Phaseolus Species Research Articles

Phaseolus novoleonensis, a New Species (Leguminosae, Phaseolinae) from the Sierra Madre Oriental, Nuevo León, Mexico

A new species of Phaseolus occurs on the eastern slope of Sierra Madre Oriental of Mexico, in the state of Nuevo León, at the northeastern corner of the Chihuahuan desert. Because of its coriaceous leaflets, Phaseolus novoleonensis belongs to section Coriacei Freytag. Although morphologically related to P. maculatus subsp. maculatus and subsp. ritensis, it differs by its having trifoliolate leaves at first nodes, small rounded leaflets, few-noded racemes, and flattened cream-colored seeds.

New Flavanol−Anthocyanin Condensed Pigments and Anthocyanin Composition in Guatemalan Beans (Phaseolus spp.)

It has long been considered that the pigments resulting from direct condensation between anthocyanins and flavanols were formed exclusively during storage and processing, both in plant-derived foods and in drinks. Recently, however, the minor presence of this type of pigment has been shown in different plant extracts, among them beans. In this work we have studied this family of pigments in beans from Guatemala belonging to two distinct species of the genus Phaseolus, confirming the presence of (epi)gallocatechin carbon-carbon linked to the aglycone of delphinidin and (epi)catechin-cyanidin-3-glucoside. Furthermore, for the first time in this foodstuff another three flavanol-anthocyanin condensed pigments formed by the linkage of (epi)catechin with the corresponding aglycones of cyanidin, petunidin, and peonidin have been described. Also, the natural occurrence of anthocyanidins in this matrix has been confirmed in some of the samples analyzed in this work, the majority being components of their anthocyanin composition. The corresponding aglycones of delphinidin, cyanidin, petunidin, pelargonidin, and malvidin have been identified as well as two aglycones with identical mass to those of peonidin and petunidin, but with different retention times, which leads to the supposition that they are probably methylated derivatives of cyanidin and delphinidin, respectively.

Induced plant uptake and transport of mercury in the presence of sulphur‐containing ligands and humic acid

The induced accumulation of mercury (Hg) by plants was investigated for the species Phaseolus vulgaris (Bush bean), Brassica juncea (Indian mustard), and Vicia villosa (Hairy vetch). All plants were grown in modified Hg-contaminated mine tailings and were treated with sulphur-containing ligands to induce Hg accumulation. The effects of varied substrate Hg concentration and humic acid (HA) level on the induced plant-Hg accumulation for B. juncea were examined. Thiosulphate salts (ammonium and sodium) mobilised Hg in the substrates and caused an increase in the Hg concentration of roots and shoots of all tested plant species. Root Hg accumulation was positively correlated to extractable Hg for (NH4)2S2O3-treated B. juncea plants grown in HA-amended substrates. However, shoot Hg translocation for this species was inhibited at 1.25 g HA kg(-1) of substrate. Mercury-thiosulphate complexes could be translocated and accumulated in the upper parts of the plants up to 25 times the Hg concentration in the substrate. We conclude that shoot Hg accumulation in the presence of thiosulphate salts is dependent upon plant species characteristics (e.g. root surface area) and humic acid content.

Revisión taxonómica de los géneros Phaseolus y Vigna (Leguminosae-Papilionoideae) en Cuba

We present a taxonomic study of the Cuban species of Phaseolus L. and Vigna Savi. It includes keys, descriptions, illustrations of selected taxa, chromosome numbers, palinology, phenology, ecology, distribution maps, notes on chemical composition and ethnobotany for each of the eleven species known from Cuba. We also present a discussion on the value of the characters used in the taxonomy of the complex.

A fragment of chloroplast DNA was transferred horizontally, probably from non-eudicots, to mitochondrial genome of Phaseolus

