Symbiotic performance of some modified Rhizobium etli strains in assays with Phaseolus vulgaris beans that have a high capacity to fix N2

Rhizobium etli and R. tropici form nitrogen-fixing nodules on Phaseolus vulgaris (common bean). In the hope that R. etli strains with additional citrate synthase genes have better carbon economies, merodiploid strains were constructed. Previously, one such construct was shown to have an increased nodulation capacity in the standard bean cultivar Negro Xamapa. In the present work, derivatives from different R. etli strains carrying the R. tropici plasmid-borne or chromosomal citrate synthase gene were constructed and tested for nodulation in bean cultivars selected for their high capacity to fix nitrogen. Nodule numbers were dependent on the strain and the cultivar used. Differences in nodule number were not reflected in plant biomass.     

Phylogenetic relationships and host range of Rhizobium spp. that nodulate Phaseolus vulgaris L.

We determined the nucleotide sequences of 16S rRNA gene segments from five Rhizobium strains that have been isolated from tropical legume species. All share the capacity to nodulate Phaseolus vulgaris L., the common bean. Phylogenetic analysis confirmed that these strains are of two different chromosomal lineages. We defined the host ranges of two strains of Rhizobium etli and three strains of R. tropici, comparing them with those of the two most divergently related new strains. Twenty-two of the 43 tested legume species were nodulated by three or more of these strains. All seven strains have broad host ranges that include woody species such as Albizia lebbeck, Gliricidia maculata, and Leucaena leucocephala.     

Nitrogen fixation and nodule occupancy by native strains of Rhizobium on different cultivars of common bean (Phaseolus vulgaris L.)

A field experiment under rainfed conditions was conducted in Durango, México, to assess N2-fixation of three cultivars of common bean (Phaseolus vulgaris L.) using ¹⁵N-methodology. In addition, diversity of rhizobial isolates obtained from nodules of the different plant genotypes was evaluated by intrinsic antibiotic resistance (IAR), PCR using enterobacterial repetitive intergenic consensus (ERIC) primers, PCR-RFLP analysis of the 16S rRNA gene and multilocus enzyme electrophoresis (MLEE). Selected isolates were used to determine acetylene reduction and competitive ability under greenhouse conditions. The three cultivars tested did not show high variation in N2-fixation, the %Ndfa values ranged from 19 to 26%. Variability in N2-fixation efficiency among various native rhizobial isolates was very high and our results indicate that differences in competitive abilitiy exist also. PCR-RFLP of the 16S rRNA gene and MLEE revealed that most of the isolates belong to the species Rhizobium etli. Intrinsic antibiotic resistance analysis and ERIC-PCR showed high diversity among isolates. In contrast, our results using MLEE show low genetic diversity (H = 0.105).     

Comparison of geographically distant populations of Rhizobium isolated from root nodules of Phaseolus vulgaris

Seventy-two rhizobial strains were isolated from the root nodules of french beans ( Phaseolus vulgaris ). They were sampled from two geographically distant field populations and 18 additional different sites in France. They were characterized by a) plasmid profiles, (b) RFLP analysis of total cellular DNA using various chromosomal and symbiotic gene probes (including nif H from Rhizobium etli bv. phaseoli ) and c) their ability to nodulate a potential alternative host, L. leucocephala. Over half of the isolates were ascribed to Rhizobium leguminosarum bv. phaseoli on the basis of the hybridization analysis, the possession of multiple copies of nif H and their inability to nodulate L. leucocephala. The remaining isolates belonged to 2 groups which were shown to be genomically distinct from R. leguminosarum bv. phaseoli, R. etli bv. phaseoli and R. tropici. Most members of these two groups shared with R. tropici the ability to nodulate L. leucocephala and, for isolates of only one of these groups, the presence of one copy of nif H. Members of each of the 3 taxa were widely distributed in France and circumstantial evidence of pSym transfer between them was shown. R. leguminosarum bv. phaseoli and one of the two novel groups co-occurred within the two geographically distant populations. Individual genotypes were conserved between them. The finding of a third taxon at various other locations indicated additional diversity among rhizobia nodulating beans.     

Characterization of a Rhizobium etli chromosomal gene required for nodule development on Phaseolus vulgaris L.

