• TABLE OF CONTENTS
HIDE
 Front Cover
 Front Matter
 Table of Contents
 Introduction
 Plan of the investigation
 Greenhouse experiments
 Field experiments
 Discussion of results
 Summary and conclusions














Group Title: Bulletin - University of Florida. Agricultural Experiment Station ; no. 417
Title: Legume inoculation
CITATION THUMBNAILS PAGE IMAGE ZOOMABLE
Full Citation
STANDARD VIEW MARC VIEW
Permanent Link: http://ufdc.ufl.edu/UF00015129/00001
 Material Information
Title: Legume inoculation
Series Title: Bulletin University of Florida. Agricultural Experiment Station
Physical Description: 32 p : ill. ; 23 cm.
Language: English
Creator: Smith, F. B ( Frederick Burean )
Blaser, R. E ( Roy Emil ), 1912-
Thornton, G. D ( George Daniel ), 1910-
Publisher: University of Florida Agricultural Experiment Station
Place of Publication: Gainesville Fla
Publication Date: 1945
 Subjects
Subject: Legumes -- Field experiments   ( lcsh )
Legumes -- Inoculation   ( lcsh )
Genre: government publication (state, provincial, terriorial, dependent)   ( marcgt )
bibliography   ( marcgt )
non-fiction   ( marcgt )
 Notes
Bibliography: Includes bibliographical references.
Statement of Responsibility: F.B. Smith, R.E. Blaser and G.D. Thornton.
General Note: Cover title.
Funding: Bulletin (University of Florida. Agricultural Experiment Station)
 Record Information
Bibliographic ID: UF00015129
Volume ID: VID00001
Source Institution: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: aleph - 000925227
oclc - 18237306
notis - AEN5875

Table of Contents
    Front Cover
        Page 1
    Front Matter
        Page 2
        Page 3
    Table of Contents
        Page 4
    Introduction
        Page 5
    Plan of the investigation
        Page 6
    Greenhouse experiments
        Page 7
        Page 8
        Page 9
        Page 10
        Page 11
        Page 12
        Page 13
    Field experiments
        Page 14
        Page 15
        Page 16
        Page 17
        Page 18
        Page 19
        Page 20
        Page 21
        Page 22
        Page 23
        Page 24
        Page 25
        Page 26
        Page 27
        Page 28
    Discussion of results
        Page 29
        Page 30
    Summary and conclusions
        Page 31
        Page 32
Full Text



November, 1945


UNIVERSITY OF FLORIDA
AGRICULTURAL EXPERIMENT STATION
HAROLD MOWRY, Director
GAINESVILLE, FLORIDA








LEGUME INOCULATION

F. B. SMITH, R. E. BLASER and G. D. THORNTON


Fig. 1.-Field of inoculated Sweet clover on Arredondo fine sand.


Bulletin 417










BOARD OF CONTROL

J. Thos. Gurney, Chairman, Orlando
N. B. Jordan, Quincy
Thos. W. Bryant, Lakeland
M. L. Mershon, Miami
J. Henson Markham, Jacksonville
J. T. Diamond, Secretary, Tallahassee



EXECUTIVE STAFF

John J. Tigert, M.A., LL.D., President of the
University3
H. Harold Hume, D.Sc., Provost for Agricul-
ture
Harold Mowry, M.S.A., Director
L. O. Gratz, Ph.D., Asst. Dir., Research
W. M. Fifield, M.S., Asst. Dir., Admin.4
J. Francis Cooper, M.S.A., Editor3
Clyde Beale, A.B.J., Associate Editors
Jefferson Thomas, Assistant Editor3
Ida Keeling Cresap, Librarian
Ruby Newhall, Administrative Managers
K. H. Graham, LL.IY., Business Managers
Claranelle Alderman, Accountants



MAIN STATION, GAINESVILLE

AGRONOMY

W. E. Stokes, M.S., Agronomist'
Fred H. Hull, Ph.D., Agronomist
G. E. Ritchey, M.S., Agronomist2
G. B. Killinger, Ph.D., Agronomist
R. W. Bledsoe, Ph.D., Agronomist
W. A. Carver, Ph.D., Associate
Roy E. Blaser, M.S., Associate5
H. C. Harris, Ph.D., Associate
Fred A. Clark, B.S., Assistant



ANIMAL INDUSTRY

A. L. Shealy, D.V.M., An. Industrialist1
R. B. Becker, Ph.D., Dairy Husbandmans
E. L. Fouts, Ph.D., Dairy Technologists
D. A. Sanders, D.V.M., Veterinarian
M. W. Emmel,' D.V.M., Veterinarian3
L. E. Swanson, D.V.M., Parasitologist4
N. R. Mehrhof, M.Agr., Poultry Husb.3
G. K. Davis, Ph.D., Animal Nutritionist
R. S. Glasscock, Ph.D., An. Husbandman
T. R. Freeman, Ph.D., Asso. in Dairy Mfg.
D. J. Smith, B.S.A., Asst. An. Hush.4
P. T. Dix Arnold, M.S.A., Asst. Dairy Husb.3
C. L. Comar, Ph.D., Asso. Biochemist
L. E. Mull, M.S., Asst. in Dairy Tech.4
J. E. Pace, B.S., Asst. An. Husbandman4
C. B. Reeves, B.S., Asst. Dairy Tech.
Katherine Boney, B.S., Asst. Chem.
Ruth Taylor, A.B., Asst. Biochemist
Peggy R. Lockwood, B.S., Asst. in Dairy Mfs.


ECONOMICS, AGRICULTURAL

C. V. Noble, Ph.D., Agri. Economist1 3
Zach Savage, M.S.A., Associate3
A. H. Spurlock, M.S.A., Associate
Max E. Brunk, M.S., Associate5

Orlando, Florida (Cooperative USDA)
G. Norman Rose, B.S., Asso. Agr. Economist
J. C. Townsend, Jr., Agr. Statistician2
J. B. Owens, Agr. Statistician2


ECONOMICS, HOME

Ouida D. Abbott, Ph.D., Home Econ.1
R. B. French, Ph.D., Biochemist


ENTOMOLOGY

J. R. Watson, A.M., Entomologist'
A. N. Tissot, Ph.D., Associate3
H. E. Bratley, M.S.A., Assistant


HORTICULTURE

G. H. Blackmon, M.S.A., Horticulturist1
A. L. Stahl, Ph.D., Asso. Horticulturist
F. S. Jamison, Ph.D., Truck Hort.
Byron E. Janes, Ph.D., Asso. Hort.
R. J. Wilmot, M.S.A., Asst. Hort.
R. IY. Dickey, M.S.A., Asst. Hort.
Victor F. Nettles, M.S.A., Asst. Hort.4
J. Carlton Cain, B.S.A., Asst. Hort.4
F. S. Lagasse, Ph.D., Asso. Hort.2


PLANT PATHOLOGY

W. B. Tisdale, Ph.D., Plant Pathologist1 '
Phares Decker, Ph.D., Asso. Plant Path.
Erdman West, M.S., Mycologist
Lillian E. Arnold, M.S., Asst. Botanist

SOILS

F. B. Smith, Ph.D., Chemist1 3
Gaylord M. Volk, M.S., Chemist5
J. R. Henderson, M.S.A., Soil Technologist
J. R. Neller, Ph.D., Soils Chemist
L. G. Thompson, Ph.D., Soils Chemist
C. E. Bell, Ph.D., Associate Chemist
L. H. Rogers, Ph.D., Associate Biochemist4
R. A. Carrigan, B.S., Asso. Biochemist
G. T. Sims, M.S.A., Associate Chemist
T. C. Erwin, Assistant Chemist
H. W. Winsor, B.S.A., Assistant Chemist
Geo. D. Thornton, M.S., Asst. Microbiologist5
R. E. Caldwell, M.S.A., Asst. Soil Surveyor4
Olaf C. Olson, B.S., Asst. Soil Surveyor4

1 Head of Department.
2 In cooperation with U. S.
3 Cooperative, other divisions, U. of F.
SIn Military Service.
5 On leave.













