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Reprinted from Sorin ScteNcE
Vol. XIII, No. 4, April, 1922

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EFFECT OF DIFFERENT REACTIONS ON THE GROWTH AND
NODULE FORMATION OF SOYBEANS!

O. C. BRYAN

Wisconsin Agricultural Experiment Station

Received for publication July 20, 1921

Although it is well known that soil acidity and alkalinity have a marked
effect on cultivated plants, especially certain legumes, yet, it has never been
definitely determined at what reaction the legumes grow best and become
inoculated most abundantly. Whether the favorable range of reaction is wide
or narrow, and what this range is for each of the legumes are very important
questions from both a practical and a scientific standpoint. In order to gain
further information on these questions, the present investigation was under-
taken.

The soybean was used in the beginning because of its adaptation to water
cultures and because of its importance as a crop. Several different strains of
soybean bacteria were studied. For comparison, some results with corn and
cowpeas obtained under similar conditions are reported herein.

An exhaustive discussion of the literature on this subject does not seem
necessary and only a brief review of some of the more important papers is
given. The magnitude of the hydrogen- and hydroxyl- ion concentration of a
number of soil solutions and suspensions have recently been determined by
Gillespie (15), Sharp and Hoagland (35), Plummer (32), Truog (37), and
others. These investigations have shown that soil solutions have a wide
range of reactions, namely, from pH 3 to pH 9.

The importance of the effect of the soil reaction on the soil bacteria is well
recognized. Gruzit (14) reported that an alkaline reaction was more favorable
for the growth of the bacteria than an acid, or even neutral reaction. Salter
(33) reported that red clover bacteria do best at a neutral or slightly acid reac-
tion while the alfalfa bacteria do best at a slightly alkaline reaction. Fred
and Loomis (11) reported that an alkaline reaction (pH 7.72) produced maxi-
mum growth of the alfalfa bacteria. Fred and Davenport (12) reported that a
correlation exists between the acid resistance of the nodule bacteria and the

1 Part I of a thesis submitted at the University of Wisconsin in partial fulfillment of the
requirements for the degree of Doctor of Philosophy.

Published with the permission of the Director of the Wisconsin Agricultural Experiment
Station. 3

The writer wishes to express his appreciation for the helpful suggestions and criticisms
tendered by Professors E. Truog and E. B. Fred under whose direction the work has been
done. “h

271

272 O. C. BRYAN

acid resistance of the higher plants. Bewley and Hutchinson (2) reported
that some of the legume bacteria are either killed in definitely acid soils, or at
least lose their activity, while Hiltner (21) claimed that liming has an in-
jurious effect on lupine bacteria.

The sensitiveness of plants to the reaction of dilute solutions of acids,bases
and salts was noted by Kahlenberg and True (26), Heald (20) and Loew (29).
These investigators studied primarily the direct chemical effect of reaction on
seedlings and not the influence on growth, and hence did not use balanced
nutrient solutions. Because of this condition definite conclusions can not be
drawn from their data regarding influence on growth. Their data show, how-
ever, that seedlings differ widely in their behavior toward reaction.

Cameron and Breazeale (4) reported that corn was much more acid tolerant
than wheat or clover. Hartwell and Pember (18) claimed that some of the
cereals are more sensitive to acidity than others but no statement was made
as to the relative sensitiveness. Hoagland (23) reported that barley seedlings
grew better in a slighty acid reaction than in a neutral or alkaline reaction.
This was also reported by Salter and MclIlvane (34) in working with corn,
wheat, soybeans and alfalfa. The data of the latter indicate that alfalfa is
more sensitive to acidity than corn, wheat or soybeans. Joffe (25) reported
that alfalfa produced good growth in soil cultures which were made very acid
(pH 3.8) by the addition of sulfuric acid. Hixon (22) noted that there was a
difference in water content, organic matter, and total ash in the roots of
16-day-old wheat seedlings grown at different reactions.

Hartwell and Pember (18) and Dachnowski (10) claimed that the hydrogen
ion was more toxic to plant growth than the hydroxyl ion. This question was
investigated by Hoagland (23) who reported the opposite results with barley
seedlings. These contradictory results are possibly due to different methods
of determining the reactions. Whether total acidity or hydrogen ion con-
centration is used as a criterion for indicating reactions is of great importance.
Other factors, e. g., kind of nutrient solution used and proper maintenance of
the desired reaction, are also important.

The change in reaction of the nutrient solution in contact with growing plant
roots was noticed by Hartwell and Pember (18) who reported that an acid
solution tended to become alkaline. This was also reported by Breazeale and
LeClerc (3) who claimed from their work with wheat seedlings that the change
in reaction of the nutrient solution was due to the selective absorption of the
ions by the plants. The buffer condition of the nutrient solution and the fre-
quency of renewing the solution are no doubt important factors in maintaining
a constant reaction. The change in reaction of the nutrient solution in
contact with plant roots during 1-day periods was found by Hoagland (23)
to be quite considerable provided the initial reaction was not favorable.

Salter and MclIlvane’s (34) results indicate that their nutrient solution
remained fairly constant over a period of 4 days when in contact with grow-
ing plants. Duggar (9) concluded that, in general, an acid or an alkaline

EFFECT OF REACTION ON NODULE FORMATION 273

solution tended toward neutrality when in contact with growing plants.
However, this change depended on the nutrient solution used and the rate of
plant growth. The most favorable reaction also varied with the. kind of
nutrient solution used.

