DISSOLVING ENZYMES. 8 1 



always gives a very strong iodoform reaction. The precipitate remaining in the flask is dissolved 

 in part by acetic acid with evolution of gas; and with ammonium oxalate an abundant precipitate 

 of oxalate of lime is obtained. It seems then that in the fermentation of the calcium metapectate, 

 a portion of the lime is used to form carbonate of lime. The solution still contains some lime. When 

 the solution was acidified with hydrochloric acid only the least trace of substance could be extracted 

 with ether. No considerable acid fermentation, such as occurs in sugar solutions inoculated with 

 the bacteria, takes place in the pectate flasks, since the liquid always remains neutral. 



Thus the part played by the enzym in the parasitism of this organism is as follows: It makes 

 possible a rapid penetration to the deeper plant tissues of the poison secreted by the bacteria; by 

 the resulting death of the protoplasm the cell-sap which is rich in nutrient substances is made accessi- 

 ble to the bacteria, insuring their more rapid multiplication, so that the plant is unable to bring 

 its natural means of protection into play quickly enough. On the other hand, the middle lamella 

 furnishes a suitable source of carbon to the organism, and finally the carbonate of lime liberated in 

 the fermentation of the calcium pectate and perhaps also of the pectates themselves combines with 

 the acids which are always produced by the bacteria in fluids containing sugar, and which would 

 otherwise restrict the development of the organism. 



Further experiments were carried on as to the manner of the poisoning of the protoplasm of 

 the plant cell by the bacteria. If sections of carrots immersed in the expressed juice of rotted potatoes 

 or carrots to which no disinfectant has been added, are observed under the miscroscope, one sees in 

 ten minutes a slow separation of the protoplasm from the cell-wall the former at the same time 

 becoming plainly granular. This process takes place slowly. In 30 minutes the protoplasm is still 

 further contracted and apparently dead; in 40 minutes it is rolled up into a ball, deformed, dark 

 brown, dead, in the middle of the cell. The same phenomena occur with sections of potato, onions, 

 and cattle beets (Futteriiben). 



If the juice from the decayed plants is boiled, its poisonous action seems to be completely lost. 

 When sections of carrot are immersed in it nearly all the cells are living after 36 hours, as is shown 

 by the production of plasmolysis with saltpeter solution. However, one finds that in such sections 

 some of the surface cells are dead and the protoplasm therein shows the same phenomena as that 

 in those sections which have been immersed in the fresh juice. Furthermore, it appears that the 

 poisonous substance diffuses through membranes only with the utmost difficulty, Thus filtrate 

 from juice which has been passed through a bougie is usually at first not at all poisonous, and only 

 slightly poisonous after the filtration of 400 cc., being similar in this respect to boiled juice. And 

 the solution of the middle lamella and the action of the poison go hand in hand as can be easily 

 observed under the microscope. The poisoning never precedes. If one uses sections which vary 

 in thickness, then one sees very plainly that plasmolysis sets in at the same instant that the cells 

 are separated from one another by the solution of the middle lamella. The sound united cells and 

 the diseased cells which are becoming separated are sharply differentiated. The poisonous action 

 never extends beyond the decayed area, as is so often observed in fungous diseases. 



There is apparently no labile toxine present. No oxalic acid is present, at least not in the neutral 

 to alkaline potato and carrot juice, and it was not detected in the acid fermentation produced by the 

 bacteria in sugar, glycerin, and mannit solutions, a test which excludes free oxalic acid and various 

 of its compounds. 



The poison also is precipitated from the juice of decayed potatoes, carrots, and onions, together 

 with the slime and the pectine dissolving enzym, by the use of alcohol. If one dissolves the pre- 

 cipitate in an equal amount of sterile water, one obtains a neutral solution which in the activity 

 and manner of its poisoning differs in no way from the original juice. On boiling a granular pre- 

 cipitate is obtained and the boiled solution still retains after many days a slight poisonous action 

 on the protoplasm. 



The most recent extensive contribution to the subject is by L- R. Jones (1910). A 

 summary of this paper follows: 



A detailed study was first made of the enzym produced by Bacillus carotovorus, both living 

 cultures and the enzym isolated from them being used in the experiments Later, comparative 

 studies were made with enzyms secreted by other soft-rot organisms, other classes of bacteria, fungi 

 and germinating seeds. 



The same strain of the carrot-rot organism was used throughout. It is one isolated from decay- 

 ing carrot tissues in 1899 and since* grown on artificial media (practically all of the time iff beef 

 broth), at room temperature 16 to 22 C. Jones believes that there has been a considerable decrease 

 in pathogenicity accompanied by a corresponding decline in the amount of enzym production. 



*The studies here reported upon were carried on during the years 1901-1904. 



