Il8 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 remainedfree 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 lyCguminosae. 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 : 



"In 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 baciUi and the infection 

 threads from the cytoplasm. He states that one minute is usually long enough in the gentian violet. 



