ROOT-NODULES OF LEGUMINOSAE. 115 
The membrane of the root-hair is not completely dissolved by this process, but becomes swollen 
and thus penetrable by the bacteria. 
The above results lead to the conclusion that the nodule bacteria of different species do not 
entirely agree, at least in their physiological peculiarities, and hence to the question whether they 
comprise one or more species. 
Hiltner obtained nodules on the roots of the East Indian Acacia lophanta, grown in sterilized 
nitrogen-free sand, by inoculating them with bacteria from peas and locust. ‘This shows that at least 
not every genus of Leguminosae requires a special species of bacteria for the formation of nodules. 
On the other hand, Kirchner observed that, among about 100 species of Leguminosae grown yearly 
in the garden at Hohenheim, only the soy-beans were free from nodules. When these were inoculated 
with earth from Japan in which soy-beans had grown, large active nodules appeared. Hiltner 
at Tharandt in experiments covering several years also found Soja hispida always nodule free. 
Although morphologically the bacteria of these nodules could not be distinguished from native nodule 
bacteria, Kirchner considered the physiological and biological differences sufficient to warrant their 
recognition as a new species, Bacterium (Rhizobacterium) japonicum. ‘This conclusion is not shared 
by Cohn or Naudin, who found nodules on soy-beans which had not been inoculated. 
Hiltner states that in his experiments in recent years no nodules were formed on his uninocu- 
lated plants, except in some of the water cultures which are extremely difficult to keep pure, and that 
hence, when inoculations produced nodules, the bacteria used must have been the nodule-forming 
bacteria. 
He found the bacteria of the most various Leguminosae, even of the Mimosae and Caesalpineae, 
extraordinarily alike morphologically. He could find no constant difference, nor could he regularly 
demonstrate the two groups of Beyerinck, much less the differences described by Gonnermann. On 
the other hand he thinks that the artificial media generally used for the culture of nodule bacteria, 
and on which bacteria from peas, clover, vetch, and Robinia flourish, is extremely unsuitable for 
serradella and lupin bacteria. Such medium consists of gelatin weakly acidified with malic acid, 
and containing an extract of Leguminosae and asparagin and grape-sugar. Concerning other dif- 
ferences Hiltner says: z 
“The investigations at Tharandt, however, have plainly shown a great difference, both bio- 
logical and physiological, in the bacteria isolated from nodules of different leguminous plants. It 
was evident, from the most carefully conducted inoculation experiments that the bacteria from 
nodules of a definite species of leguminous plant, were most active upon this same species, both as 
regards rapidity in the formation of nodules, and their nitrogen-gathering activity. On other species 
of the same genus, or of the same group, however, they generally showed a materially decreased 
activity, and finally, as a rule, such activity disappeared completely in genera which were farther 
removed systematically.” 
Within the Trifoliaceae a mutual transfer of bacteria was less successful than between Pisum 
and Vicia. Very active bacteria from Trifolium pratense failed to produce nodules on Medicago 
sativa grown in a sterilized mixture of sand and earth. 
The lupins showed to a marked degree this difference between species of the same genus. Even 
the bacteria from Lupinus luteus and L. angustifolius behaved very differently and Kirchner found 
among 14 species of lupin grown for several years in close proximity, and even mingled, that while 12 
formed nodules from the start, Lupinus hirsutus and L. subcarnosus were completely free from them. 
For the most part Robinia pseudacacia appeared to be very exclusive. It required much time to 
produce nodules even when bacteria from most closely related genera, e. g., Caragana, were employed. 
In contrast to Robinia the genus Phaseolus was easily infected by bacteria from the nodules of 
other legumes. When inoculated with bacteria from Piswm and Robinia it produced nodules, which, 
however, were inactive or nearly so as regards nitrogen fixation. Bacteria, from Phaseolus on the 
other hand, formed weakly active nodules on Pisum, but had no effect on Robinia. 
All these results lead to the probable conclusion that the different nodule-bacteria of the Legu- 
minosae are really only adaptation forms of the same species. Laurent’s experiments in which he 
obtained nodules on peas by needle-prick inoculations with bacteria from 30 different (unnamed) 
species of Leguminosae, seems to support this conclusion, although he had no check plants, and 
Zinsser’s experiments are contrary. 
A study of the bacteriods from the nodules of different species gave the same result. Although 
sometimes very different in form, the transformations which took place permitted the conclusion 
that the nodule bacteria of the Leguminosae represent a single, extraordinarily polymorphic species, 
a conclusion which agrees with Beyerinck’s observations on the bacteroids in an old culture from 
Phaseolus. 
Hiltner states also that he and Nobbe succeeded in transforming pea-bacteria into forms iden- 
tical with bean-bacteria in their activity on peas and beans, as well as in the form of their bacteroids. 