A chromosomal gene, required for nodule development on Phaseolus bean, was characterized from Rhizobium etli strain TAL182. MLC640 is a Tn5 insertion mutant of TAL182 which shows decreased motility in soft TY agar and is defective in nodule development. The site of Tn5 insertion in MLC640 mapped to a 3.6-kb EcoRI chromosomal fragment. The 3.6-kb fragment was subcloned from the cosmid pUHR80 which complemented MLC640. Further subcloning and site-directed Tn5 mutagenesis localized the gene for nodule development to a 1.7-kb region within the 3.6-kb EcoRI fragment. Southern hybridization using the 3.6-kb EcoRI fragment as the probe against genomic DNA of several Rhizobium spp. indicated that this gene is conserved in different rhizobia.The authors are with the Department of Plant Molecular Physiology, University of Hawaii, 3050 Maile Way, Gimore 402, Honolulu, Hawaii 96822. USA;     

Rhizobium tropici nodulates field-grown Phaseolus vulgaris in France

Two hundred and eighty seven isolates of Rhizobium nodulating Phaseolus vulgaris L. were sampled in France from four geographically distant field populations. They were characterized by their colony morphology and by plasmid profiles. A representative sample was further characterized: a) by the ability of each isolate to nodulate a potential alternative host Leucaena leucocephala and to grow on specific media, and b) by RFLP analysis of PCR amplified 16S rRNA genes. On the basis of their phenotypic and genetic characteristics the isolates could be assigned either to Rhizobium leguminosarum bv phaseoli, or to R. tropici. The two species co-occurred at three sites. R. leguminosarum bv phaseoli represented 2%, 4%, 72% and 100% of the population at the four different sites. Eighteen and 22 different plasmid profiles were identified within R. tropici and R. leguminosarum bv phaseoli, respectively. Some of them were conserved between distant geographical regions. The fact that R. tropici was found in France shows that this species is not limited to tropical regions and gives additional evidence of the multi-specific nature of the Phaseolus microsymbiont, even over a geographically limited area.     

QTL mapping for nodule number and common bacterial blight in Phaseolus vulgaris L.

A recently developed bean RFLP linkage map was used to identify genetic elements affecting quantitative trait loci (QTLs) in two contrasting common bean genotypes, BAT-93 and Jalo EEP558, under two levels of mineral nitrogen: low – 0.25 mM NH4NO3 and a high – 6 mM NH4NO3. QTLs affecting nodule number (NN) and response to Xanthomonas campestris bv. phaseoli, which causes common bacterial blight (CBB) were identified and mapped. Analyses of 70 F2-derived F3 families, using the F1, the two parents, and a nodulation-defective mutant (Nod-) inoculated with R. tropici UM1899 under both levels of N showed significant differences (P#60;0.0001) among the F3 families for NN.Under low N, three genomic regions influenced both traits, with seven linked markers. In three of the six regions influencing NN, higher NN was associated with the Jalo EEP-558 allele, whereas in only two regions was the BAT-93 allele associated with higher NN. One-way analysis of variance, with each marker as the independent variable and NN as the dependent variable, and interval mapping analysis identified four QTLs, which accounted for 45% of the total variation, and two additional QTLs near to yet unassigned loci. In linkage group D7, one QTL mapped to the same region as a QTL for CBB.Under high N, three additional regions were linked to NN, one where the BAT-93 allele was closely associated with CH18 (chitinase), and the others where the Jalo EEP-558 allele was associated with CHS (chalcone synthetase) and PAL-1 (phenylalanine ammonia lyase). Four regions for CBB were mapped adjacent to or in the same region as a QTL for NN. Thus, N showed dual and opposite effects on the expression of NN and CBB. Analysis of these RFLP markers revealed these hidden favorable alleles and can serve as an indirect selection tool to increase NN and resistance to CBB.     

The Phototropic Pulvinus of Bean Phaseolus vulgaris L. - Functional Features

Abstract: The laminar pulvinus of bean ( Phaseolus vulgaris L.) is a highly specialized organ for reorienting the lamina, and exhibits positive phototropic curvature. Structural and ultrastructural features of the pulvinus were studied to determine their possible role in its phototropic response. The vascular tissue forms a flexible, relatively inextensible central core enclosed by a starch sheath and surrounded by a multi-layered motor tissue. Phototropic curvature is a result of opposite anisotropic changes in volume of the cells in the exposed and opposite sectors of the motor tissue. Radial inflexibility of the epidermis and axial plasticity constrain these changes to the pulvinar axis. Anisotropic changes in volume of motor cells reduce the osmotic work involved. Motor cells exhibit features that are associated with high synthetic activity: thick cytoplasm with numerous ribosomes, polysomes, RER and SER, well-developed mitochondria and a large nucleus. Numerous, well-developed chloroplasts, with little starch, are increasingly abundant toward the periphery. The intercellular system is limited and partially filled with matrix. Stomata are absent and the motor tissue lacks vascularization. These features support the suggestion that the primary role of the chloroplasts in the photonastic response is photophosphorylation and photosynthetic electron transport (Koller et al., 1995^[339]).     

