466 
Journal o f Agricultural Research 
Vol. XXIX, No. 9 
The intercellular strands which produce 
haustoria have abundant contents. 
No hyphae filled with protoplasm have 
been found connecting the mycelium 
in the root with that outside. 
GROWTH OF THE FUNGUS IN CUL¬ 
TURE MEDIA 
As soon as the fungus was distin¬ 
guished by its morphological character 
in the plant, efforts were made to isolate 
it in pure culture by the usual labora¬ 
tory methods. It was found that in 
the smaller rootlets of alfalfa and clover 
the fungus was followed so closely by 
soil-inhabiting fungi and by bacteria 
that the apparent beginnings of growth 
of the mycorrhizal fungus were soon 
submerged and lost. In such rootlets 
surface sterilization sufficient to de¬ 
stroy the adherent soil organisms al¬ 
ways seemed to penetrate the entire 
cortex. Roots of plants having a thick¬ 
er cortex, as sweet pea and onion, 
were then chosen for isolation, and 
plants were grown in a soil held at a 
low temperature presumabh^ unfavor¬ 
able for the troublesome Fusarium 
species often found in the outer cells. 
Even in plants thus grown, fungi often 
grew out from root cortex that ap¬ 
peared uninjured, and bacteria always 
appeared whenever any fungus growth 
occurred. 
In these isolation experiments the 
only culture media which appeared 
to give any growth at all of the 
mycorrhizal fungus were agar alone 
or prune agar. When razor sections 
of roots containing mycelium with 
abundant protoplasmic contents were 
thoroughly washed in sterile water, 
stripped of the outer layers of cortical 
cells by the careful use of dissecting 
needles under the low power of the 
microscope, and placed in poured 
plates of clear agar before they had 
solidified, fungus strands which de¬ 
veloped could usually be traced to 
their origin by observation through 
the bottom of the plate with the 
microscope. The only fungus strands 
in such plates which were traced to 
the mycelium with haustoria in the 
host cells so clearly that their origin 
seemed indubitable were of a distinc¬ 
tive character, differentiating them 
clearly from most fungi in such cul¬ 
tures. These fungus strands were of 
large diameter, like those in the host 
tissue, narrowing to a blunt point at 
the end, and were thrust into the agar 
almost as straight as stiff bristles, 
rarely showing a flexuous course or 
having branches. On the third or 
fourth day after the plate was made 
the growth sometimes attained the 
length of 3 to 4 mm. After this the 
granular protoplasmic contents began 
to recede, leaving a series of thin mem¬ 
branous septa. The recession of the 
protoplasm was usually contempora¬ 
neous with the appearance of great 
numbers of bacteria in the root tissue 
and its rapid destruction. The agar 
seemed unable to support growth of 
the fungus when the host tissue failed. 
When the longer strands were severed 
from the host tissue with a sterile 
needle the protoplasmic contents oozed 
from the cut ends, and growth ceased. 
After observation of growth from thin 
sections seemed to establish the char¬ 
acter of the mycorrhizal fungus de¬ 
veloping in these culture media, many 
segments of invaded roots were given 
surface sterilization with bichloride of 
mercury and placed in agar, some 
liquid media, and sterile soil; but in 
every occasional instance where fungus 
growth was detected a bacterial growth 
also occurred, destroying the plant 
tissue and submerging the fungus. 
Thus no method of separating the 
mycorrhizal fungus from its bacterial 
associates was found, nor did any of 
the culture media tried appear capable 
of supporting the fungus growth apart 
from the host tissue. The fact that 
strands of the fungus can be found 
loosely attached to the roots has been 
regarded as indicating its ability to 
develop outside of plant tissue, and 
presumably on artificial substrata; but 
it is not impossible that this appear¬ 
ance is deceptive, and that the fungus 
is in fact an obligate parasite capable 
of growing only in or closely attached 
to root cortex. 
SPECIES OF LEGUMINOSAE IN WHICH 
THE MYCORRHIZAL FUNGUS HAS BEEN 
FOUND 
During the summer of 1923 the 
roots of all easily accessible legumes 
were examined. The fungus was found 
in the following plants: 
Falcata comosa (L.) Kuntze ( Am - 
phicarpa monoica Ell.). 
Astragalus parviflorus (Pursh) 
MacM. 
Lotus americanus (Nutt.) Bisch. 
(Hosackia americana Piper). 
Lathyrus odoratus L. 
Lathyrus tingitanus L. 
Medicago sativa L. 
Melilotus alba Desr. 
Melilotus officinalis (L.) Lam. 
Petalostemum purpureum (Vent.) 
Rydb. 
Phaseolus vulgaris L. 
Soja max (L.) Piper. 
Trifolium pratense L. 
Trifolium hybridum L. 
Trifolium repens L. 
Vida sparsifolia Nutt. 
