ROOT NODULES AND ISOLATION OF ENDOPHYTE
ON ARTEMISIA LUDOVICIAN A
RAYMOND
B. FARNSWORTH AND MAX W. HAMMOND
Brigham Young University
Introduction
Early investigations of the presence of root nodules on nonleguminous Angiospermae were contemporary with studies on
the leguminous species. In fact, studies were often conducted
by the same investigators (Allen and Allen, 1965). Because of the
greater economic value of the legumes, their uniformity as a
single plant family, and the isolation of rhizobia by Beijerinck in
1888 (Waksman, 1927), the study of nonleguminous nodules was
practically discontinued for a period of approximately sixty years.
It was largely due to the investigations of Hawker and Fraymouth
(1951) that renewed interest began to rise in the nodulation of the
nonleguminous plants. Some indication as to the extent of the
Interest in these more recent studies is shown in the reviews of
Allen and Allen, 1958; Bond, 1958, 1963; Schwartz, 1959; Norris,
1962; McKee, 1962; and Schaede, 1962 (Allen and Allen, 1965).
Nodulation has been reported in more than 100 species of 13
genera of eight families of the nonleguminous Angiospermae.
Eight of the genera, representing six widely scattered families, have
been found in the United States (Bond, 1963), (Allen and Allen,
1965), (Stewart, 1967). Plants in seven of the eight genera and five
of the six families indigenous to the United States are found in
Utah. Myrica gale of the family Myricaceae has not been found
in this region. The families and genera of the nonleguminous
nodule-bearing plants presently found in the United States and
other countries of the world are listed in Tables 1 and 2.
Nodulation ol Artemisia
Initial observations on the existence of root nodules on Artemisia ludoviciana were made on May 28, 1967, at the Milton
Cemetery about three miles northwest of Morgan in Morgan
County, Utah. Preliminary microscopic examination, in the labora182
Reprinted from Proceedings of Utah Academy of Sciences, Arts & Letters — Volume 45, Part I.
TABLE 1
Family
Genus
Species
Common Name
Betulaceae
Alims
glutinosa
Alder
Elaeagnaceae
Elaeagnus
angustifolia
Russian Olive
Shepherdia
argentea
Silver Buffalo Berry
Arctostaphylos
uva-usri
Bearberry
Myrica
(Comptonia)
gale
One of the Bayberries
Rhamnaceae
Ceanothus
velutinus
Deerbrush
(or Snowbrush)
Rosaceae
Purschia
tridenta
Bitterbrush
Cercocarpus
betuloides
Western Mt. Mahogany
Ericaceae
Myricaceae
2
'From data of: Allen and Allen 1965; Bond 1963; Holmgren 1948.
Myrica is found along the Atlantic seaboard and is not native to Utah.
2
FARNSWORTH, HAMMOND: ROOT NODULES/ENDOPHYTE
THE FAMILIES AND GENERA WITH SOME OF THE SPECIES OF PRESENTLY KNOWN
NON-LEGUME NODULE-BEARING PLANTS FOUND IN THE
UNITED STATES AND UTAH'
184
UTAH ACADEMY PROCEEDINGS, VOL. 45, PART 1, 1968
TABLE 2
TIIE DISTRIBUTION OF FAMILIES AND GENERA OF THE
PRESENTLY KNOWN NON-LEGUME NODULE-BEARING PLANTS
FOUND THROUGHOUT THE WORLD BUT NOT IN THE
UNITED STATES'
Family
Genera
Distribution
Casuarinaceae
Casuarina
Coriariaceae
Coriaria
Elaeagnaceae
IIippophae
Rhamnaceae
Discaria
Rosaceae
Dryar
(sp. drununondii)
Australia, Tropical
Asia, Pacific Islands
Mediterranean to japan,
New Zealand, Chile to
Mexico
Asia, Europe,
Himalayas to Arctic
Circle
Andes, Brazil, New
Zealand, Australia
Arctic Circle
'From data: Allen and Allen 1965; Bond 1963.
tory, of crushed nodules revealed the presence of an abundance
of small Gram Negative rods.
