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Effect of nitrate on the organic Acid and amino Acid composition of legume nodules.

Nitrate supplied to legume plants inhibits the activity of nitrogenase in Rhizobium bacteroids in root nodules. The accumulation of amino N which is known to occur in Glycine max (L.) Merr. nodules as nitrogenase activity declines was studied in more detail by analysis of changes in free amino acid composition in response to high nitrate supply. A 6-fold increase in asparagine concentration in Bradyrhizobium japonicum bacteroids was found about the time of maximum nitrogenase inhibition. However, the accumulation of amino acids in soybean nodules lagged behind the inhibition of nitrogenase. Furthermore, in studies of a second legume, Phaseolus vulgaris (L.) inoculated with two different strains of Rhizobium phaseoli, a high nitrate treatment inhibited nitrogenase but had no significant effect on amino acid composition of nodules. The possibility that nitrate may interfere with the supply of carbon substrates to bacteroids was examined by the analysis of organic acids in legume nodules supplied with nitrate. Nitrate had a small (10-20%) negative effect on the concentration of tricarboxylic acid cycle acids in P. vulgaris nodules. However, in G. max nodules, high nitrate treatment resulted in significant increases in the concentration of malate, succinate, fumarate, and citrate. Thus, carbon deprivation of bacteroids also seems unlikely as a cause of the inhibition of nitrogenase by nitrate. There was a transient increase in ammonium concentration in P. vulgaris nodules in response to high nitrate treatment. This effect was rapid relative to other effects of nitrate on nodule composition and was roughly coincident with the rapid decline in acetylene reduction activity.

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Efficiency of nodule initiation in cowpea and soybean.

When serial dilutions of a suspension of Bradyrhizobium japonicum strain 138 were inoculated onto both soybean and cowpea roots, the formation of nodules in the initially susceptible region of the roots of both hosts was found to be linearly dependent on the log of the inoculum dosage until an optimum dosage was reached. Approximately 30- to 100-fold higher dosages were required to elicit half-maximal nodulation on cowpea than on soybean in the initially susceptible zone of the root. However, at optimal dosages, about six times as many nodules formed in this region on cowpea roots than on soybean roots. There was no appreciable difference in the apparent rate of nodule initiation on these two hosts nor in the number of inoculum bacteria in contact with the root. These results are consistent with the possibility that cowpea roots have a substantially higher threshold of response to symbiotic signals from the bacteria than do soybean roots. Storage of B. japonicum cells in distilled water for several weeks did not affect their viability or efficiency of nodule initiation on soybean. However, the nodulation efficiency of these same cells on cowpea diminished markedly over a 2 week period. These differential effects of water storage indicate that at least some aspects of signal production by the bacteria during nodule initiation are different on the two hosts. Mutants of B. japonicum 138 defective in synthesis of soybean lectin binding polysaccharide were defective in their efficiency of nodule initiation on soybean but not on cowpea. These results also suggest that B. japonicum may produce different substances to initiate nodules on these two hosts.

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Nitrogen Fixation (C(2)H(2) Reduction) by Broad Bean (Vicia faba L.) Nodules and Bacteroids under Water-Restricted Conditions.

Water potentials of leaves and nodules of broad bean (Vicia faba L.) cultivated on a sandy mixture were linearly and highly (r(2) = 0.99) correlated throughout a water deprivation of plants. A decrease of 0.2 megapascal of the nodule water potential (Psi(nod)) induced an immediate 25% inhibition of the highest level of acetylene reduction of broad bean nodules attached to roots. This activity continued to be depressed when water stress increased, but the effect was less pronounced. Partial recovery of optimal C(2)H(2) reduction capacity of mildly water stressed nodules (Psi(nod) = -1.2 megapascals) was possible by increasing the external O(2) partial pressure up to 60 kilopascals. The dense packing of the cortical cells of nodules may be responsible for the limitation of O(2) diffusion to the central tissue. Bacteroids isolated from broad bean nodules exhibited higher N(2) fixation activity with glucose than with succinate as an energy-yielding substrate. Bacteroids from stressed nodules appeared more sensitive to O(2), and their optimal activity declined with increasing nodule water deprivation. This effect could be partly due to decreased bacteroid respiration capacity with water stress. Water stress was also responsible for a decrease of the cytosolic protein content of the nodule and more specifically of leghemoglobin. The alteration of the bacteroid environment appears to contribute to the decline in N(2) fixation under water restricted conditions.