In connection with the influence of reaction on plant growth, it is well to
mention the toxic effects of soluble aluminum salts as reported by several
investigators. Abbott, Conner and Smalley (1) reported that the toxic
effect of aluminum nitrate was about the same as a solution of nitric acid of
equal normality; and that water extracts from acid soils containing soluble
aluminum salts were as toxic to plant growth as a nutrient solution containing
an equal amount of aluminum salts. Hartwell and Pember (19) found that
aluminum salts were considerably more toxic to barley seedlings than to rye
seedlings. The toxic effect of acidity alone was about the same on both
plants. They concluded that lime may precipitate the aluminum and thus
be of value in this way as well as in neutralizing the acidity. Mirasol (30)
reported that aluminum salts which could be extracted from the soil with a
solution of potassium nitrate were probably the cause of the unproductiveness
of three acid soils of Illinois. Conner (6) reported that much of the harmful
effects of acidity of soils is due to soluble aluminum salts; and the presence of
abundant lime or phosphate will prevent this harmful effect. He noted that
some plants are more sensitive to aluminum salts than others. This condi-
tion was also noted by Hartwell.

Undoubtedly, in very acid soils aluminum, manganese and other toxic sub-
stances go into solution and produce injurious effects on cultivated plants.

The results of the various investigations may be summarized as follows:
The range of reaction of different soils is sufficiently wide to give conditions of
acidity and alkalinity in some cases which are unfavorable to bacteria and
higher plants. The different legume bacteria vary in their behavior toward
reaction, and the degree of the acid resistance appears to be in the same
direction as that of the host plants. In the case of plants, there are some
contradictory results, but, in general, they indicate that plants differ in their
behavior toward reaction. This is in line with field observations. In studying
a problem of this nature, there are a number of factors which are difficult
to control and the conflicting results obtained by the different investigators
are perhaps due in part to the difficulty of controlling the reactions, and in
some cases to the use of unfavorable nutrient solutions.

In regard to the toxic effects of soluble aluminum and other substances in
acid soils, it appears that plants vary in their behavior toward these toxic
substances and that the harmful effects of acidity in some soils are due in
part to soluble aluminum salts.

GROWTH OF DIFFERENT STRAINS OF SOYBEAN BACTERIA AT VARIED REACTIONS

Since some investigators (2) had noted that many of the legume bacteria are
either killed, or, at least rendered inactive in acid soils, it was thought best, as

274 O. C. BRYAN

a preliminary experiment, to determine the critical hydrogen- ion concentra-
tion of the various strains of soybean bacteria in pure cultures and also to
determine if there is any marked difference in behavior of these strains of
bacteria at different reactions. By strains of bacteria, is meant a pure culture
isolated from a known variety of soybeans.

Twenty-one? strains of soybean bacteria were grown on mannit, on sucrose
and on soil-extract agars. The mannit and sucrose agars were prepared
according to Ashby’s formula as follows:

IRRPRANTE CRIMEA oes sc inte hats tomtans cookin siete Pein ne Vacs ele Lew 15.0 gm
Ne A Po es la pc PS eke ae eT Oa ONO C oiniodig Bees tne 0.2 gm
KH2PO, OC Cece ee aes eve eee mee weet essence errr eeeernerecerene 0:2 gm
INSURES Roa Genttnce ge dale CEL, SRE) SSE OEY, Sele Lea a MSHA ae oi Lak oh 0.2 gm
CODED: «5h ihte ok 7 (craliaigic MSA 6 Seite a Wransek be o dlls AER aioe ictovas 0.1 gm
PRN ei ec kee ak lots. sh Rd teape anes ebiceta Metab baled is bia bis ohsiaieha ns 15.0 gm
DEAR LOE Mii Urol. x hs Kava Reet Pts oo eat EROS OMe Clea See REED 1000.0 cc

The soil extract agar was prepared by diluting 100 cc. of soil extract from a
silt loam soil to 1000 cc. and adding 15 gm. each of mannit and agar. The
range of reaction used was from pH 3.3 to pH 10 with the sucrose agar and
from pH 3.3 to pH 7 with the mannit and soil extract agars. The desired
reactions were obtained by adding varied amounts of sterile sulfuric acid and
sodium hydroxide, as the case required, before the media were allowed to
solidify in slants. The Clark and Lub’s method for colorimetric determina-
tion of the reactions of solutions was employed. Each culture was inoculated
with one drop of suspension containing the bacteria. ‘Triplicates of each
reaction were used in all cases. The cultures were incubated 15 to 20 days
at 28°C. before final results were recorded.

In general the mannit and soil extract agars showed a more vigorous growth
than the sucrose agar, although the three media showed only minor differences
in critical pH for the various strains of bacteria. There were small differences
in growth of bacteria between the reactions pH 7 and pH 6.5 but an increase in
hydrogen-ion concentration from pH 6.5 to the critical concentration produced
a gradual decrease in growth with each strain of bacteria. Eight strains of
the bacteria were grown in the alkaline range, all of which grew at pH 10.
The maximum growth of the eight strains grown in the alkaline range took
place at reaction of about 7.6. Table 1 gives the critical hydrogen-ion con-
centration for the different strains of soybean bacteria studied. By critical
hydrogen-ion concentration is meant the reaction at which the bacteria do not
produce any visible growth during the 15 days after inoculation. It will be
noted that there is not a great differencein the critical hydrogen-ion concentra-
tion for the various strains of soybean bacteria.

? Fourteen of the different strains of soybean bacteria studied were furnished through the
kindness of the Bureau of Plant Industry, of the United States Department of Agriculture
One strain was furnished through the kindness of Dr. A. L. Whiting, University of Illinois.