Development and use of chloroplast microsatellites in Phaseolus spp. and other legumes

Chloroplast microsatellites (cpSSRs) provide a powerful tool to study the genetic variation and evolution of plants. We have investigated the usefulness of 39 primer pairs tagging cpSSR loci on a set of eight different genera of Leguminosae (Papilionoideae subfamily) and five species belonging to the genus Phaseolus . Thirty-six 'universal' primer pairs were retrieved from the literature, one was re-designed and a further two were designed de novo . The cpSSR loci analysed were highly polymorphic across the individuals examined. Twenty-seven primer pairs were polymorphic in the overall sample, 18 within Phaseolus , and 16 in both P. vulgaris and P. coccineus . Analysis of the plastome sequences of four Leguminosae species (obtained from GenBank) showed that in the loci targeted by universal primer pairs: (i) the originally tagged cpSSRs can be lost; (ii) other cpSSRs can be present; and (iii) polymorphism arises not only from differences in the numbers of cpSSR repeats, but often from other insertion/deletion events. Multilocus linkage disequilibrium analysis suggests that homoplasy is not a major problem in our dataset, and principal component analysis indicates intelligible relationships among the species considered. Our study demonstrates that this set of chloroplast markers provides a useful tool to study the diversity and the evolution of several legumes, and particularly P. vulgaris and P. coccineus .     

Multiple paternity in common bean (Phaseolus vulgaris L., Fabaceae)

We report on two field experiments that were conducted in 1991 and 1992 at the South Coast Extension and Research Center, Irvine, CA, to study the incidence of multiple paternity in the common bean (Phaseolus vulgaris L.). Hypocotyl color and shikimate dehydrogenase (Skdh) isozymes were used as genetic markers. The white-seeded cultivar ‘Ferry Morse 53’ (FM 53) was used as the female parent. This cultivar is homozygous recessive (pp) for hypocotyl color. The pollen source parents were three homozygous dominant (PP) purple-hypocotyled, black-seeded cultivars. Three cultivars, ‘ICA Pijao,’ G4459, and the maternal parent FM 53, are of Mesoamerican origin and homozygous for the fast (F) allele at the Skdh locus. The other cultivar, Black Valentine, is of Andean origin and is homozygous for the slow (S) allele at the Skdh locus. Overall, 6 125 pods were obtained from 57 and 111 plants harvested individually in 1991 and 1992, respectively. All progeny, 28938 seeds, were scored for hypocotyl color at the seedling stage. The purple-hypocotyled seedlings were genotyped for the Skdh locus to identify their pollen parents. Multiple paternity was identified in all the pods with hybrid seeds (i.e., those of intercultivar crosses) at 5.8% and 8.1% in 1991 and 1992, respectively. All multiply sired pods produced both nonhybrid and hybrid seeds. As many as three successful fathers per pod were identified, but the number of markers limited measuring higher levels of multiple paternity. Most multiply sired pods (≈70%) were filled by nonhybrid seeds plus a single hybrid seed. Ovule position effect within multiply sired pods was inferred from the nonrandom distribution of hybrid seeds within a pod. On average, hybrid seeds occurred more frequently in ovules in position 7 (most basal) and in position 1 (most stylar) than in ovules in the middle positions of the pod.     

Reticulated and epidemic population genetic structure of Rhizobium etli biovar phaseoli in a traditionally managed locality in Mexico

We conducted a multilocus enzyme electrophoresis (MLEE) study to assess the genetic structure of the nitrogen-fixing bacteria Rhizobium etli bv. phaseoli . We analysed the genetic variation at 10 enzyme-encoding chromosomal loci of 482 isolates from root nodules of Phaseolus vulgaris and P. coccineus bean plants. The isolates were obtained from six traditionally managed agricultural plots in two localities in the State of Puebla, in Central Mexico. The total mean genetic diversity ( H _E) for the six plots was 0.531. Among the 482 isolates collected, 126 distinctive multilocus genotypes (electrophoretic types [Ets]) were obtained, and approximately half of the isolates are represented by five widespread ETs. A significant degree of genetic differentiation among the six plots ( G _ST = 0.072) and between the two localities ( G _ST = 0.022) was detected. The main part of the observed variability (70%) was found among the isolates within the plants. The cluster analysis revealed two deeply diverging lineages, separated at a genetic distance of 0.7. When a multilocus linkage disequilibrium analysis was performed at different hierarchical levels, we found significant linkage disequilibrium, but when the analysis was performed for the genotypes within the two diverging lineages, we found evidence of recombination. We propose for R. etli bv. phaseoli a reticulated and epidemic genetic structure, in which few genotypes increase in frequency to produce numerically dominant clones, and genetic exchange occurs mainly among genotypes within each lineage.     

