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The effect of rate of application of Sinorhizobium (Ensifer) fredii SMH12 or Bradyrhizobium japonicum USDA110 inoculants on grain yields of soybean [Glycine max (L.) Merrill] cv. Osumi was studied in a field experiment laid out in Southern Spain. All inoculant doses tested (104, 105, 106 and 107 rhizobia/seed) produced higher seed yields than those obtained in un-inoculated plots. Increments in nodule dry weight, seed yield and seed N content were observed when the number of rhizobia applied to seed increased from 104 up to 107. The addition of N fertilizer to inoculated soybean plants (50 kg N/ha applied at R1 or R4 stage) did not increase seed yields in comparison with treatments that were only inoculated. Survival of strains SMH12 and USDA110 was monitorized for three years in two different soils of Southern Spain after soybean inoculation. Rhizobia survival was varied dependent on the soil and the rhizobia strain used.

The symbiotic potential of fast-growing soybean rhizobia isolated from diverse provinces of China, including S. fredii SMH12 from Vietnam, has been evaluated. All of them produced nitrogen-fixing nodules with the American soybean cultivar Osumi under greenhouse and field conditions. Two years of field experiments conducted in two localities in South Spain with alkaline and moderately acid pH soils showed that seed yields of soybean plants inoculated with fast-growing strains SMH12, WH1, WH8 and WW4 were similar or higher than those obtained with soybean inoculated with the reference slow-growing Bradyrhizobium japonicum strain USDA110. Competitiveness studies between S. fredii SMH12 and B. japonicum USDA110 demonstrated that soybean cultivar and pH of soil determined the outcome of the competition. Alkaline pH (7.8) favoured SMH12 to occupied nodules with American soybean cultivars while USDA110 was more competitive in soils with a moderately acid pH (6.6). The Asiatic cultivar Peking was almost exclusively nodulated by SMH12 in both types of soils. On the other hand, the percentage of nodules occupied by fast-growing soybean inoculants applied into a soil containing indigenous Bradyrhizobium population of 4 × 10 3 rhizobia/g was inferior to 18%. SMH12 occupied a 7% and 17% of the nodules with Osumi and Kochi soybean cultivars, respectively.

Many of the microbial inoculants all over the world are based on solid peat formulations. This has been mostly true for well developed legume inoculants based on selected rhizobial strains, due to peat bacterial protection properties. Six carriers (bagasse, cork compost, attapulgite, sepiolite, perlite and amorphous silica) were evaluated as alternatives to peat. Compost from the cork industry and perlite were superior to peat in maintaining survival of different rhizospheric bacteria. Other tested materials were discarded as potential carriers for soybean rhizobia. Also, different liquid culture media have been assayed employing mannitol or glycerol as C sources. Some media maintained more than 109 cfu ml−1 of Sinorhizobium (Ensifer) fredii SMH12 or Bradyrhizobium japonicum USDA110 after 3 months of storage. Rhizobial survival on pre-inoculated seeds with both solid and liquid formulations previously cured for 15 days led to a higher bacterial numbers in comparison with recently made inoculants. An additional curing time of solid inoculants up to 120 days had a beneficial effect on rhizobial survival on seeds. The performance of different formulations of two highly effective soybean rhizobia strains was assayed under field conditions. Soybean inoculated with cork compost, perlite and liquid formulations produced seed yields that were not significantly different to those produced by peat-based inoculants.

The fast-grower Sinorhizobium fredii SMH12 has been evaluated as a soybean inoculant in alkaline soils (pH 8.2) of the Guadalquivir Valley. We also have investigated S. fredii SMH12 for the occurrence of changes after prolonged subculturing in tryptone-yeast extract media. Field experiments over three different seasons showed that soybean inoculated with S. fredii SMH12 produced seed yields that were not different from those produced by soybeans inoculated with Bradyrhizobium japonicum USDA110, a highly effective soybean inoculant. Soybeans inoculated with a mixture of S. fredii SMH12 and B. japonicum USDA110 formed nodules that only contained the S. fredii co-inoculant. All the analysed bacterial traits remained unmodified after 500 bacterial generations. These results indicate that S. fredii SMH12 appears to be genetically stable and that, at least in the soils tested, is a valuable soybean inoculant.