Further investigations were made at sites near Providence,
Utah, and in Hobble Creek Canyon, east of Springville, Utah.
Nodulation was observed on plants in each of these areas near
mid-June. These soils were becoming dry at this time, and nodulation was less evident. Nodulated plants were collected from each
of the three locations for laboratory study.
Characteristics of Nodules
The nodules when first observed in the Morgan area, as the
moist soil crumbled loosely from the roots, appeared in moderately
large clusters of 8 to 20. The individual nodules ranged in size
from about three to eight millimeters in diameter. Nodules on the
roots of plants first brought from this site are shown in Figure
1. The nodules were larger than those regularly found on alfalfa
and clovers and appeared to be somewhat similar in size and
shape to those found on soybeans. The presence of nodules on
the roots of this species seemed to be correlated very closely with
the supply of early spring moisture and the temperature. As subsequent visits were made to these sites near the end of June, it
was found that the soils had dried out and that nodules could
FARNSWORTH, HAMMOND: ROOT NODULES/ENDOPHYTE 185
FtGnat: 1. Nodules on the roots of Arterni,sia ludoviciana found near Morgan, Utah.
not be found. They appeared to have sloughed off and decomposed.
The root systems of Artemisia tridenta (subspp typica and nova —
big sagebrush and black sage) were examined in this area during
the period, June 15 to July 1; but nodules were not observed.
Preliminary Characterization of Endophyte
Nodules were washed in 0.1% HgCL for approximately sixty
seconds with subsequent washings of ethyl alcohol and sterile
water. They were transferred to fresh sterile water and crushed
with a sterile glass rod. (Exposure to HgC12 in excess of one
minute resulted in complete loss of organisms.) Streaks were made
upon plates of Yeast Extract-Mannitol Agar, regularly referred
to as Media 79 (Fred and Waksman, 1928). The yeast extract
(Difco Yeast Extract) was added in a concentration of 0.3 percent.
The growth on Media 79 was profuse and appeared as glistening,
turbid colonies resembling the growth of rhizobia. When the
yeast extract was omitted from the media, the growth was almost
negligible. Media 79 containing Congo Red was also used, and
profuse growth resulted.
186
urAil ACADEMY PROCEEDINGS, VOL. 45, PART 1, 1968
The cellular morphology of the bacterium seems to vary
with age. This variation is coccoidal to rod form as shown in
Figure 2. The coccoidal form varied in size from 0.5 to 1.0 micron
wr 0
1
10
11LB Allik
111
41110 "ra 46j
•
•.40
7,
lb
14 1" 11.
0114/400,
4
1
1.
40.
4:40.0046.1046
040.0
•
0111 01
•
•
40
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my,
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FIGURE 2. Microorganisms isolated from root mxlules of
Photograph taken using phase microscope.
A. ludoviciana.
in diameter; while the rod forms were 1.0 to 1.5 microns x 0.5
microns. The organisms were Gram Negative. They exhibited
motility, but the type of flagellation has not yet been determined.
Research is being continued to more completely characterize the
organism.
It is well to note that the endophyte isolated from the nodules
of Artemisia lucloviciana is bacterial. The organisms isolated from
the nonleguminous plants and tested by many other workers
were mostly actinomycetes and streptomyces (Bond, 1963, and
Wollum, Youngberg, Gilmour, 1966).
Discussion and Summary
The nonleguminous plant species Artemisia ludoviciana is one
of five recognized species of the genus Artemisia belonging to the
Compositae family. It is not as extensively distributed, nor as
well known, as is Artemisia tridenta. It is more commonly known
FARNSWORTII, IIAMMOND: ROOT NODULES/ENDOPHYTE 187
as herbaceous sage producing a full new top growth each season.
