WN NONNNINN

       

-=|
‘rar
nO

 

=i jae
 

SO EE 6 ee ee s— a, eee
. -= st eee
ee ee ee nw, - .

 

‘
<

>

s

THESIS
THE FIXATION OF FREE NITROGEN

BY PLANTS,

by

PERRY G. HOLDEN,
Michigan Agricultural College,

August 1, 1895.
THE FIXATION OF FREE NITROGEN BY PLANTS.
Agriculture as an art is old, as a science it is new.
Within the present century it has been elevated to the rank of a
science. Perhaps the investigation of no one subject has done
so much to place agriculture upon a scientific basis ‘as that of
the source of nitrogen ef vegetation. It is certain that no

subject has enlisted the efforts of so many eminent men, both

chemists and botanists.
The importance of the question is apparent. Nitrogen

is essential to all life, the nitrogen of animal life coming from
the nitrogen stored up by plants. Three-fourths of the weight
of the atmosphere is nitrogen. On every square inch of the
earth's surface rests 12-1/2 pounds of nitrogen. It surrowmds
. us on every side, we breathe it in at every breath, yet we are
unable to use it in its free state, If plants in their growth
can use the nitrogen of the air, there is an abundance of it
always "on hand” without importing it from the nitre beds, or
the guano fields of the far south. If on the other hand, they
must depend on the nitrogen of the soil there must somet ime come
an end to all vegetable, and consequently to all animal life.
The nitrogen of the soil is gradually but sonstant ly be ing
exhausted. The various processes of putrefaction are s low ly
turning the complex nitrogen compounds into simple ones. Some
of it 1s given off in the form of free nitrogen, but more of it

as ammonie. Again when substances are burned the nitrogen is

101412
2.

given up to the air as free nitrogen. A large part of the nitro-
gen of our food pisnts is carried by drainage water and the sewer

to the river and finally to the sea, Lastly the processes of ni-
trification annually convert large quantities of the ammonium
salts and organic nitrogen compounds of the soil into nitric acid,
whence a portion is used by pilents and moh is cerried off in the
drainage water as has been proved by Lawes and Gilbert and others |
Only 8 small fraction of this great loss will again be returned
in the rain water as anmonia end nitric acid which, as has been /
shown, falls fer short of supplying the amount necessary for
an ordinary crep. It was clear that the nitre beds of Ohili and
the F uano of the South Pacific could not long supply this great
loss, Indeed it seemed probable that the nitrogen supply of the
B0il would have been exhausted centuries ago, umless there was
some means by which plants could draw upon the unlimited supply of

the nitrogen of the air.

For more than one hundred years this important question
has been up for discussion, No scientific question has been
so meny times sett led end so persistant ly unsettled, In 1771
Dr. Joseph Priestly raised the question and finally came to the
conclusion that "plents could assimilate a small amowmt of the
free nitrogen of the air. Later, Ingenhouss confirmed the results
of Priestley's experiments, Woodhouse and Senebier came to ex-
actly the opposite conclusion, while de Saussure from his care-
fully conducted experiments decided that "plants not only did not

take up free nitrogen" from the air “but on the other hand gave
, . Se
off nitrogen during ective growth." In 1837 Boussingault began a

\

series of extensive experiments which resulted in the conclusion

that plants could not use the free nitrogen of the air in growth.
"In 1849 Ville of Paris objected to Boussingeaults meth-

od of experimentation, that plants could not make a norm growth

in such a confined body of air as that contained in a bottle or

globe. He repeated Boussingault's experiments but used a room

g lased with g less instead of a glass giobe, and announced the re-
sult, tha$ while cereals produced a crop containing only two or
three times as much nitrogen as was contained in the seed from
which they grew, colsa, cress and sunflower produced in the crop
25 to 40 times as much nitrogen as was conte ined in the seed. “{
He concluded "that while certain kinds of plants have little or
no power of taking up free nitrogen, other kinds have the power

of combining with free nitrogen and using it" in growth. “Such
contradictory results reached by two such distinguished scientists
provoxed a lively discussion and in the interests of harmony and
to establish scientific truth, a commission was appointed by the
French Aédademy, composed of such eminent men as Dumas, Regnault,
Payen, Decaise,Peligot ,and dhevreul. The commission thought
they "found evidence of some gain of nitrogen during the growth of
plants and finally reported, # "that the experiment made at the
Museum of Natural History by M. Ville is cons istant with the cone
clusion which he has drawn from his previous labors. "# :
In 1857, 20 years after Boussingault began his experi- |

