ROOT-NODULES OF LEGUMINOSAE. Ill 



washed in 90 per cent alcohol and then stained with iodine and sulphuric acid. If chitin be present 

 a beautiful pink stain is given to the hyphae while the cells of the host take on the usual blue color 

 of cellulose. 



Staining the infection tube within the root-hair shows it to consist of a chain of rodlets like those 

 found in the filaments within the nodule. The growing point of these filaments is, as Frank asserted, 

 a diffuse open end. In fresh material this open end generally shows a rosette of refringent granules, 

 suggesting the exudation of a ferment by the contained organism. 



In plants growing in ordinary soil only one infection tube was found entering each nodule, 

 while among those experimentally infected, several tubes from as many root-hairs often entered the 

 same nodule. 



The bacteroids of all species examined were the same in character, consisting of small straight 

 X-shaped or Y-shaped rodlets which stain very readily. At the close of the vegetative period the 

 older nodules are empty sac-like bodies, devoid of bacteroids, but containing a few straight rodlets 

 and some proteid bodies. This observation she thinks supports the theory that the bacteroids have 

 been absorbed by the plant along with any nitrogen contained in them. 



The characters thus far determined are opposed to the view that this organism is one of the 

 higher fungi. 



The mean size of the rodlets is given as o . 99 X 3 . sfJ,. Experiments to determine the life history 

 of the rodlets were undertaken by Miss Dawson. She secured pure cultures and by dilution isolated 

 them in drop cultures for continuous microscopic investigation. To secure pure cultures large nodules 

 were washed with mercuric chloride and alcohol, then with distilled water and cut across with a 

 sharp razor. Streak cultures were then made on slant tubes of gelatin with a sterile platinum needle 

 which had pierced the cut surface. From such streaked cultures unmixed cultures were obtained by 

 a series of plates, and slant tubes were then infected for future use. The multiplication of the rodlets 

 by division (2 to 4 hours) was successfully followed in hanging drops, but in no case was the formation 

 of bacteroids seen. The organism is aerobic. 



An attempt was made to grow the organism on dead roots. Pea seeds were germinated between 

 layers of cotton wool till the radicles were an inch long, then dropped into sterile tubes containing 

 wet plugs of cotton and steamed in a water bath for ten minutes. After cooling the roots were 

 infected with nitragin and kept in the dark. In 10 days good growth was obtained, seemingly 

 of the organism sought, but attempts to get pure cultures failed because of the presence of 

 liquefying bacteria. 



Tests were then made of the ability of nitragin to produce root-nodules. Inoculations were made 

 according to directions, both by rubbing the seeds with the nitragin rubbed up in water and by pour- 

 ing such water over the soil where the seeds were to be planted. Sterile water and utensils were 

 used. No attempt to sterilize seeds before sowing was made and check experiments, she says, 

 justified this, showing that the Leguminosae are not hereditarily infected with the nodule organism. 



Results from inoculations were in all cases positive. In 4 out of 6 experiments the controls 

 remained free from nodules, while in one of the inoculated sets the entire 20 plants developed nodules. 

 The nitragin from Pisum and Vicia was apparently identical in action and the latter when applied 

 to seeds of Lathyrus aphaca produced a considerable increase in positive results in comparison with 

 untreated plants. A similar increase resulted from the use of the nitragin supplied for Onobrychis 

 and Lupinus upon seeds of Vicia hirsuta. The appearance of nodules on controls illustrates, she 

 says, the difficulty of keeping soil or sand free for many weeks from this ubiquitous organism. [In 

 all cases an attempt at least should have been made to sterilize the surface of the seeds. If controls 

 become affected how then are we certain what caused the effects produced in the inoculated plants?] 



Miss Dawson agrees with Zinsser that the bacteroids do not occur in the aerial organs of the 

 plant or elsewhere in it, except in the nodules. Zinsser attempted direct infection of the roots under 

 conditions which could be observed, that is, by injecting the organism into the tissues and by stroking 

 the rootlets with needles dipped in the inoculating material. His results were in both cases negative. 



Since infections in nature occur always through the root-hairs Miss Dawson used external appli- 

 cations only. Seedlings whose roots were infected by drops of water containing nitragin or were 

 dipped entirely into the solution grew vigorously but gave negative results. This suggested that 

 either the organism must pass through the soil, or that infection is impossible after the root has 

 grown beyond a certain stage. Further experiments showed that the second hypothesis is the correct 

 one, since placing the bacteria on the radicle shortly after germination gave very positive results, in 

 one case fully 27 root-hairs side by side showing infection tubes. In all cases infection resulted 

 within 12 days of inoculation. 



The question is undecided as to conditions regulating the entrance of these organisms, since full- 

 grown hairs often show tubes just beginning growth, while infection of the root-hairs is perfectly easy 

 and certain if the organism is placed on roots that have not yet formed hairs. She thinks that the 