The mitochondrial genomes of some Phaseolus species contain a fragment of chloroplast trnA gene intron, named pvs-trnA for its location within the Phaseolus vulgaris sterility sequence (pvs). The purpose of this study was to determine the type of transfer (intracellular or horizontal) that gave rise to pvs-trnA. Using a PCR approach we could not find the respective portion of the trnA gene as a part of pvs outside the Phaseolus genus. However, a BLAST search revealed longer fragments of trnA present in the mitochondrial genomes of some Citrus species, Helianthus annuus and Zea mays. Basing on the identity or near-identity between these mitochondrial sequences and their chloroplast counterparts we concluded that they had relocated from chloroplasts to mitochondria via recent, independent, intracellular DNA transfers. In contrast, pvs-trnA displayed a relatively higher sequence divergence when compared with its chloroplast counterpart from Phaseolus vulgaris. Alignment of pvs-trnA with corresponding trnA fragments from 35 plant species as well as phylogenetic analysis revealed that pvs-trnA grouped with non-eudicot sequences and was well separated from all Fabales sequences. In conclusion, we propose that pvs-trnA arose via horizontal transfer of a trnA intron fragment from chloroplast of a non-eudicot plant to Phaseolus mitochondria. This is the first example of horizontal transfer of a chloroplast sequence to the mitochondrial genome in higher plants.

Isolation and characterization of polymorphic microsatellite markers in Zabrotes subfasciatus Boheman (Coleoptera: Bruchidae)

AbstractBruchid beetles of the genus Zabrotes (Coleoptera: Bruchidae) are important worldwide pests of legume seeds. Samples of Zabrotes subfasciatus and Z. sylvestris were collected from seeds of two Phaseolus species throughout Mexico to assess the role of host plant variation and plant domestication on the evolution of host use in this species. For this purpose six polymorphic microsatellite loci were isolated and characterized for the bruchid Z. subfasciatus. Cross‐species amplification tests were performed on Z. sylvestris and revealed that three loci amplified successfully and were polymorphic in this closely related species.

Microsatellite markers in a complex of Horismenus sp. (Hymenoptera: Eulophidae), parasitoids of bruchid beetles

AbstractParasitoids of the genus Horismenus (Hymenoptera: Eulophidae) are the main natural enemies of bruchid beetles that feed on several species of Phaseolus beans. Samples of Horismenus depressus, H. missouriensis and H. butcheri were collected from seeds of three Phaseolus species throughout Mexico to examine the impact of plant variability on the genetic structure of parasitoid populations. For this purpose, six microsatellite loci were isolated and characterized. These loci are of great interest in understanding the taxonomy of the genus Horismenus, the most important Eulophid genus in the Neotropics.

Morphological and phylogenetic analyses of Uromyces appendiculatus and U. vignae on legumes in Japan

Uromyces appendiculatus, inclusive of three varieties, is distinguished from U. vignae primarily by the position of urediniospore germ pores and putative host specificity. However, opinions concerning these morphological and physiological features as taxonomic characters have varied greatly, and distinction of these species has often been confused. To clarify the taxonomy of these two species, morphological features of urediniospores and teliospores of 225 rust fungus specimens on species of Phaseolus, Vigna, Apios, Lablab, and Dunbaria were examined by light microscopy and scanning electron microscopy. Forty-five specimens were subjected to molecular phylogenetic analyses. As a result, the position of germ pores in urediniospores and the teliospore-wall thickness were considered as good characters to separate three morphological groups. In molecular analyses, the specimens fell into two and three clades based on the nucleotide sequence at D1/ D2 domain of LSU rDNA and ITS regions, respectively. One of the D1/D2 clades corresponded to one morphological group whereas another D1/D2 clade included two other morphological groups. In contrast, each of the three ITS clades corresponded to a separate morphological group. Neither morphological groups nor molecular clades were host limited. It is suggested that the three morphological groups that corresponded to three distinct ITS clades constitute distinct species.

Resistance of Phaseolus species to ice crystallization at subzero temperature.

Dry bean (Phaseolus vulgaris L.) cultivars possess little or no freezing tolerance and are killed at the temperature of ice formation in their tissues. An increase in frost tolerance by 2-3 degrees C would expand dry bean production in the short growing seasons of the Canadian prairies and possibly to higher altitudes in the tropics where episodic frosts occur during the growing season. The objective of this study was to determine the differences in frost resistance of Phaseolus species in both controlled and field environments. Leaflets of dry bean cv. CDC Nighthawk, and wild relatives from the primary gene pool (P. vulgaris var. mexicanus Freytag and P. vulgaris var. aborigineus (Burkart) Baudet) and the tertiary gene pool (P. acutifolius var. tenuifolius A. Gray, P. filiformis Bentham, P. angustissimus A. Gray and P. ritensis M.E. Jones) were subjected to subzero temperatures with and without ice nucleation to determine the levels of tolerance and avoidance, respectively. The lethal temperature at which 50% of the leaflets were killed (LT(50)) was 0.5-1 degrees C lower for species of the tertiary gene pool compared to those from the primary gene pool. Leaflets of species from the tertiary gene pool were also characterized by extensive supercooling compared to leaflets of species from the primary gene pool. Resistance of Phaseolus species to spring and autumn frosts were determined on seedlings transplanted to the field. Phaseolus angustissimus, a species of the tertiary gene pool had the highest seedling survival in response to both autumn and spring frosts, when the minimum air temperatures were -5 and -7 degrees C, respectively. Frost resistance of Phaseolus angustissimus, if successfully introgressed into bean germplasm, may enable the development of frost resistant dry bean cultivars.

