32 Scientific Proceedings^ Uoijal Dnhlin Socictij. 



sexual organs with both para- and aniphigynouE antheridia were found in them, the 

 latter being decidedly in the minority. One of each type, isolated from this material, 

 is illustrated in figs. 3 and 4, Plate II. 



(c) Isolation from a single oospore. — '^msxW portions of the affected tissues which 

 had been removed from one of the original diseased apples to solidified media in 

 Petri dishes were macerated in water, and sexual organs having antheridia of the 

 amphigynous type, containing apparently ripe and viable oospores, were sought 

 out under the microscope. Several were found and were removed by capillary 

 tubes, one at a time, to films of oat-extract agar on cover-glasses, which were then 

 inverted over excavated microscope slides in the usiial way. Before removal, great 

 care was taken to ensure that no living hyphae remained attached either mechani- 

 cally or otherwise to the oogonia and antheridia. Each slide thus prepared 

 contained a single oospore ; but the films, although quite free from any other 

 funcfus, were not altogether free fi-oni bacteria. 



The slides containing the oospores were kept under close microscopical observa- 

 tion. In the majority of instances the oospores either remained in a resting 

 condition or o-erminated aird produced short germ tubes which soon died. In one 

 case, however, greater success was achieved. The inner portion of the thick wall 

 of the oospore was observed to dissolve in a manner similar to that which occurs 

 durino- the germination of the oospores of Fhytophthora erytliroseptica. A germ- 

 tube then arose which passed through the wall of the oogonium, grew for a short 

 distance, and then developed a sporangium at its distal end. This sporangiunr 

 produced a number of zoospores which, after actively swimming about for some 

 time, came to rest, developed thin walls, and proceeded to form germ-tubes. 



At this stage the cover-slip containing the film culture was carefully inserted 

 into a slit made in the flesh of a healthy apple, which was then kept for thirteen 

 days in a moist atmosphere at laboratory temperature. At the end of this time a 

 rot had developed in the apple, starting from the film side of the cover-glass, on 

 which the germinating zoospores were originally present. 



After seventeen days portions of the rotted tissues were removed (aseptic 

 precautions being still maintained) and were placed on solidified oat-extract agar 

 in Petri dishes, and from the growths which arose in the agar medium sub-cultures 

 were prepared. No sexual organs or sporangia had developed in the growths at 

 the time the sub-cultures were made ; but microscopical examination of the 

 portions of tissue themselves revealed the presence in them of sexual organs 

 with both amphi- (Plate II, fig. 5) and paragynous antheridia. 



Prom the foregoing it will be seen that pure cultures were raised by three 

 different methods, and that in all cases a single fungus was derived which produced 

 sexual organs, having both amphi- and paragynous antheridia. It might be 

 maintained that the use of raw apple tissue in the course of the work left the 

 question as to the absolute certainty of the results in some doubt ; and, in order 

 to obviate this, sterilised carrot tissue was subsequently employed. The results 

 obtained were the same as before. 



As regards pathogenicity, the fungus in pure culture has repeatedly been 

 inoculated into healthy apples, and always with the same result, viz., the produc- 

 tion of a characteristic form of rot, similar to that exhibited by the original 

 naturally infected fruits. In each case, where the attempt was made, the fungus 

 was re-isolated in pure culture from the artificially infected apples. Non-inocu- 

 lated controls, kept under similar conditions, remained unaffected in every case. 



In the diseased tissues of both naturally and artificially affected apples sexual 

 organs occur, but not abundantly. "When small portions of such tissue, however, 