ments and while he was still working upon them, Lewes and Gilbert

# R. 0. Kedzie on "The Source of Nitrogen of Plants, Mich.
Agr'l. Report, 1881-2, P. 379,
| 4,
the greatest experimenters ‘the world has ever known, began a se-
ries of experiments which resulted in the conclusion that # "plents
grown in the absence of combined nitrogen except that contained in
the seed have no power of tkking up free nitrogen end combining
it with other elements to form plant tissue," # It is not neces-
sary to give a detailed accowmt of the experiments, except to say
that the work was so carefully and thoroughly done that the re-

suits were generally accepted and the question seemed settled

beyond a doubt that the free nitrogen of the air was not in any

sense a source of plant food. The plant which was able to
breathe in carbon dioxide of the air, decompose it, and use the
carbon as plant food gust starve for the want of nitrogen which
surrouds the plant on every side, .

In 1876 M. Berthelot questioned the resuits obtainéd by
Boussingault, Lawes end Gilbert, since their experiments excluded
all micro-organisms and electrical action. The soil in its
natural condition is subject to both these influences. Berthelot
showed that free nitrogen wes fixed by various organic compounds
during the "silent electrical discharge” at ordinary temperatures

during storms, At this time organic matters would absorb both

oxygen and nitrogen. He concluded that through the influence of

electricity micro-organisms es well as higher vegetation could
fix free oxygen in the soil. I am not prepared to discuss the
results obtained by Berthelot, but if we suppose them to be cor-
rect we must admit that the nitrogen thus stored in the soil
would be available alike to all classes of plants.

# R. 0. Kedzie on “The source of Nitrogen of Plants, Mich.
Agr'l. Report, 1881-2, p. 379.
 

 
5.
The experiments of Deitzel’s and Deherains ere without
definite results. Professor Frank concludes from his ear ly experi-
ments that there are two processes going on th the soil, one lib-
erating nitrogen, the other br ing ing it into combination by the
aid of vegetation. M. Joulie found very large gains of nitrogen
in some cases, He was of the opinion that the nitrogen was first
fixed in the Boil by organisms, The nitrogen thus fixed could
subsequently be used by pieants. We ere at once doubtful of the

results since the large gains which he obtained was in the case
of a polygonum (buckwheat) and not with plants of the Veguminous

family.

This brings us dow to 1883 when Heliriegel bagan his
famous experiments which were destined to reverse the decissions
of a quarter of a century before by Boussingault, Lawes and
Gilbert. | |

#The plan of Professor. Hellriegel's experiments is briefly
as follows:- The plants experimented upon were grown in pots
of sea sand; Xa Rupply af the sand first being washed to remove
the nitrogen and then sterilized by subjecting to a temperature
of 150° C. The seeds were stertiized by dipping in a solution of
bicRhloride of mercury, then washed in boiled water and planted in
the pots with sterilized tools. The plants were watered with
distilled water in such away as to preclude the possible entrance
of living organism: into the pots. The pots were supplged
with all the elements of plant food necessary for growth except

# These experiments are described at length by Prof. W. 0.
Atwater, Exp. Sta. Rec., Vol. V, No's 8 & 9.
nitrogen. If no nitrogen was added the plants would grow for |

a time then turn yellow and finally die. An analysis of the plant
showed that inno case was there more nitrogen in the plant than
was present in the seed at the beginning. The plant had grown
until it used up the nitrogen of the seed, It was found that when
varying amounts of nitrogen were added the growth of the plants

was in proportion to the nitrogen added to the soil. This is
iliustrated by the following table giving the results of an exper-

iment with serradelia conducted in 1887.

 

Nitrogen lYield of Vine
supplied and Seed.

 

pots] Grams Grams.
1 |0.000 0.078
g |0.056 2.883
- 3 10.112 6.840

 

 

 

 

 

In no case did he find that the nitrogen in the plant
exceeded the nitrogen in the seed planted and that added to the
soil. It was evident that under these conditions there was no
assimilation of free nitrogen of the air. The conditions of

these experiments were essentially the same as those of Boussing-
ault and Lawes and Gilbert, twenty five years before and so far
the results are exact ly the same, |

Heliriegel went a step farther and inoc ulated the ster-
tlized soils with micro-organisms from rich soils.¢

# These micro-organisms were introduced by means of a "soil
infusion” prepared by mixing a small amount of a cultivated
#011 with water and allowing it to settle. The almost clear
water was poured off and used at the rate of 25 c. c. to each
pot containing 9 ibs. of sand. Analysis showed that the

85 c. c. of solution contained a small amowmt of nitrogen vary-
ing from 3/10 to 7/10 of a m. g.
 

 

 

 
7.