BRANCH STATIONS


NORTH FLORIDA STATION, QUINCY

J. D. Warner, M.S., Vice-Director in Charge
R. R. Kincaid, Ph.D., Plant Pathologist
Jesse Reeves, Asst. Agron., Tobacco
W. H. Chapman, M.S., Asst. Agron.4
R. C. Bond, M.S.A., Asst. Agronomist


Mobile Unit, Monticello

R. W. Wallace, B.S., Associate Agronomist


Mobile Unit, Milton

Ralph L. Smith, M.S., Associate Agronomist


Mobile Unit, Marianna

R. W. Lipscomb, M.S., Associate Agronomist


Mobile Unit, Wewahitchka

J. B. White, B.S.A., Associate Agronomist




CITRUS STATION, LAKE ALFRED

A. F. Camp, Ph.D., Vice-Director in Charge
V. C. Jamison, Ph.D., Soils Chemist
B. R. Fudge, Ph.D., Associate Chemist
W. L. Thompson, B.S., Entomologist
W. W. Lawless, B.S., Asst. Horticulturist
C. R. Stearns, Jr., B.S.A., Asso. Chemist
H. O. Sterling, B.S., Asst. Horticulturist
T. W. Young, Ph.D., Asso. Horticulturist
J. W. Sites, M.S.A., Asso. Horticulturist5
J. B. Redd, Ph.D., Insecticide Chemist



EVERGLADES STA., BELLE GLADE

R. V. Allison, Ph.D., Vice-Director in Charge
J. W. Wilson, Se.D., Entomologist4
F. D. Stevens, B.S., Sugarcane Agron.
Thomas Bregger, Ph.D., Sugarcane
Physiologist
G. R. Townsend, Ph.D., Plant Pathologist
B. S. Clayton, B.S.C.E., Drainage Eng.2
W. T. Forsee, Jr., Ph.D., Asso. Chemist
F. S. Andrews, Ph.D., Asso. Truck Hort.4
Robt. L. Cassell, Ph.D., Asso. Plant Path.
R. W. Kidder, M.S., Asst. An. Hush.
R. A. Bair, Ph.D., Asst. Agronomist
Earl L. Felix, Ph.D., Asst. Plant Path.


SUB-TROPICAL STA., HOMESTEAD

Geo. D. Ruehle, Ph.D., Vice-Director in
Charge
P. J. Westgate, Ph.D., Asso. Horticulturist
H. I. Borders, M.S., Asso. Plant Path.


W. CENT. FLA. STA., BROOKSVILLE

Clement D. Gordon, Ph.D., Asso. Poultry
Geneticist in Charge2


RANGE CATTLE STA., ONA

W. G. Kirk, Ph.D., Vice-Director in Charge
E. M. Hodges, Ph.D., Associate Agronomist
Gilbert A. Tucker, B.S.A., Asst. An. Husb.4



FIELD STATIONS


Leesburg
G. K. Parris, Ph.D., Plant Path. in Charge

Plant City
A. N. Brooks, Ph.D., Plant Pathologist

Hastings
A. H. Eddins, Ph.D., Plant Pathologist
E. N. McCubbin, Ph.D., Truck Horticulturist

Monticello
S. O. Hill, B.S., Asst. Entomologist2 4
A. M. Phillips, B.S., Asst. Entomologist2

Bradenton
J. R. Beckenbach, Ph.D., Horticulturist in
Charge
E. G. Kelsheimer, Ph.D., Entomologist
A. L. Harrison, Ph.D., Plant Pathologist
David G. Kelbert. Asst. Plant Pathologist
E. L. Spencer, Ph.D., Soils Chemist
Robert O. Magie, Ph.D., Hort., Glad. Inv.

Sanford
R. W. Ruprecht, Ph.D., Chemist in Charge
A. Alfred Foster, Ph.D., Asso. Hort.
J. C. Russell, M.S., Asst. Entomologist5
Ben F. Whitner, Jr., B.S., Asst. Hort.

Lakeland
E. S. Ellison, Meteorologist2 5
Warren O. Johnson, Meteorologist2

1 Head of Department.
2 In cooperation with U. S.
3 Cooperative, other divisions, U. of F.
SIn Military Service.
6 On leave.

















CONTENTS

PAGE


INTRODUCTION .................................................. ....... 5


PLAN OF THE INVESTIGATION ....................... ...... ......... ....--------- 6


GREENHOUSE EXPERIMENTS .....................------.......................- 7

Nodulation and Growth of California Bur Clover, Black Medic,
Sweet Clover and White Dutch Clover in Norfolk Loamy Fine
Sand at Different Initial Reactions, and in Acid Peat at Different
Degrees of Base Saturation ................-...--.----.-----.. ---.. 7

Effect of Moisture and Texture on the Inoculation of California
Bur Clover ............................... ...... .. ............. ......... 13


FIELD EXPERIMENTS ........................................ ... ------ --- 14

Efficiency of Different Cultures of Rhizobium and Rates and Methods
of Inoculation on the Nodulation of Clovers .................................... 14

Effect of Inoculation, Lime and Fertilizers on the Nodulation of
Lespedeza ........................ ................ ................... ............ ............. 22

Effects of Inoculation, Lime and Fertilizers on the Nodulation of
Various Clovers Grown on Different Soil Types ............-.............. 22


DISCUSSION OF-RESULTS ...................------.................................... 29


SUMMARY AND CONCLUSIONS .................- ......................


.......-... 31








LEGUME INOCULATION
F. B. SMITH, R. E. BLASER and G. D. THORNTON

All of the important leguminosae when inoculated with effi-
cient root nodule bacteria have the ability to utilize free nitrogen
of the atmosphere. The importance of this in the nitrogen
economy of the soil can hardly be overestimated. Relatively
large amounts of nitrogen are required by all crop plants. The
soil, particularly in the Southeast, is likely to be deficient in
nitrogen and this element is often the limiting factor in crop
production. Leguminous crops offer a practical method of
increasing soil nitrogen, especially when properly inoculated and
plowed under as green manure.
Legume inoculation is one of the oldest practical applications
of science. The practice was followed even before the biological
nature of the process was known. In spite of the fact that the
practice has been handed down from the ages and much experi-
mental work has been done to insure success with the growing
of legumes, much remains to be learned before definite recom-
mendations can be made to assure the desired results under all
soil and crop conditions. It has been rather definitely estab-
lished that there are different species of the legume bacteria and
that a given species of the organism will inoculate certain
legumes but will not inoculate others. For example, the bacteria
which form nodules on sweet clover will also form nodules on
California Bur clover, Black Medic clover and alfalfa, but they
do not produce nodules on White Dutch clover. Likewise, the
bacteria which produce nodules on peanuts will also inoculate
lespedeza, crotalaria and a number of legumes native to Florida,
but do not inoculate the clovers. The bacteria which inoculate
a specific legume group are referred to as a cross-inoculation
group. It has been observed that a soil which has recently
grown a well inoculated legume will also grow any legume in
the same cross-inoculation group without additional inoculation.
Under favorable conditions bacteria live in the soil for rather
long periods but recent experiments show that they disappear
from some soils in a short time. Efficiency of the bacteria varies
and in the absence of the host plant the organisms often die
or their efficiency becomes so impaired that they may even be-
come parasitic-live on the host plant without contributing
nitrogen.
It is known that these bacteria live longer, in the absence of
the host plant, in a soil well supplied with lime than in strongly







Florida Agricultural Experiment Station


acid soils. However, some of them differ greatly in their toler-
ance of acid soil conditions. The legume bacteria are sensitive
to drying. Well drained, sandy soils low in organic matter be-
come quite dry during periods of low rainfall and undoubtedly
the legume bacteria die off rapidly under these conditions. A
practice often followed in inoculation consists of supplying an
excess of inoculum to insure the survival of a sufficient number
of the organisms to produce good inoculation. It is also the
practice to treat the seed with syrup or sugar solutions to pro-
vide better conditions of survival and sticking of the bacteria
to the seed. Recently seed treatment for damping-off fungi has
been tried. The effects of such treatments on the inoculation
of various legumes have been-studied in a number of greenhouse
and field experiments and these results are presented in this
bulletin.
PLAN OF THE INVESTIGATION

A series of experiments to study factors affecting the growth
of legume root nodule bacteria and the nodulation and growth
of different leguminous plants were carried out under green-
house and field conditions. The nodulation of California Bur
clover, Black Medic, Sweet clover and White Dutch clover grown
in soils at different pH, the growth of California Bur clover in
acid peat at different degrees of base saturation, the effect of
different moisture levels, different amounts of organic matter
and the nodulation of clover in soils of different texture were
experiments conducted in the greenhouse. A description of
these experiments and the presentation of the results obtained
constitute the first part of this report.
Isolations of the root nodule bacteria from various legumes
were made from time to time and these cultures, referred to
as Station cultures, were tested in field experiments for efficiency
of inoculation in comparison with commercial cultures. The bac-
teria isolated from clovers grown in Florida were sent to a
commercial company for processing and then tested in field ex-
periments in comparison with the Station and commercial cul-
tures. The effect of treating the inoculated seeds of clovers
with sugar or syrup solutions, milk, phosphate and cottonseed
meal, the effects on nodulation of using varying amounts of
inoculum and the use of seed disinfectants were studied in field
experiments. The influence of different feretilizers on the inocu-
lation of various strains of lespedeza and different varieties of







Legume Inoculation


clovers was studied in a series of field experiments on different
soil types from 1939 to 1945. The second part of this report
deals with details of these field experiments.