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Nitrogen fixation of nodulation mutants of soybean as affected by nitrate.

It was previously reported that three soybean (Glycine max [L.] Merr.) nodulation mutants (NOD1-3, NOD2-4, and NOD3-7) were partially tolerant to nitrate when nitrate was supplied simultaneously with inoculation at the time of transplanting. The current study evaluated the effect of short-term nitrate treatment on nitrogenase activity (C(2)H(2) reduction per plant and per nodule weight) and on relative abundance of ureides when nitrate application was delayed until plants were 3 weeks old and nodules were fully developed. Nitrogenase activity of the mutants was similar to that of Williams after an initial 3-week growth period, prior to nitrate treatment. Application of 5 millimolar nitrate resulted in greater inhibition of nitrogenase activity in Williams than in the three mutants. NOD1-3 was most tolerant of nitrate among the mutants tested and showed the highest relative abundance of ureides. Although C(2)H(2) reduction activity per plant for NOD1-3 was higher than for Williams in the presence of nitrate, C(2)H(2) reduction activity per gram of nodules was lower for NOD1-3 than for Williams in the presence and absence of nitrate. Compared to Williams, NOD1-3 had higher nodule ureide concentration and had similar glutamine synthetase activity in nodule tissue, indicating its nodules have normal nitrogen assimilation pathways. Nitrate application resulted in ureide accumulation in nodule tissue as well as in all plant parts assayed. Unexpectedly, nitrate treatment also increased the rate of ureide degradative capacity of leaves in both NOD1-3 and Williams. The data confirmed that nitrogenase activity of the selected nodulation mutants was more, but still only partially, tolerant of nitrate compared with the Williams parent.

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Effect of pO(2) on Growth and Nodule Functioning of Symbiotic Cowpea (Vigna unguiculata L. Walp.).

Nodulated cowpea (Vigna unguiculata L. Walp. cv Vita 3:Bradyrhizobium CB 756) plants were cultured with their whole root system or crown root nodulation zone maintained for periods from 5 to 69 days after planting in atmospheres containing a range of pO(2) (1-80%, v/v) while the rest of the plant grew in normal air. Growth (dry matter yield) and N(2) fixation were largely unaffected by pO(2) from 10 to 40%. Decrease in fixation at pO(2) below 5% was due to lower nodulation and nodule mass and, at pO(2) above 60%, to a fall in specific N(2)-fixing activity of nodules. Root:shoot ratios were significantly lower at pO(2) below 2.5%. The effect of pO(2) on nitrogenase activity (acetylene reduction), both of whole nodulated root systems and crown root nodulation zones, varied with plant age but was generally lower at supra- and subambient extremes of O(2). H(2) evolution showed a sharp optimum at 20% O(2) but was at most 4% of total nitrogenase activity. The ratio of CO(2) evolved to substrate (C(2)H(2)+H(+)) reduced by crown root nodulation zones was constant (6 moles CO(2) per mole substrate reduced) from 2.5 to 60% O(2) but at levels below 2.5 and above 80% O(2) reached values between 20 and 30 moles CO(2) per mole substrate reduced. Effects of long-term growth with nonambient pO(2) on adaptation and efficiency of functioning of nodules are discussed.

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Activities of the pentose phosphate pathway and enzymes of proline metabolism in legume root nodules.

