Genetic diversity of common bean (Phaseolus vulgaris L.) nodulating rhizobia in Nepal

Background and Aims

This study was conducted to reveal the genetic diversity of common bean (Phaseolus vulgaris L.) nodulating rhizobia in various agroecological regions in Nepal.

Method

A total of 63 strains were isolated from common bean grown in the soils collected from seven bean fields in Nepal and characterized based on the partial sequences of 16S–23S internal transcribed spacer (ITS) regions, 16S rDNA, nodC, and nifH. Symbiotic properties of some representative strains with host plants were examined to elucidate their characteristics in relation to genotype and their origin.

Results

The isolated strains belonged to Rhizobium leguminosarum, Rhizobium etli, Rhizobium phaseoli, and one unknown Rhizobium lineage, all belonging to a common symbiovar (sv.) phaseoli. Nine ITS genotypes were detected mainly corresponding to a single site, including a dominant group at three sites harboring highly diverse multiple ITS sequences. Three symbiotic genotypes corresponded to a geographical region, not to the ribosomal DNA group, suggesting horizontal transfer of symbiotic genes separately in each region. Great differences in nitrogenase activity and nodule forming ability among the strains irrespective of their species and origin were observed.

Conclusions

Nepalese Himalaya harbor phylogenetically highly diverse and site-specific strains of common bean rhizobia, some of which could have high potential of symbiotic nitrogen fixation.     

Phylogeny and genetic diversity of native rhizobia nodulating common bean (Phaseolus vulgaris L.) in Ethiopia

The diversity and phylogeny of 32 rhizobial strains isolated from nodules of common bean plants grown on 30 sites in Ethiopia were examined using AFLP fingerprinting and MLSA. Based on cluster analysis of AFLP fingerprints, test strains were grouped into six genomic clusters and six single positions. In a tree built from concatenated sequences of recA, glnII, rpoB and partial 16S rRNA genes, the strains were distributed into seven monophyletic groups. The strains in the groups B, D, E, G1 and G2 could be classified as Rhizobium phaseoli, R. etli, R. giardinii, Agrobacterium tumefaciens complex and A. radiobacter, respectively, whereas the strains in group C appeared to represent a novel species. R. phaseoli, R. etli, and the novel group were the major bean nodulating rhizobia in Ethiopia. The strains in group A were linked to R. leguminosarum species lineages but not resolved. Based on recA, rpoB and 16S rRNA genes sequences analysis, a single test strain was assigned as R. leucaenae. In the nodC tree the strains belonging to the major nodulating groups were clustered into two closely linked clades. They also had almost identical nifH gene sequences. The phylogenies of nodC and nifH genes of the strains belonging to R. leguminosarum, R. phaseoli, R. etli and the putative new species (collectively called R. leguminosarum species complex) were not consistent with the housekeeping genes, suggesting symbiotic genes have a common origin which is different from the core genome of the species and indicative of horizontal gene transfer among these rhizobia.     

Metabolic engineering of folate and its precursors in Mexican common bean (Phaseolus vulgaris L.)

Folate (vitamin B9) deficiency causes several health problems globally. However, folate biofortification of major staple crops is one alternative that can be used to improve vitamin intakes in populations at risk. We increased the folate levels in common bean by engineering the pteridine branch required for their biosynthesis. GTP cyclohydrolase I from Arabidopsis (AtGchI) was stably introduced into three common bean Pinto cultivars by particle bombardment. Seed‐specific overexpression of AtGCHI caused significant increases of up to 150‐fold in biosynthetic pteridines in the transformed lines. The pteridine boost enhanced folate levels in raw desiccated seeds by up to threefold (325 μg in a 100 g portion), which would represent 81% of the adult recommended daily allowance. Unexpectedly, the engineering also triggered a general increase in PABA levels, the other folate precursor. This was not observed in previous engineering studies and was probably caused by a feedforward mechanism that remains to be elucidated. Results from this work also show that common bean grains accumulate considerable amounts of oxidized pteridines that might represent products of folate degradation in desiccating seeds. Our study uncovers a probable different regulation of folate homoeostasis in these legume grains than that observed in other engineering works. Legumes are good sources of folates, and this work shows that they can be engineered to accumulate even greater amounts of folate that, when consumed, can improve folate status. Biofortification of common bean with folates and other micronutrients represents a promising strategy to improve the nutritional status of populations around the world.     

