Further experiments concerning diffusion of nitrogenous compounds from healthy legume nodules or roots.

IN A previous contribution from this laboratory (15) a number of experiments were described which indicate that under a variety of conditions the diffusion of nitrogenous compounds from healthy legume nodules or roots does not occur. Three more experiments (numbered XII, XIII, XIV) in which certain conditions have been further modified are reported here. The chief specific modifications were: (1) a larger proportion of non-legumes to legumes than used in former experiments, (2) a larger adsorptive capacity of the medium, (3) variations in the proportions of certain elements in the medium, and (4) the use of various strains of rhizobia. METIrIoDs.-The procedures followed in experiments XII and XIII were similar, but differed somewhat from those used in experiment XIV. Experiments XII and XIII.-The methods were in general the same as described in previous publications (14, 15). Illumination of about 2,000 foot candles (21,500 lux) was supplied daily from midnight till six p.m. In Experiment XII the first seeds were planted on May 8, 1937, and the harvest was completed on July 24; in Exp. XIII the corresponding dates were March 10 and June 8, 1938. A solenoid valve was inserted in the cooling water line to prevent the water from running while the lights were off. This prevented the sand from cooling off as much during the period of darkness as it did with earlier cultures. Sand temperatures rose somewhat with the advance of the season but at about 2.5 cm. below the surface at the middle of the jar they were around 28 to 290C. in the "afternoon" and 2-3? lower at "night" in Exp. XII, and 18 to 220 in the "afternoon" and correspondingly low at "night" in Exp. XIII. The legume used was Nott's Excelsior garden pea (Pisum sativum L.), inoculated with a culture of Rhizobium leguminosarum Frank of Dr. A. I. Virtanen's pea strain XX. The non-legume in most of the cultures of Exp. XII was barley (Hordeum vulgare L.), seed of which also came from Dr. Virtanen. In addition, a few cultures were conducted with corn (Zea. mais L.), variety Boone County White. The non-legumes used in Exp. XIII were winter wheat (Triticum aestivum L.), and oats (Avena sativa L.). The cultures, as previously, were grown in a washed quartz sand to which enough culture solution was added to bring the water content to 12 per cent of the dry weight of the sand. The sand used in Exp. XIII included a small proportion that had been used in Exp. XII but had been later thoroughly 1 Received for publication April 09, 1940. The writers are indebted to Ellen K. Rist, of this laboratory, for the nitrogen determinations made in the course of this study. washed with hot water. However, it is doubtful if the washing removed all of the salts previously added, as will appear later in this paper and as is suggested by results since published by Dunlap (8). The culture solutions were variations of one' previously used by Thornton and associates (20). Acidities of the sand were determined colorimetrically about six or seven weeks after the beginning of the experiments. The specific variations employed are described below under "Results." Experiment XIV.-This experiment was conducted in an apparatus similar to that described previously (14) except that the only light source was a bank of 200-watt Mazda lamps that gave an illumination of approximately 600 foot candles. The illumination lasted fifteen hours daily. Each pot contained five pounds of quartz sand of the same particle size as used in the other two experiments. This was sterilized for one and one-half hours in steam at fifteen pounds pressure, but no attempt was made to keep it sterile during the experiment. All seeds were surface sterilized prior to planting. Modified Crone's nitrogen-free solution ( 11), at half the usual concentration, was added to the sand. In addition, 0.3 mg. Fe as natural humate and 0.9 mg. B as sodium borate were supplied per pot. The pots stood in a tank partially filled with water and into which a small stream of tap water flowed constantly. The legumes used were Tennessee red clover (Trifolium pratense L.), alfalfa (Medicaqo sativa L.), crown vetch (Coronilla varia L.), and Nott's Excelsior garden pea. The first three legumes had ten plants per pot and the garden pea three. All pots also contained three wheat and five to eight timothy (Phleum pratense L.) plants and were planted March 11, 1938. Each pot, except a few controls, was inoculated when planted with 5 cc. of a two-day culture of the appropriate strain of Rhizobium. The various strains of bacteria, other than those from our own stock cultures, were obtained through the courtesy of Drs. I. L. Baldwin, A. I. Virtanen, and D. W. Thorne. RESULTS.-The results are given in tables I and 2. The values in table I for nitrogen fixed and for pH are means of two cultures except as indicated. The apparent gains and losses of nitrogen in the nonlegumes, due to association in experiments XII and XIII, were obtained by subtracting the amount of nitrogen in the tops of the non-legumes grown alone from that in the tops of the corresponding plants grown in association. This procedure was followed 2 The solution contained: KCl, 0.74 gm.; K2HPO4 0.3 gmi.; KH2PO4, 0.3 gm.; MgSO4.7H20, 0.5 gin.; NaCl, 0.5 gin.; CaSO4, 0.5 gin.; water, 1 liter. Iron, usually at the rate of about 0.6 mg. Fe per culture, was supplied as humate; and boron at the rate of 1.5 mg. per culture was added as sodium borate.