CEPHALOSPORIUM WILT OF ELMS 17 



Experiment 2. 



Procedure: — One tree of each of the six varieties was inoculated, July 31, in 

 the following manner (Fig. 9): alcohol was brushed over a leaf and allowed to 

 evaporate; the surface of the leaf was then scratched with a sterile scalpel so 

 as to injure the epidermal tissue, and in some places pierce the leaf; after which a 

 suspension of spores and mycelium, prepared from an agar culture in sterile 

 water, was poured over the leaf, with care taken that some of the liquid remained 

 on the leaf. The leaf was then placed between pieces of wet cotton and rolled 

 in a celluloid cylinder which was covered with newspaper, to avoid po.ssible 

 burning of the leaf tissue, and supported by string in such a manner that the leaf 

 might remain as nearly as possible in a natural position. The cotton was kept 

 moist with sterile water for two weeks, then dried out and removed. 



The procedure was repeated on another leaf with the e.xception that the leaf 

 was not injured. Another leaf was injured and treated in the same manner with 

 the exception that a small block of agar on which the fungus was growing was 

 substituted for the spore and mycelium suspension. A fourth leaf was inoculated 

 with the agar block but was left uninjured. Careful checks with sterile water 

 and agar respectively were employed. 



Results: — The inoculated leaves fell from the trees when the cylinders were 

 removed. Tissue plantings of the leaves were made, but with very little success, 

 for it was impossible to free the cultures from contamination which overran the 

 plates. The supposedly inoculated leaves were examined carefully for any dis- 

 colorations which might indicate growth of the Cephalosporium fungus in the 

 leaf cissue. All of the injured leaves showed a darkened area around the injuries, 

 while the uninjured leaves showed no symptoms of parasitic activity. The 

 discolored portions of the leaves were sectioned longitudinally with the freezing 

 microtome. These sections, unstained, were carefully examined under the micro- 

 scope, and it was found that in all cases the fungus had entered the leaf and was 

 growing in the woody tissue of the veins (Fig. 11). Sections similarly prepared 

 from leaves which had not been injured showed no localized discolorations and 

 no fungus was found in the leaf tissue. Cross sections were also prepared from 

 the leaf with similar results. It was found more difficult, however, to discover 

 the fungus mycelium in a cross section of the leaf than in the longitudinal section. 

 The mycelium did not progress as far as the twigs and therefore did not infect 

 the rest of the tree. 



Experiment 3. 



Procedure: — Five seedlings of U. americana L. which had been collected 

 from an area of natural seeding, were inoculated July 15, 1936, as follows: two 

 trees were inoculated by the insertion method used in Experiment 1, with bark 

 from an elm twig culture as the inoculum for one and a small block of agar on 

 which the fungus was growing as the inoculum for the other. Three trees were 

 inoculated by placing the inoculum in contact with the uninjured young green 

 shoot and holding it in place with wet cotton supported by a celluloid cylinder. 

 For one tree, bark from a twig culture was used as the inoculum; for the other 

 two, the inoculum was a block of fungus-laden agar. These inoculations were 

 kept moist for two weeks, then dried out and the cotton and cylinders removed. 



Results: — The inoculated seedlings of American elm all died within five 

 months. The fungus was reisolated from those which had been inoculated under 

 the bark, but could not be isolated from the trees which had been exposed to 



