Arthur S. Horne and Eleanor Violet Horne 189 



a black centre up to I in. in diameter; these remained arrested until 

 January 31st, when the largest spot was 1 in. in diameter; the apple 

 then rotted. In over 20 varieties, rotting which could be traced to 

 "spotting" had set in before the varieties were out of season. 



3. The Fungi concerned. 



Owing to the fact that so few of the black bodies found in "spot" 

 areas proved fertile, cultural methods were adopted in the hope that 

 reproductive bodies which would facilitate the work of identification, 

 would be formed in the media employed. 



The apple was first washed in -1 per cent, mercuric chloride solution, 

 and then rinsed thoroughly in sterile water: small cubes from 1-5 mm. 

 long were cut out rapidly with a sterile knife and dropped into apple 

 extract or on the surface of apple agar — in some cases the cubes were 

 immersed for a few seconds in mercuric chloride followed by washing 

 in sterile water before they were transferred to the medium. Control 

 cubes were taken from unspotted parts of the apple and invariably 

 gave negative results. Cubes taken from bitter pit areas also yielded 

 negative results. 



The first fungi were isolated in the autumn of 1915, but owing to 

 circumstances a number of cultures were abandoned and only those 

 considered to be of importance at the time were carried on. Further 

 work was started in the autumn and winter 1917-18 and another series 

 of cultures obtained: these included all the fungi carried on from 1915, 

 with the exception of species of Alternaria and Sclerotium, and in 

 addition a number of others which were not obtained in the first 

 period. 



The total number of isolations in both periods exceeds 400 : of these, 

 140 were in the first period and 260 in the second. The actual number 

 of failures to isolate fungi in the first period exceeded 50 per cent., due 

 in some cases to the use of liquid apple extract, and in others to over- 

 sterilising in mercuric chloride the inoculation cubes. In the second 

 period out of 38 placed in extract, 19 failed — exactly 50 per cent., 

 whereas of those placed on apple agar only seven failed, being cubes 

 obtained from four varieties of apple. 



The fungi present in the original cultures were separated by plating 

 out or by transference to slant tubes; in this way several fungi whose 

 identity could be determined were obtained in pure culture ; and notably 

 Leptosphaeria vagabunda Sacc, Coryneum foliicolum Fuck, Fusarium 



Ann. Biol, vn 13 