Based on localization and high activities of pyrroline-5-carboxylate reductase and proline dehydrogenase activities in soybean nodules, we previously suggested two major roles for pyrroline-5-carboxylate reductase in addition to the production of the considerable quantity of proline needed for biosynthesis; namely, transfer of energy to the location of biological N(2) fixation, and production of NADP(+) to drive the pentose phosphate pathway. The latter produces ribose-5-phosphate which can be used in de novo purine synthesis required for synthesis of ureides, the major form in which biologically fixed N(2) is transported from soybean root nodules to the plant shoot. In this paper, we report rapid induction (in soybean nodules) and exceptionally high activities (in nodules of eight species of N(2)-fixing plants) of pentose phosphate pathway and pyrroline-5-carboxylate reductase. There was a marked increase in proline dehydrogenase activity during soybean (Glycine max) ontogeny. The magnitude of proline dehydrogenase activity in bacteroids of soybean nodules was sufficiently high during most of the time course to supply a significant fraction of the energy requirement for N(2) fixation. Proline dehydrogenase activity in bacteroids from nodules of other species was also high. These observations support the above hypothesis. However, comparison of pentose phosphate pathway and pyrroline-5-carboxylate reductase activities of ureide versus amide-exporting nodules offers no support. The hypothesis predicts that pyrroline-5-carboxylate and pentose phosphate pathway activities should be higher in ureide-exporting nodules than in amide-exporting nodules. This predicted distinction was not observed in the results of in vitro assays of these activities.

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Aspartate Aminotransferase in Alfalfa Root Nodules : III. Genotypic and Tissue Expression of Aspartate Aminotransferase in Alfalfa and Other Species.

Aspartate aminotransferase (AAT) plays an important role in nitrogen metabolism in all plants and is particularly important in the assimilation of fixed N derived from the legume-Rhizoblum symbiosis. Two isozymes of AAT (AAT-1 and AAT-2) occur in alfalfa (Medicago sativa L.). Antibodies against alfalfa nodule AAT-2 do not recognize AAT-1, and these antibodies were used to study AAT-2 expression in different tissues and genotypes of alfalfa and also in other legume and nonlegume species. Rocket immunoelectrophoresis indicated that nodules of 38-day-old alfalfa plants contained about eight times more AAT-2 than did nodules of 7-day-old plants, confirming the nodule-enhanced nature of this isozyme. AAT-2 was estimated to make up 16, 15, 5, and 8 milligrams per gram of total soluble protein in mature nodules, roots, stems, and leaves, respectively, of effective N(2)-fixing alfalfa. The concentration of AAT-2 in nodules of ineffective non-N(2)-fixing alafalfa genotypes was about 70% less than that of effective nodules. Western blots of soluble protein from nodules of nine legume species indicated that a 40-kilodalton polypeptide that reacts strongly with AAT-2 antibodies is conserved in legumes. Nodule AAT-2 immunoprecipitation data suggested that amide- and ureide-type legumes may differ in expression and regulation of the enzyme. In addition, Western blotting and immunoprecipitations of AAT activity demonstrated that antibodies against alfalfa AAT-2 are highly cross-reactive with AAT enzyme protein in leaves of soybean (Glycine max L.), wheat (Triticum aestivum L.), and maize (Zea mays L.) and in roots of maize, but not with AAT in soybean and wheat roots. Results from this study indicate that AAT-2 is structurally conserved and localized in similar tissues among diverse species.

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Adaptation of Nodulated Soybean (Glycine max L. Merr.) to Growth in Rhizospheres Containing Nonambient pO(2).