Quantitative analyses of fast- and slow-growing soybean rhizobia populations in soils of four different provinces of China (Hubei, Shan Dong, Henan, and Xinjiang) have been carried out using the most probable number technique (MPN). All soils contained fast- (FSR) and slow-growing (SSR) soybean rhizobia. Asiatic and American soybean cultivars grown at acid, neutral and alkaline pH were used as trapping hosts for FSR and SSR strains. The estimated total indigenous soybean-rhizobia populations of the Xinjiang and Shan Dong soil samples greatly varied with the different soybean cultivars used. The soybean cultivar and the pH at which plants were grown also showed clear effects on the FSR/SSR rations isolated from nodules. Results of competition experiments between FSR and SSR strains supported the importance of the soybean cultivar and the pH on the outcome of competition for nodulation between FSR and SSR strains. In general, nodule occupancy by FSRs significantly increased at alkaline pH. Bacterial isolates from soybean cultivar Jing Dou 19 inoculated with Xinjiang soil nodulate cultivars Heinong 33 and Williams very poorly. Plasmid and lipopolysaccharide (LPS) profiles and PCR-RAPD analyses showed that cultivar Jing Dou 19 had trapped a diversity of FSR strains. Most of the isolates from soybean cultivar Heinong 33 inoculated with Xinjiang soil were able to nodulate Heinong 33 and Williams showed very similar, or identical, plasmid, LPS and PCR-RAPD profiles. All the strains isolated from Xinjiang province, regardless of the soybean cultivar used for trapping, showed similar nodulation factor (LCO) profiles as judged by thin layer chromatographic analyses. These results indicate that the existence of soybean rhizobia sub-populations showing marked cultivar specificity, can affect the estimation of total soybean rhizobia populations indigenous to the soil, and can also affect the diversity of soybean rhizobial strains isolated from soybean nodules.

Growth and survival of Rhizobium leguminosarum bv. phaseoli, R. tropici, Bradyrhizobium japonicum and Bacillus megaterium in peat and perlite-based inoculants were evaluated. In general, survival was similar for all strains in both carriers. Better survival was observed when inoculants were maintained at 48C compared to 288C. Studies with two diAerent stickers suggested the existence of interactions between carriers and adhesives and showed that combination of a sucrose adhesive with the perlite carrier gave better survival of bacteria on seeds. Bean and soybean field experiments indicated that perlite-based inoculants produced similar number of nodules, nodule dry weight, crop yield and nitrogen content, as peat-based inoculants. 7 2000 Published by Elsevier Science Ltd. All rights reserved.

The purpose of this work was to study in an unsterilized acid soil the response of the tropical tree legume Mimosa caesalpiniaefolia to phosphogypsum as a P-fertilizer compared with soluble phosphorus fertilizer and to evaluate the interaction of P fertilization with Bradyrhizobium spp and vesicular arbuscular mycorrhizal fungi (VMF) (Glomus spp and Gigaspora spp) inoculations. A sandy clay red yellow latosol representative acid soil from the forest humid zone of Pernambuco, Brazil was used. P-fertilizer treatments equivalent to 40 kg P ha−1, were applied as triple superphosphate (TSP), phosphogypsum (GP) or a mixture of both. Plants were harvested 80 d after seed germination. P-sources increased mycorrhizal infection only when plants were not inoculated with rhizobia strains, suggesting that native mycorrhizal fungi are not able to develop efficiently in this soil, and the VMF-used groups did not significantly affect any of the factors analysed. In summary, it was concluded that in unsterilized acid soil without P-addition Badyrhizobium strain NFB 577 seemed to be more effective, but when P-fertilizer was used both strains were similarly effective. TSP showed the best results, but PG can be used as a P-fertilizer when combined with a suitable rhizobia strain.

