TEXAS AGRICULTURAL EXPERIMENT STATIONS.

BULLETIN NO. 83

Botanical S6CtiOn.——JANUARY, 1906.

NITRO-CULTURE.

BY

O.M.BALL,

MYCOLOGIST.

 

POSTOFFICE I

COLLEGE STATION, BRAZOS COUNTY, TEXAS.

TEXAS AGRICULTURAL EXPERIMENT STATIONS.

OFFICERS.

GOVERNING BOARD.

(BOARD OF DIRECTORS A. AND M. COLLEGE.)

M. SANSOM, President . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..A1varado.

F. A. REIGI-IARDT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..Houston.

K. K. LEGGETT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..Abi1ene.

GEO. T. JEsTER . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..Corsicana.

W. J. CLAY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..Austin.

A. HAIDUSEK . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .La Grange.

L. D. AMsLER . . . . . .  . . . . . . . . . . . . . . . . . . . . . . . . . ..Hempstead.

A. J. BROWN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..Da11as-

‘STATION OFFICERS.
H. H. HARRINGTON, M. S., President of the College.

J OHN A. CRAIG . . . . . . . . . . . . . . . . . . . . . . . . . ..Dean and Director.

pM. FRANCES, D. V. M . . . . . . . . . . . . . . . . . . . . . . . . . . .Veterinarian.

F. S. JOHNSTON . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . .Agricu1turist.

E. J. KYLE, M. S . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Horticu1turist.

F. R. IVIARsHALL . . . . . . . . . . . . . . . . . . . . . . . . ..Anima1 Husbandry.

EDWARD C. GREEN, B. S . . . . . . . . . . . . . . .Assistant Horticulturist.

G. S. FRAPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..Acting Chemist.

R. L. BENNETT . . . . . . . . . . . . . . . . . . . . . . . . . . . ..Cotton Specialist-

O. M. BALL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..Botanist..

B. C. PITTUoK . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..Feed Inspector.

C. O. M0sER . . . . . . . . . . . . . . . . . . . . . . . . . ..Deputy Feed Inspector..

M. S. CHURCH . . . . . . . . . . . . . . . . . . . . . . ..Deputy Feed Inspector.

JOHN C. BURNS . . . . . . . . . . . . . . . . . . . . . ..Deputy Feed Inspector-

J. G. HARRISON . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..Bookkeeper-

C. W. CRIsLER . . . . . . . . . . . . . . . . . . . . . . . . . ..C1erk Feed Control-

MIss M. H. WATKINS . . . . . . . . . . . . . . . . . . . . . . . . ..Stenographer..

STATE SUB-STATIONS.

J. K. ROBERTSON, Superintendent . . . . . . . . ..Beevi11e, Bee County..
W. S. HoToHKIss, Superintendent... . . ...Troupe, Smith County-

NOT;E.—The main station is located on the grounds of the Agri-
cultural and Mechanical College, in Brazos County. The Post-
oﬂice address is College Station. Reports and bulletins are sent’
free upon application to the Director.

TABLE OF CONTENTS.

 

PAGE.
Cross inoculation of alfalfa with germ from bur-clover ...................... .. 8
Record of pots in experiments on artiﬁcial inoculation ................ .. 9
Inoculation with Nitro-culture ............................................  ........... .. 11
Inoculation with Nitro-culture for alfalfa.._........  12
Cultures in soil from compost heap .........  .................................. .. 12
Cultures in pure sand ................................................................ .. 13
Inoculation with Nitro-culture for bur-clover ................................... .. 13
Behavior of the check plants ........................................................... .. 13
Soil sterilized but seed not .........  .............................................. .. 13
Neither soil nor seed sterilized ................................................... .. 14
Conclusion ................................................................................... .. 14

Summary .................................................................................... .. 15

[Blank Page in Original Bulletin]

NITRO-CULTURE AND INOCULATION.

BY O. M. BALL, MYGOLOGIST.

