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Rhizobium strain 042B(s) is able to nodulate both soybean and alfalfa cultivars. We have demonstrated, by mass spectrometry, that the nodulation (Nod) factors produced by this strain are characteristic of those produced by Sinorhizobium fredii, which typically nodulates soybean; they have 3–5 N-acetylglucosamine (GlcNAc) residues, a mono-unsaturated or saturated C16, C18 or C20 fatty-acyl chain, and a (methyl)fucosyl residue on C6 of the reducing-terminal GlcNAc. In order to study Rhizobium strain 042B(s) and its nodulation behaviour further, we introduced an insertion mutation in the noeL gene, which is responsible for the presence of the (methyl)fucose residue on the reducing terminal GlcNAc of the Nod-factors, yielding mutant strain SVQ523. A plasmid (pHM500) carrying nodH, nodP and nodQ, the genes involved in sulfation of Nod-factors on C6 of the reducing-terminal GlcNAc, was introduced into SVQ523, generating SVQ523.pHM500. As expected, strain SVQ523 produces unfucosylated Nod-factors, while SVQ523.pHM500 produces both unfucosylated and unfucosylated sulfated Nod-factors. Plant tests showed that soybean nodulation was reduced if the inoculant (SVQ523.pHM500) produced sulfated Nod-factors. In the Asiatic alfalfa cultivar Baoding, SVQ523 (absence of a substitution at C6) failed to nodulate, but both 042B(s) (fucosyl at C6) and SVQ523.pHM500 (sulfate at C6) formed nodules. In contrast, SVQ523 showed enhanced nodulation capacity with the western alfalfa cultivars ORCA and ARC. These results indicate that Nod-factor sulfation is not a requisite for S. fredii to nodulate alfalfa.

Lupinus micranthus is a lupine distributed in the Mediterranean basin whose nitrogen fixing symbiosis has not been described in detail. In this study, 101 slow-growing nodule isolates were obtained from L. micranthus thriving in soils on both sides of the Western Mediterranean. The diversity of the isolates, 60 from Algeria and 41 from Spain, was addressed by multilocus sequence analysis of housekeeping genes (16S rRNA, atpD, glnII and recA) and one symbiotic gene (nodC). Using genomic fingerprints from BOX elements, 37 different profiles were obtained (22 from Algeria and 15 from Spain). Phylogenetic analysis based on 16S rRNA and concatenated atpD, glnII and recA sequences of a representative isolate of each BOX profile displayed a homogeneous distribution of profiles in six different phylogenetic clusters. All isolates were taxonomically ascribed to the genus Bradyrhizobium. Three clusters comprising 24, 6, and 4 isolates, respectively, accounted for most of the profiles. The largest cluster was close to the Bradyrhizobium canariense lineage, while the other two were related to B. cytisi/B. rifense. The three remaining clusters included only one isolate each, and were close to B. canariense, B. japonicum and B. elkanii species, respectively. In contrast, phylogenetic clustering of BOX profiles based on nodC sequences yielded only two phylogenetic groups. One of them included all the profiles except one, and belonged to symbiovar genistearum. The remaining profile, constituted by a strain related to B. elkanii, was not related to any well-defined symbiotic lineage, and may constitute both a new symbiovar and a new genospecies.

