Laffiorty and Pkthybridgk — On a Phijtophthora Parasitic on Apples. 31 



Sub-cultures of the nine pure stocks raised were made in parallel series in 

 various media, and close study of them showed that they were identical in all 

 respects. The production of sexual organs, however, on the media employed was 

 extremely limited. Cases were found where the antheridium was undoubtedly 

 paragynous, and certain others occurred in the same cultures in which this organ 

 appeared to be ampliigynous, but absolute certainty on the matter could not be 

 arrived at. 



In order to clear up this important point it was necessary to make cultures on 

 a medium in which the sexual organs were produced in greater abundance. For 

 this purpose cylinders of tissue, cut with a sterile cork-borer from a healthy raw 

 apple under aseptic conditions, were placed in sterile plugged test tubes. One-half 

 of the number of cylinders was inoculated from a sub-culture of one of the nine 

 stocks alluded to above ; the other cylinders were left uninoculated to serve as 

 controls. 



After the lapse of about a month the cylinders were examined. The controls 

 had remained sound, and no trace of fungus or other growth could be discovered on 

 or in them when examined microscopically. The inoculated cylinders had rotted 

 and become brown, while traces of aerial mycelium were visible on them. 

 Microscopical investigation revealed the presence of no sporangia, but sexual 

 organs were present in abundance. The antheridia in the vast majority of cases 

 were paragynous ; but in two instances, at least, antheridia undoubtedly of tlie 

 amphigynous type were present in the same culture along with them. A photograph 

 of one of these is reproduced in fig. 6, PL II. 



It was thus fairly clear that only a single fungus was respoirsible for the apple 

 rot, and that this fungus produced both paragynous and amphigynous antheridia. 



(b) Isolation from a sinc/le spora7igium. — The fungus does not produce sporangia 

 at all abundantly, but in an old growth on oat-extract agar, derived from a piece 

 of tissue removed aseptically from one of the original decaying apples, a few were 

 found. The culture was in a Petri dish, and at the time was not free from bacterial 

 contamination. In the tissue from which the growth had emanated sexual organs 

 of both types were present. 



Under microscopical control six isolated sporangia were successfully removed, 

 one at a time, by means of sterile capillary tubes, and each was washed in about 

 twenty changes of sterile water to remove bacterial contamination as far as 

 possible. 



On a healthy apple, and with aseptic precautions, six small, well-isolated cut 

 surfaces were prepared, and on each a single sporangium was placed. A similarly 

 prepared healtliy apple, on which no sporangia were placed, was used as a control, 

 and both fruits were kept in a moist atmosphere under the same bell-jar. Within 

 sixteen days a rot of the first apple had started at three out of the six places where 

 sporangia had been introduced. No rot occurred at the other three places, 

 the sporangia in these cases being apparently not viable. The uninoculated apple 

 remained perfectly sound. 



Before the rot had extended from the three centres sufiiciently to become 

 coalescent, several small portions of the affected tissues were removed from one of 

 the centres to set media in Petri dishes, aseptic precautions being observed as 

 before. After a few days mycelium grew into the media from the pieces of tissue, 

 and in each case the growth was found to be free from bacterial contamination. 

 From these growths — each the product of a single sporangium — sub-cultures in test 

 tubes were prepared. 



At a later stage the pieces of tissue on the Petri dishes were examined, and 



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