Turnips, hemp, sunflower and oats derivedno benefit from the
inoe ulation and died as soon as the nitrogen of the seed had
been consumed, but with the leguminous plants such as peas, ¢ lover
and vetbhes a very great change was soon apparent. The plants
began to show a dark green color and grew repidly. From this
time on it was evident that the leguminous plants had plenty of
nitrogen at their command, and developed rapidly and normally.
An analysis of the plants showed that the nitrogen was meny times
greater than that contained in the seed planted.

The following sketch is from a photograph by Lewes and
Gilbert show ing the ef8ect of soil infection upon pear. The
soil in pots 1, 2 and 3 was sterilised nitrogen free quartz
sand, to which all the elements of plant food were added except
nitrogen. pot 1, was not infected. Pots 2 and 5 were infected
with a soil ifusion as described in foot note on page 6. Pot
4 was garden soil. The peas were planted July 10th and before
the end of July the plants in pots 2 and 3 showed a more rapid
growth than in pot > ar The plants were photographed October 22,

104 days after plant ing

 

The plents in pot 1 (not infected) were 8-1/4and 8-1/2 ins high
" 8 " © g (infected) " 14. ond 60 _
"oon " © 3 (infected) " 60» 58 "
"8 “ " 4 (garden 8011) "4h « 49 .

 

 

While the plants in the garden soil made a less extended growth
than in pots & and 3, yet they were more figorous and flowered

end produced seed which those in pots 2 and 3 did not do. Ina
 

 

 

# Popular Science, monthly, Vol. XXXV111,
Pe 49«.--Man ly Mites.
9.
similar experiment with yellow lupines, like results were obtained

except that the plants in pots 8 end 3 (infected) were more vig-

orous than in the ilupine soil. . |
The lupine plants in pot 1 (not inocculated) were 1-1/aand 2 ins.

" » " 2 (inocculated) _* 18 4,24 *
" 9 " z ( @ " ) # £0 26 e
" " " 4 (lupine so1t}) " 6.68 *

Heliriegel planted nine kinds of seed, four non- legumi-
nous, five leguminous in each of four different pots, Two of the
pots A. and B. were infected with a soil infusion froma field
where beets had been grown, the other two pots, C. and D. were
infected with a soil infusion from a lupime field. The weight

in grams of dry substance is shown in the following table:-

 

 

 

 

infected with Infeéted with
_Beet soil infusion Lupine soil infusion
Name of plant pot A. | Pot B. pot 0. | Po
Non- Turnip 0.010 0.017 0.006 0.018
Leguminous | Hemp 0.025 0.055 0.047 0.046
Sunflowen 0.305 0.493 0.330 0.644
Oats 0.257 0,153 0.140 0.238
Serradem! 0.015 0.010 8.002 2.560
Lupines 0.093 0.155 17.133 | 30.597
Leguminous/OQlover | 8.813 3.241 0.3563 1.589
Yetch 15.971 6.132 6.678 §.181
Peas 12.282 32.640 16.152 6.021

 

 

 

 

 

All four pots were sown April 18;

A. and B. were harvested Aug-

 

ust 2, 0. and D. August 20.

“Thus under absolutely the same experimental conditions,
the infus ion of beet soil was ineffective with the non-laguminous
plants, also with the serrade lia end lupines, but exerted a good
effect upon the peas, vetches and clover as will be seen by the
table. The lupine soil infusion was also ineffective on the

non- leguminous plents but exerted a beneficial influence upon the
10.
growth and assimilation of nitrogen by all the legumes employed.

Its effect being doubtful only in the case of clover."# This
painted to the important fact that the leguminous plants stood

alone in this peculair power to secure nitrogen from the air.
The non-leguminous plants in the same pot with the leguminous
plants grew until the nitrogen in the seed was consumed, then

died.
To prove beyond a doubt that this power whibh some plants

have to obtain nitrogen from the air was due to the action of the
organisms introduced in the soil infusion, Hillriegel conducted
two experiments. In the first he sterilized the infusion before
applying, by heating to 70° c. and found that there was no in-
crease in nitrogen as is shown by the following tabulated results

of an experiment with lupine in 1888.

 

 

Dry matter |Nitrogen acquired
Nos. Treatment produced from the air.
#1 Soil infusion sterilized 0.926 grat -0.007 grams
2 Soil infusion sterilized 1.008 " “0.007 "
3 Soil infusion not sterilized] 42.681 " Ll. 147 "
4 Soil infusion not sterilized] 40.574 " 1.054 "

 

 

 

 

 

In the second experiment Heliriegel grew peas in such a way that
the roots of each plant grew intwo pots, about half in one and
half in the other. The soil in both pots was sterilized by
boiling and received the same kind of treatment except that one
of the pots was inoculated with soil infusion which was not ster-
ilized, while the second pot was treated with the same amount of
the infusion but which had been previously sterilized by heating.

