112 BACTERIA IN RELATION TO PLANT DISEASES. 



infection often takes place in the root-hair just as it is emerging from the root. In one case she 

 observed several very small root-hairs, scarcely larger than the cell from which they arose, pene- 

 trated by infection tubes which had already reached and entered the outer layer of the root-cells. 



Further experiments were made to determine the possible inhibiting effect of carbon dioxide 

 collecting about the roots when seedlings were grown in tubes : These were negative. Caustic potash 

 was introduced into some of the tubes to absorb the carbon dioxide. Plants flourished equally well 

 with potash and without potash, the only positive result occurring on a seedling in a tube without 

 potash. To test the effect produced by changing conditions under which plants were growing, seed- 

 lings grown one week in sterilized sand with nutrient salts were removed, infected with nitragin and 

 fixed in tubes. Others were germinated in the same manner, but returned to fresh sand after inocu- 

 lation. Others were germinated in tubes, and when the roots were two inches long were inoculated 

 and again fixed in tubes. Three weeks after inoculation the most positive results were where condi- 

 tions had remained as far as possible unchanged. 



Examination of the nitragin and of fresh subcultures therefrom showed it to " consist of immense 

 numbers of very minute bodies, scarcely longer than broad, all non-motile and similar in size and 

 shape. No trace is found of the variety of shapes exhibited by the bacteroids." 



In 1900, Maria Dawson published "Further Investigations on the Nature and Functions of the 

 Nodules of Leguminous Plants," from which I abstract as follows: 



Phaseolus shows no nodules for at least 3 weeks after germination, and these are confined almost 

 entirely to small lateral roots. Large nodules contain a considerable quantity of starch. Situated 

 from one to three cells below the surface of the nodule is a layer containing large crystals of calcium 

 oxalate. A similar layer was found in Desmodium. Bacterial filaments strictly comparable to those 

 in Pisum, were found in small nodules, never in those larger than a pin's head, and only once was 

 an infection tube seen within a root-hair. In this genus root-hairs are few in number. 



These results suggest that in Phaseolus the germs in the absence of root-hairs can enter the host 

 directly across the piliferous layer, and that within the root they can continue their growth for a 

 while with or without the formation of a filamentous structure. 



Acacia agrees with Phaseolus in having filaments in very young nodules but not in the older. 

 In this as well as in Phaseolus it is possible that we have an intermediate stage in the adaptation of 

 the parasite. 



A detailed study was made of the nodules of Desmodium gyrans and pure cultures were made of 

 the organism concerned in their formation, since this was of an unusally large size (1.3X3 to y/t). 

 Similar large forms occur in Acacia, Flemingia, Carmichaelia, Coronilla, and Psoralea. In section, 

 the nodules of Desmodium resemble those of Lupinus and Phaseolus, but a new feature was noted. 

 This was the presence in material hardened in absolute alcohol of bright, apple-green bodies, one, 

 as a rule, in each cell. The nature of these bodies has not as yet been determined. They are promptly 

 and completely soluble in 5 per cent potash. These bodies occur also in the nodule-cells of Robinia 

 pseudacacia and in both cases digestion in gastric juice caused the green color to become more con- 

 spicuous. Gastric juice was also found useful for rendering the bacterial filaments conspicuous in 

 sections of Pisum, Vicia, and Robinia. 



Upon Cassia roots she did not observe the formation of nodules. The older roots of this genus 

 are jet black, contrasting strongly with the pale, greenish-yellow root tips. 



The author discusses further the biology of the bacteroids. The time required for the growth 

 of a lateral branch in hanging drop cultures averaged about 1.5 to 2 hours. She isolated the nodule 

 organism by a series of separations on tube and plate cultures, and from pure cultures on gelatin 

 microscopic preparations were made. A triple series of these cultures was kept under observation 

 and referred to in her descriptions as A, B, and C. They were: 



(A) Organisms from sub-cultures of commercial nitragin for Pisum sativum. 



(B) Organisms cultivated directly from the nodules of Pisum sativum. 



(C) Organisms cultivated from the nodules of Desmodium gyrans. 



Gelatin plates made from nitragin yielded what appeared to be pure cultures, the colonies looking 

 alike. Further studies led the author to consider the "Nitragin" examined by her as a bacterio- 

 logically pure culture. She says : 



" The general characters of the three organisms are alike, though small differences are noticeable 

 in aggregate cultures. They all grow readily on gelatin, or agar, containing a decoction of pea stems 

 and leaves, asparagin, and a small percentage of sugar, and giving a very faintly acid reaction." 



On broth-agar no growth occurred at 20, or at 25 to 30 C. On broth-gelatin at 20 C. growth 

 was extremely slow. Beyerinck also states that his Bacillus radicicola grows very slowly in meat- 

 juice peptone gelatin. No change occurred in milk kept for 3 weeks at 15 C. either in consistency 

 or litmus reaction. 