GREENHOUSE EXPERIMENTS
NODULATION AND GROWTH OF CALIFORNIA BUR CLOVER, BLACK
MEDIC, SWEET CLOVER AND WHITE DUTCH CLOVER IN NOR-
FOLK LOAMY FINE SAND AT DIFFERENT INITIAL REACTIONS,
AND IN ACID PEAT AT DIFFERENT DEGREES OF BASE
SATURATION

Experiment 1.-A series of plots treated in 1926 and referred
to as the "Reaction Plots," as shown in Table 1, was sampled

TABLE 1.-OUTLINE OF SOIL TREATMENTS IN REACTION PLOTS ON NORFOLK
LOAMY FINE SAND.

Plot No. Material Pounds per' 2,000,000 Pounds of Soil
_1926 1931 1935 1937 [ 1938

1 I Sulfur ........... 1,000 900 500 500 500
2 Sulfur ............ 500 450 250 250 250
3 Check ............
4 Limestone .... 1,000 450 500 500 500
5 Limestone .... 2,000 900 1,000 1,000 1,000
6 Limestone .... 4,000 1,800 2,000 2,000 2,000
7 Sulfur ............ 1,000 900 500 500 500
8 Sulfur ............ 500 450 250 250 250
9 Check ............
10 Limestone .... 1,000 450 500 500 500
11 Limestone .... 2,000 900 1,000 1,000 1,000
12 Limestone .... 4,000 1,800 2,000 2,000 2,000

for use in this experiment. A quantity of soil was taken from
Plots 1, 2, 5, 8, 9, 10 and 12, sieved and sterilized in the auto-
clave at 20 pounds pressure for 3 hours. Thirty-two pint jars
of soil from each plot were planted to California Bur clover in
December 1939. Four jars of soil from each plot were planted
with seeds inoculated with a culture of bacteria prepared from
California Bur clover root nodules and 4 jars were planted with
sterile, uninoculated seed. Similar series were planted to Black
Medic, White Dutch and Sweet clover. The moisture content
of the soils was adjusted to approximately 50 percent of the
saturation capacity and maintained at this amount by frequent
additions of sterile distilled water. A sterile nutrient solution
was added from time to time. The clovers were washed out of

SThe assistance of Messrs. David Singer and Clifford Mize with some
of these experiments is gratefully acknowledged.







8 Florida Agricultural Experiment Station

the soil and the nodules on the roots counted in January 1940.
Results obtained are presented in Table 2.

TABLE 2.-MEAN NUMBER OF NODULES ON CLOVERS AT DIFFERENT PH
(1940).

Soil Reaction Average
(pH) California Black Sweet White 4
At End of Bur Medic Clover Dutch Clovers
Initial Exp'm't _

3.71 4.34 0.20 0.02 1.7 0.43 0.59
4.26 4.77 0.25 0.25 5.4 ...... 1.97
4.59 4.77 0.25 0.15 4.6 2.55 1.89
5.94 5.58 0.33 0.43 6.7 3.50 2.74
6.33 7.03 16.60 17.58 15.0 26.98 19.04
6.50 6.59 8.33 18.78 6.3 17.50 12.75
7.05 7.33 ...... 16.43 5.9 17.68 13.34
The value of P for F test of between treatment variance is highly sig-
nificant (P<.01).

Good nodulation of California Bur clover occurred in soils of
an initial pH 6.33 and 6.50 but in soil of lower pH nodulation
was unsatisfactory. The data show excellent nodulation of
Black Medic clover in soils of initial pH 6.33, 6.50 and 7.05.
There was very little nodulation of Black Medic clover in soils
more acid than pH 6.33. The best nodulation of Sweet clover
and White Dutch clover was obtained in soils of pH 6.33. Good
nodulation of White Dutch clover was obtained in soils at initial
pH of 6.50 and 7.05 but nodule formation was scanty at initial
pH of soil 5.94 and below.
Experiment 2.-Norfolk loamy fine sand from the reaction
plots was also used for this experiment. The soil was sieved


0r


Fig. 2.-California Bur clover.
Initial pH, left to right: 3.71, 4.59, 6.33, 4.26, 5.94 and 6.50.


'''''








Legume Inoculation


Fig. 3.-Black Medic clover.
Initial pH, left to right: 3.71, 4.59, 6.33, 4.26, 5.94 and 6.50.


Fig. 4.-Sweet clover.
Initial pH, left to right: 3.71, 4.59, 6.33, 4.26, 5.94, 6.50 and 7.05.


Fig. 5.-White Dutch clover.
Initial pH, left to right: 3.71, 4.59, 6.33, 4.26, 5.94, 6.50 and 7.05.







Florida Agricultural Experiment Station


through a 2 mm. screen to remove coarse organic matter and
then treated in quart earthenware pots in duplicate according
to the outline in Table 3. The moisture content was adjusted
to 50 percent of the saturation capacity and maintained at this
amount as in the previous experiment. California Bur clover
seed was planted in January 1941 and inoculated by pouring
an excess of California Bur clover nodule bacteria over the soil
in the pots. Ten plants were removed from each pot after 23
days and the number of nodules per plant counted. The results
obtained are presented in Table 3.
The soils with initial pH 4.99 treated with basic slag, limestone
or dolomite increased in pH and in the average numbers of
nodules per plant. The average number of nodules per plant
increased in all soils as the initial pH was increased from 4.99
to 5.85. The differences in inoculation due to mineral treatment
were not significant with soils of initial pH 4.99 and 7.60. The
numbers of nodules per plant were significantly larger at initial
pH 5.85 in all soils with mineral treatments than in the untreated
soils. However, nodulation was distinctly inferior in soils treated
with rock phosphate, magnesium carbonate, magnesium sulfate
and calcium sulfate than in the other treated soils. At pH 6.42
all of the treated soils had significantly larger numbers of nodules
per plant than the untreated soils, but the differences for soil
treatment were not highly significant. Results in this experi-
ment show that fair inoculation and growth of California Bur
clover may be obtained in Norfolk loamy fine sand, provided
the pH is maintained at 6.4 and the moisture content is optimum,
but poor nodulation occurs below pH 5.85.
Experiment 3.-An acid peat from southwest Florida having
a pH of 3.85, an exchange capacity of 81.5 m.e. and 9.9 m.e.
of exchangeable bases per 100 grams was used in this experi-
ment. The peat was passed through a 5 mm. sieve, thoroughly
mixed and potted in 1-gallon earthenware pots. Duplicate pots
each containing approximately 2.5 kgs. of peat were treated
with calcium hydroxide in amounts to give the following percent
base saturation: 12.1 (check), 25, 50, 75, 100 and 200. The cal-
cium hydroxide was thoroughly mixed with the peat and then
the treated peat was replaced in the pots. After adjusting the
moisture content the peat was allowed to stand for 1 week.
Fertilizer was then applied at the rate of 100 pounds of sodium
nitrate, 1,200 pounds of superphosphate and 400 pounds of po-







TABLE 3.-EFFECT OF DIFFERENT MINERAL TREATMENTS ON THE NODULATION OF CALIFORNIA BUR CLOVER IN NORFOLK
LOAMY FINE SAND AT DIFFERENT INITIAL REACTIONS.


Treatment


Pot
No.



1

2



3



4

5


6

7

8

9


Intial pH 4.99


Final
pH


Ave. No.
Nodules
per Plant


Initial pH 5.85


Final
pH


Ave. No.
Nodules
per Plant


Intial pH 6.42


Ave. No.
Final Nodules
pH per Plant


Initial pH 7.60


Final
pH


10.9



16.3



15.2

13.3


Check ................ ......................