New SSR markers of Phaseolus vulgaris from sequence databases

AbstractTo enlarge the number of microsatellite loci of Phaseolus vulgaris (L.) (common bean), a set of primer pairs has been obtained from public databases, to amplify DNA regions including simple sequence repeats (SSR), and they have been assayed in several P. vulgaris genotypes. Publically available DNA sequence data of P. vulgaris were searched for SSR motifs. Twenty sequences containing microsatellites were identified and primer pairs were designed to amplify those microsatellites. Primers were evaluated for their ability to detect polymorphisms within a set of P. vulgaris accessions, local landraces and hybrids and in addition, two accessions of the closely related species P. coccineus. Eighteen polymorphic SSR loci were identified. Polymorphisms were found at both levels of accessions and Phaseolus species.

Paraheliotropism in two Phaseolus species: combined effects of photon flux density and pulvinus temperature, and consequences for leaf gas exchange

Phaseolus species exhibit paraheliotropic (light-avoiding) leaf movements in response to environmental stimuli. We investigated the response of two ecologically contrasting species, Phaseolus vulgaris (mesic) and Phaseolus acutifolius (xeric), to various combinations of photosynthetic photon flux density (PFD) and pulvinus temperature to compare the control and consequences, in terms of photosynthetic gas exchange, of paraheliotropism under identical conditions. Leaflet angles were measured in a cuvette that exposed the leaf to controlled PFD and pulvinus temperature. A gas exchange system was used to determine carbon assimilation, stomatal conductance and water use efficiency (WUE) in response to environmental factors and leaf movements. The effect of tilting leaves was compared with an equivalent reduction of PFD for horizontal leaves. Both species showed significant (but different) responses to PFD and temperature, with increased light avoidance at high levels of both factors. P. acutifolius exhibited greater light avoidance under equivalent conditions. The gas exchange consequences of changing leaf angles were similar but not identical to those of an equivalent modification of radiation level. The photosynthetic cost of the leaf movements was negligible, but there was a substantial increase in WUE, especially for P. acutifolius. Thus, the benefits of paraheliotropism are achieved at virtually no cost to the plant.

Purification of a trypsin-stable lectin with antiproliferative and HIV-1 reverse transcriptase inhibitory activity

A lectin, with a molecular mass of approximately 60 kDa and two different subunits exhibiting an N-terminal sequence manifesting considerable homology to phytohemagglutinin from Phaseolus species, was isolated from the ground bean ( Vigna sesquipedalis cv ground bean). The lectin was unique in hemagglutinating activity was inhibited by polygalacturonic acid and not by galacturonic acid and other simple monosaccharides. The lectin was isolated by affinity chromatography on Affi-gel blue gel, ion exchange chromatography by fast protein liquid chromatography (FPLC) on Mono S, and gel filtration by FPLC on Superdex 75. It was adsorbed on both Affi-gel blue gel and Mono S. Ground bean lectin exhibited mitogenic activity on murine splenocytes with the maximal response achieved at a concentration of 156 nM, as similar to the dose required for Con A. The viability of hepatoma (HepG2), leukemia (L1210), and leukemia (M1) cells was reduced in the presence of ground bean lectin, which also exerted an inhibitory activity toward HIV-1 reverse transcriptase IC 50 of 73 μM . The hemagglutinating activity of the lectin was unaffected by trypsinization and the presence of a number of divalent cations, but was augmented by 500 mM K + ions. The activity was unstable above 40 °C although some activity remained after heating and at 100 °C for 30 s.