EFFECT OF REACTION ON NODULE FORMATION 275

TABLE 1

The critical hydrogen-ion concentration for soybean bacteria on mannit, sucrose, and soil
extract agar

STRAIN OF BACTERIA NUMBER CRITICAL pH VALUE
| AST 51) eR Se VOL peg AERO Ws Ae pe ION Seer” WUE 118 4.2
RAMI Chie -- se pchens. Jaya o-.csseak Rac tweE Siaaas oteL LGe Cale ie eRe 293 4.2
SACD GGlsareisteraels: srsrs onctoie eienicereieiar so eiatn can rattan felons 218 4.6
eS ER ee Ee dual ont ib get as A pat ai Alc a 271 4.6
ROTO U Hes ache cee Misha bike MOREE NE Sele es Sete eae er 270 4.2
Comelleyiaut cist MAA aren Ae Meee. HAUT ss eats 150 4.5
IPA DORLAN GG: (sis ccovaneetePrsyeliee mate Romer omrcrh sto vars ore 210 4.0
Pre DeRAI baiis, titi Sse eyeamich ciauh bia etme a slab calarsah aio’ 334 4.0
ERODE Ae ee ch crass crexctourehosarattton Sra revs veee chaaete vere ra kavatetccnvetelaiejs jacakeyas 337 4.0
SUA GDh gt cl acc Mi et AA fi Ne Pa OSE Ba ea a ek 312 4.7
Manchu, Wisconsin Experiment Station............... 154 4.6
Medium early, Illinois Experiment Station............. 4.0
Mam mobcbtvell ow ateeacrs cimistonesdepsie tan ot ienen ass slatted 338 4.2
PINGS tee ee Sapo ale Pe cea ee teh Pee ci ERE OA | 275 4.2
ROOSE VE bier.) sratcta-urs tele rorereis fois erase esha neaiereie ake tastels ciel sisi * 181 4.6
BR OOGE VEL bis cisrere: Seep sir na apaie Gunde aren spe cue hed aisss Hever 187 4.0
Seyi Oey SO Sa on OA ee ee ete 152 4.6
Maliater glen fSGsae dheies CNSR Mel 0S POAT cele wl aaa s 233 4.4
TAD winnie vetth Cen uate: «atest is onpepr eestor watt «abei“a 256 4.6

PLANT CULTURES
Methods

The plants were grown in solution and sand cultures in the greenhouse from
March 1 to July 25. The culture vessels used were 500 and 600-cc. percolators.
These were provided with glass tubes and pinch-cocks at the bottom in order
that the solutions might be easily removed without disturbing the plants.
In the case of sand cultures, the percolators were supported by iron {ring
stands and wrapped in heavy brown paper to exclude the light. Those con-
taining solution cultures of soybeans were placed in boxes and surrounded
with moist sawdust to exclude light and prevent rapid changes of temperature.
The glass tubes extended through holes in the boxes in order that the solu-
tions might be removed readily. A few solution cultures of corn and cowpeas
were also set up. For holding these percolators, circular holes were cut of such
size in the top of a greenhouse bench that the percolators passed through to the
rim around the mouth which acted as a support. The light was excluded
from beneath the bench.

The most desirable nutrient solution for this type of investigation was un-
known at the start. A modified form of Shive’s three-salt solution was first
used with the soybeans. The modification consisted in reducing the amount
of Ca(NOs)2 to one-fourth that recommended by Shive, and adding an equal
amount of CaCle. This made a total reduction of calcium to about one-half

276 O. C. BRYAN

that in the regular solution. The chloride was substituted for the nitrate,
since large quantities of nitrates are known to hinder nodule formation. The
reduction of calcium was made to reduce the precipitation of Caz (POu,)2 in
the alkaline solutions. This modified solution proved unfavorable for nodule
formation of soybeans in solution cultures. Shive’s regular solution with
varied amounts of nitrates was then tried, but again no nodules developed. ©
Several other nutrient solutions, namely, Pfeffer’s, Hopkins-Pettit’s, Crone’s,
and also Mendota Lake water were tried with the result that only in Crone’s
nutrient solution and Mendota Lake water were nodules produced. Crone’s
solution being the most satisfactory, was chosen with slight modification for
the work reported herein. The modification will be given later.

The reactions used in all cases were approximately pH 3, 4, 5, 6, 7, 8, 9 and
10. For specific reactions, see table 6. The Clark and Lub’s method for
colorimetric determination of reaction of solutions was employed in making
the adjustments. The standards were checked at intervals with the hydrogen
electrode. In order to maintain the reaction of the nutrient solution, in
contact with plant roots, as constant as possible the solutions were renewed
daily.

For the sand cultures, 20-mesh (Ottawa silica) sand was used. This was
first thoroughly washed with distilled water. The sand was then placed in
the percolators and washed with the respective solutions until the reaction
remained constant on passing through the sand. The solution in sand cultures
as in solution cultures were renewed daily. This was done by means of
suction as sugested by McCall (31). The percolators were ideally adapted
for changing of solutions.

The seeds were germinated in clean quartz sand and allowed to grow 3
to 5 days before being transferred to the percolators. Two seedlings were
grown in each percolator both in sand and solution cultures. The seedlings,
in case of the solution cultures, were held in place by means of paraffined corks
and plugs of cotton. In order that the seedlings would not be subjected to a
great change in reaction at once when being transferred to the percolators, the
acid and alkaline reactions of the nutrient solution were brought to the desired
points gradually over a period of 2 days. The solutions were inoculated
3 days after the seedlings had been transferred to the percolators. This
was done by placing the inoculum in the nutrient solutions at the time the
solutions were renewed. The plant cultures were grown for 25 to 35 days.

Growth of soybeans in Shive’s nutrient solution at different reactions

As previously stated, a modified form of Shive’s nutrient solution was first
used in both sand and solution cultures. It was prepared from the following
stock solutions in which the amounts of salts are indicated on the anhydrous
basis.

EFFECT OF REACTION ON NODULE FORMATION 277

(a) PCAC HOMO A ROR ER RES SEI ted. A AR RRE 50.0 gm
Gal(NOs)ietigasats oshsconia. Eeddanatstadecies oaslds uch eather. 50.0 gm
ID ISt edi Watene ce eer i Mn Ar Whe eta a Rote bale oe 500.0 cc
GER EC chore Caw Ssaretece Sages. ¢ DA area TERM ate the aiiterate Mie tie bie We Ee 50.0 gm
Distilled! water! /MIUE RTOS Aer hae BLE AA ae Nk eo ts 500.0 cc
(@) Ge ReHiBO ps cd Seas sie onl el eee apres Uh aR eitees fit. « oacreRIe 50.0 gm.
Dustilled) water. '3.c Pesos ter aot ec cae PE ake Ste ae eh nonears 500.0 cc.
(REG eco sven c crnhicie rae aecaietorneiieie ole Tae slate rake 6, cterwicke taletay achereiataraeae 2.5 gm.
rstiled sweaters ..cie sac ane sn actoteiis wc since ceaiataiee ate a easier eee 300.0 cc.

