Enhanced Mineral Uptake by Zea mays and Sorghum bicolor Roots Inoculated with Azospirillum brasilense

Inoculation of corn (Zea mays) seeds with Azospirillum brasilense strain Cd or Sp 7 significantly enhanced (30 to 50% over controls) the uptake of NO₃⁻, K⁺, and H₂PO₄⁻ into 3- to 4-day- and 2-week-old root segments. No gross changes in root morphology were observed; altered cell arrangement in the outer four or five layers of the cortex was seen in photomicrographs of cross sections of inoculated corn roots. The surface activity involved in ion uptake probably increased, as shown by the darker staining by methylene blue of the affected area. Shoot dry weight increased 20 to 30% in inoculated plants after 3 weeks, presumably by enhancement of mineral uptake. Corn and sorghum plants grown to maturity on limiting nutrients in the greenhouse showed improved growth from inoculation approaching that of plants grown on normal nutrient concentrations. Enhanced ion uptake may be a significant factor in the crop yield enhancement reported for Azospirillum inoculation.     

Analysis of the Proteome of Common Bean (Phaseolus vulgaris L.) Roots after Inoculation with Rhizobium etli

Proteomics techniques were used to identify the underlying mechanism of the early stage of symbiosis between the common bean (Phaseolus vulgaris L.) and bacteria. Proteins from roots of common beans inoculated with bacteria were separated using two-dimensional polyacrylamide gel electrophoresis and identified using mass spectrometry. From 483 protein spots, 29 plant and 3 bacterial proteins involved in the early stage of symbiosis were identified. Of the 29 plant proteins, the expression of 19 was upregulated and the expression of 10 was downregulated. Upregulated proteins included those involved in protein destination/storage, energy production, and protein synthesis; whereas the downregulated proteins included those involved in metabolism. Many upregulated proteins involved in protein destination/storage were chaperonins and proteasome subunits. These results suggest that defense mechanisms associated with induction of chaperonins and protein degradation regulated by proteasomes occur during the early stage of symbiosis between the common bean and bacteria.     

Variation of Protein among Eleven Varieties of Common Bean (Phaseolus vulgaris)

The acylated, amidated and esterified derivatives of N-acetylglucosaminyl-α(1 → 4)-N-acetylmuramyl tri- and tetrapeptide were synthesized and examined as to their protective effect on pseudomonal infection in the mouse and pyrogenicity in the rabbit. Modifications of the terminal end function of the peptide moieties in their molecules caused enhancement of resistance to pseudomonal infection and reduction of pyrogenicity. Among the compounds tested, sodium N-acetylglucosaminyl-β(1 → 4)-N-acetylmuramyl-l-alanyl-d-isoglutaminyl-(l)-stearoyl-(d)-meso-2,6-diaminopimelic acid-(d)-amide and sodium N-acetylglucosaminyl-β(1 → 4)-N-acetylmuramyl-l-alanyl-d-isoglutaminyl-(l)-stearoyl-(d)-meso-2,6-diaminopimelic acid-(d)-amide-(l)-d-alanine were found to be advantageous and conceivably worthwhile for further investigation as immunobiologically active compounds.     

Increased Bean (Phaseolus vulgaris L.) Nodulation Competitiveness of Genetically Modified Rhizobium Strains

Rhizobium leguminosarum bv. phaseoli strain collections harbor heterogeneous groups of bacteria in which two main types of strains may be distinguished, differing both in the symbiotic plasmid and in the chromosome. We have analyzed under laboratory conditions the competitive abilities of the different types of Rhizobium strains capable of nodulating Phaseolus vulgaris L. bean. R. leguminosarum bv. phaseoli type I strains (characterized by nif gene reiterations and a narrow host range) are more competitive than type II strains (that have a broad host range), and both types are more competitive than the promiscuous rhizobia isolated from other tropical legumes able to nodulate beans. Type I strains become even more competitive by the transfer of a non-Sym, 225-kilobase plasmid from type II strain CFN299. This plasmid has been previously shown to enhance the nodulation and nitrogen fixation capabilities of Agrobacterium tumefaciens transconjugants carrying the Sym plasmid of strain CFN299. Other type I R. leguminosarum bv. phaseoli transconjugants carrying two symbiotic plasmids (type I and type II) have been constructed. These strains have a diminished competitive ability. The increase of competitiveness obtained in some transconjugants seems to be a transient property.     