Sensitivity of the common bean (Phaseolus vulgaris L.) symbiosis to high soil temperature

Summary Experiments were done to test whether N fixation is more sensitive to high soil temperatures in common bean than in cowpea or soybean. Greenhouse experiments compared nodulation, nitrogenase activity, growth and nitrogen accumulation of several host/strain combinations of common bean with the other grain legumes and with N-fertilization, at various root temperatures. Field experiments compared relative N-accumulation (in symbiotic relative to N-fertilized plants) of common bean with cowpea under different soil thermal regimes. N-fertilized beans were unaffected by the higher temperatures, but nitrogen accumulation by symbiotic beans was always more sensitive to high root temperatures (33°C, 33/28°C, 34/28°C compared with 28°C) than were cowpea and soybean symbiosis. Healthy bean nodules that had developed at low temperatures functioned normally in acetylene reduction tests done at 35°C. High temperatures caused little or no suppression of nodule number. However, bean nodules produced at high temperatures were small and had low specific activity. ForP. vulgaris some tolerance to high temperature was observed among rhizobium strains (e.g., CIAT 899 was tolerant) but not among host cultivars. Heat tolerance ofP. acutifolius andP. lunatus symbioses was similar to that of cowpea and soybean. In the field, high surface soil temperatures did not reduce N accumulation in symbiotic beans more than in cowpea, probably because of compensatory nodulation in the deeper and cooler parts of the soil.     

Sunfleck dynamics and canopy structure in a Phaseolus vulgaris L. canopy

 Photosynthetic photon flux density (PPFD) fluctuations were quantified in crops of beans (Phaseolus vulgaris L.) in the field as the canopy developed between July and October. Two different methods were used to select sunflecks and shadeflecks. Four ranges of zenith angles (60–70°, 50–60°, 40–50° and 30–40°) were selected for analysing PPFD fluctuations. At the base of the canopy, sunflecks contributed 18%, 53%, 10% and 4% during the 1st, 3rd, 5th and 7th week of growth, respectively. At a height of 20 cm above the soil surface, the respective contributions were 28% and 21% during the 6th and 7th weeks. Sunfleck lengths of 0–5 s were the most frequent, with the greatest number being found with smaller zenith angles. The proportion of short duration sunflecks increased as the growth period advanced. The number of long sunflecks decreased with time, with very few longer than 100 s by the 5th and 7th weeks. The distributions of sunfleck irradiance were similar to normal distributions and irradiance ranged in μmol m⁻² s⁻¹ from 600–900, 800–1500 and 1000–1600 respectively at zenith angles of 50–60°, 40–50° and 30–40°. A multiple regression showed that short sunflecks (<100 s) depended on zenith angle, plant height, and leaf and stem area index (L _s ), whereas long sunflecks (>100 s) depended on zenith angle and L _s. Shadefleck distributions were similar to those for sunflecks but there were fewer of the shortest examples and more of the longest. The best statistical distribution to describe sunflecks and shadeflecks was the gamma distribution, which could provide the basis for the future development of a good model for sunfleck and shadefleck distributions. Received: 10 November 1997 / Accepted: 2 April 1998     

Study of aluminum toxicity by means of vital staining profiles in four cultivars of Phaseolus vulgaris L

Aluminum toxicity is a very important factor limiting crop productivity on acid soils. Early effects of aluminum toxicity comprise inhibition of cell division and effects on root elongation. The plasma membrane can be the primary target of aluminum toxicity and thus, vital staining techniques could be a powerful tool in determining effects of metal stress on the plasma membrane.

In this paper, we discuss the effects of Al on growth and membrane integrity by staining root tips with a mixture of fluorescein diacetate and propidium iodide.

The results show a good correlation between results from growth measurement and the vital staining. From the comparison of the luminosity patterns generated by vital staining it is easy to determine Al-resistant varieties, revealing this technique as a powerful and fast method for determining tolerance to Al in different varieties.