Ten cubic centimeters of (a), (b), (c), and ten drops of (d) were added to
4 liters of distilled water. Dilute sulfuric acid or sodium hydroxide was added
to portions of this solution until the desired reactions were obtained. The
general method of procedure already outlined was followed. The plants were
allowed to remain in the solution 24 days from time of inoculation.

TABLE 2
Growth and inoculation of soybeans in sand cultures with Shive’s nutrient solution at different
reactions
REACTION OF CULTURE NODULES PER PLANT PLANT DEVELOPMENT
pH
3.32 0 Tops fair
Roots dark and stubby
4.2 5 Roots better than at pH 3.2
5.0 12 Tops good
Roots slightly dark
6.0 35 Tops good
Roots good
6.9 49 Tops good
Roots good
8.0 11 Tops good
Roots slightly brown
9.0 2 Tops small
Roots very brown
9.9 0 Very poor

The results of all plants in all experiments at different reactions will be
referred to as at pH 3, 4, 5, 6, 7, 8, 9, and 10 although the actual pH value
may have been slightly more or less. For specific reaction with Shive’s
nutrient solution, see table 2.

Only two nodules developed in Shive’s nutrient solution and these appeared
at pH 8. A poor and injured condition of the plants indicated that something
was wrong with the nutrient solution. This may have been due to the presence
of toxic impurities in the salts used. Plants at pH 3, 4 and 10 were almost

278 O. C. BRYAN

dead. Sand cultures of soybeans with Shive’s nutrient solution were also
carried on according to the regular method already described. The results
of growth and inoculation with these are given in table 2. The best growth and
inoculation were obtained at pH 6 and 7. In contrast to plants grown in
solution cultures, the plants grown in sand cultures showed no injurious effect
other than that due to reaction. The plants at pH 3, 4 and 10 had very dark -
roots and small tops. In general, a considerable increase in acidity or alka-
linity caused a decrease in plant growth. Perhaps the absence of injurious
effects in the sand cultures was due to adsorption of toxic substances from the
solution by the sand.

The effect of nitrates on nodule formation in Shive’s nutrient solution

Since practically no nodules developed in solution cultures with Shive’s
solution, it was thought that possibly the presence of nitrates prohibited
inoculation. To determine the influence of nitrates on nodule formation,
soybeans were grown in Shive’s nutrient solutions containing 2, 1, $, t, $, ze;
zs, and @; times the usual amount of nitrates recommended by Shive, and
also in one solution entirely without nitrates.

For culture vessels, 500-cc. wide-mouthed bottles were used, and two
soybean seedlings which had been germinated in clean quartz sand were
placed in each. The seedlings were held in place with paraffined corks and
plugs of cotton. The solutions were inoculated and renewed weekly. The
plants were allowed to grow for four weeks, during which time not a single
plant in any of the solutions developed any nodules. All the plants had
yellowish leaves and somewhat dark and stubby roots indicating that the
solution was toxic.

Influence of different nutrient solutions on nodule formation of soybeans

Since all attempts to get nodule formation on soybeans in solution cultures
had practically failed, it was decided to determine the effects of different
nutrient solutions on inoculation of soybeans. In addition to Shive’s solu-
tion, Hopkin-Pettit’s, Pfeffer’s and Crone’s solution, and also Mendota Lake
water were used. The cultures were carried on in exactly the same manner
as the previous ones. At the end of 4 weeks, the plants in Crone’s nutrient
solution and Mendota Lake water had developed a goodly number of nodules
and the plant growth was healthy. Those in Shive’s nutrient solution had
developed only two nodules and the roots were dark and stubby, and those in
the other two solutions did not develop any nodules at all. Their leaves were
also yellowish and the roots dark. Plate 1 shows the root development
and nodule formation in the different solutions.

EFFECT OF REACTION ON NODULE FORMATION 279

Growth and inoculation of soybeans in Crone’s nutrient solution at different
reactions

The principal experiment on the influence of reaction on growth and inocu-
lation of soybeans was next started with Crone’s nutrient solution. The
buffer condition of this solution was, however, unsatisfactory in the alkaline
range and for this reason several different substances, namely, di-basic sodium
phosphate, di-basic potassium phosphate, sodium glycero phosphate, and sodium
carbonate and bicarbonate were added and tested for their buffer effect, and
also influence on nodule formation. Of these, sodium carbonate at the rate
of 3 gm. per liter proved to be the most satisfactory, and hence was used.
The salts for Crone’s solution were ground to a fine powder and thoroughly
mixed in the following proportions.

TENA ia ssacwsdees octaves es odin Ohare Saat buat ww isiaessietula sis CHIP Ras ha wrote eee ora ok Peahvabsew 100
CAO Os agin od" oboe aie panera els aids Rivin Gared ws agai tSe pre ace a agate we he ae 25
POSTED, clawing ae Cotte same ae Maa waalels wae RIES HRW ats wale Pee o te eso 25
Ca3(PO,)2 aiatetelaieiate) ales) wid iekealaiuleiekarel eis: ola) siwlisinatal's) «felw satel svalelalviayel s\© wpaja vieiel ele spy tals\.e 25
FePQ, BOOP TC CO Meee ene eK eee need eres seeder ec ececeasees ence rescece 25

For the solution, 12 gm. of this mixture were added to 8 liters of distilled
water. The mixture was well shaken with the water and left to stand for one
day, at end of which time the solution was filtered to remove the insoluble
materials. The acid reactions were obtained by adding varied amounts of
sulfuric acid. The alkaline reactions were obtained by first adding the sodium
carbonate and then acid until the desired reaction existed.