The Effect of Cycloheximide on Absorption of Ions by Bean (Phaseolus vulgaris L) Roots

10^–7 M cycloheximide inhibited bean (Phaseolus vulgaris)root elongation by about 20 per cent but it inhibited absorptionof rubidium, sodium, and phosphate ions to a much greater extent(34–71 per cent). Tips of intact plant roots grown inthe inhibitor showed more inhibition in ion uptake than adjacentproximal portions of the same roots and this is taken to indicatethat 10^–7 M cycloheximide does not exert its effect onion uptake by any uncoupling action. Sodium uptake from 0.5 or 10 mM NaCl solutions by root tipswas inhibited by 10^–7 M cycloheximide to twice the extentthat it was in the elongating region of the root. Assuming thatthe inhibitor affects the plasmalemma more than the tonoplast,Epstein's model of parallel operation of system 1 and system2 at the plasmalemma is supported.     

Enzyme-Linked Immunosorbent Assay for Specific Identification and Enumeration of Azospirillum brasilense Cd. in Cereal Roots

The enzyme-linked immunosorbent assay is suggested as a reliable, sensitive, and highly specific method for the identification and enumeration of Azospirillum brasilense Cd. As few as 10⁵ CFU/ml can be practically identified by this method. At higher bacterial numbers, sensitivity increased linearly up to 5 × 10⁸ CFU/ml, yielding useful standard curves. No cross-reaction was found either with different closely related Azospirillum strains or with other rhizosphere bacteria. The method allows for a specific identification of A. brasilense Cd. both in pure cultures and in mixtures with other bacterial species, even when the colony morphology is variable. The method was successfully applied to assess the degree of root colonization on various cereals by A. brasilense Cd.     

Alleviation of Salt Stress in Common Bean (Phaseolus vulgaris) by Exogenous Abscisic Acid Supply

In this work the effect of abscisic acid (ABA) and 100 mM NaCl on common bean (Phaseolus vulgaris var. Coco) growth, nitrogenase activity, and nodule metabolism was studied. Experiments were carried out in a controlled environmental chamber and plants, at the vegetative growth stage (16 days old), were treated with ABA (1 μM and 10 μM) and 48 h later were exposed to saline treatment. Results revealed that plant dry weight, nodule dry weight, nitrogen fixation (acetylene reduction activity and ureides content), and most enzymes of ammonium and ureides metabolism were affected by both ABA and NaCl. The addition of 1 μM ABA to the nutrient solution before the exposure to salt stress reduced the negative effect of NaCl. Based on our results, we suggest that ABA application improves the response of Phaseolus vulgaris symbiosis under saline stress conditions, including the nitrogen fixation process and enzymes of ammonium assimilation and purine catabolism.     

Nodulation of Pole Bean (Phaseolus vulgaris L.) by Rhizobium Species of Two Cross-Inoculation Groups

Physiology and morphology of pole bean (Phaseolus vulgaris L. cv Kentucky Wonder) root nodules induced by two Rhizobium species of different cross-inoculation groups have been compared. Root nodules induced by Rhizobium sp. 127E15, which is a strain of the cowpea group Rhizobium, were pinkish, had irregular shapes, and were only partially effective. Their peak acetylene reduction activity was 4.36 μmol of C₂H₄ formed per g of fresh nodules per h at 30 days after inoculation. The effective nodules induced by Rhizobium phaseoli 127K14, which is a strain of the bean group Rhizobium, were dark red, spherical, and showed peak acetylene reduction activity of 15.95 μmol of C₂H₄ formed per g of fresh nodules per h at 15 days after inoculation. The partial effectiveness of 127E15-induced nodules was associated with fewer infected cells, a delay in the increase of bacteroid population within the host cells, abundance of cytoplasmic vesicles in the host cells, more bacteroids within a membrane envelope (peribacteroid membrane), and the inability of bacteroids to completely fill up the host cytoplasmic space. The 127K14-induced nodules were fully mature, with host cells filled with bacteroids by 12 days after inoculation. In contrast, the 127E15-induced nodules did not reach a similar developmental stage even 30 days after inoculation.     

