52 
Journal of Agricultural Research 
Vol. XXVI, No. * 
in glycerin. This served to soften the tissue to some extent, and, in gen¬ 
eral, to render it less brittle, by dissolving part of the gum. To overcome 
the tendency of many sections to break or crumble before the knife, the 
exposed surface of the material to be sectioned was covered just before 
cutting with a thin layer of collodion, which served to hold the loose 
parts together. This method is recommended by Lee (4 p . 95). By 
these methods a sufficient number of fairly good sections was obtained, 
ranging in thickness from 15 to 20 \x. 
In order to obtain thinner sections for closer and more detailed study, 
material was embedded in paraffin. Of the several killing and fixing 
reagents used chromo-acetic acid 5 gave the best results. By this 
method good sections 7 to 10 n thick were obtained. The phloem, when 
free from cork, gave the best results in sectioning. Wood tissue was 
sectioned with little difficulty by means of the wood-microtome. The 
following stains were used: Methylene blue, Ziehl’s carbol fuchsin, eosin 
in 95 per cent alcohol, and Delafield’s haematoxylin counterstained with 
the alcoholic eosin. The stain last named was recommended by Durand 
(j) as a differential stain for intercellular mycelium. 
ETIOLOGY 
ISOLATION AND INOCULATION EXPERIMENTS 
In order to ascertain what the causal agent of this disease might be, 
a fairly extensive series of culture experiments was carried out. Fresh 
galls were obtained on several occasions between January 24 and April 
7, 1916, from San Jose and Hayward, Calif. The rough surfaces of the 
galls were cut off; they were then washed in running water and placed 
in 95 per cent alcohol for five minutes, and finally soaked in an aqueous 
solution of corrosive sublimate, 1:1000, for from 20 to 30 minutes. The 
corrosive sublimate was then rinsed off with sterile water, and bits of 
tissue were removed from the interior of the galls and planted in various 
media, care being taken to observe all aseptic precautions. Tissue 
bordering the gum pockets in the bast where the latter is free from cork 
was generally used. It was soon found absolutely necessary to avoid, as 
far as possible, the carrying of gum along with the sections used in 
planting, on account of the inhibitive effect which the gum seemed to- 
have on the growth of microorganisms. The plantings were made in 
standard nutrient agar, standard nutrient broth, and sterile water. 
Bacterial dilution plates were also made in standard nutrient agar. 
The bacterial dilution plates and the plantings in standard nutrient 
broth and agar failed to give any positive results. Occasionally fungous 
mycelium developed on some of the sections in standard nutrient agar, 
but was either swamped by gum oozing out from them or failed to grow 
when transferred to other media. 
What is believed to be the causal agent of this disease was obtained 
by planting a number of sections in tubes of sterile water. These were 
left overnight. The following morning, when the water was found to 
have changed to a rich brown color, it was thought desirable to transfer 
the sections to a medium free from any pathological products. They 
were accordingly placed in flasks containing some 30 cc. of standard 
nutrient broth. In one of these flasks a thrifty fungous colony developed, 
* Chromic add. 0.05 per cent; glacial acetic add (99 per cent) o.x per cent. 
