M. Camacho

Chryseobacterium hispalense sp. nov., a plant-growth-promoting bacterium isolated from a rainwater pond in an olive plant nursery, and emended descriptions of Chryseobacterium defluvii, Chryseobacterium indologenes, Chryseobacterium wanjuense and Chryseobacterium gregarium

A novel non-motile, Gram-staining-negative, yellow-pigmented bacterium, designated AG13(T), isolated from a rain water pond at a plant nursery in Spain and characterized as a plant-growth-promoting bacterium, was investigated to determine its taxonomic status. The isolate grew best over a temperature range of 15-40 °C, at pH 5.0-8.0 and with 0-4 % (w/v) NaCl. Chemotaxonomic and molecular characteristics of the isolate matched those described for members of the genus Chryseobacterium. The DNA G+C content of the novel strain was 37.2 mol%. The strain had a polyamine pattern with sym-homospermidine as the major compound and produced flexirubin-type pigments. MK-6 was the dominant menaquinone and the major cellular fatty acids were iso-C15 : 0, C17 : 1ω9c and iso-C17 : 0 3-OH. The main polar lipids were phosphatidylethanolamine, aminolipids and several unidentified lipids. The 16S rRNA gene showed 92.0-97.2 % sequence similarity with those of the members of the genus Chryseobacterium. Based on chemotaxonomic and phenotypic traits, and DNA-DNA hybridizations with the type strains of the most closely related species, the isolate is proposed to represent a novel species, Chryseobacterium hispalense, type strain AG13(T) ( = DSM 25574(T) = CCUG 63019(T)). Emended descriptions of the species Chryseobacterium defluvii, Chryseobacterium indologenes, Chryseobacterium wanjuense and Chryseobacterium gregarium are also provided.

Bradyrhizobium pachyrhizi sp. nov. and Bradyrhizobium jicamae sp. nov., isolated from effective nodules of Pachyrhizus erosus.

Several strains isolated from the legume Pachyrhizus erosus were characterized on the basis of diverse genetic, phenotypic and symbiotic approaches. These novel strains formed two groups closely related to Bradyrhizobium elkanii according to their 16S rRNA gene sequences. Strains PAC48T and PAC68T, designated as the type strains of these two groups, presented 99.8 and 99.1% similarity, respectively, in their 16S rRNA gene sequences with respect to B. elkanii USDA 76T. In spite of these high similarity values, the analysis of additional phylogenetic markers such as atpD and glnII genes and the 16S-23S intergenic spacer (ITS) showed that strains PAC48T and PAC68T represented two separate novel species of the genus Bradyrhizobium with B. elkanii as their closest relative. Phenotypic differences among the novel strains isolated from Pachyrhizus and B. elkanii were found regarding the assimilation of carbon sources and antibiotic resistance. All these differences were congruent with DNA-DNA hybridization analysis which revealed 21% genetic relatedness between strains PAC48T and PAC68T and 46% and 25%, respectively, between these strains and B. elkanii LMG 6134T. The nodD and nifH genes of strains PAC48T and PAC68T were phylogenetically divergent from those of bradyrhizobia species that nodulate soybean. Soybean was not nodulated by the novel Pachyrhizus isolates. Based on the genotypic and phenotypic data obtained in this study, the new strains represent two novel species for which the names Bradyrhizobium pachyrhizi sp. nov. (type strain PAC48T=LMG 24246T=CECT 7396T) and Bradyrhizobium jicamae sp. nov. (type strain PAC68T=LMG 24556T=CECT 7395T) are proposed.

Characterization of Rhizobium spp. bean isolates from South- West Spain

Rhizobium spp. strains able to nodulate beans (Phaseolus vulgaris L.) were isolated from Andalusian (Southern Spain) soils with no record of recent bean cultivation (except soil 14) and no known history of bean inoculation in this area. The isolation methodology was devised to obtain an heterogeneous rhizobia population from each soil sample, by using three different bean cultivars as trap-host. No association was found between the presence of rhizobia nodulating bean and the chemical or textural properties of the soils. The isolates were grouped on the basis of their symbiotic effectiveness on bean cv. Canellini under greenhouse conditions, intrinsic antibiotic resistance (IAR), lipopolysaccharide (LPS) and protein profiles, melanin production, and by amplified rDNA restriction analysis (ARDRA). Most of the isolates were more effective than the reference strains Rhizobium leguminosarum bv. phaseoli TAL1121, R. etli type strain CFN42 and R. tropici type strain CIAT899. The symbiotic effectiveness of the isolates could not be related with other traits analyzed. Predominantly, a two bands-LPS profile was found amongst the isolates. Most of them have been assigned to R. etli by ARDRA and seem to be more competitive than R. gallicum or R. giardinii isolates. Additionally, a strong interaction between the bean cultivar and the native rhizobia populations was observed.

Perlite as a carrier for bacterial inoculants.

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.

Effect of pH and soybean cultivars on the quantitative analyses of soybean rhizobia populations

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.