Nodulated soybean (Glycine max L. Merr. cv White Eye inoculated with Bradyrhizobium japonicum strain CB 1809) plants were cultured in the absence of combined N from 8 to 28 days with their root systems maintained continuously in 1, 2.5, 5, 10, 20, 40, 60, or 80% O(2) (volume/volume) in N(2). Plant dry matter yield was unaffected by partial pressure of oxygen (pO(2)) and N(2) fixation showed a broad plateau of maximum activity from 2.5 to 40 or 60% O(2). Slight inhibition of nitrogenase activity occurred at 1% O(2) and as much as 50% inhibition occurred at 80% O(2). Low pO(2) (less than 10%) decreased nodule mass on plants, but this was compensated for by those nodules having higher specific nitrogenase activities. Synthesis and export of ureides in xylem was maintained at a high level (70-95% of total soluble N in exudate) over the range of pO(2) used. Measurements of nitrogenase (EC 1.7.99.2) activity by acetylene reduction indicated that adaptation of nodules to low pO(2) was largely due to changes in ventilation characteristics and involved increased permeability to gases in those grown in subambient pO(2) and decreased permeability in those from plants cultured with their roots in pO(2) greater than ambient. A range of structural alterations in nodules resulting from low pO(2) were identified. These included increased frequency of lenticels, decreased nodule size, increased volume of cortex relative to the infected central tissue of the nodule, as well as changes in the size and frequency of extracellular voids in all tissues. In nodules grown in air, the inner cortex differentiated a layer of four or five cells which formed a band, 40 to 50 micrometers thick, lacking extracellular voids. This was reduced in nodules grown in low pO(2) comprising one or two cell layers and being 10 to 20 micrometers thick in those from 1% O(2). Long-term adaptation to different external pO(2) involved changes which modify diffusive resistance and are additional to adjustments in the variable diffusion barrier.

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Characterization of delta-Aminolevulinic Acid Formation in Soybean Root Nodules.

Formation of the heme precursor delta-aminolevulinic acid (ALA) was studied in soybean root nodules elicited by Bradyrhizobium japonicum. Glutamate-dependent ALA formation activity by soybean (Glycine max) in nodules was maximal at pH 6.5 to 7.0 and at 55 to 60 degrees C. A low level of the plant activity was detected in uninfected roots and was 50-fold greater in nodules from 17-day-old plants; this apparent stimulation correlated with increases in both plant and bacterial hemes in nodules compared with the respective asymbiotic cells. The glutamate-dependent ALA formation activity was greatest in nodules from 17-day-old plants and decreased by about one-half in those from 38-day-old plants. Unlike the eukaryotic ALA formation activity, B. japonicum ALA synthase activity was not significantly different in nodules than in cultured cells, and the symbiotic activity was independent of nodule age. The lack of symbiotic induction of B. japonicum ALA synthase indicates either that ALA formation is not rate-limiting, or that ALA synthase is not the only source of ALA for bacterial heme synthesis in nodules. Plant cytosol from nodules catalyzed the formation of radiolabeled ALA from U-[(14)C]glutamate and 3,4-[(3)H]glutamate but not from 1-[(14)C]glutamate, and thus, operation of the C(5) pathway could not be confirmed.

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Aspartate aminotransferase in effective and ineffective alfalfa nodules : cloning of a cDNA and determination of enzyme activity, protein, and mRNA levels.

Aspartate aminotransferase (AAT) is a key plant enzyme affecting nitrogen and carbon metabolism, particularly in legume root nodules and leaves of C(4) species. To ascertain the molecular genetic characteristics and biochemical regulation of AAT, we have isolated a cDNA encoding the nodule-enhanced AAT (AAT-2) of alfalfa (Medicago sativa L.) by screening a root nodule cDNA expression library with antibodies. Complementation of an Escherichia coli AAT mutant with the alfalfa nodule AAT-2 cDNA verified the identity of the clone. The deduced amino acid sequence of alfalfa AAT-2 is 53 and 47% identical to animal mitochondrial and cytosolic AATs, respectively. The deduced molecular mass of AAT-2 is 50,959 daltons, whereas the mass of purified AAT-2 is about 40 kilodaltons as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the protein's N-terminal domain (amino acids 1-59) contains many of the characteristics of plastid-targeting peptides. We postulate that AAT-2 is localized to the plastid. Southern blot analysis suggests that AAT-2 is encoded by a small, multigene family. The expression of AAT-2 mRNA in nodules is severalfold greater than that in either leaves or roots. Northern and western blots showed that expression of AAT activity during effective nodule development is accompanied by a sevenfold increase in AAT-2 mRNA and a comparable increase in enzyme protein. By contrast, plant-controlled ineffective nodules express AAT-2 mRNA at much lower levels and have little to no AAT-2 enzyme protein. Expression of root nodule AAT-2 appears to be regulated by at least two events: the first is independent of nitrogenase activity; the second is associated with nodule effectiveness.