Ahipa is a legume of great interest for the production of raw materials (starch, sugar, oil and proteins) for industrial use. Its yield potential and ability to fix atmospheric N2 in association with rhizobia makes it an attractive option for low input agriculture systems. At present, it is cultivated on a very small scale as a food crop in a few South American countries. Little information is available on symbiotic N2 fixation in ahipa and no work has been performed on strain selection for inoculant production. Soils in southwest Europe are devoid of specific rhizobia able to nodulate on ahipa. Selecting rhizobia for symbiotic effectiveness from a collection led to the isolation of strains which provided greater shoot growth and N content under controlled conditions. In the field, inoculation at sowing with the selected strains increased significantly seed and tuberous root yield and seed protein content. The amount of N2 fixed, estimated by 15N natural abundance, reached 160-260 kg N ha-1. In previous work, ahipa appeared to be a promising alternative crop for the production of industrial raw materials. The results of the present study showed a yield increase in tuberous roots and seeds when applying effective rhizobia inoculants. Furthermore, a positive soil N balance was left after its cultivation making ahipa even more interesting for sustainable farming systems.

The survival of several Rhizobium (Hedysarum) strains on peat-based inoculants was studied. They proved to be suitable for the commercial inoculant process, as they showed good adaptation to the peat carrier under different storage conditions. Gum arabic seems to be the best protector of rhizobial strains, as deduced from the survival studies on seeds using various adhesives. Seed inoculation with selected strains, in soils with no specific indigenous rhizobia population, proved to exert a beneficial effect on sulla forage yield.

The interaction Rhizobium/Phaseolus was studied under controlled conditions using commercial bean cultivars and different Rhizobium strains. Plant growth (Shoot and pods), symbiotic parameters (nodule number and nodule mass) and N solutes (ureides; amino acids and nitrate) in xylem sap were determined. A significant variation of plant growth in response to inoculation with Rhizobium strains was observed. Inoculation with different strains affected biomass (shoot and pod growth) and N concentration in shoots, as a result of differences in N2 fixation rates, but also affected the harvest index, significant differences were observed in the concentration of N solutes in the xylem sap. The genotype of bean cultivar had an important effect on the symbiotic effectiveness of different strains, affecting variables like number and mass of nodules, and the concentration of N solutes in xylem sap. An interaction cultivar x strain effect was observed for plant growth, symbiotic parameters and ureides and aminoacids in sap. The results suggest the need to search for specific strain/cultivar combinations in order to maximize the N2 fixation capacity in beans. Because of the variation in sap ureide and amino acid concentration, the ureide method for N2 fixation assessment would require calibration for each Rhizobium/Phaseolus combination.

Several microelements (Mo, Cu, Zn, Mn, B) had some degree of influence on nodule dry weight, plant dry weightand nitrogen content of Phaseolus vulgaris cv. Canellini inoculated with Rhizobium tropici CIAT899 under greenhousehydroponic culture conditions. Nodule number was not significantly affected. The absence of boron producedthe highest degree of inhibition. The absence of microelements affected uptake and assimilation of mineral nitrogento a lesser extent than biological nitrogen fixation. A similar effect of microelements was observed on Phaseolus vulgaris(cvs. Canellini and Mutin)/Rhizobium etli ISP23 symbiosis. En condiciones de cultivo hidropónico en invernadero se observó que varios oligoelementos (Mo, Cu, Zn, Mn, B) tuvieron algún grado de influencia sobre el peso seco de los nódulos y el peso seco y contenido en nitrógeno de la parte aérea de Phaseolus vulgaris cv. Canellini inoculado con Rhizobium tropici (CIAT899). El número de nódulos no se vió afectado de una forma significativa. La ausencia de boro provocó la mayor reducción en los parámetros simbióticos. La ausencia de microelementos afectó menos a la absorción de nitrógeno mineral que a la fijación biológica de nitrógeno. Se observó un efecto similar de los microelementos sobre Phaseolus vulgaris cvs. Canellini y Mutin inoculados con Rhizobium etli ISP23

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 degrees C), or (b) at room temperature (25 degrees 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.