The plants were grown in percolators according to the general method
already outlined. Good plant growth took place at the favorable reactions.
Tn all cases duplicates agreed as to nodule formation and root development.
Plate 2 serves to indicate the relative size of plants and nodules per plants
in solution cultures. It will be noted from plates 3 to 10, inclusive, that plants
at pH 3, 4 and 10 had no nodules. Plants at pH 4.5 which is not given in
figure 2 developed a few nodules, but at this reaction the roots were dark and
stubby. Plants at pH 5 had fair inoculation. The tops of the plants at
this reaction were as tall as those at pH 6, but were yellow and much less
vigorous. Plants at pH 6 and 7 were decidedly the best, both as to growth and
inoculation. Those at pH 6 were perhaps a trifle better than those at pH 7.
Plants at pH 8 had some nodules but the roots were slightly brown. The
roots at pH 9 and 10 were still browner and did not become inoculated. It
should be noted that plants at pH 9 were a little taller than at pH 8, but of a
very poor color. In all other cultures at pH 8 the plants were better than at
pH 9. Table 3 gives dry weight of plants and number of nodules per plant.

The experiment just described was repeated using sand cultures according
to general method already given. The same solution and reactions, were
used as in the previous experiment. The extent of nodule formation was
about the same as that in the solution cultures. The limits of nodule forma-

280 O. C. BRYAN

tion were pH 4 and 8. There was less injury from an unfavorable reaction
at pH 3, 4, 5, 9 and 10 than in the solution cultures at these reactions. This
was perhaps due to a smaller amount of nutrient solution actually in contact
with the plant roots in the sand cultures than in the solution cultures, and
hence a lesser amount of acid or alkali, making it easier for the plants to
change the actual reaction to a more favorable one. Plate 13 gives the
comparative growth in sand cultures.

TABLE 3
Growth and inoculation of soybeans in Crone’s nutrient solution at different reactions

DRY WEIGHT PER} NODULES PER

REACTION PLANT PLANT PLANT DEVELOPMENT
pH gm.
3.30 0.24 0 Tops dead
Roots poor
3.97 0.40 0 Tops about dead
Roots poor
4.95 1.00 30 Tops fair
Roots dark and stubbed
6.50 1.42 77 Tops good
Roots good
7.40 1.42 68 Tops good
Roots good
8.20 1.15 21 Tops good
Roots slightly brown
8.70 1.00 3 Tops fair
Roots brown
9.60 0.60 0 Tops poor and yellow

Roots very brown

Comparative growth of soybeans, corn and cowpeas at different reactions

For a comparison with the soybean, corn and cowpeas were grown in both
solution and sand cultures with Crone’s solution in exactly the same manner
as the previous experiment with soybeans, except that nitrates instead of
chlorides were used in the solutions for corn.

Plates 11, 12 and 14 serve to indicate the general growth which the corn
and cowpeas made. ‘The corn in the alkaline range of the solution cultures
did not make good growth and the leaves were yellow. This was perhaps due
to the lack of iron in solution, which was caused by precipitation under the
alkaline conditions. The corn in the sand cultures grew better than in solu-
tion cultures and none of it became yellow. Corn plants at pH 5 in the sand
cultures were inferior to the other plants at the beginning of the experiment
and never grew as well as plants at pH 4. The maximum growth of corn
took place at pH 6 and 7 in the sand cultures, and at pH 5 and 6 in the solu-
tion cultures, Plate 15 indicates that corn can grow at a considerably more

EFFECT OF REACTION ON NODULE FORMATION 281

acid reaction than the cowpeas and soybeans. Apparently, as indicated in
table 4 cowpeas have a wider range of inoculation than the soybeans. The
corn was not grown in duplicate cultures and hence any conclusion with
corn must be tentative.

TABLE 4

Growth and inoculation of cowpeas in sand cultures at different reactions

REACTION NODULES PER PLANT PLANT DEVELOPMENT
pH
SES 0 Tops fair
Roots stubby and brown
3.97 20 Tops fair
Roots slightly dark
4.95 22 Tops and roots good
6.5 32 Good plants, roots, and tops
7.4 33 Good plants, roots, and tops
8.2 26 Tops good
Roots slightly brown
8.7 19 Tops fair
Roots brown
9.6 14 Tops fair

Roots very brown

The influence of the reaction of the culture medium on the reaction of the plant
juices

The influence of the reaction of the culture medium on the reaction of the

plant juices has been investigated by several workers (17, 38); and it was

thought desirable to obtain additional information along this line. The reac-

tion of the juices of the roots and leaves of some of the plants grown in the

previous experiments was determined electrometrically by the method de-

TABLE 5

The reaction of the juices of plants grown in media of different reactions

SOYBEANS COWPEAS CORN
REACTION OF

MEDIA Se | eT

Leaves Roots Leaves Roots Leaves Roots
Ss Eig) ee ig a a Re pH PH Geel? gue one
3.30 5.60 4.68 Basi) 4.89 5.19 4.99
3.97 5.90 5.09 esi) 5.37 5-20 5.46
4.95 6.08 5.29 5.38 Sari 5.18 5:55
6.50 6.11 5.61 5.41 5.95 SrA 5.71
7.40 6.12 Sas 5.47 6.07 5.20 5.90
8.20 6.11 5.85 5.50 6.14 5.19 5.90
8.70 6.14 6.29 Se55 6.25 RevAl 6.10

282 O. C. BRYAN

scribed by Clevenger (8) and Haas (17). The results are recorded in table 5.
In general, the reactions of the plant juices as had previously been suggested
(38) became more acid as the culture medium rose in acidity. The corn tops,
however, showed no appreciable change. The reactions of the root juices
were more nearly like those of the culture medium than were those of the
leaves.