Superphosphate, magnesium
carbonate, manganese sulfate,
calcium chloride ......................

Superphosphate, magnesium
carbonate, manganese sulfate,
calcium sulfate ....... ...........

Superphosphate .....................

Superphosphate, dolomite,
manganese sulfate ................

Basic slag ..............................

Rock phosphate ............

Superphosphate and dolomite ..

Superphosphate, magnesium
sulfate, manganese sulfate,
calcium carbonate .....................


Value of P for F test of be-
tween treatment variance ........


.< .01


.17.3 7.19


Ave. No.
Nodules
per Plant


> .05


< .01


----~----


i-


'


6.35



7.67








Florida Agricultural Experiment Station


tassium chloride per 500,000 pounds of peat. Inoculated Cali-
fornia Bur clover seed was planted in each pot in April and the
clovers were photographed in May 1942 (Fig. 6). Ten plants
were taken from each pot for nodule counts and green weight
determinations in May 1942. A sample of soil was taken from
each pot at this time for pH determinations. The results are
given in Table 4.

TABLE 4.-EFFECT OF LIME ON THE NODULATION AND GROWTH OF
CALIFORNIA BUR CLOVER IN AN ACID PEAT.


Treatment


Average
Number of
Nodules
per Plant


Green
Weight of
10 Plants,
Grams


Check ..................................... .................... 3.74 0 4.52
1/ amount of lime necessary to saturate 4.77 0 18.99
% amount of lime necessary to saturate 5.31 7.0 25.21
% amount of lime necessary to saturate 6.21 11.2 20.14
Lime in amount necessary to saturate.... 6.41 4.1 16.14
Twice the amount of lime necessary to
saturate --.................. ............................ 7.69 0 2.40


Good growth of clover was obtained in all pots, except in the
unlimed peat and in the peat treated with twice the amount of
lime required to completely saturate the exchange capacity,
Fig. 6. Growth in duplicate pots was fairly uniform. The pH
of the unlimed peat after harvesting the clover was 3.74, whereas
the peats with sufficient lime to saturate 50, 100 and 200 per-


Fig. 6.-Effect of

Pot No. pH
1 3.74
2 5.31
3 6.41
4 7.69


lime on the growth of California Bur clover
in an acid peat.
Approximate Degree of Base Saturation

1
2










Legume Inoculation


cent of the exchange capacity was 5.31, 6.41 and 7.69, respec-
tively. The best growth of clover was obtained in the peat
adjusted to pH 5.31 but good growth and the best nodulation
were obtained in the peat of pH 6.21. The poor growth of clover
in the peat at pH 7.69 was due, in part at least, to waterlogging.
This was not noticeable until the clovers were taken out of the
pots for examination. There was no nodulation of clover in the
peat with less than sufficient lime for 50 percent saturation or
more than 100 percent saturation. The best nodulation was
obtained at 75 percent base saturation.

EFFECT OF MOISTURE AND TEXTURE ON THE INOCULATION
OF CALIFORNIA BUR CLOVER
Experiment 1.-Arredondo fine sand, pH 6.01, was sieved into
a series of 2-gallon earthenware pots and treated with ground
limestone at the rate of 2,000 pounds per acre, superphosphate
at the rate of 600 pounds per acre and muriate or sulfate of
potash at the rate of 100 pounds per acre. The moisture content
of the soils was adjusted to form 4 different series on the basis
of its water-holding capacity as shown in Table 5, and maintained
at those levels throughout the experiment. California Bur clover
seed inoculated with a culture of the organisms isolated from a
vigorous bur clover plant were planted in each pot in December
1940. The plants were carefully removed from the soil by wash-
ing 27 days after planting and the number of nodules counted on
10 representative plants taken at random. Results are presented
in Table 5.

TABLE 5.-EFFECT OF MOISTURE ON THE NODULATION OF CALIFORNIA
BUR CLOVER.

Percent Water-holding Capacity Average Number Nodules per Plant

25 15.6
50 19.3
75 19.3
100 12.8
The value of P for F test of between treatment variance is significant
(P < .05).

The plants indicated no appreciable difference in nodulation
when potassium sulfate and potassium chloride were compared.
There was a smaller number of nodules per plant in the saturated
soil than in the soils at lower moisture contents. The moisture







Florida Agricultural Experiment Station


content of this soil for optimum nodulation is about 50 to 75
percent of the water-holding capacity.
Experiment 2.-Four Bladen soils varying in textures from
1.4 to 27 percent clay were used in this experiment.- The soils
were air-dried and passed through the 2 mm. sieve and treated
in duplicate 1/-gallon earthenware pots with the same fertilizer
application as that made in Experiment 1 above. The moisture
content was adjusted to 60 percent of the water-holding capacity
and maintained at this amount throughout the experiment.
Inoculated California Bur clover seed was planted in February
1941 and the nodule counts were made in March 1941. Results
are given in Table 6.
TABLE 6.-EFFECT OF SOIL TEXTURE ON THE NODULATION OF CALIFORNIA
BUR CLOVER.
Average Number
Treatment Percent Silt Percent pH of Nodules
Number and Clay Clay per Plant'
1 9.7 1.4 5.4 5.5
2 15.6 3.9 5.5 9.0
3 18.7 8.9 5.2 9.3
.4 39.5 27.0 5.3 12.0

Mean differences are not significant according to "F" test (P > .05).

The data show some tendency for the number of nodules per
plant to increase with increasing amounts of clay. There was
a smaller number of nodules per plant in the soil which contained
1.4 percent clay than in the soil containing 27 percent clay.
These results were apparently complicated by pH effects, since
lime applied had not had time to neutralize fully the acidity of
the soil, or was not applied in sufficient amount to decrease the
acidity.
FIELD EXPERIMENTS
EFFICIENCY OF DIFFERENT CULTURES OF RHIZOBIUM AND
RATES AND METHODS OF INOCULATION ON THE
NODULATION OF CLOVERS
Experiment 1.-The efficiency of various cultures for the
inoculation of different clovers was studied in 2 field experiments
near Gainesville during 1941-42. One field of this experiment
was on Norfolk fine sand, pH 6.50, and the other on Plummer
fine sand, pH 4.26. There were 4 separate inoculation tests with
3 replications on each field. The treatments made are given in
Tables 7, 8, 9 and 10.





TABLE 7.-MEAN NUMBER OF NODULES PER PLANT AND YIELD OF CALIFORNIA BUR
CULTURES ON TWO SOILS.


CLOVER INOCULATED WITH DIFFERENT


Norfolk Fine Sand Plummer Fine Sand
Culture Host Plant I Yield Yield
Nodules Pounds (Green Wt.) Nodules Pounds (Green Wt.)
per Plant per Acre per Plant per Acre


Check -.............

"N" 3EOb2(b)*

"N" 3DOc2 ........

"N" 3DOd3 ......

"N" 3DOel(a) ..

"N" 3DOc6 ......

"N" 3DOc3 ........

"N" 3DOh7 ......

"N" 3DOe2 ........

"N" 3DOel(d)..

"N" 3DOa10 ....

"N" 3DOc5 ........

"N" 3DOd2 ......

"N" Mixture of
organisms ....


Fenugreek ...........................

Giant southern bur clover ....

California bur clover** ..........

Manganese bur clover ............

Giant southern bur clover ....

Giant southern bur clover ....

Biennial White Sweet clover..

Manganese bur clover ............

Manganese bur clover ............

Alfalfa ........................ ........

Giant southern bur clover**..

California bur clover ..............


* "N" Commercial cultures.


* "N"-Commercial cultures.
**Isolations from Florida-grown clovers.
The P value of F test of between culture variance is


3,110

6,970

6,740

4,460

3,900

5,720

5,990

2,230

4,410

6,920

4,180

3,810

4,930

5,580


766

1,910

1,810

980

2,330

2,370

2,300

1,810

1,810

2,720

910

1,810

1,670


1,250


highly significant on both soils (P < .01).







Florida Agricultural Experiment Station


The soils were disked and treated in October 1941 with 1,000
pounds of 18 percent superphosphate and 200 pounds of 50 per-
cent muriate of potash per acre. Applications of 1,000 pounds
of ground limestone per acre on Norfolk fine sand and 3,000
pounds on the Plummer fine sand were made. The limestone
and the fertilizers were applied broadcast and disked in. The
clover seed was inoculated and planted about 6 weeks later.
The plots were raked by hand to cover the seed. Sufficient water
was applied to the plots on Norfolk fine sand to wet the ground
thoroughly. Twenty-five plants were taken from each plot in
January 1942 for a determination of the number of nodules per
plant. The average pH of Norfolk fine sand was 6.70 and that
of Plummer fine sand was 6.68 at that time. Results obtained
are shown in Tables 7, 8, 9 and 10.