In view of the extravagant claims now bei.ng published regard-
ing the wonderful beneﬁts t0 be derived from artiﬁcial inoculation
of the soil, or of the seeds of leguminous plants, with bacteria
which ﬁx the free nitrogen of the atmosphere, a careful investiga-
tion of the question was thought advisable. It is true that some
of these extravagant expressions have lately been modiﬁed to a
certain extent, but the numerous requests for information and
advice which are being received by the Experiment Station of this
State show that the public interest has been aroused to a high
pitch by the sensational announcements of vast proﬁts to accrue
from artiﬁcial inoculation of leguminous crops. Many seedsmen
and dealers are pushing the sale of a commodity which is guar-
anteed to produce these marvelous results at a price of from $1.50
to $2.00 per acre, when the actual cost of the stuff has been shown
to be less than 1M0 cents, and is worthless, in most cases, at that.
Moreover, the process of artiﬁcial inoculation, in essentials, is not
a new one, but is merely a revival of an attempt ﬁrst made by
Nobbe, a German scientist, in 1898. Owing to questionable re-
sults, Nobbe’s method quickly fell into disfavor. Nobbe distrib-
uted his inoculating material in a solution containing nitrogen;
Carron, both as ﬂuid and dry, and Moore sends his dessicated on
cotton. It appears, however, that undue haste has been exercised
by the last named investigator in announcing his results. Proper
veriﬁcation of results, before publication, has been lacking.

Claims Advanced for Nitro-Oulture.—Essentially, the claims ad-
vanced for Nitro-Culture are based on the following considera-
tions: '

1. The presence of nitrogen-ﬁxing bacteria on the roots of
legumes is requisite for the production of good crops, especially
where the soil is lacking in nitrates.

2. The various" legumes are affected by a species of bacterium
which has so adapted itself to its host plant that each legume has
its own variety of that species.

3. If soils are naturally deﬁcient in nitrogen-ﬁxing bacteria,
these may be introduced by inoculation of the soil, or of the seed
to be sown. .

4. In order to obtain the best results, it is necessary to inocu-
late a given legume with its own variety of bacterium.

5. By cultivation of these laacteria under suitable conditions,
for example, in solutions of food stuff in which no nitrogen is
present, their “virility” or greed for nitrogen may be greatly in-
creased.

6. After having been inoculated with these greedy bacteria, the
crop of given legume will be vastly enlarged, because the bacteria

6 TEXAS AGRICULTURAL EXPERIMENT STATIONS.

will ﬁx a quantity of nitrates corresponding to their enhanced
appetites, thus affording a greatly enlarged food supply for the
legume. _

In regard to these claims, the ﬁrst is to some extent true. It 1s
a matte-r of common observation that most legumes are more
thrifty when their roots show a copious supply of the nodules pro-
duced by the activities of these bacteria. Their presence does not
seem to be requisite, however, when the soil contains a plentiful
supply of available nitrates, which fact has l.ed to the statement
that the bacteria will not thrive in such soil.

Secondly, in the light of recent experiments*, it is certain that i

in the case of the bacteria which produce nodules on the roots
of sweet melilot, the same nodules will appear upon the roots of
alfalfa, after “cross-inoculation.” As will hereafter appear in
this paper, the same is true of those bacteria infecting the roots of
‘bur-clover. It will also appear that the bacteria sold by certain
parties for the exclusive inoculation of alfalfa will sometimes in-
fect the roots of bur-clover, and the bacteria designed for the ex-
clusive use of bur-clover will in turn sometimes infect alfalfa.

