Emilio Cervantes

nodSU, two new nod genes of the broad host range Rhizobium strain NGR234 encode host-specific nodulation of the tropical tree Leucaena leucocephala.

Rhizobium species strain NGR234 nodulates at least 35 diverse genera of legumes as well as the nonlegume Parasponia andersonii. Most nodulation genes are located on the 500-kilobase pair symbiotic plasmid, pNGR234a. Previously, three plasmid-borne host range determinants (HsnI, HsnII, and HsnIII) were identified by their ability to extend the nodulation capacity of heterologous rhizobia to include Vigna unguiculata. In this study, we show that HsnII contains two new nod-box linked hsn genes, nodS and nodU.nodS controls nodulation of the tropical tree Leucaena leucocephala, while the nodSU genes regulate nodulation of the pasture legume Desmodium intortum and the grain legume V. unguiculata. Regulation of the nod-box upstream of nodSU by the flavonoid naringenin was shown using a fusion with a promoterless lacZ gene. Determination of the nucleotide sequence of the nodS gene did not reveal homology with any gene in the EMBL library, although Bradyrhizobium japonicum USDA110 contains both nodS and nodU (M. Göttfert, S. Hitz, and H. Hennecke, Molecular Plant-Microbe Interactions 3:308-316, 1990). We suggest that broad host range in NGR234 is controlled in part by a nodD gene which interacts with a wide range of flavonoids, and in part by host-specific nod genes such as nodS.

Differential effects of coinoculations with Pseudomonas jessenii PS06 (a phosphate-solubilizing bacterium) and Mesorhizobium ciceri C-2/2 strains on the growth and seed yield of chickpea under greenhouse and field conditions

In the course of a project carried out in two regions of Spain, Castilla y Leon and Andalucia, aiming to find useful biofertilizers for staple grain-legumes, an efficient rhizobia nodulating chickpea (termed as C-2/2) and a powerful in vitro phosphate-solubilizing bacterial strain (termed as PS06) were isolated. Analyses of their 16S rDNA sequence indicated that they belong to the bacterial species Mesorhizobium ciceri and Pseudomonas jessenii, respectively. Greenhouse and field experiments were carried out in order to test the effect of single and dual inoculations on chickpea (ecotype ILC-482) growth. Under greenhouse conditions, plants inoculated with Mesorhizobium ciceri C-2/2 alone had the highest shoot dry weight. The inoculation treatment with P. jessenii PS06 yielded a shoot dry weight 14% greater than the uninoculated control treatment, but it was not correlated with shoot P contents. However, the co-inoculation of C-2/2 with PS06 resulted in a decrease in shoot dry weight with respect to the inoculation with C-2/2 alone. Under field conditions, plants inoculated with M. ciceri C-2/2, in single or dual inoculation, produced higher nodule fresh weight, nodule number and shoot N content than the other treatments. Inoculation with P. jessenii PS06 had no significant effect on plant growth. However, the co-inoculation treatment ranked the highest in seed yield (52% greater than the uninoculated control treatment) and nodule fresh weight. These data suggest that P. jessenii PS06 can act synergistically with M. ciceri C-2/2 in promoting chickpea growth. The contrasting results obtained between greenhouse and field experiments are discussed.

Mesorhizobium chacoense sp. nov., a novel species that nodulates Prosopis alba in the Chaco Arido region (Argentina)

Low-molecular-weight RNA analysis was performed for the identification and classification of 20 Argentinian strains isolated from the root nodules of Prosopis alba. SDS-PAGE of total cellular proteins, determination of the DNA base composition, DNA-DNA reassociation experiments and physiological and biochemical tests were also carried out for these strains and the whole 16S rRNA gene was sequenced from one of the strains, strain LMG 19008T. Results of the genotypic and phenotypic characterization showed that the strains isolated in this study belong to a group that clustered in the genus Mesorhizobium. The results of DNA-DNA hybridizations showed that this group is a novel species of this genus. The name Mesorhizobium chacoense sp. nov. is proposed for this species. The type strain is LMG 19008T (= CECT 5336T).

The Rhizobium meliloti host-range nodQ gene encodes a protein which shares homology with translation elongation and initiation factors

The Rhizobium meliloti nod region lib is involved in host‐range determination: (i)the presence of region lib is necessary for transfer of alfalfa root hair curling ability to Rhizobium legumlnosarum biovar trifolii; (ii) a mutation in region llb extends the R. meliloti infection host range to Vicia sativa nigra; (iii) dominance of R. meliloti nod genes over R. leguminosarum biovar viciae nod genes is abolished by mutations in region llb. The nucleotide sequence of this region has been determined. Genes corresponding to the two open reading frames identified are designated nodP and nodQ. The predicted amino acid sequence of the NodQ protein shows homology with translation initiation and elongation factors. The consensus sequence involved in the GTP‐binding domain is conserved.

Ethylene regulates the expression of a cysteine proteinase gene during germination of chickpea (Cicer arietinum L.)

