W. F. Bewlby 123 



when the supcrmitant ulcohol was syphoned oft' and the precipitate dried. 

 After 24 hours the precipitates were dissolved in 3000 c.c. sterile water, 

 and placed in flasks, 300 c.c. per flask. 



Series 5. Five flasks with 300 c.c. each of solution of precipitate from 



culture filtrate. 

 „ 6. Five flasks with 300 c.c. each of solution as in series 5 



heated for 5 minutes at 100° C. 

 ,, 7. Five flasks with 300 c.c. each of solution of precipitate from 



the sterile medium. 



Tomato seedhngs severed from their roots were placed in these flasks 

 as above described and a definite wilt was obtained in series 5 in 72 

 minutes. Series 6 yielded a shght wilt in 3 hours, but no Avilt appeared 

 in series 7 during 24 hours. 



C. An attempt was made to isolate the exo- and endo-enzymes of 

 V. albo-atrmn by the technique devised by Brown (3), but difficulties arose 

 in the process owing to the comparatively small size of the Verticillium 

 spores. A much greater number of spores than was used by Brown 

 had to be treated and the difficulty of obtaining a clean separation by 

 centrifuging vitiated any attempt at quantitative determination. 



The method adopted was as follows : 



An abundant supply of spores was obtained by cultivating the fungus 

 on Dox's agar with 1 per cent, saccharose, at 25° C. for 14 days, six 

 petri-dishes of 6 inches diameter being used in each determination. The » 

 plates were covered by a thin layer of sterile water and the spores re- 

 moved by gently scraping the fungal growth with a knife. Any rubbing 

 with the finger as recommended by Brown for Botrytis had to be avoided, 

 as such treatment rubbed the spores into the medium. The mixture of 

 water, mycelium and spores was filtered through fine muslin and the 

 filtrate consisting of water, spores and fine pieces of mycelium was 

 centrifuged. It was almost impossible to separate much of the finer 

 pieces of mycelium from the spores by this process, because of the light- 

 ness of the latter, but a good number of the larger pieces were removed. 

 The spores were next germinated in strong turnip juice; 0-2 c.c. centri- 

 fuged spores being added to 21 c.c. turnip juice. Plates seven inches in 

 diameter were each sown with 3 c.c. of the spore suspension, which was 

 carefully spread over the surface. After 72 hours the mat of germinated 

 spores was removed, and the turnip juice in which it had developed 

 collected and tested for the presence of an exo-enzyme. The mat of 

 mycelium was carefully washed to remove any spores, etc., and dried 