As regards the third proposition, there are certain conditions .

under which it is conceivable that a given soil will be deﬁcient in
nitrogen-ﬁxing bacteria; for instance, when a ﬁeld has been
cropped to excess with a growth which does not serve as host to
these bacteria, such as wheat or corn. Under such circumstances,
if the germs can not preserve their vitality for a year or two in the
soil, they most inevitably disappear. These conditions are not
apt to occur in a natural state of aﬁairs, since, in most localities,
one or other form of legume will be found abundant. On the
barren sands of the uplands near the Brazos river, in l.and which
has never been under cultivation, isolated legumes (Lupinous sub-
carnosus, L. Texensis, bur-clover, Japan clover and others) have
been examined by the writer, and in every case show a plentiful
supply of the nodules. The same fact was observed on the lately
excavated embankment of the I. & G. N. Railroad, where the stiﬂ’
and poor sub-soil of the “post-oak” country had been exposed to
the gradual encroachment of vegetation, with scattered specimens
(L. subcarnosus, L. Texensis, etc.). The writer has also noted,
during four or ﬁve years, the gradual spread of Japan clover on
the mountains of certain parts of Virginia. This plant has spread
slowly over the Blue Ridge and the adjacent hills, on land never
cultivated in any manner. From year to year, the plants appear-
ing for the ﬁrst time, even at elevations of 2500 feet, invariably
show roots well supplied with nodules. The bacteria causing these
nodules have either been obtained from other legumes, or the in-
fection has been carried with Japan clover as it spread. Even
isolated plants, on remote “knobs,” have invariably been found to
be supplied with tubercules. In another case, on land which had
received a top dressing of stable manure, but which had not been

*Hopkins, Bulletin Illinois Experiment Station, 94, 1904. .LeWis and
Nicholson, Oklahoma Experiment Station, Bulletin No. 68, 1905, page 14
gives a table showing several other cases of cross-inoculation.

‘NITRO-GULTURE AND INOCULATION. 7

cultivated for many years, both alfalfa and bur-clover developed
nodules, apparently to suﬂiciency. In fact, it would be difﬁcult
to ﬁnd a soil in which a wild legume would show no nodules.
Hence, the need for artiﬁcial inoculation would be a very rare
occurrence.

The claim put forward in the fourth of the foregoing considera-
tions has been directly contradicted by results obtained by the
writer, working in the laboratory, as will appear below. In fact,
it will appear, that in no case of artiﬁcial inoculation was the sup-
ply of nodules on the roots of the plants inoculated so copious as
where the plants grew in the same soils but which had not been
previously sterilized.

As regards the claims put forward in the ﬁfth statement, the
writer has seen no adequate statement of the methods by which the
alleged increased “virility” has been obtained. Though great cau-
tion should always be exercised in reasoning by analogy, it may be
urged against such doctrine that other like organisms do not show
an increased appetite for their natural food after having been de-
prived of this food for a time. An aerobic bacterium (one that re-
quires oxygen) may be, at another time, an anaerobe (requiring no
oxygen), but no one has ever observed an increase in the “virility”
of such anaerobes after having been reared again in the presence of
oxygen; at least, to the writer’s knowledge, no one has reported
such observation. There are other so-called facultative bacteria,
organisms which have the power of living with or without certain
articles of food. There has been no recorded observation, in the

knowledge of the writer, of an increase in “virility” of such organ- .

isms after having been changed to a medium containing a normal
food substance which was lacking in a previous culture. Against
this doctrine, the facts observed in the laboratory have even
greater weight. In no case of artiﬁcial inoculation with “Nitro-
culture” or other like preparations was there an observable increase
in thriftiness in the inoculated plant, whether in good soils or in
pure sand or water cultures in which all traces of nitrogen had
been removed except that of the atmosphere.

Experiments t0 Test These Claims.—In ampliﬁcation of these
objections to the claims which have been made for the “Nitro-
culture” and other substances of like nature, a detailed statement
will follow of a series of experiments which were made i.n the labor-
atory of the Department of Botany and Mycology during the winter
and spring of 1904-5, and the fall of 1905. Circumstances pre-
vented immediate publication of these results. Further detailed
and quantative work has been planned for the ﬁeld.

The work here described was wholly by means of pot cultures
and falls into two groups. In the ﬁrst series, the object was to
determine whether nodules will appear on the roots of alfalfa
when this plant is grown in soils where none had ever before been
cultivated, but which produced a ﬁne growth of bur-clover (Medi-
cago denticulata), and further, whether the nodules already pres-
ent on the roots of bur-clover could be carried over to alfalfa, when
grown in soil that had previously been sterilized; in short, whether
artiﬁcial inoculation of alfalfa with the germs from bur-clover is

8 TEXAS AGRICULTURAL EXPERIMENT STATIONS.

feasible. In the second series, the question was to determine the
value, if any, of the so-called Nitro-culture and other like “cul-
tures.”

