S. O. Paine and H. Stansfield 33 



formation of the gummy substance had only just begun, hence the con- 

 tents were so little refractory that a tolerable section of this cell was 

 obtained. 



In spots on young leaves the yellow material is accompanied bv a 

 bright vermilion pigment. This is insoluble in water and in alcohol, even 

 after 24 hours steeping of the tissue in boiling alcohol; it is slightlv 

 soluble in ether; it is insoluble in hydrochloric acid but dissolves slowlv 

 in cold concentrated sulphuric acid; it is readily soluble in ammonia, 

 from which it is precipitated as a red powder by hydrochloric acid. These 

 reactions^ with the exception of sparing solubility in alcohol, are those 

 of phlorotannin red, but solutions of the red precipitate in caustic soda 

 do not show the fluorescence characteristic of this substance (Beilstein, 

 3rd ed., vol. 2 B, p. 1919). A substance of this character would probably 

 be sufficiently toxic to account for the death of the bacteria ; their sub- 

 sequent disappearance is, however, a matter of pure conjecture. 



After extraction of the red pigment by ammonia the yellow substance 

 remains apparently unaltered. It stains black with ferrous sulphate and 

 may perhaps be of the nature of a resino-tannin. 



Isolation of the Parasite. 



The surface of diseased leaves was sterilised by immersion in hydrogen 

 peroxide for one hour, after which the leaves were allowed to dry and 

 the epidermis was removed from a young blister-spot by means of a 

 flamed scalpel. A little of the exposed soft brown tissue was crushed in 

 sterile water and dilution plates in bouillon gelatine and bouillon agar 

 were poured. Several attempts at isolation in this way failed to give any 

 result; this may have been the effect of the treatment with hydrogen 

 peroxide, but it is considered more probable that the organisms in the 

 spots selected had been destroyed in the manner suggested above. 

 Eventually, however, success was attained; numerous colonies appeared 

 on the plates after three days incubation at 20° C. and were apparently 

 all of one type. Repeated platings yielded pure cultures without much 

 difficulty. 



Infection Experiments. 



Through the courtesy of the Director of Kew Gardens two small 

 plants of Protea cynaroides were available for inoculation. These were 

 sturdy young seedlings six and eight inches in height, and had several 

 young leaves which were quite free from the disease. In all, fourteen 

 experiments were made upon seven separate occasions, using seven 



Arm. Biol, vi 3 



