30 Scientific Proceedings, Roijal Dublin Society. 



In microscopical preparations it was found tliat rather coarse, non-septate niyceliiun 

 permeated the tissues, being present both in and between the cells. White aerial 

 mycelium was present in the cavities of the core, but it bore neither sporangia nor 

 sexual organs. The latter, however, were found embedded in the soft tissues 

 above and below these cavities ; and antheridia of both the amphi- and paragynous 

 types were present, the latter in much greater number than the former. 



Eighteen small pieces of affected tissue were removed aseptically, and each was 

 placed in the centre of a Petri dish, in which a sterile nutrient agar medium had 

 previously been allowed to set. In all cases mycelium grew from the tissue into 

 the medium; but, in the early stages at least, with one' exception, the mycelium 

 remained barren. This exception was Quaker Oat agar. On this medium the 

 fungus developed freely, and sexual organs with both types of antheridia were 

 produced on the mycelium in close proximity to the original piece of tissue, whilst 

 they were also present in the tissue itself .x Photographs of each type are reproduced 

 on PI. II, figs. 1 and 2, and the two types are figured on PL I, figs. 1 and 2. 



Sub-cultures were prepared from the growth on Quaker Oat agar, and from 

 them inoculations were made into healthy apples, the necessary controls being also 

 provided. A rot identical with that found in the original material was produced in 

 every case where the inoculum was introduced, whilst the controls remained sound. 



It appeared, therefore, that the rot was caused either by a single species of 

 fungus having sexual organs of two types or by one or both of two associated 

 species, each producing its own particular type of antheridium. In any case, the 

 matter was of more than usual interest, and demanded further study. 



II. — Isolation of the Fungus and Proof of Pathogenicity. 



An attempt was first made to discover by direct microscopical observation 

 whether the two types of sexual organs were borire on one and the same portion of 

 mycelium, but this attempt gave only uncertain results. It was therefore decided 

 to raise pure cultures. 



(a) Isolation from portions of single hyphae. — When I'adiating growth had 

 proceeded for some time on the Quaker Oat agar Petri dish cultures, mentioned 

 above, a narrow circular band of the medium was removed just beyond the limit 

 of growth. When the tips of the growing hyphae reached the inner margin of this 

 annular space, they proceeded to cross it; but it was found possible, by careful 

 manipulation with sterile needles under the low power of the microscope, to 

 prevent the crossing of this space by all hyphae in a given region save by a single 

 selected one. When the latter had crossed and had become well established in a 

 completely isolated condition in the medium on the other side of the annular space, 

 it was removed, together with a small portion of the medium in which it was 

 embedded, and placed on a suitable medium slanted in a test tube. In this way 

 nine pure cultures were raised, each derived from an isolated portion of a single 

 hypha. 



As a means of isolating both members of a pair of fungi intimately associated 

 with each other during growth, the above described technique is undoubtedly open 

 to criticism. If, for example, the rate of growth of one of the pair were greater 

 than that of the other, the former would invariably reach the annular space first, 

 and the cultures derived, as described above, would almost certainly represent only 

 one of the two organisms. Nevertheless, to obtain even one of them in pure 

 culture was a step in advance, and, as the sequel will show, the method adopted 

 served its purpose. 