The method of procedure was very similar in the two series. A
variety of soils was used in the ﬁrst series, as will appear in the
following notes of pot cultures. Soil of the best quality, obtained
from the compost heap of the College Horticultural Department
and pure crushed quartz sand, were used in the second series. The
pots were of white clay, of the the ordinary six-inch pattern.

CROSS-INOCULATION OF ALFALFA WITH GERM FROM
BUR-CLOVER.

Soils of several varieties were obtained from the College lands,
from points where no alfalfa had ever been cultivated. Some
samples were from the parade ground directly in front of the
buildings and others from the cultivated ﬁelds. The seed were
selected from samples sent by various commercial houses in the
State. These seeds were carefully washed through many changes
of water, but chieﬂy by placing them under the tap from the 700-
foot well, and allowing them to remain for several hours. They
w-ere then placed in a 2 per cent solution of corrosive sublimate
and vigorously stirred for from several minutes to a half hour.
In this connection, it may be of interest to state that the seed does
not absorb the sublimate, since it was a matter of frequent obser-
vation that the young sprouts would appear from the seed acci-
dentally left in the solution, "and would grow to a length of half
an inch before being killed by the poison. The careful washings
and treatment with sublimate were designed to get rid of any bac-
teria which might adhere to the outer coats of the seed, if such
were possible, and was simply a precautionary measure, the utility
of which is doubtful. However, we were never troubled with the
appearance of injurious fungi, “damping off,” or other disagree-
able contingencies of this kind.

In all, about forty pots were used in this series, of which sev-
eral were used as checks. The soil in the others was sterilized by
heating for six to eight hours in an Arnold’s sterilizer at a steam
heat of 206 to 208 degrees F. They were then left in the ster-
ilizer over night and the heat again applied for a like period.
This resulted, in all but one case, in a complete sterilization of the
soil. A sufficient quantity of the seed, treated as above outlined,
was now sown in each pot. After germination, the pots were
carefully examined at intervals to ascertain if nodules were pres-
ent. This was done by means of thoroughly sterilized section lift-
ers. One or two seedlings would be lifted out of the soil and ex-
amined. With one exception, the young plants when four inches
high and ﬁve weeks old showed no nodules. The sterilization was
then considered complete.* If no nodules were found, the re-

 

*It had previously been observed on many young bur-clover plants which
make their appearance above the ground at College during the month of
November, that nodules are very evident when the seedling is two Weeks
old and from one to two inches high.

NITRoJOULTURE AND INOOULATION. 9

maining plants were then inoculated and set aside, together with
the check pots, in. an isolated section room, and were watered with
distilled water. A careful record of each pot was kept. A con-
densed statement of this record follows:

Record of Pots in Experiments on Artiﬁcial Inoculatioru-Pot I.
Seeded February 9, 1905, in sterilized soil. On March 11th the
plants were about four inches high, and their roots showed no

nodrules. The pot was then inoculated with a “solution” from ~

carefully selected and thoroughly washed nodules from the roots
of bur-clover found growing on the grounds of the Agricultural
Building. These nodules were crushed in sterilized water, in steril-
ized glass beakers, and the “solution” was poured around the roots
of the young alfalfa plants. Six weeks later, on April 28th, the
plants were thrifty and ﬂourishing, and their roots were plenti-
fully supplied with nodules. The check pot showed no tubercules,
although there was no observable difference in the appearance of
the plants.

Pot II. The soil in this pot was sterilized by heating in porce-
lain crucibles for several hours, by which means the organic mat-
ter was thoroughly burned out and all forms of life destroyed.
Seeded February 9th, and inoculated March 11th with the same
“solution” used in No. I. April 28th plants, as was to be expected,
from the burning of all organic matter in the soils, were not so

‘thrifty as in No. I, but rootlets showed a fair supply of nodules,

though by no means so many as in No. I.