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Mathematical modeling of oxygen diffusion and respiration in legume root nodules.

The O(2) permeability of legume root nodules is under physiological control; decreases in permeability are triggered by various forms of stress. Two linked mathematical models were used to explore several hypotheses concerning the physical nature of the variable diffusion barrier in nodules. Respiration and diffusion of dissolved O(2) and oxygenated leghemoglobin were simulated for the nodule cortex and the nodule interior. Measured nodule permeabilities were shown to be inconsistent with the hypothesis that large numbers of air-filled pores penetrate the diffusion barrier. Changes in the affinity of leghemoglobin for O(2) or in the rate of cytoplasmic streaming in diffusion barrier cells did not result in the large changes in O(2) permeability reported for real nodules. The presence or absence, but not the thickness, of aqueous plugs in radial pores through the cortex was found to have a large effect on permeability. Flooding of intercellular spaces, either between layers of cells in the cortex or in the nodule interior, also caused large changes in simulated permeability. The unsteady-state O(2) method for determining nodule permeability was tested using data generated by the model. The accuracy of the method was confirmed, provided that certain assumptions (full oxygenation of leghemoglobin under pure O(2) and uniform conditions in the nodule interior) are met.

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Suppressive effect of the angiogenesis inhibitor TNP-470 on the development of carcinogen-induced hepatic nodules in rats.

Tumor metastasis can be prevented by inhibiting angiogenesis. In the present study, we have demonstrated that the angiogenesis inhibitor TNP-470 also suppresses the development of primary hepatic nodules. Hepatocarcinogenesis was performed by the feeding of 2-acetylaminofluorene to hepatectomized rats during 8-14 weeks of age. Predominantly arterial-to-portal circulation and sinusoidal capillarization were determined by the staining of nodules with arterially infused ink and immunostaining for factor VIII-related antigen, respectively. Intraperitoneal administration of 30 mg/kg b.w. of TNP-470 twice a week significantly reduced the number of hepatic nodules. Among the nodules, hyperplastic nodules stained with ink, atypical hyperplastic nodules and hepatocellular carcinoma, all of which possess structurally altered sinusoidal endothelial cells or capillary-type endothelial cells, were dramatically decreased in number. Suppression was observed equally in nodules of all sizes. TNP-470 was more effective when administered during 8-20 weeks than during 14-26 weeks. In contrast, ink-non-stained hyperplastic nodules, which have normal sinusoidal endothelial cells, were not affected at all. The present results indicate that TNP-470 suppresses the development of primary hepatic nodules whose microvessels are capillaries or transitional forms from sinusoids to capillaries.

2-Acetylaminofluorene↗

Arterial elements and perisinusoidal cells in borderline hepatocellular nodules and small hepatocellular carcinomas.

Borderline hepatocellular nodule in the human cirrhotic liver is considered a preneoplastic lesion of hepatocellular carcinoma (HCC). However, the angiogenetic process and changes in perisinusoidal cells (fat-storing cells or Ito cells) during the borderline nodule-HCC sequence have not been investigated. We have investigated intraparenchymal arterial elements and perisinusoidal cells in normal livers, chronic hepatitis, borderline nodules and small HCC, using an immunohistochemical staining for alpha-smooth muscle actin. In normal livers, chronic hepatitis, cirrhotic nodules and large regenerative nodules, no or few arterial elements were present in the parenchyma, and alpha-smooth muscle actin-positive perisinusoidal cells were increased further. These data suggest that angiogenesis first occurs in borderline hepatocellular nodules and it gradually proceeds during the nodule to HCC sequence along with an increase in perisinusoidal cells. The demonstration of arterial elements and perisinusoidal cells may be useful for the differential diagnosis of large regenerative nodule, borderline hepatocellular nodule and small HCC.