Heritable variation in the phaseolin protein of nondomesticated common bean,Phaseolus vulgaris L.

Summary Crude protein extracts from single seeds of nondomesticated Mexican bean accessions were analysed by SDS polyacrylamide gel electrophoresis for variability in phaseolin protein. Six new phaseolin types; M1, M2, M3, M4, M5, M6, which contained polypeptides within the same range of molecular weights (51,000 to 45,000 daltons) as occur in the S, T and C phaseolin types of cultivated beans were identified. No T and C types were found among the non-domesticated Mexican accessions, and the S type occurred in less than 7% of the seeds screened. Genetic analyses of F₂ progenies from crosses between Sanilac (S), and five of the M types showed that each M phaseolin phenotype was allelic to the S type and expressed codominantly.     

Monosomics in common bean,Phaseolus vulgaris

Summary Two monosomics of Phaseolus vulgaris (2n = 22) were found among selfed progeny of plants treated with colchicine. The monosomic chromosomes involved were identified as chromosomes H and J according to the previously suggested Giemsa karyotype. Both monosomic plants had slower growth rate and smaller size as compared with their respective euploid sibs. However, no apparent morphological characteristics distinguished the two monosomics. The frequencies of transmission through selfing of monosomics H and J were 9% and 10 % respectively.     

Genetic diversity of rhizobia nodulating common bean (Phaseolus vulgaris L.) in the Central Black Sea Region of Turkey

We have analyzed 30 rhizobial isolates obtained from common bean (Phaseolus vulgaris L.) root nodules grown in the Middle Blacksea Region of Turkey, using ARDRA and nucleotide sequence data. ARDRA analysis with enzymes CfoI, HinfI, NdeII, MspI and PstI revealed three patterns. Based on sequence data from 16S rDNA, the patterns were identified as, Rhizobium leguminosarum bv. phaseoli (n = 16), R. etli bv. phaseoli (n = 8) and R. phaseoli (n = 6). On the other hand, nucleotide sequence phylogenies of housekeeping genes (recA, atpD and glnII) selected to confirm the 16S rDNA phylogeny revealed different evolutionary relationships. These results suggested the possibility of lateral transfers of these genes amongst different rhizobial species (including R. leguminosarum, R. etli and R. phaseoli) sharing the same ecological niche (nodulating P. vulgaris) which also indicates that there may be no true genetic barier among these species. Phylogenetic analysis based on DNA sequence data from the nodA and nifH genes showed that all rhizobial species obtained in this study were carrying nodA and nifH haplotypes which were the same or similar to those of CFN42 (R. etli type strain), suggesting a further support for the lateral transfer of CFN42 Sym plasmid, p42, amongst Turkish common bean nodulating rhizobial isolates.     

Five primary trisomics from translocation heterozygote progenies in common bean,Phaseolus vulgaris L.

Summary Twelve distinct phenotypic groups of plants were isolated from nondisjunction progenies of 11 translocation heterozygote stocks. All the plants in these phenotypic groups originated in the light weight seed class. Five of the 12 phenotypic groups of plants have been verified as primary trisomics. They are all phenotypically distinguishable from each other and from disomics. One of the five primary trisomic groups, puckered leaf, was directly recovered as a primary trisomic from the original translocation heterozygote progenies. Three of the five trisomics — weak stem, dark green leaf, and convex leaf — originated first as tertiary trisomics. The related primary trisomics were isolated later from progenies of selfed tertiary trisomics. The fifth group, chlorotic leaf, originated at a low frequency among the progenies of three other trisomics: puckered leaf, convex leaf, and dark green leaf. The chlorotic leaf did not set seed under field conditions. The remaining four groups — puckered leaf, dark green leaf, convex leaf, and weak stem — are fertile, though sensitive to high temperature conditions. The transmission rate of the extra chromosome on selfing ranges from 28% to 41%. Physical identification of the extra chromosome has not been achieved for any of the five trisomic groups. Two trisomic groups, dark green leaf and convex leaf, have produced tetrasomics at low frequency. The phenotypes of these two tetrasomics are similar to the corresponding trisomics but more exaggerated.Fla. Agr. Expt. Stn. Journal Series No. 7137     

