Botanical Gazette 82: 438-442 (1926)
[Accepted for publication March 23, 1926]



1WHITING, A. L., and HANSEN, R., Cross inoculation studies with the nodule bacteria of lima beans, navy beans, cowpeas, and others of the cowpea group. Soil Science 10: no. 1, 1920.

That the nodule bacteria of Phaseolus lunatus are distinct from those of P. vulgaris has been shown by WHITING and HANSEN.1 This probably is the first case on record in which all the species within a given plant genus are not inoculated by the same nodule organism. As yet no fundamental relationship has been reached upon which a satisfactory grouping of the nodule organism for inoculation purposes can be made.

As it is possible to grow to maturity a lima bean top on a navy bean root, or a navy top on a lima root, an entirely untouched method of investigating the relation of the host plant to its nodule-producing organism is opened for study. The answer to such questions as follows might throw light upon the relationship of the host plant to its nodule organism: (1) Which organism, that specific to the lima or to the navy bean plant, will produce nodules upon a navy root when grafted with a lima top? (2) Will the nitrogen content of a lima top upon an inoculated navy root be the same as that of a normally inoculated lima plant? (3) Will the processes of photosynthesis and metabolism be the same in a grafted plant as in the normal plant? (4) Will the nitrogen compounds found in the seeds from uninoculated, normally inoculated, and grafted plants be the same? (5) Will the seeds produced by grafted plants produce plants that still retain the power of selection between the nodule organisms?

Preliminary work

In most of the following work sterilized seeds were planted in quartz sand and allowed to grow to a height of 6-8 inches before washing out and making the desired grafts. Grafting was performed by cutting half the stem away for a distance of about inches. Lima and navy seedlings thus treated were placed so that the two cut surfaces were together and bound firmly to each other with string. After the seedlings were securely fastened, the united stem was punctured in several places with a needle. The combined plants were then replanted in quartz sand in a gallon. jar and tied to a support. In about a week's time the stem of one of the two beans was nearly cut in two between the root and bottom of the graft, and at the same time the top of the other plant was cut off. In a few days' time the cut root was severed and removed from the jar. This would then leave a lima bean root with a navy bean top, or a navy root with a lima bean top, as the graft happened to be made. In all cases the plants were watered with tap water, which was found to be not only free of the nodule organism, but also contained sufficient plant food elements, with the exception of nitrogen, to grow plants to maturity. After a successful graft had been made, the nodule organism to be used was added to the jar and the plant allowed to grow for the required time.

EXPERIMENT 1.—The first graft made was a lima top upon a navy root. This was successful and the nodule organisms of the lima bean added to the jar June 1, 1924. Planted in this same jar were sterilized seeds of both lima and navy beans, which were allowed to grow as normal plants in order to test the purity of the nodule organism used. The grafted plant grew well, and upon examination of the check plants in July it was found that the navy bean was uninoculated while the lima bean was well inoculated. This indicated that the lima bean culture used caused nodulation upon lima, but not upon navy bean, and was therefore a pure culture of the lima bean organism.

The grafted plant grew to a height of several feet, and produced well developed pods with several beans in each (fig. 1). This plant was washed out September 2, 1924, and it was found that the root, while free from nodules, had been stimulated to a marked degree. The dry weight and total nitrogen determination for this plant were as follows:

Oven-dry weight of top (except seeds) 2.40 gm.
Oven-dry weight of roots 1.62 gm.
Weight of nitrogen in top 49 mg.
Weight of nitrogen in roots 34 mg.

As this plant had matured several beans, and still had a nitrogen content of 83 mg., the fact is evident that it must have been able to obtain nitrogen even though no nodules were formed upon its roots.

FIGS. 1, 2.—Fig. 1, lima bean top-navy bean root, treated with lima bean nodule organism; leaves removed from part of stem; fig. 2, lima bean top growing upon an inoculated navy bean root.

The nitrogen content of the original bean, as determined by the analysis of similar beans, was about 12-15 mg. The normal navy bean plant grown in the same jar, as a check upon the organisms used, was dying of nitrogen starvation when removed for examination of its roots for nodules, showing that the nitrogen added in the bacterial culture and tap water was not the source of nitrogen available to the grafted plant.

EXPERIMENT 2.—In this trial, grafted plants were inoculated with the organism common to the root of the graft, and it was found in every case in which the navy bean organism was added to a jar in which a lima top was growing upon a navy root, that inoculation occurred and the grafted plant grew to maturity and produced seed. The reciprocal grafts and inoculations with lima bean cultures also produced nodules in all cases.

EXPERIMENT 3.—In this case a lima bean top was grafted upon a navy bean root that already had nodules upon its roots. This graft was successful and the plant grew well. The graft was made June 1, and washed out August 8, 1924 (fig. 2). The plant had several mature beans and was normal in all respects. The root was in good shape and the nodules active. This lima bean top had been able to obtain nitrogen through the inoculated navy bean root.

EXPERIMENT 4.—The question of the behavior of beans grown upon grafted plants toward the nodule organism was next investigated. Navy beans which had grown upon lima roots, and lima beans which had been grown upon navy roots were used in this experiment. The sterilized beans were planted in quartz sand and inoculated June 9, 1924, as indicated:

Modified navy bean seed plus navy bean bacteria +
Modified navy bean seed plus lima bean bacteria +
Modified lima bean seed plus navy bean bacteria +
Modified lima bean seed plus lima bean bacteria +
Normal lima bean seed plus navy bean bacteria -
Normal navy bean seed plus lima bean bacteria -

These plants all grew well and were washed out July 18. Nodules were found upon the roots of all the plants except the checks, but it was at once apparent that the nodules upon the navy bean roots inoculated with lima bean organisms and those upon the lima beans inoculated with navy bean organisms were quite different from normal nodules. They occurred only upon the main root, which appeared to be enlarged and cracked at the point of union, and were whitish in color and flat. The plants, however, gave no appearance of nitrogen starvation.

As normal lima and navy beans inoculated and grown in this same way gave no signs of nodule formation, it appears that the beans grown upon grafted plants have in some way been modified so that they are no longer able to differentiate between the nodule organisms of the lima and navy bean.


  1. When a lima bean top is growing upon a navy bean root, in quartz sand, a pure culture of lima bean bacteria will not cause the formation of nodules upon the roots of the grafted plant, but the roots are stimulated, the plant grows to maturity, and appears to be able to obtain atmospheric nitrogen.
  2. When such a graft is inoculated with a navy bean culture, nodules are formed and the plant grows to maturity. The navy bean organisms in the roots apparently are able to furnish nitrogenous compounds to the lima bean tops, in exchange for carbohydrates synthesized by a top not normal to the nodule organism used.
  3. Similar results are obtained with the reciprocal grafts and inoculations.
  4. A lima bean top grafted upon an inoculated navy bean root grew to maturity and developed seeds.
  5. When seeds are produced by a grafted plant, either lima or navy, they are so modified that plants grown from such seeds no longer have the power of selective adaptation for the specific nodule organism common to it, but are inoculated by either the lima or navy bean organism.