Relative Efficacy of Different Alfalfa Cultivar-Rhizobium meliloti Strain Combinations for Symbiotic Nitrogen Fixation.

Five Rhizobium meliloti isolates known to have different capabilities for expression of nitrogenase activity under symbiotic conditions were used to inoculate four representative Medicago sativa cultivars under aseptic conditions. Nitrogenase activities and respiratory activity were measured for whole plants and excised nodules. Dry weights and nodule numbers were also recorded after 4 weeks of growth in plastic pouches on a nitrogen-free nutrient medium. Hydrogen evolution and acetylene reduction rates were used to calculate the fraction of reducing power allocated to dinitrogen reduction. Although the efficiency of the system defined in this way was poorly correlated with plant yield, a very high linear correlation was obtained between yield and the algebraic product of nitrogenase activity and efficiency. High correlation (r > 0.78) was obtained between respiration and nitrogenase activity for whole plants as well as for excised nodules. Nodular respiration accounted for between 10 and 20% of the total plant dark respiration. The four test cultivars exhibited significantly different symbiotic responses to the inocula, although trends in potential for expression of the nitrogenase system by the five R. meliloti strains were evident. There was significant interaction between the host plant and symbiont in determining nitrogenase activity and yield. This screening method allows quantitative discrimination between effective and ineffective host-inoculum combinations.

Potential effects of Thiram on Medicago - R. meliloti symbiotic association.

The effects of Thiram and 2 commercial Thiram formulations on the growth and respiration of rhizobia were tested to compare the extent of bacteriostasis under controlled conditions. Although bacteriostasis was measurable at all concentrations tested, liquid cultures grew to maximum optical density in Thiram suspensions containing less than 10 micrograms/ml. Percentage germination, root elongation, and subsequent nodulation by R. meliloti of 2 cultivars of alfalfa, were determined in thiram suspensions to determine potential physiological effects of the fungicide on the host plant. Conditions were identified which produced enhancement or inhibition of germination, root elongation and development of nodular nitrogenase activity. At concentrations of the fungicide recommended for seed application, only minor, temporary bacteriostasis was observed as a possible negative effect while germination rates of fungi-contaminated seed were markedly increased.

Uptake and Growth-promoting Activity of Indoleacetic Acid in Segments of Cold-hardened Wheat Coleoptiles.

Seedings of winter wheat (Triticum aestivum L. cv. Kharkov MC 22) were grown at 24 C (unhardened) and 4 C (hardened). Indoleacetic acid (IAA) was added to excised coleoptile segments after lengthy incubation and their responses were determined by photometric auxanometry at both 25 C and 5 C. The segments' rates of uptake of (14)CIAA were also compared at both temperatures. Cold hardening had no significant effect on the rates of elongation and uptake in a saturating concentration of IAA (2 to 10 muM) at either temperature. Elongation was more sensitive to temperature of measurement than was uptake. At suboptimal concentrations of IAA and 25 C, hardened coleoptiles took up [2-(14)C]-IAA twice as fast but elongated half as fast as unhardened coleoptiles. This and the lack of effect of cold hardening on apparent uptake of [1-(14)C]-IAA raised the possibility that a higher rate of IAA-decarboxylation was coupled with the higher rate of uptake of IAA by hardened coleoptiles. Homeostatic hormonal regulation was also evident in the same endogenous rates of elongation of segments of cold-hardened and unhardened coleoptiles.

Importance of Time after Excision and of pH on the Kinetics of Response of Wheat Coleoptile Segments to Added Indoleacetic Acid.

Segments of coleoptiles of 3-day-old wheat (Triticum x Aestivum L. cv. Kharkov M.C. 22) grown at 24 C were strung on a glass rod and the kinetics of their elongation in 0.01 m K-phosphate buffer was examined photometrically. Measured rates of elongation in response to treatments were corrected by subtraction of endogenous rates. The customary practice of testing the effects of growth regulators added between the two endogenous surges of growth, that is, up to 3 hours after segments were excised from coleoptiles, gave erroneous kinetic data. Rates of response were then limited by the passive penetration of added auxin and the second endogenous surge interfered with late responses. It was necessary to wait for a phase of more rapid but more steady elongation after the second endogenous surge was over, about 4 hours for wheat at 25 C, to attain the active uptake required for nearly synchronous response through the segment. The more active uptake in this steady phase was confirmed with beta-[2-(14)C]indoleacetic acid and it was greater at pH 5 than at pH 7. The degree of dissociation of indoleacetic acid added at pH 7 was an impediment to penetration that could be compensated for by removal of intercellular air. The pH did not influence the endogenous rate of elongation. The dependence of the rate of elongation on the concentration of indoleacetic acid added at pH 5 was bell-shaped with maximum rate at 10 mum indoleacetic acid, in confirmation of previous measurements made over long intervals of time. The relation between the response and suboptimal concentrations was not sigmoid but was indicative of greater binding affinity than previously reported.