espanolLa esparceta (Onobrychis viciifolia Scop.) es una leguminosa forrajera de creciente interes en distintos agroecosistemas debido principalmente a la buena palatabilidad, tolerancia a la sequia y presencia de polifenoles, entre ellos los taninos. Se estudio el efecto de la inoculacion con dos cepas Rhizobium de la semilla de esparceta del ecotipo ‘Reznos’ y de la fecha de siembra (primavera y otono) sobre la produccion de forraje y valor nutritivo en Asturias, durante 2014 y 2015. Las mayores producciones de materia seca por corte se encontraron en los tratamientos con esparceta inoculados frente a los no inoculados. Igualmente los tratamientos de esparceta inoculados presentaron mayores contenidos en proteina bruta que los no inoculados. En ambos casos no hubo diferencias entre las dos cepas de Rhizobium. La siembra primaveral presento valores mas altos de produccion por corte que la siembra otonal. La nodulacion de la esparceta resulto eficaz, tanto en su influencia en la produccion de materia seca como por sus valores mas altos de proteina bruta EnglishSainfoin (Onobrychis viciifolia Scop.) is a forage legume of growing interest in different agroecosystems due mainly to good palatability, drought tolerance and presence of polyphenols, including tannins. The effect of inoculation with two strains of Rhizobium on seed of a sainfoin ecotype ‘Reznos’ and the sowing date (spring and autumn) on forage yield and nutritive value was studied in Asturias during 2014-2015. The largest production of dry matter per harvest was found in treatments with sainfoin inoculated compared to un-inoculated sainfoin. Similarly, inoculated sainfoin treatments had higher crude protein content than un-inoculated treatments. In both cases, there were no differences between the two Rhizobium strains. Spring sowing showed higher values of dry matter yield per harvest than autumn sowing. Nodulation of sainfoin was effective both in its influence on dry matter production and its higher crude protein values.

Lupinus mariae-josephi is a recently described endemic Lupinus species from a small area in Eastern Spain where it thrives in soils with active lime and high pH. The L. mariae-josephi root symbionts were shown to be very slow-growing bacteria with different phenotypic and symbiotic characteristics from those of Bradyrhizobium strains nodulating other Lupinus. Their phylogenetic status was examined by multilocus sequence analyses of four housekeeping genes (16S rRNA, glnII, recA, and atpD) and showed the existence of a distinct evolutionary lineage for L. mariae-josephi that also included Bradyrhizobium jicamae. Within this lineage, the tested isolates clustered in three different sub-groups that might correspond to novel sister Bradyrhizobium species. These core gene analyses consistently showed that all the endosymbiotic bacteria isolated from other Lupinus species of the Iberian Peninsula were related to strains of the B. canariense or B. japonicum lineages and were separate from the L. mariae-josephi isolates. Phylogenetic analysis based on nodC symbiotic gene sequences showed that L. mariae-josephi bacteria also constituted a new symbiotic lineage distant from those previously defined in the genus Bradyrhizobium. In contrast, the nodC genes of isolates from other Lupinus spp. from the Iberian Peninsula were again clearly related to the B. canariense and B. japonicum bv. genistearum lineages. Speciation of L. mariae-josephi bradyrhizobia may result from the colonization of a singular habitat by their unique legume host.

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 isolation and characterisation of nitrogen-fixing root nodule bacteria from Medicago marina, a tolerant legume species, were studied in two areas from southwest Spain. A total of 30 out of 82 isolates with distinct ERIC-PCR fingerprints were analysed on the basis of molecular (PCR-RFLP of the 16S-23S rDNA intergenic spacer region (IGS) with two endonucleases, analysis of the 16S rDNA and symbiotic nodC gene sequences, plasmid profiles and SDS-PAGE of LPS, including the partial sequence of the housekeeping gene glnII and the symbiotic gene nodA of some representatives), physiological (utilisation of sole carbon sources, tolerance to antibiotics, NaCl, heavy metals, temperature and pH) and symbiotic parameters (efficacy on M. marina, M. minima, M. murex, M. orbicularis, M. polymorpha, M. sativa and M. truncatula). All the bacteria isolated from M. marina nodules belonged to Ensifer meliloti, except for one strain that belonged to E. medicae. To determine the nodulation range of M. marina, 10 different Ensifer species were tested for their ability to nodulate on this plant. E. kummerowiae CCBAU 71714 and the E. medicae control strain M19.1 were the only Ensifer species tested that developed nitrogen-fixing nodules on this plant. Most of the M. marina-nodulating strains showed tolerance to stress factors and all of them shared the presence of a gene similar to cadA, a gene that encodes for a PIB-type ATPase, which is a transporter belonging to the large superfamily of ATP-driven pumps involved in the transport of metals across cell membranes.

