120 Rayner.—Obligate Symbiosis in Calluna vulgaris. 
hoped might favour the growth of the endophyte, as compared with that 
of its more epiphytic competitors; neutral and slightly acid sugar solu¬ 
tions, soil extracts, peat extract, Calluna extract, were all used for this 
purpose. 
In many of these drop cultures the endophyte was obviously active, 
but on transferring to plate cultures, the growth, if present, was always 
masked by that of one or several of the Fungus species mentioned above. 
Attempts were also made, using similar hanging-drop cultures, to 
extract the Fungus from the seed-coat of the resting seed, and from seedlings 
soon after infection. 
In several cases interesting results were obtained, throwing light on the 
formation of bacterial colonies on the roots of seedlings, as described in an 
earlier paper ( 3 ), but the results were negative in so far as the isolation of 
the endophyte was concerned. 
The discovery of mycelium within the ovary, and of its distribution 
throughout the plant, provided a fresh starting-point. Unripe capsules were 
sterilized by passing them through a flame, or by immersion in i per cent, 
mercuric chloride. The seeds and internal tissues were then removed and 
transferred with aseptic precautions to agar plates. 
Colonies of a non-sporing Fungus developed on several of these plates, 
both from seeds and from pieces of ovary tissue, and were subcultured on 
various media. The Fungus isolated in this way was used for the inocula- 
; tion of sterile seedlings. These seedlings, immediately after infection, 
developed a root-system and grew vigorously in a perfectly normal manner 
under aseptic conditions in closed tubes (PI. VI, Fig. 14). 
The agar medium in which they were planted was similar to that 
used previously, without success, for the cultivation of sterile seedlings 
( P . 106). 
As in former cultures, uninfected controls remained rootless, and 
in other ways showed more or less complete inhibition of growth (PI. VI, 
Fig. 15, a and b). 
Demonstration of an obligate relation between the plant and the Fungus 
which besets its roots, and of the identity of this Fungus with that isolated 
from the ovary, is therefore complete. 
Uninfected seedlings, grown under strictly aseptic conditions, or 
subjected to casual infection by Fungi and Bacteria from the air, fail to 
form roots. 
Similar seedlings, infected with a Fungus isolated from the ovary of the 
flower morphologically identical with that in the cells of the root, develop 
a root-system, and continue to grow normally under aseptic conditions, 
the rooting medium being alike in the two cases (cf. PL VI, Figs. 14 
and 15). 
Only as an embryo in the resting seed does the heather plant retain an 