Growth, gas exchange, water relations, and ion composition of Phaseolus species grown under saline conditions

We examined the effects of salinity on four wild ( Phaseolus angustissimus, Phaseolus filiformis, P. microcarpus, and P. vulgaris) and two cultivated ( P. acutifolius and P. vulgaris L.) Phaseolus species. Relative growth rate (RGR, g g −1 per day), unit leaf rate (ULR, g m −2 per day), leaf area ratio (LAR, m 2 g −1), specific leaf area (SLA, m 2 g −1), leaf weight ratio (LWR, g leaf g −1), and rate of ion uptake were calculated for the period between 10 and 20 days after planting. Salinity significantly reduced RGR, ULR, LAR, and SLA whereas LWR showed no definite trend. In all species, except in P. filiformis, ULR, but not LAR, was significantly correlated with RGR, indicating that ULR was an important factor underlying the salinity-induced differences in RGR among species. In P. filiformis, high salinity reduced SLA, and consequently LAR. The significant correlation of SLA and LAR with RGR suggested that growth components affecting leaf area expansion were the primary factors explaining the inhibition of growth in this species. Increasing salinity progressively decreased leaf water vapor conductance. The rate of CO 2 assimilation decreased gradually with salinity, showing significant reductions only at the highest salt level (80 mM NaCl). Approximately two-thirds of the reduction in CO 2 assimilation rate at high salinity was attributable to reduced stomatal conductance. In P. filiformis, however, neither stomatal conductance nor CO 2 assimilation were affected by salt stress. Leaf water and osmotic potentials declined significantly as stress intensified. However, osmotic adjustment permitted the maintenance of positive turgor throughout the growth period. Salinity had a significant effect on tissue concentrations of Na +, K +, Ca 2+, and Cl − and on the uptake rate of Na +, K +, Ca 2+, and Cl −. Thus, in addition to the toxic effects of high concentrations of Na + and Cl − in plant tissue, saline-induced changes in mineral nutrient uptake likely contributed to the reduction of plant growth. It appears that salt tolerance in P. filiformis is associated with Na + exclusion and organ Na + compartmentation in roots and stems as well as sustained K + concentration in leaves and better stomatal control through osmotic adjustment. All other Phaseolus species are Na + excluders, and maintained turgor-driven extension growth by accumulating Cl − (osmotic adjustment), but subsequent weight gain reductions suggest that this led to ion toxicity.

Microsatellite Repeats in Common Bean (Phaseolus vulgaris)

ABSTRACTPhaseolus beans are distributed worldwide and are cultivated in the tropics, subtropics, and temperate zones. The common bean is the most important grain legume for direct human consumption in the world. The objectives of this study were to isolate microsatellite repeats and to establish their discriminatory power (DL) to be used in bean diversity characterization and mapping. We isolated, cloned, and sequenced genomic DNA fragments that contained microsatellite loci from three genomic libraries of Phaseolus vulgaris L. The polymorphism of the microsatellites was evaluated in a panel of 21 P. vulgaris genotypes made up of cultivated and wild beans from the Mesoamerican and Andean pools, and nine genotypes from four Phaseolus species. The number of alleles per microsatellite locus ranged from 1 to 14, with an average of 6 alleles per primer pair. Almost all the microsatellite loci showed high levels of discriminatory power, with the highest value being 0.94. These results indicate that microsatellites can be valuable genetic markers for assessing genetic diversity in the P. vulgaris. The high levels of polymorphism of these new bean microsatellites and their wide cross‐species transportability make these new markers useful for mapping and molecular characterization of Phaseolus species.

Salinity Tolerance in Phaseolus Species during Early Vegetative Growth

ABSTRACTThe genus Phaseolus includes important cultivated species as well as wild species with diverse ecological adaptations. Characterization of the ecological adaptations of the wild species would be useful for improved understanding, conservation, and utilization of these genetic resources. Salinity tolerance during vegetative growth was evaluated for 132 accessions for 14 wild Phaseolus species (P. acutifolius A. Gray, P. angustissimus A. Gray, P. carteri Freytag & Debouck, P. filiformis Bentham, P. glabellus Piper, P. leptostachyus Bentham, P. lunatus L., P. micranthus Hook & Arnold, P. microcarpus Mart, P. mcvaughii A. Delgado, P. oligospermus Piper, and P. vulgaris L.) and 11 accessions representing five cultivated species (P. acutifolius, P. coccineus L., P. lunatus L., P. polyanthus Greenman, and P. vulgaris) in nutrient solution containing 0 and 180 m M sodium chloride for 21 d. When plants were salinized after the emergence of the first trifoliate leaf, wild accessions of P. acutifolius, P. filiformis, P. lunatus, and P. vulgaris showed a wide range of variation in their salinity tolerance as defined by total dry weight reduction (PR) as a percentage of the unsalinized controls, salt susceptibility index (SSI), and root: shoot ratio (RSR). SSI and PR were correlated positively, indicating either trait could be used to select salt‐tolerant accessions. Cluster analysis revealed substantial intraspecific and interspecific variation in salinity tolerance. Salinity tolerance was observed in wild P. micranthus, P. mcvaughii, P. lunatus, cultivated P. coccineus, and several accessions of wild P. filiformis, and P. vulgaris Of these, P. filiformis was noteworthy in having 9 of 11 accessions rated as highly tolerant. Wild P. vulgaris was more salinity tolerant than the three cultivated P. vulgaris accessions included in the study. Many tolerant accessions originated in arid, coastal, or saline areas. We conclude that the genus Phaseolus has substantial diversity in salinity tolerance.

