With Plate X and Six Text-figures) ASSOCIATED cultures of legumes and non-legumes have been employed in ordinary farming practice since ancient times, as in such cultures the non-legumes have generally been found to develop better than when grown alone. However, the underlying reason for this beneficial effect has not been clearly understood. Lipman (1912) was probably the first worker to attempt an elucidation of this problem by direct experiments with pot cultures. His results, though in part conflicting, led him to the conclusion that inoculated legumes do somehow furnish nitrogenous food for the non-legumes. Lipman's findings were, however, not conclusive as proof for an excretion. Virtanen's (1929) first experiments with pot cultures showed that oats grew excellently in association with inoculated peas in N-free quartz sand. An excretion of nitrogen was therefore held to be probable. However, in ordinary pot cultures, where the action of various foreign micro-organisms is not excluded, the results may always be rendered valueless through the presence of free-living nitrogen-fixers which thrive particularly well in the rhizosphere. Beijerinck (1908) and others have shown long ago that bacterial life is indeed greatly enhanced in the immediate proximity of the roots of leguminous plants. Under such conditions the nitrogen taken up by the non-legumes is not necessarily derived from the legume nodules but might be fixed by the non-symbiotic organisms. The literature (up to 1934) on the derivation of the nitrogen in associated growth has been reviewed by Nicol (1934).The first conclusive proof for the excretion of considerable quantities of nitrogenous compounds from the nodules 1 of inoculated legumes was presented by Virtanen & v. Hausen (1931a). The work necessitated the use of "sterile" cultures containing only the desired nodule bacteria, 1 The excretion of the nitrogenous compounds has been definitely shown to occur from the nodules and not from the roots (Virtanen et al. 19366).

1. It has been shown experimentally that the excretion of nitrogen noted by us in cultures of inoculated legumes takes place from the nodule bacteria, probably from the intranodular ones, and not from the roots. No excretion of amino acids occurs in cultures of uninoculated legumes growing on nitrate nitrogen.2. Our earlier hypothesis that the legumes receive their nitrogen nutrition from the nodules in the form of organic nitrogen compounds, particularly amino acids, is in perfect accord with our new observations concerning the process of excretion. All facts indicate that the amino acids concerned are primary products of the nitrogen fixation, and not breakdown products of proteins. Bond's valuable work along quite different lines produced results which support this conclusion. He, however, did not study the chemical nature of the nitrogen compounds in question.3. The excretion of nitrogen occurs in media capable of absorbing the excreted nitrogen compounds (cellulose, kaolin, sand, soil). The demonstration of the excretion is not possible in water cultures except when very large quantities of water are used. On the basis of these facts a hypothesis is advanced to explain the nature of the excretion.4. The term total fixed nitrogen has been used as an expression for the extent of nitrogen fixation, while the term extent of excretion is employed to indicate that percentage of the total fixed nitrogen which is excreted from the nodules.5. The extent of excretion depends largely on the strain used for inoculation. With strains of apparently equal effectiveness in nitrogen fixation, the extent of excretion may vary considerably, so that actually such strains differ in their effectiveness.

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The effect of the air content of the medium on the development and function of the nodule was studied with sterile cultures of peas inoculated with their appropriate strain of nodule bacteria.In water cultures the plant grows well without added nitrogen, even though all nodules are submerged, if air is bubbled through the medium during the period of growth. Abundant nodulation occurs also in nonaerated liquid cultures; the nodules, however, are much smaller than in aerated cultures, and the plant grows poorly. Subsequent aeration of such cultures results in a distinct increase in the size of the nodules and improvement in the growth of the plant.Nodulation is completely prevented by passing a stream of gaseous N through the medium. That oxygen is indispensable for the function of the nodules is shown also by the fact that the peas grow excellently on added nitrogen in non-aerated cultures.

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