Pot III. Sterilized as before and seeded February 2d. On
March 16th no nodules were present; whereupon the plants were
moistened with water, in which the roots of bur-clover had been
allowed to lie in the dark for forty-eight hours. April 29th the
rootlets showed abundant nodules. The plants were no better than
their checks, grown in sterilized soil and which showed no nodules.

Pot IV. Sterilized, and seeded February 1st. By March 16th
no nodules had appeared on the roots, and water which had been al-
lowed to ﬁlter through soil containing bur-clover roots was poured
around the young plants. On April 25th there was a very ﬁne
growth of nodules on the roots.

Pot V. Unsterilized soil, of very poor quality and containing
but little organic matter; taken from a spot where bur-clover was
growing, but very sparsely. This soil became very compact in the
pots, and was with great diﬂiculty kept from packing. Seeded
February 8th with sterilized seed and allowed to grow until May
15th. At this time the plants were about one-eighth as large as
those grown in sterilized rich soil. Nevertheless, the rootlets
showed a very large number of nodules. .

Pot VI. Seeded February 5th, in soil taken from the compost
bin of the Horticultural Department, and which was very rich,
both in humus and in nitrates. The alfalfa seed sown in this pot
were sterilized with extra precaution. On March 16th the root-
lets showed very ﬁne nodules. Thereafter, the plants throve well.
but made no better growth than the check alongside in the same
soil, but which had been previously sterilized by steam heat.

10 TEXAS AGRICULTURAL EXPERIMENT STATIONS.

Pot VII. Soil taken from parade ground, near ﬂagpole, and
was dense and unpromising. There are no legumes at this point.
Seeded February 15th, and examined May 23d, but no nodules
made their appearance.» The plants were of about the same size
and appearance as those described in No. V.

Pots VIII-IX. Soil from compost bin, sterilized as above and
sown with alfalfa seed which had been allowed to lie for a few
minutes in “soil solution” used in No. 1. Four weeks later the
plants in these two pots showed a large supply of nodules on the
rootlets. When two months old the difference between these plants
and their checks, of same age, growing in sterilized soil, of the
same kind without inoculation, and never showing nodules, was so
slight as to be negligible.

Pots X-XI. Soil from compost bin, sterilized as above, sown
with alfalfa seed which had received treatment from a solution of
crushed nodules from rootlets of young bur-clover plants. These
plants all showed the presence of nodules when examined three
weeks later. If there was any difference between these plants and
their checks, growing in sterilized soil of the same kind, and never
showing nodules, such was not observable.

In one case, after the usual sterilization, the young plants were

_ found to be infected, and in still another this infection was found

four weeks after the young plants had been inspected and found
to contain no nodules; at least none were present o-n the rootlets of
those taken up. These were the only irregular occurrences in the
whole series of forty pots, and may be explained as the result of
incomplete sterilization or of accidental inoculation. Unfortu-
nately, the department possesses no green house where conditions
may be rigidly controlled and all visitors excluded. It is possible
that infection may have been caused by some visitor with good in-
tentions but poor judgment.

The experiments above outlined were repeated quantitatively,
and the work was carried on by two different parties, working sepa-
rately, and whose results were not compared until the end of the
series. From a survey of the results, it will appear that the bac-
terium which affects the roots of bur-clover will produce nodules
on those of alfalfa with equal readiness. Further, in good soil,
as in No. VI, it is not necessary for the welfare of the plant that
nodules be present. This, however, has long been known to be
true. In poor, stiff soil, the presence of nodules did not appear to
materially affect the growth of the plant, there being no noticeable
difference in the plants in pots in Nos. V and VI, in which the
soil was of very similar character.