NolR Regulates Diverse Symbiotic Signals of Sinorhizobium fredii HH103

We have investigated in Sinorhizobium fredii HH103-1 (=HH103 Str(r)) the influence of the nolR gene on the production of three different bacterial symbiotic signals: Nod factors, signal responsive (SR) proteins, and exopolysaccharide (EPS). The presence of multiple copies of nolR (in plasmid pMUS675) repressed the transcription of all the flavonoid-inducible genes analyzed: nodA, nodD1, nolO, nolX, noeL, rhcJ, hesB, and y4pF. Inactivation of nolR (mutant SVQ517) or its overexpression (presence of pMUS675) altered the amount of Nod factors detected. Mutant SVQ517 produced Nod factors carrying N-methyl residues at the nonreducing N-acetyl-glucosamine, which never have been detected in S. fredii HH103. Plasmid pMUS675 increased the amounts of EPS produced by HH103-1 and SVQ517. The flavonoid genistein repressed EPS production of HH103-1 and SVQ517 but the presence of pMUS675 reduced this repression. The presence of plasmid pMUS675 clearly decreased the secretion of SR proteins. Inactivation, or overexpression, of nolR decreased the capacity of HH103 to nodulate Glycine max. However, HH103-1 and SVQ517 carrying plasmid pMUS675 showed enhanced nodulation capacity with Vigna unguiculata. The nolR gene was positively identified in all S. fredii strains investigated, S. xinjiangense CCBAU110, and S. saheli USDA4102. Apparently, S. teranga USDA4101 does not contain this gene.

Sinorhizobium fredii HH103 has a truncated nolO gene due to a -1 frameshift mutation that is conserved among other geographically distant S. fredii strains.

Strain SVQ121 is a mutant derivative of Sinorhizobium fredii HH103 carrying a transposon Tn5-lacZ insertion into the nolO-coding region. Sequence analysis of the wild-type gene revealed that it is homologous to that of Rhizobium sp. NGR234, which is involved in the 3 (or 4)-O-carbamoylation of the nonreducing terminus of Nod factors. Downstream of nolO, as in Rhizobium sp. NGR234, the noeI gene responsible for methylation of the fucose moiety of Nod factors was found. SVQ121 Nod factors showed lower levels of methylation into the fucosyl residue than those of HH103-suggesting a polar effect of the transposon insertion into nolO over the noel gene. A noeI HH103 mutant was constructed. This mutant, SVQ503, produced Nod factors devoid of methyl groups, confirming that the S. fredii noeI gene is functional. Neither the nolO nor the noeI mutation affected the ability of HH103 to nodulate several host plants, but both mutations reduced competitiveness to nodulate soybean. The Nod factors produced by strain HH103, like those of other S. fredii isolates, lack carbamoyl residues. By using specific polymerase chain reaction primers, we sequenced the nolO gene of S. fredii strains USDA192, USDA193, USDA257, and 042B(s). All the analyzed strains showed the same -1 frameshift mutation that is present in the HH103 nolO-coding region. From these results, it is concluded that, regardless of their geographical origin, S. fredii strains carry the nolO-coding region but that it is truncated by the same base-pair deletion.

Phenotypic and genotypic characterization of rhizobia from diverse geographical origin that nodulate Pachyrhizus species.

Legumes from the genus Pachyrhizus, commonly known as yam bean, are cultivated in several countries from the American continent and constitute an alternative source for sustainable starch, oil and protein production. The endosymbionts of these legumes have been poorly studied although it is known that this legume is nodulated by fast and slow growing rhizobia. In this study we have analyzed a collection of strains isolated in several countries using different phenotypic and molecular methods. The results obtained by SDS-PAGE analysis, LPS profiling and TP-RAPD fingerprinting showed the high diversity of the strains analyzed, although all of them presented slow growth in yeast mannitol agar (YMA) medium. These results were confirmed using 16S-23S internal transcribed spacer (ITS) region and complete sequencing of the 16S rRNA gene, showing that most strains analyzed belong to different species of genus Bradyrhizobium. Three strains were closely related to B. elkanii and the rest of the strains were related to the phylogenetic group constituted by B. japonicum, B. liaoningense, B. yuanmingense and B. betae. These results support that the study of rhizobia nodulating unexplored legumes in different geographical locations will allow the discovery of new species able to establish legume symbioses.

Molecular phylogenetic analysis shows that Amanita ponderosa and A. curtipes are distinct species

Amanita curtipes and A. ponderosa are two Mediterranean taxa sharing a number of morphological features as well as their habitat. Their synonymy or variety status has been proposed by several authors. To clarify this taxonomic issue we have sequenced the D1-D2 domains of the 28S rRNA gene as well as the complete ITS1-5.8S-ITS2 region of several specimens of the two species collected in Spain, and aligned these sequences with those from other Amanita species. Molecular phylogenetic analysis based on the two regions revealed that A. ponderosa and A. curtipes are clearly distinct species. The distribution of Amanita species in the phylogenetic trees was consistent with the division of the genus in subgenera and sections as proposed by previous authors. Sequences of A. ponderosa and A. curtipes were grouped in a monophyletic cluster together with other species of the section Amidella. However, A. ponderosa was closer to other species in the section, such as A. peckiana and A. volvata, than to A. curtipes. We also indicate the macromorphological characters that are most useful to reliably distinguish A. ponderosa and A. curtipes.

Field assessment and genetic stability of Sinorhizobium fredii strain SMH12 for commercial soybean 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.