Salinity Tolerance ofPhaseolusSpecies during Germination and Early Seedling Growth

Salinity tolerance during germination and early seedling growth was evaluated for 24 accessions representing four wild Phaseolus species (P. angustissimus A. Gray, P. filiformis Bentham, P. leptostachyus Bentham, and P. microcarpus Mart.) and four accessions of cultivated common bean (P. vulgaris L.) at 0, 60, 120, and 180 mM NaCl. Salinity stress delayed germination in all accessions to varying degrees. Eight accessions of P. filiformis germinated fastest under high salinity (120 mM NaCl). Additional wild accessions exhibiting rapid germination at 120 mM NaCl were P. angustissimus, PI535272, P. leptostachyus, PI535336, and P. microcarpus, PI430196. Among accessions, median germination time (days to 50% germination, T50) at 120 mM NaCl was correlated positively (r2 = 0.55, P less than or equal to 0.01) with germination in the control treatments. Seeds that germinated rapidly at 60 mM NaCl also germinated rapidly at 120 mM NaCl. At 180 mM NaCl, several accessions reached 50% germination by 6 d, demonstrating high genetic potential within Phaseolus for salinity tolerance during germination. The biomass of radicles plus hypocotyls decreased with increasing salinity. Cluster analysis separated the accessions into three groups. Group I included salt sensitive accessions with late germination, high sensitivity index (ratio of median germination time at 120 mM NaCl versus control), and reduced seedling growth. Group II included salt tolerant accessions with rapid germination, high sensitivity index, and enhanced seedling growth. Group III included cultivated accessions corresponding to the Mesoamerican and Andean gene pool with rapid germination, low sensitivity index, and intermediate seedling growth. The results confirm that wild Phaseolus species, and in particular P. filiformis, represent a genetic resource for improvement of salinity tolerance in common bean.

Molecular Evolution and Phylogenetic Implications of Internal Transcribed Spacer Sequences of Nuclear Ribosomal DNA in the Phaseolus–Vigna Complex

Molecular phylogeny based on internal transcribed spacer (ITS) sequences was studied to resolve the taxonomic contradiction in Vigna and its relation to Phaseolus. The ITS region of the 18S–26S nuclear ribosomal DNA repeat was sequenced for 29 Vigna species, selected from five of the nine subgenera, and 9 species of Phaseolus. The length of ITS-1 ranged from 187 to 243 bp and 217 to 290 bp, and that of ITS-2 from 187 to 219 bp and 225 to 243 bp, within Vigna and Phaseolus species, respectively. Phylogenies derived from ITS sequences based on maximum-parsimony and neighbor-joining methods gave trees essentially of similar topology. The ITS phylogeny was generally congruent with recent classifications based largely on morphological, biochemical, cytogenetical, and palynological features, except that subgenus Plectotropis of Neotropical origin was revealed to be closely related to subgenus Vigna instead of forming a link between African (subgenus Vigna) and Asiatic (subgenus Ceratotropis) vignas, and subgenus Sigmoidotropis, featuring morphological characters of both Vigna and Phaseolus, was placed as the sister group to the Phaseolus taxa. The ITS sequences were shown to be useful for identifying wild progenitors of V. mungo, V. radiata, V. umbellata, and V. unguiculata and for clarifying taxonomy-related problems in many previously controversial cases. This study also affirms that V. umbellata and V. angularis are the diploid progenitors of the only tetraploid species (V. glabrescens) known in the genus.

Natural Infection of Vicia faba by Bidens mottle virus in Florida.