The plant rhizosphere is an important soil ecological environment for plant–microorganism interactions, which include colonization by a variety of microorganisms in and around the roots that may result in symbiotic, endophytic, associative, or parasitic relationships within the plant, depending on the type of microorganisms, soil nutrient status, and soil environment. Rhizosphere competence may be attributable to the differences in the extent of bacterial attachment to the root surface. We present results of the effect of various factors on the attachment to bean ( Phaseolus vulgaris ) and soybean ( Glycine max ) roots of some bacterial species of agronomic importance, such as Rhizobium tropici, Rhizobium etli, Ensifer fredii (homotypic synonym Sinorhizobium fredii ), and Azospirillum brasilense ; as well as the attachment capability of the plant growth promoting rhizobacteria Pseudomonas fluorescens and Chryseobacterium balustinum . Additionally, we have studied various bacterial traits, such as autoaggregation and flagella movements, which have been postulated to be important properties for bacterial adhesion to surfaces. The lack of mutual incompatibility between rhizobial strains and C. balustinum has been demonstrated in coinoculation assays.

nodD1 of Sinorhizobium fredii HH103, which is identical to that of S. fredii USDA257 and USDA191, repressed its own expression. Spontaneous flavonoid-independent transcription activation (FITA) mutants of S. fredii HH103 M (=HH103 RifR pSym::Tn 5-Mob) showing constitutive expression of nod genes were isolated. No differences were found among soybean cultivar Williams plants inoculated with FITA mutants SVQ250 or SVQ253 or with the parental strain HH103M. Soybean plants inoculated with mutant SVQ255 formed more nodules, and those inoculated with mutant SVQ251 had symptoms of nitrogen starvation. Sequence analyses showed that all of the FITA mutants carried a point mutation in their nodD1 coding region. Mutants SVQ251 and SVQ253 carried the same mutation, but only the former was symbiotically impaired, which indicated the presence of an additional mutation elsewhere in the genome of mutant SVQ251. Mutants SVQ251 and SVQ255 were outcompeted by the parental strain for nodulation of soybean cultivar Williams. The symbiotic plasmids of mutants SVQ251 and SVQ255 (pSym251 and pSym255, respectively) and that (pSymHH103M) of the parental strain were transferred to pSym-cured derivatives of S. fredii USDA192 and USDA193 (USDA192C and USDA193C, respectively). Soybean responses to inoculation with S. fredii USDA192C and USDA193C transconjugants carrying pSym251 and pSymHH103M were not significantly different, whereas more nodules were formed after inoculation with transconjugants carrying pSym255. Only transconjugant USDA192C(pSym255) produced a significant increase in soybean dry weight.

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.

The genetic diversity of 45 bradyrhizobial isolates that nodulate several Lupinus and Ornithopus species in different geographic locations was investigated by 16S rDNA PCR-RFLP and sequence analysis, 16S-23S rDNA intergenic spacer (IGS) PCR-RFLP analysis, and ERIC-PCR genomic fingerprinting. Reference strains of Bradyrhizobium japonicum, B. liaoningense and B. elkanii and some Canarian isolates from endemic woody legumes in the tribe Genisteae were also included. The 16S rDNA-RFLP analysis resolved 9 genotypes of lupin isolates, a group of fourteen isolates presented restriction-genotypes identical or very similar to B. japonicum, while another two main groups of isolates (69%) presented genotypes that clearly separated them from the reference species of soybean. 16S rDNA sequencing of representative strains largely agreed with restriction analysis, except for a group of six isolates, and showed that all the lupin isolates are relatives of B. japonicum, but different lineages were observed. The 16S-23S IGS-RFLP analysis showed a high resolution level, resolving 19 distinct genotypes among 30 strains analysed, and so demonstrating the heterogeneity of the 16S-RFLP groups. ERIC-PCR fingerprint analysis showed an enormous genetic diversity producing a different pattern for each but two of the isolates. Phylogeny of nodC gene was independent from the 16S rRNA phylogeny, and showed a tight relationship in the symbiotic region of the lupin isolates with isolates from Canarian genistoid woody legumes, and in concordance, cross-nodulation was found. We conclude that Lupinus is a promiscuous host legume that is nodulated by rhizobia with very different chromosomal genotypes, which could even belong to several species of Bradyrhizobium. No correlation among genomic background, original host plant and geographic location was found, so, different chromosomal genotypes could be detected at a single site and in a same plant species, on the contrary, an identical genotype was detected in very different geographical locations and plants.