TABLE 8.-MEAN NUMBER OF NODULES PER PLANT ON BLACK MEDIC CLOVER
INOCULATED WITH VARIOUS CULTURES ON TWO SOILS.
S Nodules per Plant
Culture Host Plant Norfolk Plummer
SFine Sand Fine Sand

Check ...... ............... ............ .... .............. 2.2 3.t)
"S" 3DObl* .......... Black Medic clover.... 23.2 11.4
"S" 3DOb2 ............. Black Medic clover.... 22.7 11.5
"S" 3DOb5 -.......... Black Medic clover.... 17.8 12.2
"S" 3DOb5-Su-P04 I Black Medic clover.... 11.8 12.3
"N"* ....-..................... I.-.................. 11.5 6.3
"N Su** .................. ............... .. 14.4 8.0
"N Su-Pt ........-..... ...... ...... ..1............. 23.4 8.1
*"N"-Commercial culture; "S"--Station culture.
** Su-Sucrose.
t Su-P-Sucrose and peanut meal.
S Su-PO--Sucrose and phosphate solution.
The P value of F test of between culture variance is highly significant
(P < .01).
Twelve cultures from individual strains of bacteria were used
alone and mixed together for inoculating California Bur clover
seed. As shown in Table 7, there were significant differences
in the efficiency of the various cultures in producing nodules on
California Bur clover. Most effective cultures were "N"2
3DOel(d), "N" 3EOb2(b), "N" 3DOc2, "N" 3DOc6, "N" 3DOd2,
and "N" 3DOel(a). Similar results were obtained on both soil
types.
Results with various cultures of Black Medic clover organisms
are presented in Table 8, showing significant differences in effi-

SIn this discussion "N" refers to commercial cultures and "S" to
Station cultures.











TABLE 9.-MEAN NUMBER OF NODULES PER PLANT AND YIELDS OF SWEET CLOVER INOCULATED WITH DIFFERENT CULTURES.


Culture



Check ..........................

"S" 3DOj2* .................

"SI 3DOj3 ...................

"S" 3DOj6 ...................

"N "* ................................

"N" Su** ......................

"N" Su-Pt ......................

"S" 3DOj6 Su-P04 ....


Host Plant




..Sweet clover...... ......

Sweet clover......

Sweet clover ......




Sweet clover......
..............................

..............................

.J. ................ ..........

Sweet clover ......


Norfolk Fine Sand


Yield
Nodules Pounds (Green Weight)
per Plant per Acre

3.1 2,460

11.9 4,180

11.6 4,180

14.2 3,390

10.1 3,620

7.8 4,550

10.7 5,300

13.8 4,090


Plummer Fine Sand


Yield
Nodules Pounds (Green Weight)
per plant per Acre


5.2 1,720

18.0 2,970

8.5 1,860

14.2 2,280

8.1 3,000

11.6 2,930

11.9 2,560

15.8 2,090


"N"-Commercial cultures; "S"-Station cultures.
** Su-Sucrose.
Su-P-Sucrose and peanut meal.
SSu-P04--Sucrose and phosphate solution.
The P value of F test of between culture variance is highly significant (P < .01) on Plummer fine sand and significant
(P < .05) on the Norfolk fine sand.


Norfolk Fine Sand







Florida Agricultural Experiment Station


ciency of the different cultures. Cultures "S" 3DObl, "S" 3DOb2
and "S" 3DOb5 w6re superior to the others. The use of sucrose
and.peanut meal on the clover seeds was effective in increasing
efficiency of the commercial culture on Norfolk fine sand and
improved nodulation slightly on plants grown on Plummer fine
sand. Sucrose and phosphate solution was without significant
effect on culture "S" 3DOb5 on either soil.
Results of various seed treatments and the inoculation of
Sweet clover with different cultures are presented in Table 9.
In Norfolk fine sand there was no significant difference in effi-
ciency between the various cutures, except that "S" 3DOj6 was
superior to the commercial culture. Nodulation with commer-
cial culture was not improved by supplemental seed treatment.
On the Plummer fine sand culture "S" 3DOj3 and the commercial
culture were significantly less efficient than cultures "S" 3DOj2
and "S" 3DOj6. Sucrose and phosphate seed treatment with
culture "S" 3DOj6 did not improve inoculation significantly on
Plummer fine sand. Sugar and peanut meal seed treatments
slightly increased the nodulation of Sweet clover.
Results obtained on the inoculation of White Dutch clover are
given in Table 10. Differences in efficiency of cultures used for
producing nodules on this clover were marked. Cultures "S"

TABLE 10.-MEAN NUMBER OF NODULES PER PLANT AND YIELDS OF WHITE
DUTCH' CLOVER INOCULATED WITH DIFFERENT CULTURES.

Norfolk Plummer Fine Sand
Fine Sand
Culture Host Plant Nodules Nodules Yield, Lbs.
per per (Green Wt.)
Plant Plant per Acre
Check ........... -...... -- ...... 4.7 1,160
"S" 3DOm3* ........ White Dutch clover 3.9 3.3 1,950
"S" 3DOm4 .......... White Dutch clover 10.6 7.9 2,830
"S" 3DOm5 .......... White Dutch clover 18.7 10.6 2,000
"S" 3DOm6 ......... White Dutch clover 8.7 1,490
"S" 3DOm7 ........ White Dutch clover 18.4 14.2 1,490
"S" 3DOm5-
Su-PO .-.----..- 18.8 15.9 2,790
"N"* ...................... 11.3 10.3 2,090
"N" Su** ..........-... 9.3 8.4 2,040
"N" Su-Pt .....--...-- 9.0 11.5 2,040
"N"-Commercial culture; "S"-Station culture.
** Su-Surcrose.
t Su-P-Sucrose and peanut meal.
Su-PO4--Sucrose and phosphate solution.
The P value of F test of between treatment variance is highly significant
.(P < .01) for both soil types.







Legume Inoculation


3DOm5 and "S" 3DOm7 were distinctly superior to all others
on both soils. Culture "S" 3DOm3 was decidedly inefficient for
the nodulation of White Dutch clover. The supplemental seed
treatments were without beneficial effect on increasing the
nodulation of White Dutch clover, except sucrose and phosphate
improved nodulation on Plummer fine sand. The data show
that there was
a tendency for
yields to be cor-
related with nod- $&
ulation.
Experiment 2.-
Two field experi-
ments similar to
those conducted
in Experiment 1
were established
on Norfolk fine .
sand and Plum-
mer fine sand in
November 1942
in the vicinity of
the old plots. Fer- .
tilizers were ap-
plied and inocu-
lated seed planted -
as in the previous
year. Nodule
counts were made
as usual but the
differences be -
tween cultures Fig. 7.-Effect of inoculation on the growth of Cali-
and methods of fornia Bur clover. (1) Uninoculated; (2) inoculated.
inoculation were
not significant. However, appearance and growth of clovers
were striking. Plants from several plots of California Bur
clover were taken up and placed in 1-gallon earthenware pots
for photographing (Figures 7, 8 and 9). These pictures show
the increased growth of California Bur clover brought about
by inoculation, the effect of increasing the amount of inoculum
over that recommended by the producer of the culture and the







Florida Agricultural Experiment Station


superiority of processed cultures isolated from clovers grown
in Florida.


.7. 3


3-l


Fig. 8.-Effect of amount of ii
growth of California Bur clover. (1
with recommended amount of culture
ulated with 10 times the recommend
of culture.