Genetic diversity of rhizobia in a Brazilian oxisol nodulating Mesoamerican and Andean genotypes of common bean (Phaseolus vulgaris L.)

Brazil is the largest producer and consumer of common bean worldwide, and the crop can benefit from its symbiosis with a variety of rhizobia by means of biological nitrogen fixation in root nodules. In this study, the role of Mesoamerican and Andean genotypes of common bean in trapping rhizobia directly from a Brazilian oxisol in the field or in pots in greenhouse conditions with unaltered or diluted soil solutions was investigated. Genetic diversity was evaluated by the profiles of BOX-PCR obtained, and by estimates of Shannon and Abundance-based Coverage Estimator (ACE) indices. Rhizobia trapped by Mesomaerican genotypes had greater diversity, reinforcing the hypothesis of an important and long-time contribution of this genetic center to the establishment of common bean in Brazil. Greater diversity was also seen in rhizobia trapped straight from the soil than from plants inoculated with diluted soil solutions, emphasizing a highly diverse and competitive rhizobial indigenous population. Studies on genetic diversity of common bean rhizobia are important not only for helping to understand the evolution of the legume-rhizobia symbiosis, but also to devise strategies to increase the contribution of the biological nitrogen-fixation process.     

Auxin-induced assimilate translocation in the bean stem (Phaseolus vulgaris L.)

Decapitation of the fully-elongated fourth internode of Phaseolus vulgaris plants resulted in the disappearance from the internode of soluble acid invertase (EC 3.2.1.26). This loss was prevented by local applications to the internode of indol-3yl-acetic acid (IAA) and, at the point of IAA application, the specific activity of the enzyme increased by up to 3 times its initial value within 48 h of treatment. IAA applications stimulated the acropetal translocation to the internode of ¹⁴C-sucrose applied to the subtending (second) trifoliate leaf 30 h after decapitation and the start of the auxin treatment. Labelled assimilates accumulated in the IAA-treated region of the internode. Following decapitation the concentration of hexose sugars in the internode fell and that of sucrose rose substantially, but these trends were reversed by IAA treatment. However, small local accumulations of sucrose occurred at the point of auxin application where tissue concentrations of IAA were greatest (determined using [1-¹⁴C] IAA).Considerable quantities of starch were present in the ground parenchyma of the internodes at the start of the experiment but, in the absence of IAA, this was remobilised within 48 h of decapitation. IAA prevented starch loss at and below its point of application to the internode, but not from more distal tissues. Cambial proliferation, radial growth and lignification were stimulated in and below IAA-treated regions of the internode. These observations are discussed in relation to the hormonal regulation of assimilate translocation in the phloem.     

Characteristics of the common bean, Phaseolus vulgaris, associated with resistance to the leafhopper Empoasca kraemeri

Experiments were conducted using the common bean, Phaseolus vulgaris, and its most important pest in Latin America, Empoasca kraemeri, in order to assess the value of insect counts and various plant characteristics as selection criteria in a plant breeding programme. The combination of insect counts with measurements of damage symptoms should make it possible to distinguish tolerance from the resistance mechanisms of antibiosis and non-preference. By this means, and by a knowledge of the plant characteristics associated with resistance, different forms of resistance may be combined in the progeny of crosses. In a comparison of two contrasting genotypes the more severely damaged genotype was the more heavily infested at early stages of plant growth, but had the lower level of infestation at later stages. The association of high early counts with high subsequent damage was not confirmed in an experiment on six genotypes, but the association of high damage with low late counts was partially confirmed. These results indicate that the level of early infestation of E. kraemeri is not consistently reflected in the subsequent levels of damage which P. vulgaris genotypes display. This suggests that these two criteria represent distinct forms of resistance which can be combined in hybrid progeny. Resistance was also associated with late maturity, indeterminate growth habit, purple flowers and black or beige seeds. However, consumer preferences and the requirements of agricultural systems place constraints upon the use of these relationships in resistance breeding.     