# W. @. Atwater, Exp. Sta. Rec., Vol. V. No. 9.
# E. 8. Rey Vol. Ve No. 9, P. 844 by ¥. 0. Atwater.
 

 

11.
This experiment was repeated several times and in every instance

but one the half of the roots grown in the soil affected with
living organisms were well supplied with tubercles while the other
half of the roots were destitute of tubercles in every case.

The plant which failed to develope tubercles died.

It was now apparent to Hellriegel that there was a
direct relation between the assimilation of a&smospheric nitrogen
and the formation of tubercles. Where no organisms were present
no tubercads were formed,and where no tubercles were formed no
atmospheric nitrogen was fixed by the plant. It still remained
for Hillriegel to determine whether the source of the nitrogen
thus acquired was the combined or the free nitrogen of the air.

He grew plants in a closed giass vessel so arranged that only free
nitrogen was admitted to the plant.# , The nitrogen free sand was
inoculated and on June 6, one pea was planted. The rapidity of
growth and gain of nitrogen will be seen from the following fig-
ures which show the weight of the plant dried at 100° c.

Seed 0.376 grams
First cutting, August 31

 

Vines 6.173"

Vines 2.320 grams
Second cutting, October 4

 

Roots 1,290
Total 10. 189 "

# In this experiment, the air in the vessel at the begin-
ning was not analyzed or purified and therefore contained
@ small amount of combined nitrogen. The amount of com-
bined nitrogen in 100 liters of air (the amount which the
vessel held) has never exceeded 1 mg. at the station.
This error was avoided in the subsequent experiments and
like results were obtained.
Nitrogen Ba lance.#

Combined nitrogen in the air of the vessel at .
beginning, less than,.........--ceee.- or.-eeee QO.0001 grams

In the BONG... ccccccccc ccc cece cree cece ccc cceeee 0.0000 "
In nutritive solutions and twice distilled "

WOTEP ccc ccc cccrvcccccccevcsescecvccccscse Ve0000

In the s0il Infusion... .cccccceresesercssces Je0008
In the BECA,. cece ccc ccc ccccccccvvccsccsccee Os008L "
Total nitrogen supplied.........--.cseeee. 000084 "

Nitrogen found at the end of the experiment.

In the pea plant... ... cc ee cecccccccece0eBO0D grams
In the BOLL. cc ccc ww ccc ec cccce seers esscecs 0.0807 "
Total found..............5.-. ese voetcerne 0.2542

TOtal Supplied. .ccccsssccsvevccccsvscceer 0.0084 "
Gain. cccccccccccccccevrecceeeccceee 0.2458 "

The gain was 0.2458 grams of combined nitrogen, for which
the free nitrogen of the air was the oniy source, Leurent and
Schiloessing’ recent experiments fully confirm these results.

In their experiments a known amount of free nitrogen was allowed
for the plant. At the end of the experiment it was found that

the plants gain in nitrogen was the air's loss. Thus after
years of"patient scientific thoroughness" Professor Hellriegel,

Director of Bernberg Experiment Station announced the results
of “certainly the most important discovery for agricultural
science."

The one fundamental truth that some plents under certain
conditions could utilize atmospheric nitrogen has never been dis-
puted by the many experimenters who have entered the field since
Heliriegel announced his results, Meny subsidiary questions,
though questions (of much importarice:. have arisen and been widely
discussed. The discussions though active have not been bitter

# P. 847, BE. 8. Re, VOl. V. NOW 9-
13
and all agree that much remains to be worked out.

Where does the fixation of nitrogen take place? Is it
in the soil or in the plant? It has been claimed by Berthe lot
and Andre and others that the fixation of nitrogen must first
take place in the soil by means of organisms and electrical ac-
tion . Without denying the statement of Berthelot and Andie
that nitrogen may be fixed in the soil, we have abundant evidence
thet nitrogen thus fixed is not the only source of nitrogen for

plants, if indeed it is the source of any considerable amount of
it. The results of analyses of the soils by Lawes and Gilbert

end SchAiocessing and others where plants had accumulated large
amounts of nitrogen showed no gain of nitrogen in most cases.#

If the nitregen was first accumulated in the soil it must have
been taken up by the plant eas fast as formed which is not a rea-
sonable supposition, | Again in Hilitriegeil’s experiments where

he grew 9 sorts of plants, 4 non- leguminous end 5 leguminous,

the nitrogen if formed in the soil would have been available alike
to both classes of plants, but the non-leguminous plants in the
same pot with the leguminous pilents failed to secure any nitrogen.
It can hardly be supposed that if the nitrogen was formed in the
soil that the leguminous plants were better able to secure it than
the non- leguminous. This is contrary to all experience.