Experiment 3.-
The efficiency of va-
rious cultures and
f* the effect of different
F seed treatments on
t the inoculation of
Black Medic clover
were studied again in
a field experiment on
Leon fine sand near
*Gainesville during
S1944-45. Results ob-
tained in earlier tests
on inoculation indi-
cated that a larger
A* amount of inculum
S* h than is ordinarily
used might be neces-
sary for good inocu-
lation. Preliminary
tests have shown
that cultures obtain-
ed from local strains
--- of the bacteria are
superior to other
strains; also results
Sof former tests on
noculum on
) Inoculated seed treatment were
e; (2) inoc- erratic, indicating an
ded amount
apparent beneficial
effect in some in-


stances and no effect in others. This experiment was designed
to test the efficiency of local strains of the organisms and ordi-
nary commercial cultures when both cultures were processed
alike by the manufacturer, the rates of inoculation and the effect
of different seed treatments. The "Special" culture was pre-
pared from a strain of bacteria isolated locally and processed
in the same manner as the commercial culture. The "soil
formula" used was made by mixing 100 parts of soil, 10 parts
of finely pulverized manure and 2 parts of 18 percent super-







Legume Inoculation


phosphate. This mixture was applied at the rate of 400 pounds
per acre mixed with the seed. Fourteen plots consisting of
12-foot rows were ran-
domized in 4 replicated @ 2 "". :
blocks. The soil had an
initial pH of 4.30. Lime- r'
stone at the rate of 11/2
tons and an 0-10-10 fer-
tilizer at the rate of 500
pounds per acre were
applied broadcast and
disked in lightly. At the
time of seeding the pH
of the soil was 5.37 and
when the samples were
taken for nodule counts
the pH varied from 5.59
to 6.50. Sterilized and Fig. 9.-Effect of different cultures on
subsequently inoculated growth of California Bur clover. (1) Regu-
lar culture; (2) special culture.
Black Medic clover seed
were planted in December 1944. Ten plants were taken at ran-
dom from each plot in March 1945 for nodule counts. Results
obtained are presented in Table 11.
The results show a highly significant increase in the nodula-
tion of Black Medic clover plants inoculated with the local strain
of organisms over that obtained with the commercial strain,
especially where no supplemental seed treatment was made. A
highly significant increase in number of nodules per plant was
also obtained on both cultures when 5 or 10 times the standard
amounts of inoculum were used. However, 5 times the standard
rate gave as good nodulation as 10 times the standard rate.
Treatment of the seed with "soil formula," milk, syrup, cot-
tonseed meal and ceresan gave significant increases in the nodu-
lation of Black Medic clover inoculated with the commercial
culture; whereas only the "soil formula" and syrup seed treat-
ments gave significant increases in nodulation with the "special"
culture. Apparently seed treatment with phosphate was with-
out significant effect on nodulation with either culture. The
numbers of nodules per plant were significantly lower on plants
which received seed treatments with arasan and spergon when
inoculated with the "special" culture. Treatment of the seed
with ceresan was beneficial on plants inoculated with the com-







Florida Agricultural Experiment Station


mercial culture but without significant effect on plants inocu-
lated with the "special" culture.

TABLE 11.-EFFECT OF CULTURE AND SEED TREATMENT ON INOCULATION OF
BLACK MEDIC CLOVER ON LEON FINE SAND (1945).

Mean Number of Nodules
per Plant
Treatment Seed Inoculated Seed Inoculated
with Commer- with Special
cial Culture Culture
Standard rate of application .................... 1.6 4.3
Standard rate x 5 ...... ............ ........... .... 4.5 14.7
Standard rate x 10 ..................................... 5.1 9.0
Standard rate and soil formula .............. 10.1 8.8
Standard rate and milk ..................... 4.6 1.1
Standard rate and syrup .................. .... 4.3 9.2
Standard rate, syrup and cottonseed meal 6.5 4.7
Standard rate and phosphate ................ 0.2 3.4
Standard rate and arasan .................... 2.7 1.2
Standard rate and ceresan ...................... 5.2 3.2
Standard rate and spergon ....................... 1.3 0.5
Check .................. ............ ......................- 0.01 0.01
The P value of F test of between culture and treatment variance is
highly significant (P < .01).

EFFECT OF INOCULATION, LIME AND FERTILIZERS ON
THE NODULATION OF LESPEDEZA
Effects of inoculation, lime and fertilizers on the nodulation
of lespedeza were studied in 2 field experiments in 1939. One
field was planted on Leon fine sand and the other on Portsmouth
fine sand in April 1939. Treatments and results obtained are
listed in Table 12.
These data show that the average number of nodules per
plant was low in all treatments on the Portsmouth fine sand.
The largest mean number of nodules per plant was obtained
with inoculation and with lime, superphosphate and muriate of
potash. Significant increases in nodulation were obtain by inocu-
lation on both soils. A mixture of lime, phosphorus and potash
increased nodulation of lespedeza on the Leon fine sand greatly
when compared with untreated soil. The omission of either
lime, phosphorus or potash also decreased nodulation appreciably.

EFFECTS OF INOCULATION, LIME AND FERTILIZERS ON THE
NODULATION OF VARIOUS CLOVERS GROWN ON
DIFFERENT SOIL TYPES
A series of 5 field experiments was conducted on Leon fine
sand at widely separated locations in Florida. These experiments







Legume Inoculation


TABLE 12.-EFFECT


OF INOCULATION, LIME AND FERTILIZERS ON THE
NODULATION OF LESPEDEZA.


Soil Treatment Mean Number of Nodules per Plant*
Pounds per Acre Portsmouth
Fine Sand** Leon Fine Sand**

N one .....................---------------. 3.21 2.6
Superphosphate 450 lbs.
Muriate of potash 100 lbs. 1.70 2.1
Lime 1,000 lbs.
Superphosphate 450 lbs.
Muriate of potash 100 lbs. 2.83 6.7
Lime 2,000 lbs.
Superphosphate 450 lbs.
Muriate of potash 100 lbs. 4.88 10.2
Lime 3,000 lbs.
Superphosphate 450 lbs.
Muriate of potash 100 lbs. 1.49 7.8
Lime 2,000 lbs.
Muriate of potash 100 lbs. 4.18 3.6
Lime 2,000 lbs.
Superphosphate 450 lbs. 4.08 4.9
Lime 2,000 lbs.
Superphosphate 450 lbs.
Muriate of potash 100 lbs.
Not inoculated ...................... .71 2.7


Mean number of nodules of 10 plants
lespedeza.


each of Common, Kobe and Tennessee No. 76


** The initial pH value of the Leon fine sand was 4.73 and for the Portsmouth fine
sand, 5.6.
The P value of F test of between treatment variance is highly significant
(P < .01).

were designed to test the effects of lime and various fertilizers
on the inoculation and growth of a number of clovers used for
pastures. The soil treatments were designed to study the value
of limestone, the comparative value of rock phosphate and super-
phosphate, and the value of rock phosphate as a source of cal-
cium for clover. The effect of a number of the minor elements
on nodulation of clovers was studied in 1 field for a period of
2 years. The clovers used in these experiments were California
Bur, Black Medic, Sweet, White Dutch, Red, Hop, Persian,
Alsike and Crimson.
In all cases, except as noted, the fertilizer was broadcast by
hand and disked in lightly. The seeds were inoculated with a
commercial culture in the amount of 10 times the producer's
recommended amount of inoculum. The seed were planted and







Florida Agricultural Experiment Station


the soil rolled immediately after the fertilizer treatments were
made. Approximately 6 weeks after planting 10 plants from
each plot were taken into the laboratory for nodule counts in
the usual manner. (The number of plants to take the nodule
counts was decided after a study of technique was made to de-
termine how many plants were necessary to give a representative
sample. Rank of the different clovers arranged in order of the
number of nodules per plant was the same when 10 plants were
taken as when 20 plants were used for the counts.)
Largo Field, 1941.-The effect of various soil treatments on
the nodulation of different clovers was studied in the field near
Largo, Florida, on Leon fine sand. The initial pH value of this
soil was 4.7. All plots were treated with muriate of potash at
the rate of 150 pounds per acre. The clovers were planted in
duplicate blocks and the soil treatments were randomized on
each clover in November 1940. The clovers included Crimson,
Hop, Alsike, Red, Sweet, White Dutch, Giant Southern Bur,
California Bur and Black Medic, and the soil treatments are
given in Table 13. Ten plants were taken from each plot in
February 1941 and the nodules counted. The data showing the
average number of nodules per plant for each clover under each
treatment are given in Table 13.
Results of this experiment show the need for lime and phos-
phate fertilizers for clovers on this soil type. Rock phosphate
did not supply adequate calcium for the species of Medicago-
Melilotus, but some of the Trifoliums were as well nodulated
when rock phosphate was the sole source of calcium and phos-
phorus as when limestone and superphosphate were used. There
were some exceptions. For example, Crimson clover was better
nodulated when fertilized with limestone at the rate of 4,000
pounds per acre and superphosphate at the rate of 600 pounds
per acre than when 3,000 or 6,000 pounds per acre of rock phos-
phate were applied. The addition of 750 pounds of limestone
per acre in addition to the 3,000 pounds of rock phosphate in-
creased nodulation of Crimson clover significantly. Rock phos-
phate without lime at the rate of 6,000 pounds per acre was
slightly more effective in stimulating the nodulation of Crimson
clover than limestone and superphosphate, 2,000 and 600 pounds
per acre, respectively, but less effective than limestone and
superphosphate at the rate of 4,000 and 600 pounds per acre,
respectively. The largest average numbers of nodules per plant
for both groups of clovers were obtained in this experiment with












TABLE 13.-EFFECT OF LIME AND PHOSPHATES ON THE NODULATION OF SEVERAL INOCULATED CLOVERS ON LEON FINE
SAND, LARGO, FLORIDA, 1941.