Mechanism of Zinc Uptake in Bean (Phaseolus vulgaris) Tissues

Uptake of micronutrient zinc by intact leaves, enzymically isolated leaf cells, leaf disks, excised roots, and stem-callus tissue of two field bean cultivars 'Saginaw’ and ‘Sanilac’) was studied using radio-isotope tracer technique. Radio-phosphorus absorption by these tissues was also followed under comparable experimental conditions. A rapid absorption of the micronutrient and strong dependency on external zinc concentration and pH were revealed. Absorption of zinc was not inhibited by respiratory inhibitors (dinitrophenol, azide, cyanide, and amytal), and was not light or temperature dependent. Q₁₀ values for zinc uptake ranged between 1 and 1.2. Uptake of phosphate, on the other hand, was temperature and light dependent and drastically reduced by the presence of metabolic inhibitors. Differences in responses to respiratory inhibitors, temperature, pH, light and darkness, and kinetic data, strongly suggest that zinc uptake in bean tissues occurs primarily by a passive mechanism, involving possibly a physical or physiochemical binding of the micronutrient ions to the cell wall and free space components, and a passive diffusion into the interior of the cell.     

Absorption and translocation of Cd in bush beans (Phaseolus vulgaris)

A series of experiments was conducted to examine some factors affecting the absorption and translocation of Cd in young bean plants ( Phaseolus vulgaris L. cv. Bulgarian). Absorption of Cd by roots was reduced in the presence of other cations of increasing valency or ionic radii. Reduced absorption was also found in the presence of EDTA. Concentration of Cd in exudates from excised stems increased with increased passage of Cd solutions and approached the concentration in the external medium (4.5 μ M Cd). This was apparently associated with saturation of adsorption sites in the stems. The stem behaved as a cation exchange column resulting in a chromatographic distribution of Cd towards the top of the plant. These experiments indicate that Cd existed in the xylem fluid as a free or weakly complexed cation. Additional experiments showed that the total amount of Cd absorbed by bean plants was elevated by inducing higher transpiration rates. The effect of water flux on Cd transport indicated apoplastic flow to the stele.     

Selenate and Selenite Uptake and Translocation in Bean Plants (Phaseolus vulgaris)

After 3 h, selenate uptake by roots of Phaseolus vulgaris L.cv. Contender resulted in more than 50% of the Se absorbed beingconveyed to the aerial organs. This distribution was sensitiveto respiratory inhibitors and when roots were soaked in a solutionsupplied with hydroxylamine, the level of Se decreased by about80% in the whole plant, suggesting that selenate uptake requiresenergy. Addition of glucose to the nutrient medium resultedin slightly decreased uptake and distribution. Under the same growth conditions and 3 h incubation with selenite,a major part of the Se had accumulated in the roots, while asmall fraction was conveyed towards the aerial organs. Thispercentage was decreased by about 20% when plants were transferredto a solution supplied with hydroxylamine, suggesting that partof the selenite entered the roots passively. Addition of glucoseto the nutrient solution, resulted in enhanced levels of Sein the whole plant. Application of plant growth substances affected Se transport.When roots were incubated in abscisic acid (ABA), selenate uptakewas affected, while foliar spraying of gibberellin A₃ (GA₃)enhanced selenite uptake and translocation. Key words: Phaseolus vulgaris, selenate, transport, selenite, glucose, harmones     

Amitrole Absorption by Bean (Phaseolus vulgaris L. cv ;Red Kidney') Roots : Mechanism of Absorption