Synthetic oligonucleotides corresponding to conserved regions of cysteine proteinases were used as primers in the RT-PCR amplification of a fragment of cDNA corresponding to a region of a cysteine proteinase gene expressed during germination of chickpea (cac for Cicer arietinum cysteine proteinase). The identity of the PCR-amplified fragment was confirmed by sequencing and the fragment used as a probe to investigate the pattern of cac gene expression during germination and its hormonal regulation. The corresponding transcript is undetected in the seed during embryogenesis and before imbibition, being detected 24 h after imbibition. Ablation of the embryonic axis before imbibition results in a dramatic decrease in the amount of transcript detected. Expression of the cac transcript in excised cotyledons is restored in the presence of aqueous extracts from embryonic axes and also by incubating the excised cotyledons in 1 mM ethephon. Experiments with various known inhibitors of ethylene action indicate that ethylene activates the expression of cac gene in the cotyledons of chickpea during normal germination.

22 Relationships between the Mycorrhizal and Actinorhizal Symbioses in Non-legumes

Publisher Summary This chapter discusses relationships between the mycorrhizal and actinorhizal symbioses in non-legumes. Some non-leguminous, nitrogen-fixing plants carry dual symbiotic infection involving both mycorrhizal fungi and actinorhizal actinomycetes. The characterization of the genus Frankia, the microsimbiont in the actinorhizal symbiosis, is approaching maturity but the optimal application of methods already developed requires the consideration of the real contribution of mycorrhiza associated with the actinorhizal symbioses. The chapter discusses mycorrhiza in actinorhizal plants. Mycorrhizal species are found in most actinorhizal plant genera so far examined in both ectomycorrhizal and vesicular-arbuscular mycorrhizal symbioses. The presence of a mycorrhizal symbiosis in a plant species depends largely on many soil characteristics. Thus, infection of Myrica gale by both ecto- and vesicular-arbuscular mycorrhiza is restricted to well-drained soils, being absent in wet soils. The actinorhizal symbioses are also highlighted in the chapter.

Analysis of LMW RNA profiles of Frankia strains by staircase electrophoresis.

An optimized technique of polyacrylamide gel electrophoresis, Staircase Electrophoresis (SCE), was applied to determine the stable Low Molecular Weight RNA (LMW RNA) profiles of 25 Frankia strains from diverse geographic origins and host specificity groups as well as species from other actinomycete genera. Application of the technique permits the rapid identification of Frankia strains and their differentiation from other actinomycetes. The isolates used in this study were grouped in eight clusters, each comprising strains with identical LMW RNA profiles. Comparison of these results with others obtained from DNA sequences or DNA hybridization methods suggest a high degree of complexity in the genus Frankia. Application of SCE to profile LMW RNA should in the future facilitate biodiversity studies of Frankia and discrimination of new species.

Modeling the Arabidopsis seed shape by a cardioid: Efficacy of the adjustment with a scale change with factor equal to the Golden Ratio and analysis of seed shape in ethylene mutants

A new model for the description of Arabidopsis seed shape based on the comparison of the outline of its longitudinal section with a transformed cardioid is presented. The transformation consists of scaling the horizontal axis by a factor equal to the Golden Ratio. The elongated cardioid approximates the shape of the Arabidopsis seed with more accuracy than other figures. The length to width ratio in wild-type Columbia Arabidopsis dry seeds is close to the Golden Ratio and decreases over the course of imbibition. Dry seeds of etr1-1 mutants presented a reduced length to width ratio. Application of the new model based on the cardioid allows for comparison of shape between wild-type and mutant genotypes, revealing other general alterations in the seeds in ethylene signaling pathway mutants (etr1-1).

The effects of aluminium on nodulation and symbiotic nitrogen fixation in Casuarina cunninghamiana Miq.

In order to investigate the effects of Al on nodule formation and function in the Casuarina-Frankia symbiosis, inoculated plants were grown in sand culture at five nominal Al concentrations (0-880 μM Al) at pH 4.0. There was an Al-free control at pH 6.0 to assess the effects of pH 4.0 treatments. Mean N concentration of nodules was significantly less at pH 4.0 (1.83%) than at pH 6.0 (2.01%). There were nodulated plants at all Al levels, though there were fewer nodulated plants at 440 and 880 μM Al. Dry weights of nodules, shoots and roots were not reduced by Al concentrations at or below 220 μM Al, but were decreased by Al concentrations at or above 440 μM Al. Nodule weight expressed as a percentage of total weight did not differ significantly with respect to an Al-free control at pH 4. N concentrations of shoots and whole plants were significantly reduced at 440 μM Al. Nodular specific acetylene reduction activity (ARA) did not differ significantly among Al treatments. However, N2-fixation efficiency was decreased from 0.20 to 0.10 mg N fixed mg nodule dry weight−1 at 880 μM Al.

Cellulase isoenzyme profiles in Frankia strains belonging to different cross-inoculation groups

Carboxymethyl cellulase activities were evaluated in eight strains of Frankia from diverse host specificity groups and geographical origins. Cellulase activity was detected in culture supernatants of all strains in the absence of CMC (Carboxymethylcellulose) as an inducer using both double-layer plate and reducing sugar assays, indicating a constitutive production of CM-cellulases by Frankia. CM-cellulase isoenzyme profiles were visualized using activity gel electrophoresis of concentrated culture supernatants. Different electrophoretic profiles were observed among the eight strains tested, which correlate with the host specificity and taxonomic grouping of Frankia.