizobia in cultures diluted 1,000-fold or less multiplied and stored satisfactorily in the presence of postinoculation contaminants, as determined by plate counts, membrane filter immunofluorescence, and plant infection procedures. All strains of rhizobia used in both the methods showed various degrees of population decline in the inoculants when stored at 28°C. Fast- and slow-growing rhizobia in matured inoculants produced by the two methods showed significant (P < 0.01) decline in viability when stored at 4°C, whereas the viability of some strains increased significantly (P < 0.01) at the same temperature. The plant effectiveness of inoculants produced with diluted cultures and autoclaved peat did not differ significantly from that of inoculants produced with undiluted cultures and gamma-irradiated peat.     

Growth of Rhizobium japonicum Strains at Temperatures Above 27°C

A study was conducted to examine the growth responses of different Rhizobium japonicum strains to increasing temperatures, determine the degree of variability among strains in those responses, and identify temperature-related growth characteristics that could be used to select temperature-tolerant strains. Each of 42 strains was grown in liquid culture for 96 h at 19 incubation temperatures ranging from 27.4 to 54.1°C in a temperature gradient apparatus. Growth was estimated by measuring the change in optical density over time. Strains differed in their responses to increasing temperatures. Three characteristic temperatures were determined for each strain: the temperature giving the maximum optical density at 96 h (optimum temperature), the maximum temperature allowing a continuous increase in optical density during the 96-h period (maximum permissive temperature), and the maximum temperature allowing growth of the cultures after they were transferred to a uniform incubation temperature of 28°C (maximum survival temperature). The three characteristic temperatures varied among strains and had the following ranges: optimum temperature, from 27.4 to 35.2°C; maximum permissive temperature, from 29.8 to 38.0°C; and maximum survival temperature, from 33.7 to 48.7°C. Significant positive correlations were found between maximum permissive temperature and optimum temperature and between maximum permissive temperature and maximum survival temperature. Eight strains which had the highest maximum permissive temperature, optimum temperature, and maximum survival temperature were considered tolerant of high temperatures and were able to grow at temperatures higher than those previously reported for the most tolerant R. japonicum strains. The strains were of diverse geographical origin, but the response to high temperatures was not related to their origin. Evaluation of the temperature responses in pure culture may be useful in the search for R. japonicum strains better suited to environments in which high soil temperature is a limiting factor.     

Bradyrhizobium japonicum Survival in and Soybean Inoculation with Fluid Gels

The utilization of gels, which are used for fluid drilling of seeds, as carriers of Bradyrhizobium japonicum for soybean (Glycine max (L.) Merr.) inoculation was studied. Gels of various chemical composition (magnesium silicate, potassium acrylate-acrylamide, grafted starch, and hydroxyethyl cellulose) were used, although the hydroxyethyl cellulose gels were more extensively investigated. Gel inocula were prepared by mixing gel powder with liquid cultures of B. japonicum (2% [wt/vol]). The population of B. japonicum USDA 110 did not change in each gel type during 8 days of incubation at 28°C. These fluid gels were prepared with late-exponential-growth-phase cells that were washed and suspended in physiological saline. Mid-exponential-growth-phase B. japonicum USDA 110, 123, and 138 grew in cellulose gels prepared with yeast extract-mannitol broth as well as or better than in yeast extract-mannitol broth alone for the first 10 days at 28°C. Populations in these cellulose gels after 35 days were as large as when the gels had originally been prepared, and survival occurred for at least 70 days. Soybeans grown in sand in the greenhouse had greater nodule numbers, nodule weights, and top weights with gel inoculants compared with a peat inoculant. In soil containing 10³ indigenous B. japonicum per g of soil, inoculation resulted in increased soybean nodule numbers, nodule weights, and top weights, but only nodule numbers were greater with gel than with peat inoculation. The gel-treated seeds carried 10² to 10³ more bacteria per seed (10⁷ to 10⁸) than did the peat-treated seeds.     