Microsatellite marker diversity in common bean (Phaseolus vulgaris L.)

A diversity survey was used to estimate allelic diversity and heterozygosity of 129 microsatellite markers in a panel of 44 common bean (Phaseolus vulgaris L.) genotypes that have been used as parents of mapping populations. Two types of microsatellites were evaluated, based respectively on gene coding and genomic sequences. Genetic diversity was evaluated by estimating the polymorphism information content (PIC), as well as the distribution and range of alleles sizes. Gene-based microsatellites proved to be less polymorphic than genomic microsatellites in terms of both number of alleles (6.0 vs. 9.2) and PIC values (0.446 vs. 0.594) while greater size differences between the largest and the smallest allele were observed for the genomic microsatellites than for the gene-based microsatellites (31.4 vs. 19.1 bp). Markers that showed a high number of alleles were identified with a maximum of 28 alleles for the marker BMd1. The microsatellites were useful for distinguishing Andean and Mesoamerican genotypes, for uncovering the races within each genepool and for separating wild accessions from cultivars. Greater polymorphism and race structure was found within the Andean gene pool than within the Mesoamerican gene pool and polymorphism rate between genotypes was consistent with genepool and race identity. Comparisons between Andean genotypes had higher polymorphism (53.0%) on average than comparisons among Mesoamerican genotypes (33.4%). Within the Mesoamerican parental combinations, the intra-racial combinations between Mesoamerica and Durango or Jalisco race genotypes showed higher average rates of polymorphism (37.5%) than the within-race combinations between Mesoamerica race genotypes (31.7%). In multiple correspondance analysis we found two principal clusters of genotypes corresponding to the Mesoamerican and Andean gene pools and subgroups representing specific races especially for the Nueva Granada and Peru races of the Andean gene pool. Intra population diversity was higher within the Andean genepool than within the Mesoamerican genepool and this pattern was observed for both gene-based and genomic microsatellites. Furthermore, intra-population diversity within the Andean races (0.356 on average) was higher than within the Mesoamerican races (0.302). Within the Andean gene pool, race Peru had higher diversity compared to race Nueva Granada, while within the Mesoamerican gene pool, the races Durango, Guatemala and Jalisco had comparable levels of diversity which were below that of race Mesoamerica.     

Light-stimulated cell expansion in bean (Phaseolus vulgaris L.) leaves

The quantity and quality of light required for light-stimulated cell expansion in leaves of Phaseolus vulgaris L. have been determined. Seedlings were grown in dim red light (RL; 4 micromoles photons m-2 s-1) until cell division in the primary leaves was completed, then excised discs were incubated in 10 mM sucrose plus 10 mM KCl in a variety of light treatments. The growth response of discs exposed to continuous white light (WL) for 16 h was saturated at 100 micromoles m-2 s-1, and did not show reciprocity. Extensive, but not continuous, illumination was needed for maximal growth. The wavelength dependence of disc expansion was determined from fluence-response curves obtained from 380 to 730 nm provided by the Okazaki Large Spectrograph. Blue (BL; 460 nm) and red light (RL; 660 nm) were most effective in promoting leaf cell growth, both in photosynthetically active and inhibited leaf discs. Far-red light (FR; 730 nm) reduced the effectiveness of RL, but not BL, indicating that phytochrome and a separate blue-light receptor mediate expansion of leaf cells.     