It may possibly be urged against the results that the time of
experimentation was not sufficiently long to permit the plants to
derive any beneﬁt from the presence of the nodules, which had
been formed on the rootlets in plants in Nos. I, III and VI. To
this it may be replied that the plants were large and well grown,
being in most cases from one and one-half to two feet high and
about two and one-half-month old, when the work was brought to a
close by the ending of the session of the College. During this

NITRO-CULTURE AND INOCULATION. 11

time, it seemed that some difference between the infected and non-
infected plants would have appeared, if such were ever to occur.

INOCULATION WITH “N ITRO-CULTURE.”

To determine the value of the claims put forward for “Nitro-
culture,” and, further, whether “Culture,” intended for alfalfa
would affect bur-clover and vice versa, one package of the “Cul-
ture” for alfalfa and one for bur-clover were obtained from the
National Nitro-Culture Co, West Chester, Pa., and one for alfalfa
from the Texas Seed & Floral Co, Dallas, Texas. The latter was
obviously the product of the former concern, since the packages
and instructions in all were alike. All were marked “Acre Pack-
age.” The solutions were made precisely and carefully according
to directions found in the packages; not all of the materials being
used in the ﬁrst preparation. The remainder was carefully en-
closed in the original papers and laid aside for future use. After
about twenty-four hours, a milky color appeared in the bottles, ac-
cording to schedule. This cloudy appearance increased during the
following day and was so decided that yeasts were suspected, and
a microscopic inspection of a drop showed the presence of a yeast;
but this examination was carried no farther.* A second and third
solution was prepared from the remainder of the materials of the
packages at intervals of three days. Each behaved as the ﬁrst,
and hence was considered to be good, since there was no observable
difference in the solutions. The soil used in the experiments was
taken from the compost heap of the Horticultural Department, and
was carefully sterilized, as above described, by steam heat, in
Arnold’s sterilizers.

In one series, alfalfa seed was inoculated with the solutions and
sowed in pots carefully marked, so that there would be no chance
of confusing the bur-clover with the alfalfa cultures. In the other
series, young alfalfa plants about four weeks old were watered
with these solutions, after having been scrutinized in the manner
above explained, to determine if nodules were already present.
These cultures were likewise carefully kept apart. A number of
pots of the same soil were kept alongside of the above as check pots.
One-half of these had been sterilized previous to seeding; the
other was of native, unsterilized soil, fresh from the compost heap.
As far as was possible, the seed in any one series was sown at the
same time, in order to keep the plants of the same age and develop-
ment. ~

To resume, these experiments were intended to determine:

I. If the solution made from the materials sold as “Nitro-cul-
ture” will in reality produce nodules on the roots of legumes in
soil which had previously been thoroughly sterilized.

2. Whether “N itro-culture” for alfalfa will inoculate bur-

 

*This observation of the presence of yeasts in the cultures so prepared
has been fully veriﬁed by Harding & Prucha, New York Experiment Sta-
tion, Bulletin No. 270, November, 1905.

12 TEXAS AGRICULTURAL EXPERIMENT STATIONS.

clover, and whether the culture for bur-clover will in turn affect
alfalfa.

3. To compare the results of inoculation, if such occurred, with
conditions found in plants growing naturally in the same soils and
aﬁected with tubercle-producing bacteria.

IN OCULATION WITH NITRO-OULTURE FOR ALFALFA.

Cultures in Soil from Compost H cap-In the ﬁrst series, eight-
een pots were used. Twelve of these were sterilized, and were
sown with alfalfa seed which had been thoroughly washed and al-
lowed to lie for a short time in corrosive sublimate solution. The
seed planted in six of these pots was treated with the solution of
“Nitro-culture” for alfalfa, according to directions found in the
packages; the second set of six received no “culture,” while the

third set served as controls and were ﬁlled with the same soil, but ,

unsterilized, and-the seed were also unsterilized. From ten to twelve
seeds were sown in each pot.

After two weeks, when the young plants were about two inches
high, two or three were carefully removed with sterilized section
lifters and their rootlets examined for tubercles. Not a single
plant of those inoculated showed tubercles, and none were found
on those in the sterilized uninoculated pots. Every pot of natural
soil showed tubercles on the rootlets of the alfalfa plants. This
result was so surprising and so unexpected that the remaining
plants were allowed to grow for two weeks longer, after which they
were again examined, with the same results. There were no
tubercules to be found, either in inoculated or in the sterilized pots,
while the check pots continued to develop a good crop.