Inhibition of Soybean Cell Growth by the Adsorption of Rhizobium japonicum

Soybean cells in suspension culture were inhibited in their growth by mixed culture with Rhizobium japonicum 5033. Rhizobium cells had the ability to adsorb on the surface of soybean cells. Cell envelope prepared from Rhizobium by sonic oscillation inhibited the growth of soybean cells. The growth-inhibiting activity of the cell envelope was depressed by β-glucosidase, KIO₄, urea, sodium cholate, and Triton X-100, but was stable on heating at 120 C for 15 minutes. Adsorption of the cell envelope on soybean cells was depressed by only β-glucosidase. The sodium cholate-soluble fraction of the cell envelope had the growth-inhibiting activity. Results in this paper suggest that components of the Rhizobium cell surface cause the inhibition of soybean cell growth after the adsorption of the Rhizobium cell to the soybean cell.     

Genomic insight into the taxonomy of Rhizobium genospecies that nodulate Phaseolus vulgaris

Due to the wide cultivation of bean (Phaseolus vulgaris L.), rhizobia associated with this plant have been isolated from many different geographical regions. In order to investigate the species diversity of bean rhizobia, comparative genome sequence analysis was performed in the present study for 69 Rhizobium strains mainly isolated from root nodules of bean and clover (Trifolium spp.). Based on genome average nucleotide identity, digital DNA:DNA hybridization, and phylogenetic analysis of 1,458 single-copy core genes, these strains were classified into 28 clusters, consistent with their species definition based on multilocus sequence analysis (MLSA) of atpD, glnII, and recA. The bean rhizobia were found in 16 defined species and nine putative novel species; in addition, 35 strains previously described as Rhizobium etli, Rhizobium phaseoli, Rhizobium vallis, Rhizobium gallicum, Rhizobium leguminosarum and Rhizobium spp. should be renamed. The phylogenetic patterns of symbiotic genes nodC and nifH were highly host-specific and inconsistent with the genomic phylogeny. Multiple symbiovars (sv.) within the Rhizobium species were found as a common feature: sv. phaseoli, sv. trifolii and sv. viciae in Rhizobium anhuiense; sv. phaseoli and sv. mimosae in Rhizobium sophoriradicis/R. etli/Rhizobium sp. III; sv. phaseoli and sv. trifolii in Rhizobium hidalgonense/Rhizobium acidisoli; sv. phaseoli and sv. viciae in R. leguminosarum/Rhizobium sp. IX; sv. trifolii and sv. viciae in Rhizobium laguerreae. Thus, genomic comparison revealed great species diversity in bean rhizobia, corrected the species definition of some previously misnamed strains, and demonstrated the MLSA a valuable and simple method for defining Rhizobium species.     

Stability of Bradyrhizobium japonicum Inoculants after Introduction into Soil

Bradyrhizobium japonicum USDA 125-Sp, USDA 138, and USDA 138-Sm had been used as inoculants for soybean (Glycine max (L.) Merr.) in soils previously free of B. japonicum. At 8 to 13 years after their release, these strains were reisolated from soil samples. A total of 115 isolates were obtained through nodules, and seven colonies were obtained directly by a serological method. The stability of the inoculants was confirmed by comparing the reisolated cultures with their respective parental strains which had been preserved by being lyophilized or stored on a yeast extract-mannitol agar slant at 4°C. Comparisons were made on morphological and serological characters, carbon compound utilization (8 tested), intrinsic antibiotic resistance (9 tested), and enzymatic activity (19 tested). Mucous and nonmucous isolates of serogroup 125 were analyzed for symbiotic effectiveness and restriction fragment hybridization with a DNA probe. Our data suggest that the B. japonicum inoculants have survived for up to 13 years in the soils without significant mutation except for two reisolates with a slightly increased kanamycin resistance level.     