The mechanism of transport of the herbicide 3-amino-1,2,4-triazole (amitrole) into Phaseolus vulgaris roots appears to be passive, as judged by the effect of temperature (Q₁₀ = 1.3 between 15 and 25°C) and the lack of sensitivity to metabolic inhibition afforded by 2,4-dinitrophenol and NaN₃. Amitrole absorption is a linear function of external concentration over several orders of magnitude and, thus, is not facilitated by a carrier mechanism. The absorption of amitrole is sensitive to external pH, being stimulated under acid conditions. This stimulation of amitrole absorption is seen at low (≤1 millimolar) amitrole concentrations, but not at high (50 millimolar) amitrole levels. While the apparent octanol-water partition coefficient varies with the pH of the aqueous phase, there is no clear correspondence between absorption and the apparent partition coefficient. Roots do not accumulate amitrole above concentration equilibrium; however, at a time when the net amitrole content of the root tissue begins to saturate, amitrole can be detected in the xylem stream. On a fresh-weight basis, amitrole absorption by roots is equal to that accomplished by trifoliate-leaf tissue. An estimate of the permeability coefficient (according to the analysis of Tyree et al. 1979 Plant Physiol 63: 367-374) suggests that amitrole possesses near-optimal permeability for an ambimobile solute, on the order of 2.12 (± 0.47) × 10⁻⁹ meters per second.     

The Effect of Combined Nitrogen on the Growth and Nodulation of Excised Roots of Phaseolus vulgaris L.

Isolated roots of Phaseolus vulgaris were grown in aseptic cultureusing a modified Raggio technique in which the organic nutrientsare fed to the cut (basal) end of the excised root while theroot tip grows into a mineral-salt solution inoculated withRhizobium. Treatments were applied to investigate the effectsof varying the level of sucrose in the organic medium and ofadding combined nitrogen as nitrate and urea to both media. There was a marked effect of sucrose on the growth and nodulationof the roots. Increasing the concentration increased the numberof nodules both per root and per unit fresh weight of tissue. The growth of the roots was not affected by the level of combinednitrogen. Nitrate in the mineral-salt solution markedly reducednodule numbers but in the agar medium it had only a slight delayingeffect on nodulation. Urea in either the mineral-salt solutionor the organic medium reduced nodule numbers though to a lesserextent than nitrate in the mineral-salt solution. The number of nodules formed was inversely correlated with thelevels of soluble and inorganic nitrogen compounds in the tissues. It is suggested that the adverse effect of combined nitrogenon nodulation is due to the accumulation of unsequestered nitrogencompounds and a depletion of carbohydrates generally withinthe root tissues rather than the local effect of any particularnitrogen compound.     

Nodulation and Nitrogen Fixation Efficacy of Rhizobium fredii with Phaseolus vulgaris Genotypes

Phaseolus plant introduction (PI) genotypes (consisting of 684 P. vulgaris, 26 P. acutifolius, 39 P. lunatus, and 5 P. coccineus accessions) were evaluated for their ability to form effective symbioses with strains of six slow-growing (Bradyrhizobium) and four fast-growing (Rhizobium fredii) soybean rhizobia. Of the 684 P. vulgaris genotypes examined, three PIs were found to form effective nitrogen-fixing symbioses with the R. fredii strains. While none of the Bradyrhizobium strains nodulated any of the genotypes tested, some produced large numbers of undifferentiated root proliferations (hypertrophies). A symbiotic plasmid-cured R. fredii strain failed to nodulate the P. vulgaris PIs and cultivars, suggesting that P. vulgaris host range genes are Sym plasmid borne in the fast-growing soybean rhizobia.     

Genome-Wide Association Studies of Anthracnose and Angular Leaf Spot Resistance in Common Bean (Phaseolus vulgaris L.)