It is well known that the application of nitrogenous fertilizers
produce a much greater gain in the case of non- leguminous, than
with leguminous crops.

# In several instances there is unmistakable evidence of a
small gain in the soil which will be spoken of later. ,
14.
The question erises if the fixation of free nitrogen
takes piece in comection with the plant as we must believe that
it does, where is the important function performed? No less an
authority than Professor Frank of Berlin who hes written more upon

this subject than any other scientist oleims that the fixation
takes place in the cell protoplasm through"a powerful act of the.j
machinery of the leguminous plant , urged to the necessary expen-
diture of energy by the stimulating action of the organisms in

}

the roots." This view is also held by Prasmowski, Hil'riegel and

others. Frank claims that other plants than legumes. are able
to assimilate nitrogen, but that the leguminous plants have the

power in a greater degree than non-leguminous plents, which is due
to the stimulating action of the orgen isms in the tuberctes of

the roots. To show that the assimilating action is not due to
the tubercles, professor Frank gives the results of experiments
which in his opinion show that other plants such as oats, potatoes,
mustard, spurry, turnips, buck beans end norway maple are capable

of fixing free nitrogen
We cannot avoid a feeling of doubt as to the reliability

of Professor Frank's results ,since his experiments were mostly
conducted in the open air. The plants were simply sheltered

from rain, and were accessable to the combined nitrogen of the air.
Professor Prank gives us the general conelusions but fails to
support them by giving an account of methods, and results obteined
by ene lysis. Not only are the results of Boussingault and Lawes
and Gilbert's experiments of thirty five years ago,entirely con-
trary to Frank's results, but the more recent and" exceed ing ly"

carefully conducted experiments of Laurent and Sch ioess ing, show
 

=<

15.
no fixation of nitrogen in oats, tobacco, cress, mustard, cabbage,
Spurry and potatoes. Many of these are the very same plants
Frenk experimented with.

Schicessing and Laurent went further, By an ingenious
contrivance they managed to grow leguminous plants so that no
nitrogen was accesshbie to the roots of the plants, The leaves
were left exposed to the free nitrogen of the air. The soil
was inoculated with organisms. The cell protoplasm of the
plants had every opportunity to fix the free nitrogen of the air,
put in every case the plants died for the want of nitrogen. on
the other hand, where the roots had access to atmospheric nitrogen,
tuber les were formed and the plants fixed nitrogen. Again when
the conditions were reversed and the atmosphere about the leaves
was deprived of its nitrogen, hydrogen being substituted in its
place, the plants developed normally showing that the nitrogen was
assimilated in connection with the roots. The results of
Schicessing Sons and Laurent are confirmed by Kosch and Kossowitsch

later.

professor Frank answers by saying that plants must be
very vigorous and near the "maturing point before they have power
to energetically seize and fix the atmospheric nitrogen.” But
as we have already seen, this will not apply to leguminous plents
which have the power to fix free nitrogen at an early stage in
their development. In the case of peas, Lawes and Gilbert found

that within twenty days after plenting, nitrogen was being
_ . . - ~~ ——-

16.
assimilated. It is further shown by the analysis of Lawes and
Gilbert that during cerfein stages of their development, the tuber-
cles contain a much higher percent of nitrogen than the other
parts of the plant, and in some cases higher than the highly
nitrogenous seeds.

Professor Frank admits this, but claims that the ine
creased amount in the tubercies is not sufficient at any time to _
account for the large gaim in the plant, which is taken from the
air. He further contends that if the nitrogen fixation takes
place in the tubercles alone, they must yield a gradual supply
to the plant, a supposition which he claims has no advocates.

We will grant for the present that no nitrogen is formed
in the tubercles wntil they have reached a certain stage in their
deve lopment. But Professor Frank's argument looses much of its

force when we remember that the tubercles on the growing jpleant, .are
in various stages of development. I found the tubercles very .

much more uiform in development during the month of May and ear ty
gune than later, but even then in most cases there were plenty of

\

young tubercles just forming by the side of those which were

-_~

'
three and four weeks old. At the present time (July 25) it

would be difficult to find a plant from April seeding ,that does
not contain both the newly formed tubercles and those which are
being absorbed.