Since there was a possibility that some error in preparation of
the “culture” had occurred, a fresh solution was prepared with the
utmost care, exactly according to the directions given. A portion
of the package previously used was made up, reliance being placed
upon the statements found in the sheet of directions, that “The
package accompanying this direction will keep for one vear from
date, if it is not wet or overheated”; which, to say nothing of the
grammar, is certainly ambiguous. A new set of twelve pots was
prepared, as before, by careful sterilization. Six were sowed with
alfalfa seed inoculated with the new solution, and six were sowed
with sterilized but not inoculated seed, and a third set of six were
ﬁlled with natural soil, sown with unsterilized seed. At the same
time the six which had previously been inoculated, and had failed
to catch, were watered copiously with this new solution, to obtain,
if possible, a “setting” of the tubercles. In both cases, absolute
failure to obtain nodules resulted, though tubercles were uniformly
formed in the unsterilized soils. Later, forty-eight pots were thus
inoculated, and in this number we succeeded in inoculating nine
pots, at least tubercles were found on the rootlets of plants in that
many pots. In this series, twelve pots were used as checks, being
ﬁlled with unsterilized soil. In none of the inoculated pots were
the tubercles abundant, the plants showing few and scattered nod-

NrTRo-OULTURE AND INOCULATION. 13

ules, which did not seem to thrive, nor did the plant once affected
seem to be able to develop the tubercles in increasing numbers.
All of the check pots developed abundant nodules.

Cultures in Pure Sand.——Six pots were ﬁlled with pure crushed
quartz sand, which had ﬁrst been washed by tap water for several
hours before sterilization. The tap water was from a cistern under
the roof of the Agricultural Building, and was pure rain water,
falling on and collected from a slate roof. This water was used
also in making up the Nitro-culture solutions. These pots were
sown with inoculated seed, and were watered with a von der
Krone’s nutrient solution, in which the nitrate of potash had been
omitted. These plants throve remarkably well, considering the
lack of nitrates, but never at any time showed tubercles, though
they received frequent waterings with the “Nitro-culture” after
they had reached a height of several inches and had developed no
tubercles from the ﬁrst inoculation.

One alfalfa plant, grown in a water culture of v. d. Krone’s
solution without nitrates, developed, six days after receiving “Nitro-
culture,” three tubercles on the rootlets. This plant was kept under
observation for more than four months. It grew fairly well, but
never developed more than the three nodules which ﬁrst appeared.

INOOULATION WITH NITRO-OULTURE FOR BUR-
CLOVER.

A parallel series of thirty-six pots ﬁlled with compost was pre-
pared at the same time as the foregoing and placed in another
room. Eighteen of these received alfalfa seed inoculated with
Nitro-culture for bur-clover, prepared and sent out by the ﬁrms
above mentioned. Of the remaining eighteen, nine were sterilized
and planted with sterilized seed without previous inoculation, and
nine were not sterilized and were planted with unsterilized seed.
Four of the inoculated pots developed tubercles, but as in the case
of alfalfa culture, the number of nodules appearing was very small,
being only three or four on each‘ plant. The check series of nine
pots all showed an abundant supply of nodules.

BEHAVIOR OF THE CHECK PLANTS.

Soil Sterilized, But Seed N 0t.—Twenty-eight pots, in all, were
used as checks, in which the soil was sterilized, and the seed not. Of
this number, only two showed the presence of tubercles, the re-
mainder being at all times free from nodules. These two cases
may have resulted from imperfect sterilization in the ﬁrst place,
and this imperfect sterilization may reasonably account for some
of the cases observed of infection after inoculation, though cer-
tainly not in the case of the water culture where the nodules were
observed to form within a few days after the rootlets of the plant
had been treated with the solution.