The common bean (Phaseolus vulgaris L.) is the world’s most important legume for human consumption. Anthracnose (ANT; Colletotrichum lindemuthianum) and angular leaf spot (ALS; Pseudocercospora griseola) are complex diseases that cause major yield losses in common bean. Depending on the cultivar and environmental conditions, anthracnose and angular leaf spot infections can reduce crop yield drastically. This study aimed to estimate linkage disequilibrium levels and identify quantitative resistance loci (QRL) controlling resistance to both ANT and ALS diseases of 180 accessions of common bean using genome-wide association analysis. A randomized complete block design with four replicates was performed for the ANT and ALS experiments, with four plants per genotype in each replicate. Association mapping analyses were performed for ANT and ALS using a mixed linear model approach implemented in TASSEL. A total of 17 and 11 significant statistically associations involving SSRs were detected for ANT and ALS resistance loci, respectively. Using SNPs, 21 and 17 significant statistically associations were obtained for ANT and angular ALS, respectively, providing more associations with this marker. The SSR-IAC167 and PvM95 markers, both located on chromosome Pv03, and the SNP scaffold00021_89379, were associated with both diseases. The other markers were distributed across the entire common bean genome, with chromosomes Pv03 and Pv08 showing the greatest number of loci associated with ANT resistance. The chromosome Pv04 was the most saturated one, with six markers associated with ALS resistance. The telomeric region of this chromosome showed four markers located between approximately 2.5 Mb and 4.4 Mb. Our results demonstrate the great potential of genome-wide association studies to identify QRLs related to ANT and ALS in common bean. The results indicate a quantitative and complex inheritance pattern for both diseases in common bean. Our findings will contribute to more effective screening of elite germplasm to find resistance alleles for marker-assisted selection in breeding programs.     

Chlorophyll Fluorescence Induction in Leaves of Phaseolus vulgaris Infected with Bean Rust (Uromyces appendiculatus)

To our knowledge, this report describes the first application of video imaging of Chl fluorescence to the study of light utilization in photosystem II of attached leaves of Phaseolus vulgaris infected with the obligate biotrophic fungus Uromyces appendiculatus (race 38). The video-based detection system produced a spatially resolved, quantifiable signal that was highly specific for chlorophyll fluorescence. Video images of spatial variation in the initial stage of the fluorescence induction (dark-light) transient revealed discreet regions of intense emission coinciding with centers of subsequent lesion development and accompanying chlorosis. Incipient lesions were visible by this procedure 3 d following inoculation, fully 3 to 4 d prior to visible symptoms. Fluorescence emission patterns in infected areas during the induction transient were heterogeneous with radial distance from the point of invasion and varied with the length of the time delay between re-illumination and image capture. During later ([greater than or equal to]1 min) stages of the induction transient, fluorescence emission in incipient lesions was quenched compared to surrounding tissue. These essential features of the induction transient observed in video images were also noted when individual lesions were examined using pulse modulation fluorimetry.     

Common Bean (Phaseolus vulgaris L.) Mutants Defective in Root Nodule Formation: II GENETIC ANALYSIS

Genetic analysis of crosses between two induced, ineffectivelynodulating mutants of common bean, NOD238 and NOD109, revealedthat their mutated nodulation phenotype is under the controlof the same locus in both mutants. The two mutants also resultedallelic for poor pod fertility, the other trait common to themutants. F₁ plants from crosses with their wild types nodulatedeffectively and had wild type pod fertility. Ineffective nodulationand poor pod fertility traits co-segregated in the F₂generationin which plants with the mutant nodulation and pod fertilityphenotypes represented 12.5% of the total population. Analysisin F₃confirmed that these plants were homozygous for both mutatedcharacters. The results indicated both mutant traits studiedare determined by a single recessive allele, named sym-2, whoseinheritance is negatively affected by its pleiotropic effecton pod fertility determining a deficit of ineffectively nodulatingcombinations. In an allelism test with the non-nodulating mutantof common bean NOD125 it was found that ineffective nodulationis controlled at a different locus and that the two loci arenot linked. Key words: Phaseolus valgaris, nitrogen fixation, nodulation mutants, genetics     

Calcium,Calcium Oxalate Crystals,and Leaf Differentiation in the Common Bean (Phaseolus vulgaris L.)*

Young plants of Phaseolus vulgaris were grown in nutrient solutions at different levels of calcium concentration. When the calcium concentration was low more palisade parenchyma and less extended bundle sheath was formed at the adaxial side of minor veins of the leaves as compared to leaves of plants grown with higher calcium supply. The number of calcium oxalate crystals in the bundle sheath extensions was positively correlated to the amount of calcium fed to the plants. The ion induces additional cell divisions in the bundle sheath extensions. A high supply of calcium leads to the formation of a second type of crystal in the bundle sheath.