The strain Bacillus sp. CECT 450 increased nodulation on bean (Phaseolus vulgaris L.) when co-inoculated with Rhizobium tropici CIAT 899. This positive effect occured under controlled conditions on perlite–vermiculite, sand, or in a mixture of soil and sand. This increase was also observed in a field assay. Nodulation kinetic studies suggested that the synergistic effect is pronounced during the latter stages of cultivation. In contrast, the same bacteria co-inoculated with Bradyrhizobium japonicum USDA 110 reduced nodulation on soybean (Glycine max (L.) Merr.). Inoculation with Bacillus sp. CECT 450 alone had no effect on bean plants, but reduced root growth in soybean. The survival of Bacillus sp. CECT 450 on inoculated seeds was high, even when inoculated seeds were maintained for several months at room temperature.Key words: Rhizobium spp., Bacillus sp., Phaseolus vulgaris, nodulation, co-inoculation.

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.

Homologues to Sinorhizobium meliloti insertion sequences ISRm3 and ISRm4 were found within Rhizobium leguminosarum bv. viciae field populations. Similarly, homologous sequences to S. meliloti ISRm4 were found in S. fredii strains. Based on the polymerase chain reaction, we cloned and partially sequenced some of these putative insertion sequence elements. DNA sequence comparisons indicate that S. meliloti and R. leguminosarum ISRm3-type elements are closely related whereas the ISRm4 homologues show distinct relationships. In addition, specific primers for PCR recognition of ISRm3 and ISRm4 elements in rhizobia species and S. meliloti ISRm6 were designed.

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.

Rhizobium (now Sinorhizobium) fredii is a symbiotic nitrogen-fixing bacterium that can nodulate soybean in a cultivar-specific manner. This process is governed by a set of negatively acting nodulation genes termed noIXWBTUV. These genes prevent R. fredii strain USDA257 from infecting soybean cultivars such as McCall, but they do not block nodulation of cultivar Peking. R. fredii strain USDA191 contains DNA sequences that hybridize to noIXWBTUV, yet it forms normal nitrogen-fixing nodules on both McCall and Peking soybean. These sequences were isolated and their structure and function examined in comparison to noIXWBTUV of strain USDA257. Restriction maps of the two loci are identical, as is a 2∙4 kb DNA sequence that corresponds to noIX and its promoter region. Expression of noIX by strain USDA191 is flavonoid-dependent in culture and readily detectable in nodules. The gene is not inducible in a mutant of strain USDA191 that lacks the regulatory nodD1 gene, and its expression is greatly attenuated in a nodD2 mutant. noIX is also present and flavonoid-inducible in HH103, a second R. fredii strain that nodulates McCall soybean normally. Inactivation of noIX in strain HH103, USDA191 or USDA257 leads to retardation of initial nodulation rates on soybean cultivars such as Peking and to acquisition of the capacity to form nitrogen-fixing nodules on two species of Erythrina. noIX is thus of symbiotic significance in all three strains, even though it regulates soybean cultivar specificity only in strain USDA257.