Survival of several <Emphasis Type="Italic">Rhizobium/Bradyrhizobium</Emphasis> strains on different inoculant formulations and inoculated seeds

The effect of a variety factors on the survival of several rhizobia strains on inoculants and inoculated seeds has been evaluated. Since the rhizobia strains showed different cell-density-evolution patterns on peat-based inoculants and on inoculated seeds, several inoculant formulations with highly effective Rhizobium/Bradyrhizobium strains (for Lupinus, Hedysarum, Phaseolus and Glycine max.) were monitored under the following storage conditions: (a) the inoculants were kept refrigerated (at 4 °C), or (b) at room temperature (25 °C). The effect of water content (30–50%, w/w) in the inoculants as well as that of several seed-coating adhesives were also investigated. Alternative carriers including perlite and vermiculite were tested. For all of the strains, survival on sterile peat-based inoculants was higher than on the corresponding unsterile peat formulation; for the latter, refrigerated storage conditions are recommended to ensure high bacterial densities. The water content of the inoculants had a differential effect on strain survival depending on the sterility of the peat, such that a high water content was more detrimental when unsterilized peat was employed. The best adherent for rhizobia survival was a gum arabic/water solution. Perlite was as effective as peat in maintaining a high population of rhizobia, at least for 6 months of storage. Electronic Publication     

An Ouchterlony double diffusion study on the interaction between legume lectins and rhizobial cell surface antigens

Acidic exopolysaccharides and O-antigen containing lipopolysaccharides were isolated from Rhizobium japonicum, R. leguminosarum, R. lupini, R. meliloti, R. phaseoli, cowpea Rhizobium sp. and a non-nodulating soil bacterium. Lectins from seeds of soybean (Glycine max), garden pea (Pisum sativum), lentil (Lens culinaris), alfalfa (Medicago sativa), field bean (Phaseolus vulgaris), jackbean (Canavalia ensiformis) and from wheat germ were tested for their capacity to precipitate rhizobial exopolysaccharides and lipopolysaccharides in the Ouchterlony double diffusion test. Soybean lectin precipitated exclusively with the exopolysaccharide of R. japonicum, whereas the lectins from pea and lentil precipitated exopolysaccharides from all the fast growing strains of Rhizobium. Host range specific interactions between lipopolysaccharides and lectins were observed in the pea/lentil-R. leguminosarum and in the alfalfa-R. meliloti systems. Concanavalin A precipitated the exopolysaccharides of all fast growing strains of Rhizobium, the exopolysaccharide of the cowpea strain and several lipopolysaccharides of different Rhizobium species and thus did not show any correlation between polysaccharide binding and symbiotic specificity. Non-leguminous wheat germ agglutinin did not precipitate any of the rhizobial polysaccharides tested and the lipopolysaccharide of the soil bacterium did not precipitate with any of the lectins examined.Abbreviations Con A Concanavalin A - CPC cetylpyridinium chioride - EPS exopolysaccharide - FITC fluorescein isothiocyanate - KDO 2-keto-3-deoxyoctonic acid - LPS lipopolysaccharide - PBS phosphate-buffered saline - PS polysaccharide     

Semienclosed Tube Cultures of Bean Plants (Phaseolus vulgaris L.) for Enumeration of Rhizobium phaseoli by the Most-Probable-Number Technique

The semienclosed tube culture technique of Gibson was modified to permit growth of common bean (Phaseolus vulgaris L.) roots in humid air, enabling enumeration of the homologous (nodule forming) symbiont, Rhizobium phaseoli, by the most-probable-number plant infection method. A bean genotype with improved nodulation characteristics was used as the plant host. This method of enumeration was accurate when tubes were scored 3 weeks after inoculation with several R. phaseoli strains diluted from aqueous suspensions, peat-based inoculants, or soil. A comparison of population sizes obtained by most-probable-number tube cultures and plate counts indicated that 1 to 3 viable cells of R. phaseoli were a sufficient inoculant to induce nodule formation.     