Mean Number of Nodules per Plant
Treatment White Crim- Black Calif. Giant
Pounds per Acre* Dutch Alsike Hop son Red Medic Bur So. Hubam Tri- Medicago-
Clover Clover Clover Clover Clover Clover Clover Bur folium Melilotus

Limestone 4,000 11.4 11.8 17.8 23.6 17.2 13.8 17.3 16.2 17.1 16.36 16.10
Superphosphate 600

Limestone 2,000 9.7 8.8 15.8 12.3 14.4 12.3 15.7 12.5 15.1 12.20 13.95
Superphosphate 600

Rock phosphate 3,000 14.4 12.5 12.8 11.4 18.7 5.9 6.2 8.1 11.8 13.96 8.00

Rock phosphate 3,000 14.3 19.1 14.2 17.3 16.7 9.4 17.5 12.7 11.2 13.50 12.70
Limestone 750

Rock phosphate 6,000 11.6 16.3 14.1 19.4 17.3 6.1 12.6 6.2 7.0 15.70 7.97

Muriate of potash at the rate of 150 pounds per acre applied to all plots.
The P value of F test of between treatment variance is highly significant (P < .01).
The P value for F test of between clovers is significant (P < .05).








Florida Agricultural Experiment Station


the application of limestone and superphosphate at the rates of
4,000 pounds and 600 pounds per acre, respectively.
Farm Colony Field 1 1941.-A field experiment similar to
the one conducted at Largo was established near Gainesville
on Leon fine sand with pH 5.6. Results obtained in this experi-
ment 3 were similar to those obtained in the Largo field, except
that lime was more effective in stimulating nodulation. The
nodulation of species of Medicago-Melilotus was unsuccessful
when rock phosphate was used without lime. Nodulation of
Trifolium was also improved when lime was added to the rock
phosphate.

TABLE 14.-EFFECT OF LIME AND FERTILIZERS ON THE NODULATION OF
INOCULATED CALIFORNIA BUR AND WHITE DUTCH CLOVER ON LEON FINE
SAND, GAINESVILLE, 1941.

Average Number of Nodules
Treatment Pounds per Plant
per Acre California White
Bur Dutch

Limestone ............ 2,000
Superphosphate ....... 600 23.0 40.3
Potash ........................ 100
Rock phosphate ....... 3,000 0.9 7.8
Potash ......................- 100
Rock phosphate ....... 3,000
Limestone .................. 2,000 20.1 53.3
Potash ...................... 100
Rock phosphate ....... 6,000 0.9 17.9
Potash .......................... 100
Colloidal phosphate.... 3,000 2.3 7.3
Potash .......................... 100
Colloidal phosphate .... 3,000
Limestone ............... 2,000 30.8 43.4
Potash .......................... 100
Limestone .................... 2,000
Basic slag .................... 750 19.9 30.9
Potash ......................... 100
Basic slag .................... 2,000 16.8 36.9
Potash .......................... 100

The P value of F test of between treatment variance is highly significant
for both clovers (P < .01).

"Blaser, R. E., G. M. Volk and F. B. Smith. The Yield, Composition
and Nodulation of Several Clover Varieties as Affected by Sources of
Calcium and Phosphorus in Combination with Other Fertilizers on Several
Soils. Proc. Soil Sci. Soc. Amer. 6: 298-302. 1941.







Legume Inoculation


Farm Colony Field 2- 1941, 1942 and 1943.-In 1941 a test
was established to measure the value of phosphate from 4 sources
and lime from 2 sources for California Bur and White Dutch
clover. In 1942 an additional experiment was set up to evaluate
lime and phosphates from 2 sources. Three clovers belonging
to each of the Trifolium and the Medicago-Melilotus groups were
used. Both tests were located on Leon fine sand near Gainesville.
Some of the fertilizer treatments used and nodulation responses
are shown in Tables 14 and 15.
The nodulation responses of California Bur and White Dutch
clover differed greatly. California Bur clover plants treated
with 3,000 pounds of rock phosphate per acre and potash had
an average of 0.9 nodules per plant, as compared with 20.1
when 2,000 pounds of lime were added. Nodulation at the 6,000-
pound rate of rock phosphate did not exceed that obtained at
the 3,000-pound rate. Colloidal phosphate gave similar nodula-
tion responses. Nodulation of California Bur clover grown in
soils treated with 2,000 pounds of lime, 600 pounds of super-
phosphate and 100 pounds of muriate of potash; 2,000 pounds
of lime, 750' pounds basic slag and 100 pounds of muriate of
potash; and 2,000 pounds basic slag and 100 pounds of muriate
of potash per acre was satisfactory. The nodulation responses
with the White Dutch clover were similar to those of California
Bur, except that more nodules per plant were present on un-
limed soils.
The mean nodulation of 3 Trifoliums and species of Medicago-
Melilotus as affected by treatments is shown in Table 15. Two
tons of lime per acre produced better nodulation than the 1-ton
rate of lime for the species of Medicago-Melilotus. These 2 lime
rates did not affect the number of nodules significantly in the
Trifoliums. The rock phosphate treatments without lime again
retarded nodulation of all clovers. When 1,000 pounds and 3,000
pounds of rock phosphate per acre were used with lime and
potash, nodulation did not differ appreciably.
Farm Colony Field 3 1942 and 1943.-An experiment cbm-
paring the effects of various minor elements and fertilizers on
the nodulation of California Bur clover and White Dutch clover
grown on virgin Leon fine sand was established in November
1941. The fertilizers were applied broadcast by hand and left
on the surface to avoid mixing. The treatments made are shown
in Table 16. The area was planted immediately after treatment
with inoculated seed and rolled. A seeding mixture of 4 pounds














TABLE 15.-EFFECT OF LIME AND SOURCES OF PHOSPHATES ON NODULATION OF INOCULATED CLOVERS ON LEON
FINE SAND (1942-1943).

Mean Number of Nodules per Plant
TIit4il So il ^Tt4 il I


n a 0 reatmen
November 1941,
Pounds per Acre


4,000 lbs. limestone
600 lbs. 0-16-12

2,000 lbs. limestone
600 lbs. 0-16-12

3,000 Ibs. rock phosphate
600 lbs. 0-0-12

2,000 lbs. limestone
3,000 lbs. rock phosphate
600 lbs. 0-0-12

2,000 lbs. limestone
1,000 lbs. rock phosphate
600 lbs. 0-0-12


OUli r1ea4tmenll
November 1942,
Pounds per Acre


400 Ibs.


400 lbs.


400 lbs.


400 lbs.


0-14-10


0-14-10


0-0-10


0-0-10


400 lbs. 0-0-10


Trifoliums*
.943 1 Mean 1942-43


1942


Medicago-Melilotus
Species Clovers**
1943 Mean 1942-43


* Mean for White Dutch. Crimson and Red clovers.
** Mean for California Bur. Black Medic and Sweet clovers.
The P value of F test of between treatment variance of the two clover groups is highly significant (P < .01).







Legume Inoculation


of White Dutch clover and 8 pounds of California Bur clover
per acre was used. Nodule counts on 10 plants of each clover
from each plot were made in January 1942.