Host plant nodule parameters associated with nitrogen fixation efficiency in French bean (Phaseolus vulgaris L) cultivars

Two cultivars of French bean (Phaseolus vulgaris L.) viz. contender and arka komal were planted in polythene bags containing sand and grown under glasshouse conditions. The nodulation status, shoot/root biomass, activities of several nodule enzymes, total soluble protein and leghaemoglobin contents were monitored over the entire growth period. Allantoinase activity in leaves was measured to monitor the ureide degrading capacity. Significant genotype difference was observed in both the cultivars. All the parameters showed a decline after flowering except uricase, which declined before flowering. Malate dehydrogenase and isocitrate dehydrogenase showed a constant decline throughout the growth period. Degree of decline varied with the genotype for all the parameters. Leghaemoglobin content, PEP carboxylase activity and ureide degrading capacity of leaves did not show an appreciable decline in contender and were significantly higher than in arka komal. These factors can be used to increase nitrogen fixation in French bean.     

Light-stimulated cell expansion in bean (Phaseolus vulgaris L.) leaves

Cell expansion in dicotyledonous leaves is strongly stimulated by bright white light (WL), at least in part as a result of light-induced acidification of the cell walls. It has been proposed that photosynthetic reactions are required for light-stimulated transport processes across plasma membranes of leaf cells, including proton excretion. The involvement of photosynthesis in growth and wall acidification of primary leaves of bean has been tested by inhibiting photosynthesis in two ways: by reducing chlorophyll content of intact plants with tentoxin (TX) and by treating leaf discs with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). Exposure to bright WL stimulated growth of intact leaves of TX-treated plants. Discs excised from green as well as from TX-or DCMU-treated leaves also responded by growing faster in WL, as long as exogenous sucrose was supplied to the photosynthetically inhibited tissues. The WL caused acidification of the epidermal surface of intact TX-leaves, but acidification of the incubation medium by mesophyll cells only occurred when photosynthesis was not inhibited. It is concluded that light-stimulated cell enlargement of bean leaves, and the necessary acidification of epidermal cell walls, are mediated by a pigment other than chlorophyll. Light-induced proton excretion by mesophyll cells, on the other hand, may require both a photosynthetic product (or exogenous sugars) and a non-photosynthetic light effect.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1 -dimethylurea - OC osmotic concentration - RL red light - TX tentoxin - WL white light We thank Dr. G.E. Templeton, University of Arkansas, Fayetteville, USA, for initially supplying us with TX, and also Dr. Stephen O. Duke, Southern Weend Science Laboratory, Stoneville, Miss., USA, for suggesting this compound for our experiments. We are grateful to Professor E. Ballio for his generous gift of fusicoccin.     

Ability of selected accessions of Phaseolus vulgaris L. to restrict nodulation by particular rhizobia

Plant genotypes that limit nodulation by indigenous rhizobia while nodulating normally with inoculant-strain nodule occupancy in Phaseolus vulgaris. In this study, eight of nine Rhizobium tropici strains and six of 15 Rhizobium etli strains examined, showed limited ability to nodulate and fix nitrogen with the two wild P. vulgaris genotypes G21117 and G10002, but were effective in symbiosis with the cultivated bean genotypes Jamapa and Amarillo Gigante. Five of the R. etli strains restricted in nodulation by G21117 and G10002 produced an alkaline reaction in yeast mannitol medium. In a competition experiment in which restricted strains were tested in 1:1 mixtures with the highly effective R. etli strain CIAT632, the restricted strains produced a low percentage of the nodules formed on G2117, but produced over 40% of the nodules formed on Jamapa. The interaction of the four Rhizobium strains with the two bean genotypes, based on the percentage of nodules formed, was highly significant (P<0.001).     

Inheritance of partial resistance to bean rust in the common bean (Phaseolus vulgaris L.)

The inheritance of partial resistance within eight bean cultivars to a single-pustule isolate of bean rust was studied by means of a F₁ diallel test. General combining ability (GCA) and specific combining ability (SCA) were very highly significant over two seasons and in interaction with seasons. The partitioning of the sums of squares indicated the greater importance of GCA in the inheritance of the resistance. Reciprocal effects were not significant. The estimates of narrow-sense heritability in the two seasons were 0.899 ± 0.056 and 0.603 ± 0.065.