DISCUSSION

In making a comparison of the data presented, it should be clearly under-
stood that pH 3 signifies an acidity ten times greater than pH 4, and pH 4 ten
times greater than pH 5, etc. Hence, a small change in pH means a great
difference in hydrogen-ion concentration. It will be noted from the data
that the most favorable reaction for plant growth and nodule formation with
soybean was from pH 6 to 7, which is a condition of slight acidity. This fact

TABLE 6

Change in reaction of Crone’s nutrient solution in contact with soybean roots for 24 hours,
as determined by the hydrogen electrode

CULTURE NUMBER INITIAL REACTION FINAL REACTION

pH
3.30
3.95
4.80
6.90
7.80
8.70
9.50
9.70

>
ay

SOMNICUMN Bw
CoOIWNTA ue
PoODOrRrF OW

SSS8SS88S

—"
oS

was indicated more clearly in the solution cultures than in the sand culture.
The reactions at which maximum growth took place agrees with those reported
by Hoagland (23) and Salter and MclIlvane (34). Corn grew at a much
stronger acidity than soybeans. This conforms with field observations.

The unsatisfactory results obtained in solution cultures with the modified
form of Shive’s nutrient solution are not readily explained since the plants
grew well and became inoculated fairly well in the sand cultures. It was
thought that possibly the salts used contained toxic substances which the
sand adsorbed and thus prevented their toxic effect on the plants. In order
to test this possibility some of the solution was thoroughly shaken with
activated charcoal before being used. ‘This appreciably lessened the injurious
effects on the roots, but still no nodules appeared. Wilson (39) failed to
obtain any nodules with soybeans in solution cultures using Pfeffer’s nutrient
solution even after varying the amount of nitrates. This same thing was
found to be true with Shive’s nutrient solution in the present investigation.

EFFECT OF REACTION ON NODULE FORMATION 283

German and Didlake (13) reported inoculation of soybeans in solution cultures,
using a nitrogen free commercial fertilizer for the nutrient salts. The writer
believes that the unsatisfactory results obtained with Shive’s nutrient solution
in solution cultures were possibly due to toxic impurities in the salts used.

Crone’s nutrient solution was not entirely satisfactory in the alkaline range
because of the difficulty of maintaining a constant reaction when in contact
with growing plants. This was even true to some extent after the addition of
sodium carbonate as is indicated in table 6. The acid range was much more
constant than the alkaline range, although it changed some. This same
difficulty exists, of course, to some extent with all nutrient solutions, especially
if the plants grow well in them,

The carbon dioxide excreted by the plant roots and that absorbed from the
atmosphere were no doubt large factors in changing the reaction in the alkaline
range.

It was found in the present investigation that when sodium glycero phos-
phate was added to Crone’s solution the reaction remained almost constant
for a daily period, but no nodules were formed in solution cultures. The
addition of dibasic potassium phosphate to the alkaline range of Crone’s solu-
tion was also found to hold the reaction of the solution fairly constant for
daily periods, but nodule formation was again poor. In general, the reaction
of a solution which was fairly favorable to plant growth so that considerable
growth took place, would not remain constant very long in contact with the
growing plants, unless the initial reaction of the solution was exactly the one
most favorable for the plants. The change in reaction of the solution due
to growing plants was always in the direction of a more favorable reaction.
Salter and MclIlvane’s data (34) indicate that the reaction of their nutrient
solution in contact with growing plants remained nearly constant during
four-day periods. This was possibly due to the slow rate of growth of their
plants.

The data show that pure cultures of soybean bacteria are able to grow at an
acid reaction almost as great as that of the host plant. These results are
not exactly in line with those of Bewley and Hutchinson (2) who reported that
definitely acid soils would finally kill the nodule bacteria of lupines, red
clover, and broad bean. However, they did not state the exact degree of
acidity of the soil used and hence it is possible that they worked with very
strongly acid soils. It will be seen from tables 2 and 3 that the best inocula-
tion took place at a slightly acid to neutral reaction, although some inocula-
tion took place at a reaction of pH 5 and even pH 4. The information secured
thus far does not indicate that the critical hydrogen-ion concentration of pure
cultures of the soybean bacteria is different from what it is in the soil.

The results show distinctly that the reaction of the media in which the
soybeans are grown has a direct influence on growth and inoculation. The
reactions which produced injury and poor inoculation were within the range
of reactions of actual soil solutions and suspensions as reported by Gillespie

284 O. C. BRYAN

(15) Sharp and Hoagland (35), Plummer (32) Truog (37) and others. Differ-
ent plants vary considerably in their ability to grow at reactions of pH 4, 5,
and 6. Thus it will be seen that a proper adjustment of the reaction of
the soil for different plants is of prime importance for the best growth and
inoculation.

The reaction of the media in which the plants are grown often has a direct |
influence on the reaction of the plant juices. Further studies on the influence
of reaction on legumes and legume bacteria are in progress.

SUMMARY

A study was made of the influence of acidity and alkalinity on growth
and inoculation of soybeans in solution and sand cultures. The plants were
grown in 500- and 600-cc. percolators. A modified form of Shive’s nutrient
solution was used at first, but with unsatisfactory results in solution cultures.
Three other nutrient solutions were tested for influence on inoculation in
solution cultures. Of these, Crone’s nutrient solution proved to be the most
satisfactory, and was selected with slight modification for the principal work
reported herein. The reactions of the solutions were adjusted by adding
varied amounts of acid or alkali as the case required. The reactions were
kept as constant as possible by renewing the solutions daily. The old solu-
tions were removed from the sand with suction before the new solutions were
added. The cultures were allowed to grow from 25 to 35 days after inocula-
tion. A few cultures of corn and cowpeas were grown under similar condi-
tions to that of the soybean for comparison. Twenty-one different strains of
soybean bacteria were grown on pure cultures of different reactions in order to
compare the critical hydrogen-ion concentration of the soybean bacteria with
that of the host plant.

