292 SYMBIOTIC NITROGEN FIXATION 



regarded as normal products of the roots, but were certainly infec- 

 tions from the soil. In a series of researches, undertaken with the 

 assistance of Wilfarth these results were thoroughly confirmed. 

 They showed that in sterilized soil the legume behaves the same as 

 the non-legume and dies of nitrogen hunger if not supplied with 

 suitable forms of nitrogen. When the sterilized soil was inoculated 

 with fresh soil on which legumes had made a normal growth they 

 then made a vigorous growth in sterilized soil. Under similar 

 conditions non-legumes did not recover. The recovery of the 

 starving legume was found to coincide with the formation of root 

 tubercles. 



Wigand (1887) found that the tubercles contained true bacteria 

 and the following year these were obtained in pure cultures by 

 Beijerinck. He found further that there were bacteria associated 

 with all tubercles, and although the bacteria differed somewhat in 

 the tubercles of different species of plants, still there were certain 

 constant characteristics to be seen in them all. He, therefore, 

 regarded the tubercles as the result of the action of bacteria and gave 

 to the organism producing the tubercles the name of Bacillus 

 radicicola. Beijerinck regarded the so-called bacteroids of Woronin 

 as degenerate forms of the bacteria-involution forms, which appeared 

 only after the bacteria had lost their vigor. In a later investigation , 

 after isolating the bacteria and keeping them in pure cultures for 

 many months, he was able to produce the tubercles at will by inocu- 

 lating soils in which his plants were grown with the pure cultures of 

 the organisms. 



Prazmowski (1890) published researches which confirmed all of 

 HellriegePs results, showing conclusively that if sufficient precau- 

 tions were taken to sterilize the soil in which leguminous plants were 

 grown no tubercles were ever produced. He further showed that 

 the tubercles grow on plants developing both in the light and in the 

 dark, but are larger on plants growing in the light; that they only 

 appear on healthy plants; that there are very few on plants growing 

 in well-washed sand; that if plants growing in sterilized soil be 

 watered with brook or river water, tubercles occasionally develop 

 but never in abundance; and that the infection of the roots occurs 

 early in the germination of the plant and cannot take place in the 

 older roots. 



Two years later Schlosing and Laurent demonstrated the fixation 

 of atmospheric nitrogen through the joint activities of leguminous 

 plants and Pseudomonas radicicola by the actual diminution of the 

 amount of elementary nitrogen in the inclosed atmosphere sur- 

 rounding the plants. 



Species. Whether the different varieties of legume bacteria are 

 distinct species is a perplexing question which today cannot be 

 definitely answered. It is known that certain legumes are readily 