Model legumes such as Lotus japonicus have contributed significantly to the understanding of symbiotic nitrogen fixation. This insight is mainly a result of forward genetic screens followed by map-based cloning to identify causal alleles. The L. japonicus ecotype 'Gifu' was used as a common parent for inter-accession crosses to produce F2 mapping populations either with other L. japonicus ecotypes, MG-20 and Funakura, or with the related species L. filicaulis. These populations have all been used for genetic studies but segregation distortion, suppression of recombination, low polymorphism levels, and poor viability have also been observed. More recently, the diploid species L. burttii has been identified as a fertile crossing partner of L. japonicus. To assess its qualities in genetic linkage analysis and to enable quantitative trait locus (QTL) mapping for a wider range of traits in Lotus species, we have generated and genotyped a set of 163 Gifu × L. burttii recombinant inbred lines (RILs). By direct comparisons of RIL and F2 population data, we show that L. burttii is a valid alternative to MG-20 as a Gifu mapping partner. In addition, we demonstrate the utility of the Gifu × L. burttii RILs in QTL mapping by identifying an Nfr1-linked QTL for Sinorhizobium fredii nodulation.

The stability of industrial polymeric sulfur is a key parameter in the characterization of this material. In the present work, a method based on differential scanning calorimetry (DSC) for the determination of the stability of polymeric sulfur is proposed as an alternative to the gravimetric “classical” method. This calorimetric method is simple and allows estimation of the stability in a short time and with adequate precision, and is thus suitable for plant control purposes.

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 reaction of the singlet oxygen with 9-methyl-1,2,3,4-tetrahydrocarbazole has been undertaken to give a benzazonine derivative in good yield. Rose-bengal supported on an anionic resin was used as heterogeneous photosensitizing agent. Kinetic evaluation has been made for the additon of singlet oxygen to the indole ring at room temperature.

Summary We have analysed 198 fast-growing soybean-nodulating rhizobial strains from four different regions of China for the following characteristics: generation time; number of plasmids; lipopolysaccharide (LPS), nodulation factors (LCOs) and PCR profiles; acidification of growth medium; capacity to grow at acid, neutral, and alkaline pH; growth on LC medium; growth at 28 and 37 °C; melanin production capacity; Congo red absorption and symbiotic characteristics. These unbiased analyses of a total subset of strains isolated from specific soybean-cropping areas (an approach which could be called “ strainomics ”) can be used to answer various biological questions. We illustrate this by a comparison of the molecular characteristics of five strains with interesting symbiotic properties. From this comparison we conclude, for instance, that differences in the efficiency of nitrogen fixation or competitiveness for nodulation of these strains are not apparently related to differences in Nod factor structure.

Summary Biodiversity studies of native soybean-nodulating rhizobia in soils from the Chinese Hubei province (Honghu county; pH 8, alluvial soil) have been carried out. Inoculation of an American (Williams) and an Asiatic (Peking) soybean cultivar with eleven soil samples led to the isolation of 167 rhizobia strains. The ratio (%) of slow-/fast-growing isolates was different depending on the trap plant used. All isolates were able to nodulate both cultivars, although the N 2 -fixation efficiency (measured as plant-top dry weight) was different among them. A total of thirty-three isolates were selected for further characterisation on the basis of physiological parameters, PCR-RFLP of symbiotic genes and Low Molecular Weight RNA, lipopolysaccharide, protein and plasmid profiles. Low Molecular Weight RNA profiling indicates that all the isolates belong to species Sinorhizobium fredii . The dendrogram obtained with the physiological parameters has been useful to classify the isolates at strain level, although plasmid profiling was the most discriminating technique to detect differences among the analysed soybean-rhizobia isolates, showing there is not two isolates identical each other. Plasmid profile analyses also revealed that some of the investigated strains contain low molecular weight plasmids (7–8-kb). They are, to our knowledge, the smallest ever found in rhizobia and they could be the starting point for the construction of the first group of vectors based on a native rhizobia replicon.

