118 BACTERIA IN RELATION TO PLANT DISEASES. 
The view that the nitrogen-fixing activity of the nodules is due to an enzym excreted by the 
bacteroids was first advanced by Stoklasa, who said that lupin plants from which nodules had been 
carefully removed, continued to fix nitrogen. His results were not confirmed by Hiltner who doubted 
whether the roots remained free from nodules. In Hiltner’s own water-culture experiments, Robinia 
plants, when freed from numerous active nodules, lost their power to assimilate free nitrogen shortly 
afterwards, but soon formed new nodules where the others had been. The same results were obtained 
with Alnus several years running. Uninoculated plants in nitrogen-free solutions plainly suffered 
from hunger. A sudden greening of the younger leaves followed the formation of a few nodules from 
spontaneous infection. When these were removed, starvation again set in until other nodules 
appeared. -Stoklasa’s conclusion must, therefore, be regarded as wholly erroneous. 
Nevertheless Hiltner agrees with Stoklasa that the bacteroids do secrete an enzym-like substance 
which is absorbed by the plant and used as food. This substance may be seen, especially in glycerin 
mounts, in the form of greenish, spherical bodies of varying size, behaving like soluble albumen. He 
thinks that it is this substance which affords immunity by penetrating to all parts of the roots. It 
has not yet been determined whether it is identical with that contained in the slime of the bacteria, 
which exerts such a peculiar influence on the membrane of the root-hairs. 
Root-nodules are active only under favorable conditions of temperature and moisture—condi- 
tions above those required for the plant. Hiltner found that beans planted in a mixture of sand and 
earth and inoculated in May, showed results by the end of the thirty-third day and a very strong 
growth by the end of September. The same experiment started the last of August produced plants 
that in 25 days were plainly suffering from nitrogen-hunger, which they did not overcome in spite 
of the presence of numerous large nodules. These nodules, therefore, remained wholly inactive 
during the unfavorable weather of September and October. On the other hand check plants in 
nitrogenous soil continued moderate growth to the end. 
Different varieties of nodule bacteria may produce nodules varying in size and form on the same 
species of Leguminosae. Nobbe produced on Robinia, nodules like those of the pea by inoculating 
with Pisum bacteria. Hiltner states that he has obtained irregular forms of nodules on Acacia 
lophanta by inoculating with bacteria from pea or locust. 
By altering his nutrient media, Hiltner states, he has obtained a better growth of bacteria than 
formerly. Cultures of lupin-bacteria obtained from the Farbwerken at Hochst formed luxuriant 
colonies but were not virulent. This was not due to loss of virulence from cultivation on artificial 
media. ‘The bacteria were probably taken not from active nodules on the tap-root but from nodules 
on side roots caused by unadapted forms, and hence weak in virulence from the start. On this sub- 
ject Hiltner says: 
“Tn the future, therefore, when we wish to obtain really active, virulent, inoculating material 
for the lupin, we must take the cultures from nodules which appeared as early as possible, and give 
evidence of this by the fact that they are situated on the main root. Also, with all the other Legumi- 
nosae, it would be a great mistake to secure pure cultures from unselected nodules, or even from such 
nodules as occur deep down and at the ends of lateral roots.” 
The studies made on the nodules of leguminous plants of many species kept in botanical gardens 
do not hold good in all situations, where plants are subjected to varying conditions of soil, fertili- 
zation, virulence of bacteria, and climate. Thus the soy-bean which produced no nodules in Germany 
unless inoculated with soil from Japan, in France regularly produced nodules. On the other hand, 
Phaseolus which in Germany produces such large numerous nodules and seems to be most subject 
to infection with non-active bacteria, forms only small ones in France. 
Hiltner is inclined to believe that the size of nodules is influenced by the rapidity with which the 
plant changes the bacteria into bacteroids. When this process is rapid, the nodules remain small but 
active. When it is slow they grow large and inactive. 
Many have thought that the benefit to the plant was derived by the absorption of the bacteroids, 
since at the time when fruit was ripening the nodules were emptied. Nobbe and Hiltner claim, how- 
ever, that this is not true. They found the greatest activity at a time when no sign of this emptying 
had appeared. Besides this, the amount of nitrogen assimilated by the nodules of a plant during a 
season exceeds a hundred fold that contained in the nodules. Hiltner thinks that the bacteroids are 
not absorbed, but simply regain their original form when the sap gradually loses the properties which 
transformed them into bacteroids in the first place, and that only such bacteroids are dissolved as 
had been so thoroughly changed as to lose the power of retransformation, 
In 1902 Peirce in California published a monograph on the root-nodules of Bur clover. He 
stained by means of Flemming’s stain, followed by Ehrlich’s method of staining cover-glass prepara- 
tions of bacteria. The sections, which must be left in the gentian violet for a minute or two only, were 
then placed a half hour or longer in Gram’s iodine solution to differentiate the bacilli and the infection 
threads from the cytoplasm. He states that one minute is usually long enough in the gentian violet. 