Simple photometric auxanometers of high sensitivity.

The elongation phase of growth of plant parts, as 1-cm segments excised from wheat coleoptiles here, is very simply recorded photometrically. One or more aligned segments submerged in aerated buffer pushed an Al foil shutter over a slit of light incident on a photodetector such as a solar or photronic cell, connected directly to a recorder, or a photomultiplier tube in a commercial photometer. Convenient kinetic records were obtained at 5 mm per min chart speed when one segment was combined with a 1-mm long slit, or more segments and longer slits, for which a 1-mm chart unit usually exceeded noise and was equivalent to 4 mum growth per cm coleoptile. In the presence of 10 cm of coleoptile segments no replenishment of solution was necessary during kinetic measurements of response in a 50-ml reservoir of IAA more concentrated than 30 nm.

1,2-Benzanthracene in soil.

Samples from three cultivated soils and one from a roadside, all in Ontario, were found to contain less than 1 to 68 ppb of 1,2-Benzanthracene (BA). Two plots subjected to stubble (residue of wheat crop) burning annually for 15 years did not contain significant amounts of BA, although polyaromatic hydrocarbons, including BA, result from pyrolysis of most organic matter.

The reaction of coumarins with horseradish peroxidase.

The peroxidase catalyzed oxidation of indole-3-acetate is inhibited by naturally occurring coumarins such as scopoletin. This inhibition is due to the preferential reactivity of the coumarins with the peroxidase compounds I, II, and III. In view of the possible growth regulatory role of coumarins in plants, the mechanism of oxidation of scopoletin by horse-radish peroxidase has been investigated.Peroxidase catalyzed coumarin oxidation requires either an electron donor and molecular oxygen or hydrogen peroxide. If peroxide is present, the reaction is mediated by peroxidase compound II which reacts rapidly and stoichiometrically with scopoletin. Different oxidation products are formed, depending on whether IAA or hydrogen peroxide promotes the reaction. A scopoletin-free radical intermediate has been isolated from the peroxide reaction mixture but was not detected in the peroxide-free system.When indole-3-acetate is the electron donor, reduced peroxidase combines with molecular oxygen to give peroxidase compound III. Added scopoletin is cooxidized with indole-3-acetate. Compared to the scopoletin peroxidation, this reaction is slower and yields fewer coumarin oxidation products. The differences observed between the two scopoletin oxidation pathways reflect: (a) the competition between indole-3-acetate and scopoletin for peroxidase compounds; (b) the lower reactivity of scopoletin with peroxidase compound III compared with peroxidase compound II. The peroxide-promoted reaction is eliminated by catalase, while the indole-3-acetate initiated oxidation is not affected by excess quantities of either catalase or superoxidase dismutase.

The Mechanism of the Scopoletin-induced Inhibition of the Peroxidase Catalyzed Degradation of Indole-3-acetate.

The naturally occurring coumarin, scopoletin, has been found to modify horseradish peroxidase rapidly to give a stable, spectroscopically distinguishable form of the enzyme. Peroxidase treated with scopoletin is less active in reactions with molecular oxygen and indole-3-acetic acid. Kinetic data for the degradation of this growth regulator were obtained with a continuously monitored fluorometric procedure. Lineweaver-Burk plots of the reciprocal rate of degradation against the reciprocal substrate concentration were markedly curved in the presence of the inhibitor, scopoletin. Excess indole-3-acetate restored the scopoletin-treated enzyme to a reactive state. In the presence of molecular oxygen, concentrations of indole-3-acetic acid which were at least 10-fold greater than the inhibitor concentration led to the rapid oxidation of the coumarin and converted peroxidase to compound III as expected from previous studies. This form of the enzyme is the catalytically active species in the oxidative degradation of the growth regulator. The kinetically preferential reaction of scopoletin or related coumarins with peroxidase and the suppression of indole-3-acetic acid degradation may provide a possible control mechanism over the oxidative degradation of indole-3-acetate by this plant enzyme.

Studies on growth regulators. I. Improved Avena coleoptile elongation test for auxin.

A modified procedure is presented for the bioassay of auxin using Avena coleoptile segments. The modifications introduced result in a substantial improvement of the commonly used coleoptile elongation tests.The proposed test retains the simplicity of physical requirements and of manipulation characteristic of all elongation tests. The main feature of the proposed test is that it permits the measurement of the elongation of coleoptile segments as being directly proportional to the concentration rather than to the logarithm of the concentration of indoleacetic acid. With an accuracy and a range comparable to those of the standard Avena curvature test, the proposed bioassay overcomes the limitations for the quantitative assessment of auxin inherent in other coleoptile elongation tests. An additional advantage is provided by the present procedure: two 5 mm segments from each coleoptile can be utilized thus doubling the number of assays hitherto possible with a given number of coleoptiles.