520 Journal of Agricultural Research voi.xx.No. 7 



fairlv large pieces under as sterile conditions as possible, rinsing these 

 through 5 to 10 sterile water blanks, transferring to a sterile Petri dish, 

 where they were further cut up into small pieces and transferred to 10 cc. 

 of potato agar in a Petri dish acidified with two to three drops of 25 

 per cent lactic acid. Out of hundreds of pieces plated out in this manner 

 apparently pure cultures of the causal organism were rapidly secured in 

 practically all cases. Mercuric chlorid treatment apparently resulted 

 in part of the chlorid entering the bundles, from which it was not readily 

 washed out, and consequently did not prove useful for plating out in 

 this case. 



Single spore isolations were made from the Maryland Fusarium, and 

 these have been used in some but not in all infection experiments, cul- 

 tural studies, and spore measurements. 



Infection experiments during the summer of 1917 consisted chiefly 



in inoculating large plants in the field with pure cultures of the Fusarium 



through wounds in the stalk. No infection was secured except in one 



instance which was questionable. In the fall of 1917 sterilized soil was 



inoculated with mycelium from pure cultures of the wilt Fusarium, and 



very young White Burley tobacco plants were transplanted into it. 



After about five weeks several of the plants wilted and died. Infection 



thereafter was intermittently secured on White Burley through the 



medium of the soil. The inoculum was usually grown on a mixture of 



100 gm. of sand, 10 gm. of corn meal, and about 1 gm. of glucose to 50 cc. 



of water in i-pint milk bottles or mason jars. This culture medium 



was cooked for one hour in the autoclave, then stirred up so as to render 



the medium "spongy," and again sterilized. After being cooled, the 



medium was inoculated with the Fusarium and incubated at 25 to 30 



C. for four or five weeks, after which the inoculum was allowed to dry 



sufficiently to permit pulverizing, when it was thoroughly mixed with the 



soil. Good infection was also secured from mycelium and spores directly 



from potato agar tubes and also from a suspension of conidia alone. 



The latter method was usually not so successful as the former. Failure 



to secure as high precentage of infection at one time as at another led to 



a preliminary study of environmental and other conditions favoring the 



disease, and these will be reported upon briefly in this paper. It should 



be stated here, however, that as soon as the plants were intentionally 



wounded more uniform results in infection were secured. Ordinarily 



this consisted simply in pulling or pinching off one or two of the lower 



leaves and setting the plant deep enough to bring the resulting wound 



below the surface of the soil. Although it can not be said with certainty 



that infection would never occur in a plant perfectly free from wounds of 



any sort on the root or stem, it is quite certain that infection is 



greatly enhanced by wounding. The first signs of infection on leaves 



have been secured in as short a time as two days after exposing wounded 



stems to heavily infested soil. 