In a study to evaluate the potential of Vicia faba (faba bean) as a cover and forage crop for Florida, 60 accessions of faba bean with diverse genetic backgrounds and geographic origins were acquired from the USDA Germplasm Repository in Pullman, WA. The beans were grown south of Lake Okeechobee in Belle Glade, FL, from December 2000 to April 2001. Reddish-brown elliptical lesions first appeared on the leaflets of two of the faba bean plants 10 to 12 weeks after planting. Within 2 weeks of initial symptoms, a mosaic pattern was expressed on the newly emergent leaves of the same plants. After disease expression, new pods aborted, while developing pods became stunted, distorted, and blistered. Potyvirus cylindrical inclusions (CI) were found in leaf strips (1) of the original plants. Viral symptoms were expressed in manually inoculated plants of Chenopodium amaraticolor, C. quinoa, Lactuca sativa, Nicotiana benthamiana, Petunia × hybrida, Verbena × hybrida, Vicia faba, and Zinnia elegans. Inoculated species of Phaseolus and Pisum were not infected. The virus causing the disease was identified as Bidens mottle virus (2) based on host range, characteristic CI in Z. elegans, and homologous lines of precipitation in SDS-immunodiffusion using antiserum to Bidens mottle and a known antigen. Both the primary host of this virus Bidens mottle virus and its aphid vectors are ubiquitous throughout Florida. To our knowledge, this is the first report of Bidens mottle virus infecting a member of the Leguminosae.

Resistance to Common Bacterial Blight of Bean Introgressed from Phaseolus coccineus

Common bacterial blight, incited by Xanthomonas campestris pv. phaseoli (Smith) Dye, is a major bacterial disease of dry bean (Phaseolus vulgaris L.). Resistance to common bacterial blight has been identified in other Phaseolus species and resistance genes have been introgressed into P. vulgaris. The objective of this study was to characterize in dry bean the inheritance pattern of common bacterial blight-resistance genes derived from P. coccineus. Two common, bacterial blight-susceptible, dry bean cultivars were crossed with different common, bacterial blight-resistant dry bean lines with resistance derived from P. coccineus. F2 progeny were inoculated with Xanthomonas campestris pv. phaseoli strain F19-W and were scored for disease reaction. The ratio of resistant to susceptible plants for F2 populations did not differ significantly from a 1 resistant: 3 susceptible ratio. The F3 segregation was obtained for only one cross and did not differ significantly from a 1 resistant: 2 heterozygous: 1 susceptible ratio, suggesting that the resistance introgressed from P. coccineus into dry bean was controlled by one recessive gene. Additionally, the range of symptom expression within the susceptible class provided evidence of other genes modifying the expression of resistance.

Nitrate reductase activity, distribution, and response to nitrate in two contrasting Phaseolus species inoculated with Rhizobium spp.

The nitrate reductase activity distribution and response of two nodulated species of Phaseolus ( Phaseolus vulgaris-common bean, and Phaseolus lunatus-lima bean) to different exogenous nitrate levels were studied during the vegetative period. These Phaseolus species showed to be very contrasting in respect to the pattern of nitrate reductase (NR) activity distribution thought the plant. The highest level of NR activity in P. vulgaris was clearly shown to occur in leaves in contrast with the lowest one detected in roots and nodules as widely seen for other tropical species of the Phaseoleae tribe. Conversely, P. lunatus had higher NR activity in the nodules, whereas its leaves exhibited a steadily decrease during the plant development. Indeed, at 32 days after emergence (pre-flowering stage), the nodulated P. vulgaris had approximately 95% of the total NR activity localized in its leaves, whereas in P. lunatus it was equally distributed in the nodules and in the leaves. Under long-term exposure to increasing exogenous level of nitrate, the leaf-NR activity of nodulated P. vulgaris presented a positive response, whereas the enzyme activity was very low and unresponsive in P. lunatus. In contrast, the nodule-NR activity showed a reverse response to the increasing NO 3 − level. The nodule-NR activity of P. lunatus significantly increased whereas in the P. vulgaris nodules it was very low and unresponsive. This present study suggests that P. lunatus inoculated with Rhizobium tropici presents a singular pattern of nitrate reduction distribution among leaves and nodules during the vegetative development. It is speculated that the nodulated Phaseolus lunatus may have different NR isoforms in their leaves (at least a constitutive type) and an inducible form in their nodules, responsive to long-term exposure to nitrate.