ROOT-NODULES OF LEGUMINOSAE. 127 



Frank in his first paper on the dimorphism of the pea nodules stated that the albuminous nodules 

 contain 6.936 per cent nitrogen while those containing the " amylodextrin " only 4.828 per cent 

 nitrogen in the dry substance. 



Hiltner denies the existence of a dimorphism of pea nodules in the sense used by Frank, but says 

 we might speak of a dimorphism of the bacteroids. He also takes issue with Moller that the end of 

 the bacteroids in all nodules is fatty degeneration. Specimens examined by him showed plainly all 

 degrees of transition between the two sorts of nodules described by Frank. The contents of normal 

 pea nodules flow out readily when the nodule is cut, and are easily mixed with water. On the contrary 

 the nodules in question which are distinguished outwardly by a somewhat darker color show in 

 section a chalky consistency; even from cut cells the contents do not flow out into the water, but 

 adhere so firmly together that it is difficult to separate the single elements from each other. Normal 

 nodules, even when in decay, show no sign of this chalky consistency. The cells of the bacteroid 

 tissue in these chalky nodules are filled with large starch-like grains which, however, stain red-brown 

 with iodine instead of blue. An examination of these chalky nodules showed that almost without 

 exception the septate mycelium of a fungus was present in the nodule or on that part of the root from 

 which the nodule grew. The subsequent investigations at Dahlem verified the assumption that the 

 aggregation of a fatty substance was to be referred to this fungus. Hiltner verified Frank's statement 

 that these abnormal nodules are likely to occur on land where the pea has been cultivated several 

 years. This may, he says, be due to early exposure of the roots to soil fungi, or to the lack of a 

 physiologically important nutrient substance. 



At Dahlem, in soil used for the first time for pea culture, often after 6 to 8 weeks' growth, in a 

 portion of the plants, the upper leaves of pea vines formerly green and healthy took on a pale color 

 and a cessation of growth occurred. In another such field sickly plants appeared among the healthy 

 ones. Investigation showed that healthy plants possessed normal nodules while great numbers of 

 abnormal nodules were found on the roots of the sickly plants, so that one could tell by the appear- 

 ance of the plants before uprooting them, whether the roots bore normal or abnormal nodules. From 

 these facts it is easy to explain why abnormal nodules form on the pea, for the pea is known to 

 experience Bodenmiidigkeit very rapidly and even to fail on some soils in its first year of cultivation 

 after passing successfully the earlier stages of growth. Hiltner considers this a valuable addition to 

 our knowledge of the causes of soil sickness. 



A close study of the waxy substances of bacteroids from artificial cultures, which substance is 

 similar to that in the abnormal nodules, led Hiltner to agree with Moller in regard to its solubility 

 but to disagree with Frank in that he found it unchanged by concentrated sulphuric acid. It can be 

 shaken out of nodules or old cultures by means of chloroform and has rather the consistency of gutta- 

 percha than of wax. The red-brown color caused by iodine is not a reaction of the waxy substance 

 but of a soluble substance present with it. When sections of waxy nodules were left in water for 2 

 days the granules stained pure yellow instead of red-brown, without having otherwise changed in 

 appearance. From his experiments Hiltner found no reason why the red-brown color should not be 

 considered as a glykogen reaction. 



A qualitative analysis of this waxy substance from abnormal nodules showed that it was abso- 

 lutely nitrogen-free. This was at first a disappointing discovery since Hiltner had considered that the 

 waxy substance, so constant in its appearance in artificial cultures and in abnormal nodules repre- 

 sented an accumulation of the products of nitrogen assimilation which the plants had lost the power 

 to absorb. To test this point further, cultures of nodule bacteria from pea, soy-bean and Robinia 

 were allowed to grow for 4 months in seven different nutrient solutions and then tested for increase 

 in nitrogen. Though in suitable solutions the large outgrowths containing waxy inclusions had been 

 formed, in no case was there a trace of nitrogen increase. These solutions contained i per cent grape- 

 sugar, 0.02 per cent monopotassium phosphate and variable amounts of nitrogen in the form of 

 peptone, asparagin and saltpeter in all 50 experiments. 



It is further interesting to note that while, ordinarily, nodule bacteria stain very little or not at 

 all by Gram's method, the waxy outgrowths or germ-plasma of the bacteroids, arising in nutrient 

 solutions containing carbon compounds, stain intensively by Gram soon after their differentiation. 

 As this stage passes, the ability to retain the stain is gradually lost. The substance which holds this 

 stain evidently passes largely into the bacterial slime for this stains readily while the almost colorless 

 bacteroids show then only a few deeply colored granules which somtimes stick to their exterior. 



The following observations were made on the soy-bean, grown in pots for several years. 



In this plant the hunger stage lasts uncommonly long. The plants suffer from it even after 

 numerous large nodules have formed on the roots. On plants showing nitrogen hunger for about 

 8 days, the bacteriods of these inactive nodules were small and only beginning to show plasma 

 differentiation. In the nodules of other plants which the authors knew would in a few days show 

 greening from the beginning of nitrogen assimilation, all the bacteroids possessed large, roundish 