Effect of temperature on nitrogenase functioning in cowpea nodules

Nitrogenase (EC 1.7.99.2) activity of a cowpea (Vigna unguiculata (L.) Walp cv Caloona) symbiosis formed with a Rhizobium strain (176A27) lacking uptake hydrogenase and maintained under conditions of a 12-hour day at an air temperature of 30°C (800-1000 microeinsteins per square meter per second) and a 12-hour night at an air temperature of 20°C showed a marked diurnal variation in ratio of nitrogen fixed to hydrogen evolved. As little as 0.3 micromole nitrogen was fixed per micromole hydrogen evolved in the photoperiod versus up to 0.6 in the dark period. In plants maintained under the same diurnal illumination regime but at constant (day and night) air temperature (30°C), this difference was abolished and a relatively constant ratio of nitrogen fixed to hydrogen evolved (around 0.3 micromole per micromole) was observed day and night. Exposure of nodulated roots to a range of temperatures maintained for 2 hours in a single photoperiod indicated that, whereas hydrogen evolution increased with increasing temperature from 15°C to a maximum around 35°C, nitrogen fixation was largely unaffected over this temperature range. Both functions of the enzyme declined sharply at temperatures above 38°C. A similar general response of nitrogen fixation to root temperature was observed in glasshouse-grown, sand-cultured plants maintained under a range of temperatures (from 15 to 35°C) for a 14-day period in mid vegetative growth. The effect of temperature on the proportion of electrons allocated to proton reduction compared with nitrogen reduction showed a linearly increasing relationship (correlation coefficient = 0.96) between 15°C and 47°C.     

Differences Among Cowpea Rhizobia in Tolerance to High Temperature and Desiccation in Soil

Strains of cowpea rhizobia grew in mannitol-amended, nonsterile soil at 29 to 35°C but not at 40°C. Little decline in numbers of these bacteria occurred in dry, nonsterile soil incubated at 42°C for 7 days. Strains of cowpea rhizobia differed widely in their tolerances to drying at 30°C in nonsterile and sterile soil, and from less than 1 to 50% of the bacteria were still viable after 11 days. No relation was evident between tolerance to desiccation and the degree of aridity of the site from which the bacteria were isolated or their growth rates in culture, but strains not producing extracellular polysaccharide were often more tolerant than those producing extracellular polysaccharide. It is suggested that desiccation-tolerant rhizobia be used for the production of legume inoculants.     

Effect of Conditioning Treatments on the Survival of Radopholus similis at High Temperatures

Heat treatments are an environmentally safe method for eliminating quarantine pests from tropical foliage. Conditioning heat treatments can induce thermotolerance against subsequent and otherwise phytotoxic temperatures in tropical foliage, allowing heat treatments to be even more effective. However, if thermotolerance is also induced in nematodes of quarantine significance like Radopholus similis, heat treatments would be rendered ineffective. A lethal thermal death point (LT_99.9) was established for R. similis by recording mortality at 25 (control temperature), 43°C, 45°C, 47°C, or 49°C after a 0, 1-, 2-, 4-, 6-, 8-, 10-, 12-, or 15-minute exposure. In a second experiment, nematodes were conditioned at 35, 40, or 45°C for 0, 15, 30, 60, 120, and 180 minutes, allowed to rest for 3 hours, and then challenged at 47°C for 5 minutes. No nematodes survived the challenge heat treatment; rather, nematode mortality was hastened by the conditioning treatment itself. In a third experiment, R. similis inside anthurium roots were conditioned at 25°C or 40°C for 15 minutes and then treated at 45°C for up to 8 minutes. Mortality of conditioned and unconditioned nematodes was similar (P > 0.1). Conditioning treatments increase plant thermotolerance but do not induce thermotolerance in R. similis. Heat treatments have promise as disinfection protocols for quarantines.     

Degradation of aromatic compounds byRhizobium spp.

Summary The ability of rhizobia to utilize catechol, protocatechuic acid, salicylic acid, p-hydroxybenzoic acid and catechin was investigated. The degradation pathway of p-hydroxybenzoate byRhizobium japonicum, R. phaseoli, R. leguminosarum, R. trifolii andRhizobium sp. isolated from bean was also studied.R. leguminosarum, R. phaseoli andR. trifolii metabolized p-hydroxybenzoate to protocatechuate which was cleaved by protocatechuate 3,4-dioxygenasevia ortho pathway.R. japonicum degraded p-hydroxybenzoate to catechol which was cleaved by catechol 1,2-dioxygenase.Rhizobium sp., a bean isolate, dissimilatedp-hydroxybenzoate to salicylate. Salicylate was converted to gentisic acid prior to ring cleavage. The rhizobia convertedp-hydroxybenzoate to Rothera positive substance. Catechol and protocatechuic acid were directly cleaved by the species.R. japonicum converted catechin to protocatechuic acid.