Arteries↗

Automated detection of lung nodules in CT scans: preliminary results.

We have developed a fully automated computerized method for the detection of lung nodules in helical computed tomography (CT) scans of the thorax. This method is based on two-dimensional and three-dimensional analyses of the image data acquired during diagnostic CT scans. Lung segmentation proceeds on a section-by-section basis to construct a segmented lung volume within which further analysis is performed. Multiple gray-level thresholds are applied to the segmented lung volume to create a series of thresholded lung volumes. An 18-point connectivity scheme is used to identify contiguous three-dimensional structures within each thresholded lung volume, and those structures that satisfy a volume criterion are selected as initial lung nodule candidates. Morphological and gray-level features are computed for each nodule candidate. After a rule-based approach is applied to greatly reduce the number of nodule candidates that corresponds to nonnodules, the features of remaining candidates are merged through linear discriminant analysis. The automated method was applied to a database of 43 diagnostic thoracic CT scans. Receiver operating characteristic (ROC) analysis was used to evaluate the ability of the classifier to differentiate nodule candidates that correspond to actual nodules from false-positive candidates. The area under the ROC curve for this categorization task attained a value of 0.90 during leave-one-out-by-case evaluation. The automated method yielded an overall nodule detection sensitivity of 70% with an average of 1.5 false-positive detections per section when applied to the complete 43-case database. A corresponding nodule detection sensitivity of 89% with an average of 1.3 false-positive detections per section was achieved with a subset of 20 cases that contained only one or two nodules per case.

Adult↗

Computer-aided diagnostic scheme for the detection of lung nodules on chest radiographs: localized search method based on anatomical classification.

We developed an advanced computer-aided diagnostic (CAD) scheme for the detection of various types of lung nodules on chest radiographs intended for implementation in clinical situations. We used 924 digitized chest images (992 noncalcified nodules) which had a 500 x 500 matrix size with a 1024 gray scale. The images were divided randomly into two sets which were used for training and testing of the computerized scheme. In this scheme, the lung field was first segmented by use of a ribcage detection technique, and then a large search area (448 x 448 matrix size) within the chest image was automatically determined by taking into account the locations of a midline and a top edge of the segmented ribcage. In order to detect lung nodule candidates based on a localized search method, we divided the entire search area into 7 x 7 regions of interest (ROIs: 64 x 64 matrix size). In the next step, each ROI was classified anatomically into apical, peripheral, hilar, and diaphragm/heart regions by use of its image features. Identification of lung nodule candidates and extraction of image features were applied for each localized region (128 x 128 matrix size), each having its central part (64 x 64 matrix size) located at a position corresponding to a ROI that was classified anatomically in the previous step. Initial candidates were identified by use of the nodule-enhanced image obtained with the average radial-gradient filtering technique, in which the filter size was varied adaptively depending on the location and the anatomical classification of the ROI. We extracted 57 image features from the original and nodule-enhanced images based on geometric, gray-level, background structure, and edge-gradient features. In addition, 14 image features were obtained from the corresponding locations in the contralateral subtraction image. A total of 71 image features were employed for three sequential artificial neural networks (ANNs) in order to reduce the number of false-positive candidates. All parameters for ANNs, i.e., the number of iterations, slope of sigmoid functions, learning rate, and threshold values for removing the false positives, were determined automatically by use of a bootstrap technique with training cases. We employed four different combinations of training and test image data sets which was selected randomly from the 924 cases. By use of our localized search method based on anatomical classification, the average sensitivity was increased to 92.5% with 59.3 false positives per image at the level of initial detection for four different sets of test cases, whereas our previous technique achieved an 82.8% of sensitivity with 56.8 false positives per image. The computer performance in the final step obtained from four different data sets indicated that the average sensitivity in detecting lung nodules was 70.1% with 5.0 false positives per image for testing cases and 70.4% sensitivity with 4.2 false positives per image for training cases. The advanced CAD scheme involving the localized search method with anatomical classification provided improved detection of pulmonary nodules on chest radiographs for 924 lung nodule cases.