While there is diversity of opinions among those who
have given the matter much attention, yet the evidence at hand

strongly indicates that the fixation process takes place in the
17.
tuberec les. But whether the protoplasm or the organism is the
chief factor in the process is by no means settled. HH. Marstiiat™ .
Ward favors the protopasm theory of Frenk, but does not agree with
him that the protoplasm in the cella outside the tubercles and
in non-leguminous flowering plants have this power. Werd's modi-
fication of Frank's theory briefly stated is to the effect that
the protoplasm: in some way fixes the free nitrogen through the
stimulating effect of the organisms. In proof of this theory
M. Ward cites the wonderful powers which protoplasm is adinitted
to have of disorganizing and reorganizing the materials of fiant
food. He thinks if not wreasonabile to go a step farther and
suppose that the protoplasm can in some way force this"notor ious ly
inert"element (nitrogen) into combination with other substances,
especially when urged to such greet activity as isshown by the
a lka Line
reaction of the tubercle contents.

M. Gonnerman in a recent article on the probable number
of organisms capable of forming tubercles says ,"it seems probable
that the plant itself without symbiosis can take up and assimilate
free nitrogen; the bacteria may, however, assist the plant in
contributing to its higher nitrogen content, "#

On the other hand there is some evidence that the organisms when
not in contact with the protoplesm can fix free nitrogen.
Berthelot claims to have established beyond a doubt that several
species of soil bacteria,as well as the organisms of leguminous
tubercles cultivated separately ,have this power. In one case
there was an increase of 50 percent. Beyerinck while regarding

# E. 8. R. Vol. V1. No. 9, P. 784.
 

18.
it as probable that the nodule organisms fix atmospheric nitrogen
admits that he does not prove it. Laurent and Immendorf both

failed to satisfy themselves that the organisms can flourish

without organic compounds of nitrogen. Lawes and Gilbert think
that the fixation is probably due directly to the organisms, end
"af this should eventually be established, we have to recognize
a new power of tiving organisms--that of assimilating an elemen-
tary substance."# "Neither experience in practical agriculture,
nor the nitrogen statistics of soils and crops, points to the
to any material extent

conclusion that there is a gain of nitrogen, under the agency of
microbes within the soil independently of leguminous growth. "#

It is known that the organisms do not fix nitrogen in
the nitrogen-free sand cultures, but this is not proof that they
might not fix nitrogen in rich soils where other substances could
perhaps take the place of the protoplasm in the tubercies. on
the one side, the fact that the pisnt az the absence of the organ-
isms fixes no free nitrogen, and on the. eide, the evidence that
little if any is fixed by the organisms in the soil, strongly
indicates that the organisms and the protoplasms are both dssen-
tial factors in the process, but the part played by each is un-
known.

and the plant

The relation between the organism,seeme to be one of
true symbissis. However during the early stages of nodule for-
mation the action is large ly parasitic, the organisms deve loping
at the expense of the plant, as is shown by the pale yellow

appearance and arrested growth of the plants in the sand cultures.

# Jr. Royal Agricultural Soc. P. 695-692.
19.

From recent experiments it would seem that leguminous
plants are not the only ones which can assimilate free nitrogen.
H. Marshall Werd in an article on Fixation of nitrogen "¥ seys,
“the experiments of Nobbe, Schmid, Hiltner, and Hott er show that
Eleagnus plants, the roots of which develope nodules due to the
invasion of a funguss totally differnet from the one causing the
leguminous nodules, also fix and assimilate the free nitrogen
of the air, as shown by their growing and fiourishing much better
and more rapidly than Eleagnus plants side by side with them,

but not infected with the root organisms." "It will be inter-

esting to see if further research shows similar resuits with any
of the physiologically similar root-growths, due to very different
fimgi, met with in Taxodium, Podecarpus. ainus, Juncus and many
other plents.” In reviewing the more recent works of Nobbe and
Hiltner, Walter H. Evans who is at the head of the Department

of Botany and Diseases of Plants, says # "the ability to assimilate
the free nitrogen of the air as possessed by tubercle -bear ing
plants such as legumes, Alders, Eleagnus, Podocarpus etc. is
recognized." Nobbe and Hiltner further claim that only those
plants which show an increased nitrogen content in the leaves

end stems above ground are able to assimilate free nitrogen.

There is now little doubt that some of the alg&ut have
the power to fix free nitrogen when affected with the proper
bacteria. It has been observed by Hilliriegel and others that
when the nitrogen free sand cultures became affected by an algug

# Nature Vol. 49, P. 613.
# E. S.R. Vol. V1, P. 381 1895.
£0.
growth there was ea small gain of nitrogen in the soil. professor

Frank has held this view for some years and the recent experiments

of Laurent and Schioessing, shows that not only were the green
algee able to fix gasseous nitrogen but that some of the mosses

possessed this power in a marked degree. The still more recent

experiments of Kosch and Kossowétsch who repeated this work with
green .and blue-green alg&éé, using purely inorganic solutions con-
firm the results of Laurent and Schioessing. Later than this
Kossowitsch arrives at a somewhat differnet cone lusion. This
time he was able to separate the alguw from all bacteria and se-
cure pure cultures. There was no gain of nitrogen in any of
the entire series of experiments, but when they were mixed with
8011 bacteria and fungi there was in some cases a considerable
increase of nitrogen.

