128 BACTERIA IN RELATION TO PLANT DISEASES. 



outgrowths like those which had been observed repeatedly in nutrient solutions. These outgrowths 

 stained strongly with carbol fuchsin. However, when these plants actually became green there was 

 no longer a trace of the outgrowths on the bacteroids. The outgrowths, therefore, formed when the 

 plants began to hunger and disappeared as the nitrogen hunger stage passed away. From this it 

 appears that nitrogen assimilation must stand in some sort of relation to the absorption of the out- 

 growths of the bacteroids which in active tubercles are continually formed but not easily demon- 

 strated. Nitrogen assimilation is made possible not by the bacteroid formation per se, but by the 

 process of plasma differentiation therein, in that from the nuclear plasma or through its action a 

 nitrogenous substance is formed, the nitrogen for which is drawn from the atmosphere. 



Bacteroid formation and nitrogen assimilation do not always go hand in hand. This showed 

 plainly in the cultures of pea bacteria in saltpeter solutions previously referred to, where, though 

 bacteroid formation took place, plasma outgrowths did not appear, no staining was obtained with 

 Gram's stain, with iodine the color became yellow, not red-brown, and no nitrogen assimilation 

 occurred. These circumstances explain the fact previously mentioned by Nobbe and Hiltner that 

 nitrogen assimilation in Leguminosae begins only when the available nitrogen in the soil has been 

 exhausted. This is true also in Alnus. 



Hartleb's idea of the sporangial nature of the bacteroid is not new but originated with Brunchorst 

 and Moeller and was confirmed by Hiltner prior to the appearance of Hartleb's paper. Later experi- 

 ments by the authors have not altered this view. The bacteroids of the alder bacteria are even more 

 strikingly sporangia-like. All observations point to the conclusion that in certain solutions and under 

 certain circumstances the outgrowths from the bacteroids break up into pieces of varying size which 

 directly or after further division may grow into bacteria or bacteroids. Some of Hiltner's figures 

 suggest certain figures published by Gino de Rossi. The bacteroids within the nodules show less 

 plainly their sporangial nature. 



According to these observations the relation between host-plant and bacteroids is much more 

 intimate than was previously supposed. In sharp contrast to the opinion that the nitrogen assimi- 

 lation is concerned with an absorption of the bacteroids, Hiltner claims that his observations support 

 the view that under normal circumstances this absorption does not take place, but rather that some 

 substance produced by the nitrogen assimilation which goes on within the bacteroid is absorbed. 

 This opinion is based on the fact that within the root-nodules the bacteria have a tendency to form 

 sporangia to protect themselves against the influence of the host-plant, but that this does not succeed 

 so long as the plant is active, because the indispensable building material obtained by nitrogen 

 assimilation is constantly drawn away from them by the plant. 



Beyerinck also has found that soil bacteria known as true nitrogen fixers (his Granulobacter, 

 Radiobacter, Aerobacter) are able to use very little of the product of their assimilation, but that this 

 is used chiefly by a species living with them (Azotobader chroococcum), the nitrogen fixers (other 

 then Granulobacter) being stimulated to action only by the symbiotic life. 



The effectiveness of pure cultures of nodule bacteria depends on a number of factors, viz., 

 genuineness, nutrition, virulence, and nitrogen-fixing power. 



That true nodule bacteria should be used is self evident, yet that extreme care is required to 

 obtain them is not fully realized. Nodules in which decay has begun may contain all manner of 

 strange species and it is not out of the question that completely sound nodules may contain intruders. 

 Beyerinck showed this to be the case. Though Hiltner did not find the same intruding species 

 except in decaying nodules, he did find in 1902 another contaminating species in cultures from 

 several distinct sources. This intruding species developed very slowly on gelatin compared with the 

 true nodule bacteria, and differed from it quite materially in the appearance of its colonies. On the 

 other hand it formed bacteroids and behaved very similarly to the nodule bacteria in grape-sugar 

 solutions. This organism was not able to produce nodules and seemed able to penetrate the nodules 

 only when the real nodule producer had prepared the way. An especially characteristic peculiarity 

 of this species, which does not liquefy gelatin, is that the individuals of a colony are held together 

 by such an extraordinarily viscid slime that even when they are allowed to lie for a week in water 

 they do not separate. Hiltner suspects that they are able to fix nitrogen, but there is no possibility 

 of confusing them with the true nodule bacteria. However, there are, as Beyerinck observed, several 

 species of bacteria resembling the true nodule bacteria in the appearance of their colonies and in their 

 general behavior which can give much trouble in the securing of pure cultures because of their fre- 

 quent appearance in the nodules. Only a test of the nodule producing ability of a culture can here 

 prevent mistakes. Additional certainty is obtained by determining under what circumstances bac- 

 teroids are formed. 



But even when it is known that a culture consists of the true nodule bacteria, one does not 

 know with any certainty whether he has the desired adaptation form. It seems indeed at first sight 

 as if it would be sufficient to know the immediate source of the cultures. Any one who has not 