1. Shive’s nutrient solution was favorable for growth and inoculation of
soybeans in sand cultures, but not in solution cultures. It seems possible
that there were toxic impurities in the nutrient salts used which were adsorbed
by the sand.

2. Crone’s nutrient solution was favorable for growth and inoculation in
both solution and sand cultures. The alkaline range of this solution has a
very poor buffer action, which is improved markedly by the addition of
2 gm. of sodium carbonate per liter.

3. The reaction of the nutrient solution in contact with growing plants does
not remain constant very long, unless the initial reaction of the solution is the
most favorable one for the plants. The rate of change in reaction is greater
in the alkaline range than in the acid range. Plants growing rapidly appar-
ently influence this rate of change more than slow growing plants.

4, The most favorable reaction for growth and inoculation of soybeans
was pH 6.5. The limits for which inoculation took place were about pH 4.6
and 8. The limits for growth of soybeans were about pH 3.9 and 9.6. Reac-
tions pH 4.95 and 8.2 are injurious to the growth of soybeans, but do not
entirely prevent inoculation.

EFFECT OF REACTION ON NODULE FORMATION 285

5. The hydrogen-ion concentrations which were markedly injurious to
plant growth and inoculation in this investigation were not any greater and
in some cases considerably less than the hydrogen-ion concentration of very
acid soil solutions and suspensions as reported by recent investigators.

6. The critical hydrogen-ion concentration for nodule formation of soybeans
was slightly less than that for plant growth. The different strains of
soybean bacteria showed small differences in regard to critical hydrogen-ion
concentration.

7. Corn grew at a greater acidity and alkalinity than the soybean or cowpea.
The cowpea apparently has a greater range of reaction at which nodules are
formed than the soybean.

8. The reaction of plant juices varied with the degree of acidity or alkalinity
at which the plants were grown, except in the case of the juice of the corn
leaves which showed little change. The juices of the roots followed the reac-
tion of the media more closely than did the juices of the leaves.

REFERENCES

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and the toxicity of soluble salts of aluminum. Ind. Agr. Exp. Sta. Bul. 170.
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nodule organism (PS. Radicicola) passes under cultural conditions. In Jour.
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(3) BREAZEALE., J. F., AND LeCterc, J. A. 1912 The growth of wheat seedlings as
affected by acid or alkali conditions. U.S. Dept. Agr. Bur. Chem. Bul, 149.
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on seedlings. In Jour. Phys. Chem., v. 8, no. 1, p. 1-13.
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(6) Conner, S. D. 1921 Liming and its relation to injurious inorganic compounds in
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1903, p. 167-173.
(8) CLEVENGER, C. B. 1919 MHydrogen-ion concentration of plant juices. In Soil Sci.,
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(9) Duccar, B. M. 1920 Hydrogen-ion concentration and the composition of nutrient
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(10) Dacnnowsut, A. 1914 The effect of acid and alkaline solutions upon the water
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(13) Garman, H., anp Diprakr, Mary 1914 Six different species of nodule bacteria.
Ky. Agr. Exp. Sta. Bul., 184.
(14) Gruzir,O.M. 1917 The effects of some acids and alkalies on soil bacteria in solution.
In Soil Sci., v. 3, p. 289-295.

286 O. C. BRYAN

(15) Gittespre, L. J. 1916 The reaction of the soil and measurement of hydrogen-ion
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(16) Gittespre, L. J. 1920 Colorimetric determination of hydrogen-ion concentration
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(17) Haas, A. R. C. 1920 Studies on the reactions of plant juices. J Soil Sci., v. 9,
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(18) HartwEtt, B. L., AND PEMBER, F.R. 1907 The relation between the effect of liming
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(19) Hartwett, B. L., anD PEMBER, F. R. 1918 The presence of aluminum as a reason
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(20) Heatp, F.D. 1896 On the toxic effects of dilute solutions of acid and salts on plants.
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(22) Hixon, R. M. 1920 The effects of reaction of a nutrient solution on germination
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(23) Hoactanp, D. R. 1918 Effect of hydrogen- and hydroxyl-ion concentration on
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(24) Hopxins, C. G., anp Pettit, J. H. 1910 Soil Fertility Laboratory Manual, p. 22
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(25) JorFre, J.S. 1920 The effects of soil reaction on the growth of alfalfa. In Soil Sci.,
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(26) KAHLENBERG, L., AND TRUE, R. O. 1896 On the toxic action of dissolved salts and
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(27) Kappen, H. 1918 Untersuchangen und Wurzelsiften. Jn Landw. Vers. Stat.,
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(28) Kapren, H. 1920 Uber die Azidititsforms des Bodens und ihre Phlanzenphysiolo-
gische Bedeutung. Jn Landw. Vers. Stat., v. 96, p. 306-307.

(29) Lorw, F. A. 1903 Toxic effects of hydrogen- and hydroxyl-ions on seedlings of
Indian corn. Jn Science, n.s., v. 18, no. 453, p. 304-308..

(30) Mrrasot, J. J. 1920 Aluminum asa factor in soil acidity. J Soil Sci., v. 10, no. 3,
p. 153-219.

(31) McCatt, A. G. 1916 Physiological balance of nutrient solution for plants in sand
cultures. J Soil Sci., v. 2, p. 207-254.

(32) Prummer, J. K. 1918 Studies on the soil reaction as indicated by the hydrogen
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(33) Satter, R. C. 1916 The behavior of legume bacteria on acid and alkali media. Jn

\ Proc. Iowa Acad. Sci., v. 23, p. 309-313.

(34) Satter, R. M., anp McIivane, F. C. 1920 The effect of reaction of solution on
germination of seed and growth of seedlings. Jn Jour. Agr. Res., v. 19, no. 2,
p. 73-96.