Are there few or many tubercles forming organisms?
Bearing upon this question, are the interesting observations ofr
Professor Bolley # relative to the distrébution of tubercles on
native and introduced leguminous plants of the Dakotas. He ex-

amined a great number of plants and everywhere found the native
plants well supplied with tubercles, while many of the introduced

plants bore no tubercles, This was particularly the case with
é¢ommon red clover. He claims, however, that red clover thrives
and forms tubercles when it is preceded by white clover, which
does well in Dakota and never fails to be supplied with many
tubere les.

In the case of two tropical legumes grown in France by

# Agricultural Science 7, 58, 1893.
21.

C. Naudin, no tubercles were formed while several species of
Australian plants bore tubercles in profusion, My own observa-
tions in 1892 and again during the present year confirm the attove
results in a general way. For examplegainfoin which thrives
well in England does very poorly here and seldom forms any tuber-
cles. Also tubercles are rarely found on lupine plants here
while they are very abundant in Engdand. On the other hand
several introduced famts as lathyrus silvestris and.hopse. bea
produce tubercies in great numbers.

Recently several series of experiments were conducted
by Nobbe, Schmid and Hiltner in pure quartz sand, with inocula-
tions of pure cultures from various legume: tubercles. In one
series,peas and common locust were inoculated with cultures from
peas, common locust , elfaifa, Vicia sepium, and Caragana arbo-
rescens, Tuberc les were formed on the peas from all the inocu-

lations, while on the locust only those receiving the cultures

from the tubercies of locust and Caragana produced any. In
another series lathyrus latifiolius was inoculated with pure cul-
ture from peas, vetch and locust. Only the first and second
produced tubercles. The tabulated results of a third series of
experiments by the same authors is shown below. in this exper-
iment, ‘ocust, Acacia lophantha, Yililous vetch, and peas were
inoculated by cultures of two-year-old tubercles of locust, two-
year-old tubercles of Caragana, tubercles of vetch and peas.#
The peas met with an accident in the last part of the experiment

and had to be omitted from tables 2 and 3.
# Experiment Station Record, Vol. V1, No. 6, P. 505.
time of

Transpiration from inoculation to harvest.

B22.

 

Inoculated with pure cultures from--

 

Robinia/Acacia | Vicia |pisum

Oc. ‘Cc. Ce. ‘CC.

Robinia pseudacacia........4 3,570 | 1,136] 1,425; 1,396
Acacia lophantha...........4 1,538 | 3,865 1,205; 1,611
Vicia villosa.....ccsecceeed 934 | 1,097 | 4,978) 1,277
Pisum sativum.........++..-4 1,380 1,034; 1,265; 1,849

 

 

 

 

 

 

Average Beight of plants at harvest.

 

Robinia..
ACACIMi cccccvcccvcccsececcece

 

 

 

ViCidecccccccesssrccrecvece
a

  

Inoculated with pure cultures from--
Robinia] Acacia! Vicia |Pisum
Mm. Mm. Mm. Mm.
131 50 50 $0
80 £295 6& 75
350 400 1, 126 450

 

 

 

 

Chemical analysis of plants.

 

 

Inoculated with pure cultures from--

 

 

Robinia] Acacia] Vicia | Pisum
Robinia dry substance grams 7.408 1.188| 0.858| 1.479
Robinia nitrogen..... Mg. (832.100) 16.600/| 13.800|21.100
Acacia dry substance, grams; 1.953) 6.945; 1,848; 1.817
Acacia nitrogen..... Mg. 17 .000109.800} 16.200/19.700
Vicia dry substance.. grams 883; .866/ 9.133] 1.033
Vicia nitrogen...... Mg. a 264.000 |22.600

 

 

 

 

 

"From the above table it will be seen that in all but

one case each plant was most favorably affected when it was inoc-

ulated with bacteria from the tubercles of its own species."

In a fourth series 81 different leguminous plants were inoculated

with pure culture bacteria from tubercles of peas and locust.

Of the 21 species inoculeted with bacteria from peas, only three,

the vetch, lentils and beans developed many normal tubercles.
23.
The crimson c lover and locust had a few scattering tubercles.
The remaining 16 species bore no tubercles. Of those inoculated
with locust bacteria only the locust gave good results. The
beans had many small tubercles and the red clover a very fuw
scattering ones. The other 18 species were wiaffected by the
inoculation.

These investigations, however, conclude that the “dif-
ferences between the forms is not sufficient to entitle them to

be ranked as separate species of bacteria and agree with Beyerinch
that there is but one species, Bacillus radiciola,which becomes
more or less modified by the different host pants on which the
tubercles are grown.