It is characterized by a rhizome-type root system as compared
with a tap-root system of A. tridenta. It is indigenous to much of
the western United States, from Utah northward to Canada and
from California eastward to Montana and the Dakotas.
It is important in that it has been found to produce root
nodules which are reported in other nonleguminous Angiospermae
to function in the assimilation of atmospheric nitrogen similar to
the nodules of the family Legurninoseae.
At this time the importance of the phenomenon of nitrogen
fixation by this nonlegume can only be conjectured. The many
investigators of the nonlegume genera (Bond, 1963) agree that
nodule-bearing plants have the capacity, which nodule-free plants
lack; i.e., to make satisfactory, or even vigorous, growth in a root
medium free of combined nitrogen.
The observation of nodules on this plant now adds another
genus (Artemisia) and another family (Compositae) to the growing
list of nodule-bearing nonlegumes. Should future investigations
reveal the presence of nodules on other species of Artemisia, it
could well be the means of explaining why western soils under
sagebrush have always been recognized as the more fertile and
productive soils.
Laboratory, field, and greenhouse studies are presently under
way in continuing this project. Seeds were collected during the
fall of 1967 from Artemisia ludoviciana, Artemisia trident° (typica
and nova). It can be reported that progress is being made in
technique of propagating these plants from seeds and root cuttings,
and in the inoculation by the endophyte.
The importance of nodule-bearing nonleguminous plants as
a nitrogen source is only now being appreciated. Allen and Allen
(1965) reviewed this quantitative significance. It may be concluded (Stewart, 1967) that nodulated nonleguminous plants contribute as much nitrogen to the soils in which they grow as do
leguminous crops.
It is envisioned that in the future, proper management of
much of our existing wild lands, ranges, and forests would include
certain specific nodulated nonlegumes for the improvement of
the nitrogen economy of the soil. As soil scientists, foresters, and
ecologists become more alerted to the value and existence of
nonleguminous symbiosis, it is most likely that nodulated species
will be reported in other plant families and genera.
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UTAH ACADEMY PROCEEDINGS, VOL. 45, PART 1, 1968
Special Note: In further substantion of the concluding paragraph
above, on April 9, 1968, in the desert sands about
20 miles north of Delta, Utah, the authors observed
and collected nodules from the roots of the "Prickly
Pear." This plant is of the genus Opuntia of the
Cactaceae family.
References
K. AND O. N. ALLEN. 1965. Non-leguminous Plant Symbiosis.
Oregon State University: In Proc. Bio. Colloq. (April 1964), 77-106.
BOND G. 1963. The root nodules of non-leguminous angiosperms. In symbiotic associations: Soc. Gen. Microbiol., 13th Symp. 13:72-91.
FRED, EDWIN B. AND SELMAN A. WAKSMAN. 1928. Laboratory Manual of General Microbiology. New York: McGraw-Hill Book Company, Inc.
HAWKER, LILLIAN E. AND JOAN FRAYMOUTH. 1951. A re-investigation of the
root-nodules of species of Elaeagnus, Hippophae, Alnus, and Myrica,
with special reference to the morphology and life histories of the causative
organisms. J. Gen. Microbiol. 5:369-386.
HOLMGREN, ARTHUR H. 1948. Handbook of the Vascular Plants of the Northern
Wasatch. San Francisco: Lithotype Process Company.
STEWART, W. D. P. December 15, 1967. Nitrogen-Fixing Plants, Science.
158:1426-1432.
WAKSMAN, SELMAN A. 1927. Principles of Soil Microbiology. Baltimore: The
Williams & Wilkins Company.
Wouutd, A. G. II, C. T. YOUNGBERG, AND C. M. GILMOUR. 1966. Characterization of a Streptomyces sp. Isolated from Root Nodules of Ceanothus
velutinus Dougl. In Soil Science Society of America Proceedings, Vol.
30, No. 4, July, August, pp. 463-467.
ALLEN, ETHEL