We isolated a Tn 5 -induced Rhizobium tropici mutant that has enhanced capacity to oxidize N,N -dimethyl- p -phenylendiamine (DMPD) and therefore has enhanced respiration via cytochrome oxidase. The mutant had increased levels of the cytochromes c 1 and CycM and a small increase in the amount of cytochrome aa 3 . In plant tests, the mutant increased the dry weight of Phaseolus vulgaris plants by 20 to 38% compared with the control strain, thus showing significantly enhanced symbiotic performance. The predicted product of the mutated gene is homologous to glycogen synthases from several bacteria, and the mutant lacked glycogen. The DNA sequence of the adjacent gene region revealed six genes predicted to encode products homologous to the following gene products from Escherichia coli : glycogen phosphorylase ( glgP ), glycogen branching enzyme ( glgB ), ADP glucose pyrophosphorylase ( glgC ), glycogen synthase ( glgA ), phosphoglucomutase ( pgm ), and glycogen debranching enzyme ( glgX ). All six genes are transcribed in the same direction, and analysis with lacZ gene fusions suggests that the first five genes are organized in one operon, although pgm appears to have an additional promoter; glgX is transcribed independently. Surprisingly, the glgA mutant had decreased levels of high-molecular-weight exopolysaccharide after growth on glucose, but levels were normal after growth on galactose. A deletion mutant was constructed in order to generate a nonpolar mutation in glgA . This mutant had a phenotype similar to that of the Tn 5 mutant, indicating that the enhanced respiration and symbiotic nitrogen fixation and decreased exopolysaccharide were due to mutation of glgA and not to a polar effect on a downstream gene.

The initial steps of the thermal and catalytic cracking of low-density polyethylene (LDPE) at different temperatures and reaction times have been studied in batch operation. The catalysts employed were silica- alumina, USY zeolite and MCM-41. The thermal degradation follows a random scission mechanism with little contribution of isomerization and aromatization reactions; cracking over SiO 2 -Al 2 O 3 shows both products coming from random scission and isomerization processes. On the contrary, USY zeolite favours an end- chain cracking mechanism with pronounced isomerization and aromatization. LDPE degradation over MCM-41 proceeds through a random scission mechanism due to their large pores, while hydrogen transfer reactions involving high molecular weight molecules are also favoured by this catalyst.

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.

Rhizobium tropici CIAT899 has a branched respiratory chain. During aerobic growth, the transport of electrons to oxygen takes place through at least two alternative pathways with oxidases of the aa 3- and “o”- (or bb 3-) types. Cytochrome d has also been observed although only in a cytochrome c biogenesis mutant. Bacteroids express a high affinity cbb 3-type cytochrome c oxidase for microaerobic respiration in the nodule. Our goal is to isolate mutants with altered respiration and possibly increased symbiotic N2 fixation.

Reaction of 2,3-polymethylene-1H-indolylmagnesium iodides with methyl iodide gives the C-methyl derivative and the spiro [cycloalkane-1,2'-indolin]-3'-one; the selectivity and optimization of the reaction has been analysed and a mechanism for the formation of the oxidation product is proposed

The synthesis and reactivity of 1,2-dhydrocarbazol-4(3H)-one, (1), is reported. An X-ray molecular structure study of (1) provided information on the inactivity of its carbonyl group with potentially anionic reagents. 4(2- Dimethylaminoethyl)-1,2,3,4-tetrahydrocarbazole has been synthesized from 1,2,3,4-tetrahydrocarbazol-4-ylidenemethyl acetate by reduction of the exocyclic double bond, followed by transamination and reduction.

