104 Rayner.—Obligate Symbiosis in Calluna vulgaris . 
producing Fungi from the roots of various ericaceous species (of which 
Calluna vulgaris appears to have been one) and referred them to the 
genus Phoma . Complete proof of the identity of these Fungi with the 
species endophytic in roots of the respective plant-species was unfortunately 
not possible, since sterile seedlings were not obtained. Incidentally, this 
author recognized the possibility of seed-coat infection in Ericaceae, but did 
not investigate it further. 
It was claimed that five of these species of Phoma in pure culture fixed 
nitrogen from the air, although in very different degrees ; in no case was 
combined nitrogen found necessary for their development. Appreciable 
nitrogen fixation from the air has been claimed also by this author for 
Aspergillus niger and for Penicillium glaucum , and this result was corrobo¬ 
rated by Froelich ( 27 ) in 1907 for the same species. On the other hand, 
a negative result is reported for the same and additional species by Wino- 
gradski ( 28 ), Koch ( 29 ), Czapek ( 30 ), &c., and more recently by Goddard 
for a large number of soil Fungi ( 31 ). 
The fixation of free nitrogen from the air has been claimed also for the 
endotrophic Fungus of Podocarpus ( 32 ), and is suggested, but not established, 
for the Fungus which infests the seed and vegetative parts of Lolium 
temulentum ( 33 ). There is no evidence for fixation of atmospheric nitrogen 
by the Fungi concerned in the root mycorrhiza of Orchids, and attempts 
to cultivate them on nitrogen-free substrata have not been successful ( 19 ). 
The whole question of the possibility of fixation of free nitrogen by 
Fungi growing on nitrogen-free media, as determined by direct estimation of 
the combined nitrogen present at the end of the experiment, is still in an 
unsatisfactory condition, and, in spite of the positive results claimed 
by a number of observers, the balance of evidence appears to be on the 
negative rather than on the positive side. 
The sources of error are obvious. The possibility of working with 
impure cultures, the difficulty of obtaining a substratum free from traces of 
nitrogen, and the small quantities of nitrogen fixation reported by most 
observers who claim a positive result, together with the comparatively 
large margin of experimental error, demand greater uniformity of results 
before ability to fix free nitrogen can be claimed as a general property 
of Fungi in the same or even in less degree than has been finally established 
and placed on a sure scientific basis for a number of Bacteria. 
The Present Investigation. 
The purpose of the investigation described in the following pages is 
as follows : 
1. To confirm and extend the conclusions already reached with respect 
to the inability of Calluna seedlings to form roots unless infected at an early 
stage by a specific Fungus identical with that present in the cells of the root, 