(35) SHarp, L. T.,.AND Hoacrtanp, D. R. 1916 Acidity and absorption of the soil as
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(36) Saive, J. W. 1918 A study of physiological balance in nutrient media. Jn Physiol.
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(37) Truoc, E. 1918 Soil acidity: Its relation to the growth of plants. J Soil Sci..
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EFFECT OF REACTION ON NODULE FORMATION 287

(38) Truoc, E., AND Mracuam, M. R. 1919 Soil acidity: Its relation to the acidity of
plant juices. Jn Soil Sci., v. 7, no. 6, p. 469-475.

(39) Witson, J. K. 1917 Physiological studies of the Bacillus Radicicola of the soybean
(Soja Max Piper) and of the factors influencing nodule production. N. Y.
(Cornell) U. Agr. Exp. Sta. Bul. 285.

SOIL SCIENCE, VOL. XIII, NO. 4

PLATE 1

EFFECT OF REACTION ON NODULE FORMATION

0. C. BRYAN

288

Ne
i, DA

~ j

ADS ==

Pfeffer’s solution Crone’s solution Mendota lake water

Hopkin-Pettit’s solution

Shive’s so'ution

THE Root GROWTH AND INOCULATION OF SOYBEANS IN DIFFERENT NUTRIENT SOLUTIONS

STAIVA yd GLVWNIXOUddY AHL INASAUdAY SUAAWAN THI, “SNOILOVAY INDATIIG 10 SUYNLTAD NOIWLNATIOS NI NMOUD SNVAGAOS

| 4,

NVAUG “9 ‘O
¢ ALV 1d NOILVWaYOA ATNGON NO NOILOVAY JO Loaaag

289

EFFECT OF REACTION ON NODULE FORMATION PLATE 3
0. C. BRYAN

i

ibid entrance

SoyBEAN Roots GROWN IN SOLUTION CULTURES WITH pH VALUES OF APPROXIMATELY
3 AND 4

290

PLATE 4

EFFECT OF REACTION ON NODULE FORMATION

O. C. BRYAN

ee

>

SovBEAN Roots Grown IN SotuTION CULTURE WITH pH VALUE OF APPROXIMATELY 4.5

EFFECT OF REACTION ON NODULE FORMATION PLATE 5
0. C. BRYAN

SOYBEAN Roots GROWN IN SOLUTION CULTURE WITH pH VALUE OF APPROXIMATELY 5

292

PLATE 6

EFFECT OF REACTION ON NODULE FORMATION

0. C. BRYAN

eT

x No. S

SovBEAN Roots GROWN IN SOLUTION CULTURE WITH pH VALUE OF APPROXIMATELY 6

293

PLATE 7

EFFECT OF REACTION ON NODULE FORMATION

O. C. BRYAN

SOYBEAN Roots GROWN IN SOLUTION CULTURE WITH pH VALUE OF APPROXIMATELY 7

294

EFFECT OF REACTION ON NODULE FORMATION

O. C. BRYAN

x .

w)

é
ee

Sin

- SSN % ™ \
PSUS,

tx

, y Q
4
SS

S

PLATE 8

(Be av

WY,

SoyBEAN Roots Grown In SoLuTion CULTURE wiTH pH VALUE oF APPROXIMATELY 8

EFFECT OF REACTION ON NODULE FORMATION PLATE 9
0. C. BRYAN

co*

SoyBEAN Roots Grown IN SoLuTION CULTURE WITH pH VALUE OF APPROXIMATELY 9

296

EFFECT OF REACTION ON NODULE FORMATION PLATE 10
0. C. BRYAN

SOYBEAN Roots GROWN IN SOLUTION CULTURE WITH pH VALUE OF APPROXIMATELY 10

297

SHUNLIND AHL AO SANIVA

yd ALVWIxodddy FHL AATD suadWAN AHL

“SNOILOVAY INGDAGAICG JO SHYALTND) NOILNTOS NI NMOU‘) NAOD

Il ALVId

NVAUM *0 °O
NOILVNYOA ATOGON NO NOILOVaAY AO LOdsIAa

oO
N

SMaYNLTAY AHL wo
*SNOILOVAY INGAAIIG AO SHANITAD) AGNVS NI NMOU) NAO)

SHNIVA Y{d ALVNIXONddy AAIN SUAAWAN AHI,

al ALV Id

NVAWE “DO
NOLLVWUOdA AINGON NO NOILOVaa AO Loads

299

Hd @IvWixodddy AAIN suam#WAN AHL,

SHUALIAD AHL IO SANIVA
‘SNOILOVAY INANAIMIG] IO SAANLIND GNVS NI NMOUYD SNVadAOS

£l ELV Id

e NVAU ‘0 ‘°O

NOLLVNYOA ATNGON NO NOLLOVaY AO LOFdAA

300

SdUALIAD AHL Ao saAIVA Fd ALVWIXOUddy AAIH SUAMWAN ANL

tT ALVId

‘SNOILOVAY INAUAIIIG] O SHANALIAD NOILATIOS NI NMOUD SvadMoD

NVAUG “9 °O
NOILVNYOA ATAOGON NO NOILOVaa AO LOddda

301

EFFECT OF REACTION ON NODULE FORMATION PLATE 15
O. C. BRYAN

Corn, SOYBEANS AND CowPEAs GROWN IN SOLUTION CULTURES OF APPROXIMATELY pH 4.0
IN EACH CASE

302

SOIL SCIENCE
VOLUME 13, NUMBER 4, APRIL, 1922

CON TENTS

C. T. Hirst anp J. E. GreAves. Factors Influencing the Determination of Sulfates in Soil.. 231
J. E. GreAvEsS AND E. G. Carter. The Influence of Moisture and Soluble Salts on the
Bactenal Aptivitieswot ithe ‘Son 3. og.2s0s. oa chive o o-oRis scenes > nae Se ie. ae ee 251
O. C. Bryan. Effect of Different Reactions on the Growth and Nodule Formation of Soybeans. 271
Wittram M. Gress anp C. H. WrerkMaANn. Effect of Tree Products on Bacteriological Activi-
ties, insole... Ammonification and Nitrification.; 22%. st:. os ees oleic de ei Oo os Oe 303