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

Spiro[cycloalkane-1,3′-[3H]indoles] 2 can be obtained from the cycloalkanecarbaldehydes 1 by the Fischer reaction of their phenylhydrazones. These cyclizations are sensitive to the acid catalyst, solvent and temperature employed. Rearrangement of the 2 to the homologous cycloalkane derivatives 3 can occur by an acid catalyst or by thermal treatment of 2 in ethyleneglycol.

A conformational study of the title compound has been carried out in solution and solid states. The molecules are packed in the crystal forming a charge-transfer complex, where two different molecular pairs are found. Visible spectra data show two weak absorption bands.

1′-Methylspiro[3H-indole-3,n'-piperidines] 2 can be obtained from the 1-methyl-n-piperidinecarbaldehydes 1 by the Fischer reaction of their phenylhydrazones. The Fischer reaction provides different kinds of products -3H-indole, indole and oxindole- which depend on the N atom position in the piperidine ring.

The structures of cyclohexan[b]indole N-β-propionic acid (1) and cyclooctan[b]indole N-β-propionic acid (2) have been determined by X-ray methods. The conformational populations of their N-β-propionic chains have been determined in solution by 1H nmr analyses. Crystals of 1 are triclinic, space group [Formula: see text] with a = 12.3562(4), b = 11.0727(3), c = 10.2050(4) Å, α = 108.519(3), β = 83.941(3), γ = 104.826(3)°, and Z = 4. Crystals of 2 are monoclinic, space group P21/c, with a = 5.636(1), b = 18.937(3), c = 13.976(4) Å, β = 98.84(3)°, and Z = 4. In both structures, the molecules are linked forming dimers through centrosymmetric H bonds. In solution the trans conformation of the chain is the preferred one in both compounds and increases with dilution until a constant value is reached.

Reactivity of the CN bond in spiro[cycloalkane-1,3′-3H-indoles] has been analysed. Indolines were obtained in practically quantitative yield using organomagnesium reagents in the presence of copper(i) chloride in toluene at 120 °C. On the basis of an analysis of the reaction products and the e.s.r. spectra, a mechanistic pattern could be proposed. N-Alkylation of 2′-substituted spiro[cycloalkane-1,3′-indolines] has been carried out using as alkylating agent 3-chloro-N,N-dimethylpropionamide, which gave the 1′-(3-dimethylaminopropyl)-2′-substituted spiro[cycloalkane-1,3′-indolines] in good yield on reduction with LiAlH4.

2′-Substitued 1′-(2-dimethylcarbamoylethyl)-3′-spirocycloalkanoindolines (2a–f) have been obtained by a phase-transfer attack of 3-chloro-NN-dimethylpropanamide on the indolines (1a–f). 1H N.m.r. spectra of the nitrogen side chain in compounds (2a–f) show an ABCD proton system for the CH2–CH2–CON fragment. Conformational analyses of this fragment for (2a–f) are given and the conformer populations discussed in terms of the bulky ring and the 2′-substituent. Conformational analysis of the CH–CH2–Ph fragment in the indoline (1h) is also given.

1′-Methylspiro[3H-indole-3,n'-piperidines] 2 can be obtained from the 1-methyl-n-piperidinecarbaldehydes 1 by the Fischer reaction of their phenylhydrazones. The Fischer reaction provides different kinds of products -3H-indole, indole and oxindole- which depend on the N atom position in the piperidine ring.

Reactivity of the CN bond in spiro[cycloalkane-1,3′-3H-indoles] has been analysed. Indolines were obtained in practically quantitative yield using organomagnesium reagents in the presence of copper(i) chloride in toluene at 120 °C. On the basis of an analysis of the reaction products and the e.s.r. spectra, a mechanistic pattern could be proposed. N-Alkylation of 2′-substituted spiro[cycloalkane-1,3′-indolines] has been carried out using as alkylating agent 3-chloro-N,N-dimethylpropionamide, which gave the 1′-(3-dimethylaminopropyl)-2′-substituted spiro[cycloalkane-1,3′-indolines] in good yield on reduction with LiAlH4.

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.