On the other hand Albert Schneider, Frank and others
have gone so far as to name several different species.¢ Their
Classification is based upon a microscopical study of the organ-
isms, both in the tubercles and in pure cultures.

Other evidence such as variations in shape, size, color
and markings of tubercles on different species of legumes, as
recorded by myself and others could be presented upon this question;
but the multiplicity of forms which have been found among the
bacteroids, together with the facts that they are constantly wm-
dergoing modifications and are under abnormal conditions, makes
it very apparent that the question of whether there are few or
many species, must remain unsettied until their life history has
been worged out.

The morpho log ice 1 data is most extens ive. Over one

# Torrey Bot'l. Qlub Vol. 19, P. 213.
24.
hundred investigators in more than two humdred papers have touched
upon this phase of the question. Among these are to be found our
greatest scientists, such as Frank,# H. Marshall Ward, Schloes-
sing, Laurent, Hiltner, Atkinson, Beyerinck, Sehmeider and others.
These papers together with the extensive drawings, represent many
years of work.

In the words of George F. Atkinson## of Oornell Univer-
sity;"The record presents a discouraging volume of conflicting
testimony. It would indeed be a misfortune should ail these
pains-taking and laborious investigations be so much at variance
as appears from the examinations of the contributions." When
more is known regarding the life history of the organisms, many
of the theories and apparent facts which at present seem to be

so mich at variance with each other, will no doubt be harmonized.

# Professor Frank alone has presented over twenty papers
on the question of the fixation free nitrogen.
## Bot'l. Gazette, Vol. 18, Pp. 257.
25.

REFERENCES.

# Atkinson,Geo. F... Bot'l. Gaz., Vol. 18, 1893, P. 157, 226,& 257.

Atwater, W. 0....... Conn. Agr'l. Rep’s. 1889-91-92.

Atwater, W. 0.....0+ Exp. Sta. Rec., Vol. V, No's.8-9.

Bolley, H. Le......- Agr'l. Science, Vol. V11, NO. 2, 1892.

Comm, H. Wee.oee-eee Amer. Naturalist, Dec. 1892.

de Chalmont, Geo.... Agr'l. Science, 1895, Vol. V111, P. 471.

Evens, W. H.......+. Bxp. Rec., Articdes reviewed by
Caron, A. VOl- V1, No. ll, P. 966.
Frank B., Vol. VL, NO. 1, P. 1S.
Gonnerman, M. vol. 6, No. 9, P. 784.
Hiltner and Nobbe You. vi, No. 9, Pp. 381.
Kossowitsch, P. Vol. Vl, NO. 4, P. 278
Laurent, Es 1892-3.
Naudin, Co, Vol. Vi, NO. 5, P. 382.
Salfeld, Vol. V1, No. 6, Pe. 507-534.
Schioessing, 1892-3.
Schmid, E. Vol. V1, No. 6, P. 504.
Wilson, We Vol. Vl, No. 7, P. 616.

Frank, B.-+o-+ssceoee TeXt BOOk of Botany, 1892-3.

Gilbert, J. H......- Lecture Nov. 1, 1889, Roy. Agr'l. College.

Johnson, J. F. We... Johnson's El. of Agr'\. Chem.

Kedzie, R. C.....-.. Mich. Agr'l. Rep., 1881-2, P. 380.

Lawes, J. Beowseceoe Je ROY. Agr’. Soc., 3d Series ,Vol.11,P.657.

MileS, M.-.ccccseeee POP. Science Monthly.

Russell, H. Leesee.+ BOt'l. Gas., VOL. X1X, 1894, P. 284

Schneider, A......+- Torrey Bot’. Clubb, 1898, Vol. X1X, P. 203.
Tl. Exp. Sta. Bul. NO. 89, 1893, P.SOl.
Amer. Nat., Sept. 1893.

Ward, H. Mee.-..-ee4 Phil. Transactions Roy. Soc. 1887.
Proceedings of Roy. S8oc., 1889.
Nature, Vol. 49, 1893-4, P. 511.

Warrington, R.....+.U. S. Exp. Sta., Bul. No. 8, "1892.

Woods ,C. Desecscecves Storrs School of Agr'1., Bul. No. 5, 1889.
Conn. Agr’l. Rep., 1890, P. 44.

~ Jumelte..... cocoeee REVGU General De Botanique, 1895.

# The above references are to papers which I heave consulted
in preparing this thesis. For a complete list of ref-
erences to January 1, 1894, see Bulletin No. 29 of I1li-
nois Experiment Station, P. 311, by Albert Schneider.
—.

se | we