14 TEXAS AGRICULTURAL EXPERIMENT STATIONS.

N either Soil nor Seed SterilizedL-Each of the twenty-four pots
containing unsterilized soil gave an abundant catch of tubercles,
but the general appearance of these plants was no better at the
closeof the experiments than those growing in the same unsteril-
ized soil.

CONCLUSIONS.

The results obtained by this method of investigation were so
uniform that there could be no room for doubt but that something
was wrong with the substances sold as Nitro-culture. So directly
at variance with the claims put forward by the Department of Agri-
culture and by numerous writers, presumably inspired from the
same source, were the results of this investigation, that in May,
1905, the writer addressed a summary of these to Dr. Geo. T.
Moore. The statement of results was purposely made in a guarded
manner, since the matter was of such far-reaching importance that
it was thought necessary to make fuller and more extended experi-
ments in order to verify and extend the work already done. The
claims then put forward were essentially as follows:

1. In no case was the supply of nodules found on rootlets after
inoculation as abundant as in plants growing normally in the same
soil unsterilized.

2. It is not necessary to make a “culture” before cross-inocula-
tion can be affected.

Later experiments have not only veriﬁed the exactness of these
results but have demonstrated that only in a small percentage of
cases is there any inoculation produced at all by the cultures used.
Since the ﬁrst series of these experiments was ended in April, 1905,
bulletins from several experiment stations have appeared dealing
with the problem by other methods of procedure. All have sub-
stantiated the results here put forward to the extent of showing
that the so-called cultures are worthless for practical purposes.
Notes of warning had been given from time to time, as, for in-
stance, those af Director A. M. Soule and Dr. Meade Ferguson,
Virginia Agricultural Experiment Station, Bulletin 154, April,
1905; of Professor Bailey, in Country Calendar, June, 1905; also
of Lawrence T. Clark, in Bulletin 231, of the Agricultural Col-
lege of Michigan, June, 1905, who deprecates especially the in-
judicious enthusiasm aroused by the questionable methods of her-
alding inoculation as a “panacea for all crop failures.” The most
authoritative statements of the value of the “cultures” are as fol-
lows:

New York Experiment Station, November, 1905, appearing
January, 1906. H. A. Harding and M. J. Prucha. We quote:

“These packages of treated cotton have had a wide sale at a high
price—two dollars for a package sufficient to treat an acre--while
the cost of production was less than ten cents.

“These examinations make it very evident that the packages were
worthless for practical purposes.”

NrrRo-CULTURE AND INOCULATION. 15

Oklahoma Agricultural Experiment Station, Bulletin 68, De-
cember, 1905:

“The pot cultures proved that the tubercle-forming germ of the
alfalfa plant was present, but the results as compared with those
from pots inoculated with cultures isolated in the laboratory show
that the germ was very scarce in the ‘Nitro-culture.’

“In the plates poured from the culture, the alfalfa germ could
not be found, as the plates showed almost a pure culture of an en-
tirely different organism. This germ, when used in pure cultures
on alfalfa, gave negative results.”

The latter ﬁnding sustains the work of Messrs. Harding and
Prucha, and that of the other examinations quoted by them. In
the case of the investigations made at the Oklahoma Station, it is
possible, though scarcely probable, that in the cultivation of pure
cultures, by plates, conditions arose which were fatal to the germ.
It is also possible, in the work of this laboratory, that incomplete
sterilizatiommay account for the presence of the tubercles on the
rootlets, and that they were not due to the inoculation by the “Ni-
tro-culture” at all.

SUMMARY.

Summarizing the results obtained in this laboratory, we ﬁnd:

1. Alfalfa will become infected with the tubercle-forming
bacterium:

When grown‘ in soil which is producing a crop of bur-clover.

After being watered with a “solution” of such soil.

By making an infusion of the nodules on the roots of bur-
clover.

2. Inoculation with the so-called “Nitro-culture” is of more
than doubtful value since only a small proportion of the plants
treated developed tubercles, and these were in no observable degree
beneﬁted by their presence.

3. In no case of artiﬁcial inoculation were the number and
vigor of the tubercles so great as in that occurring by natural
means.