Databases, Factual↗

Competition Among Rhizobium spp. for Nodulation of Leucaena leucocephala in Two Tropical Soils.

The successful nodulation of legumes by a Rhizobium strain is determined by the competitive ability of that strain against the mixture of other native and inoculant rhizobia. Competition among six Leucaena rhizobial strains in single and multistrain inoculants were studied. Field inoculation trials were conducted in an oxisol and a mollisol soil, both of which contained indigenous Leucaena-nodulating rhizobia. Strain-specific fluorescent antibodies were used for the identification of the strains in Leucaena nodules. Mixtures of three recommended inoculum strains for Leucaena spp. (TAL82, TAL582, and TAL1145) were used in peat-based inocula either alone or with one of the three other strains isolated from the sites, B213, B214, and B215. Each of these latter three strains was also used as single-strain inocula to study their competition with the native rhizobia in the two soil systems. In the oxisol soil, strains B213 and B215, when used as single-strain inocula, outcompeted the native rhizobia and formed 92 and 62% of the nodules, respectively. Strain B214 was the least competitive in oxisol soil, where it formed 30% of the nodules, and the best in mollisol soil, where it formed 70% of the nodules. The most successful competitor for nodulation in multistrain inocula was strain TAL1145, which outcompeted native and other inoculum Leucaena rhizobia in both soils. None of the strains in single or multistrain inoculants was capable of completely overcoming the resident rhizobia, which formed 4 to 70% of the total nodules in oxisol soil and 12 to 72% in mollisol soil. No strong relationship was detected between the size of the rhizosphere population of a strain and its successful occupation of nodules.

Journal Article↗

Erwinia herbicola isolates from alfalfa plants may play a role in nodulation of alfalfa by Rhizobium meliloti.

Erwinia herbicola was isolated from roots of plants derived from surface-sterilized seeds of all alfalfa varieties that were tested. Some of these E. herbicola strains affected nodulation by certain strains of Rhizobium meliloti. In previously published work we presented the isolation of slow-and fast-nodulating variants from a single culture of R. meliloti 102F51. In the absence of E. herbicola, the slow-nodulating variant induced the formation of nodules on alfalfa as rapidly as the faster-nodulating strain. The rates of nodulation by the faster-nodulating variant were the same in the presence and absence of E. herbicola. All of the previously reported slower-nodulating strains derived from R. meliloti 102F51 nodulated more rapidly on sterilized plants than in the presence of certain E. herbicola isolates.

Erwinia↗

Influence of 5-Methyltryptophan-Resistant Bradyrhizobium japonicum on Soybean Root Nodule Indole-3-Acetic Acid Content.

Bradyrhizobium japonicum mutants resistant to 5-methyltryptophan were isolated. Some of these mutants were found to accumulate indole-3-acetic acid (IAA) and tryptophan in culture. In greenhouse studies, nodules from control plants inoculated with wild-type bradyrhizobia contained 0.04, 0.10, and 0.58 mug of free, ester-linked, and peptidyl IAA g (fresh weight) of nodules, respectively. Nodules from plants inoculated with 5-methyltryptophan-resistant bradyrhizobia contained 0.94, 1.30, and 10.6 mug of free, ester-linked, and peptidyl IAA g (fresh weight) of nodules, respectively. This manyfold increase in nodule IAA content indicates that the Bradyrhizobium inoculum can have a considerable influence on the endogenous IAA level of the nodule. Further, these data imply that much of the IAA that accumulated in the high-IAA-containing nodules was of bacterial rather than plant origin. These high-IAA-producing 5-methyltryptophan-resistant bacteria were poor symbiotic nitrogen fixers. Plants inoculated with these bacteria had a lower nodule mass and fixed less nitrogen per gram of nodule than did plants inoculated with wild-type bacteria.

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