TABLE 16.-EFFECT OF VARIOUS MINOR ELEMENTS ON THE NODULATION OF
INOCULATED CALIFORNIA BUR AND WHITE DUTCH CLOVER.
Mean Number of Nodules
per Plant**
Treatments*,
Pounds per Acre California White Dutch
Bur
1942 1942 1943

50 lbs. CuSO ...................................................... 16.8 12.3 23.9
10 lbs. ZnSO4 ..................................... .............. 7.8 17.6 24.2
75 lbs. MnSO4 ........................................... ....... 10.1 20.8 17.9
10 lbs. NaB40, ................................ ............ 14.9 27.2 20.7
75 lbs. FeSO, .......... ..... ..... .......... .......... 24.5 19.0 29.6
75 lbs. MgSO4 ......... .... ................ .......... 18.7 20.0 23.3
10 lbs. CoC ........................................................ 9.7 16.6 27.9
Cu, Zn, Mn, Fe, Mg in % above amounts .... 6.5 24.4 24.0
Cu, Zn, Mn, Fe, Mg in twice above amounts 4.1 15.5 18.4
Cu, Zn, Mn, Fe, Mg, Co, B in above amounts 6.2 13.5 20.3
No Minor elements .................................. 28.7 20.5 24.8

2,000 pounds ground limestone, 600 pounds 20% superphosphate and 200 pounds
muriate of potash per acre on all plots.
** The P value of F test of between treatment variance was highly significant for both
clovers in 1942, but not significant in 1943.
There were significant differences in number of nodules per
plant of both California Bur and White Dutch clover in 1942
(Table 16). The nodulation of both clovers during 1942 was
generally retarded by minor elements. The reduced nodulation
with many of the minor element treatments may be attributed
to toxic concentrations of fertilizer due to surface applications
and also perhaps to increased acidity.
The number of nodules per plant on White Dutch clover were
significantly different in 1942, but only 3 minor element treat-
ments produced more nodules per plant than untreated soils.
The reduction in number of nodules on plants in soils treated
with minor elements was not as pronounced as with California
Bur clover. The number of nodules per plant on White Dutch
clover did not differ significantly in 1943.

DISCUSSION OF RESULTS
Outstanding result of the experiments was the difference in
the efficiency of cultures for inoculation. Cultures of bacteria
isolated from clovers grown in Florida and processed by a com-
mercial company were generally superior to the cultures not
processed or to the ordinary commercial cultures.







Florida Agricultural Experiment Station


Nodulation of leguminous plants is greatly influenced by soil
acidity and the different species of Rhizobium are affected dif-
ferently. There are various ways in which Rhizobia and nodule
formation may be affected by soil acidity. The acidity may
affect the bacteria directly before they have entered the host
plant or the symbiotic equilibrium between the bacteria and the
host plant may be disturbed by the effect of soil acidity or a
lack of bases on the metabolism of the host plant. The evidence
seems to indicate a detrimental effect of acidity on the organ-
isms as well as a lack of calcium for the nutrition of both the
bacteria and the host plant. The need for limestone to correct
the acidity of the soil was emphasized. Rock phosphate even in
heavy applications could not in all cases successfully substitute
for lime. Limestone and rock phosphate together gave good
nodulation of some of the Trifoliums, but limestone and super-
phosphate were generally superior to rock phosphate alone in
stimulating the nodulation of all clovers.
Salter4 reported that the Red clover bacteria do best at a
neutral or slightly acid reaction of the soil; and Fred and Daven-
port 5 found the critical acidity of these bacteria to be pH 4.2.
Numerous greenhouse and field experiments on Florida soils show
that the Trifoliums require less calcium and can tolerate a
higher degree of soil acidity than the Medicago-Melilotus and
Trigonella groups. Members of these genera of primary in-
terest in Florida are California Bur, Black Medic and Sweet
clover. Alfalfa also belongs to this group and is of interest here
because so much of the research work done has been carried
out on the alfalfa bacteria. Salter 4 reported that alfalfa nodule
bacteria reproduce more rapidly on slightly alkaline media than
on acid media. Fred and Davenport 5 found the critical pH for
alfalfa and Sweet clover organisms to be 4.9. In greenhouse
experiments on acid mineral soils the largest number of nodules
were produced on the species of Medicago-Melilotus when the
initial reaction of the soil was adjusted to pH 6.33 and pH 6.42.
In an acid peat California Bur clover was better nodulated when
sufficient lime was applied to produce about 75 percent satur-
ation of the exchange capacity than at higher or lower pH.
The reaction of this peat was pH 6.21. The effect of soil reaction
on the nodulation of these clovers was illustrated in the 2 field

4 Salter, R. The Behavior of Legume Bacteria in Acid and Alkaline
Medium. Iowa Acad. Sci. Proc. 23: 309-313, 1916.
SFred, E., and A. Davenport. Influence of Reaction on Nitrogen As-
similating Bacteria. Jour. Agri. Res. 14: 317-336.







Legume Inoculation 31
experiments on the same soil type at Largo and at Gainesville
in 1940. The lower pH limit for the successful inoculation of
species of Medicago-Melilotus in Florida soils was about pH 6.3.
A standard rate of application of lime for all soils cannot be given
because of the variation in acidity and buffer capacity of the
different soil types.
The low organic matter content of soils of the Leon, Plummer
and Norfolk series and the poor moisture relations in the Nor-
folk soils, especially during periods of dry weather, cause failure
of inoculation of clovers. The observation has been made re-
peatedly, although no experiments have been reported, that
nodulation is uniformly successful under moderate to heavy
applications of barnyard manure. The organic matter improves
the moisture relations in these soils, preventing the rapid desic-
cation of the organisms and, no doubt, supplies nutritive and pos-
sibly accessory growth substances to both the Rhizobia and the
seedling clovers.
Results obtained in these experiments indicate that the sup-
plemental seed treatments with such materials as syrup, sugar,
cottonseed meal, peanut meal, milk and phosphate solutions are
of doubtful value on inoculation of clovers.
Field and greenhouse experiments 6 on the longevity of Rhizobia
in Florida soils indicate a rapid decrease in the numbers of viable
organisms and explain the beneficial effect of larger dosages of
inoculum for inoculation than those ordinarily recommended by
the producers of commercial cultures. This is undoubtedly re-
lated to fertility level, texture, organic matter, moisture rela-
tions and the low population of Rhizobium in most Florida soils.
Until further research reveals the cause of this rapid death of
the organisms and methods for obtaining better survival it is
recommended that sufficient inoculum be used to supply a large
excess of bacteria for the clovers.

SUMMARY AND CONCLUSIONS
Five greenhouse and 12 field experiments designed to study
the effects of soil acidity, lime and fertilizers, the efficiency of
cultures and various seed treatments on the inoculation of clovers
and lespedeza were conducted on different soil types over a
period of 6 years. The clovers studied were California Bur,
White Dutch, Giant Southern Bur, Black Medic, Annual Sweet,
6 Thornton, Geo. D. Some Factors Affecting the Longevity of Rhizobium
in Florida Soils. Proc. Soil Sci. Soc. Amer. 8: 238-240. 1943.







Florida Agricultural Experiment Station


Red, Hop, Crimson, Persian and Alsike. The lespedeza varieties
used were Common, Kobe and Tennessee 76. The experiments
were conducted on virgin Leon fine sand, Norfolk fine sand,
Plummer fine sand, Norfolk loamy fine sand, Bladen fine sand,
Bladen fine sandy loam, Bladen clay loam, Arredondo fine sand
and an acid peat. Effects on inoculation were determined by
counting the number of nodules per plant after growing inocu-
lated seed in soils variously treated. Yields of some clovers
were determined. Growth and yield of clovers were associated
with good inoculation. The lime requirement of species of
Medicago-Melilotus was found to be higher than that of the
Trifoliums. The amount of lime required also varied with the
soil type. Seventy-five percent of the base saturation capacity
appeared to be about the optimum amount in a strongly acid
peat. Rock phosphate produced satisfactory nodulation of some
of the Trifoliums on certain soil types but rock phosphate and
calcium carbonate gave superior results for all clovers. Supple-
mental seed treatment to enhance nodulation gave contradictory
results and was generally without significant effects. The use
of larger amounts of inoculum than those recommended by the
producers of commercial cultures gave significantly increased
nodulation of clovers. The efficiency of cultures was found to
vary markedly. Bacteria isolated from clovers grown in Florida
were superior to those isolated from clovers grown elsewhere.
The efficiency of the Station cultures was generally increased
by processing.
The results obtained seem to warrant the following conclu-
sions:
1. The limiting pH for the successful inoculation of species
of Medicago-Melilotus was 6.3.
2. The limiting pH for the successful inoculation of the Tri-
foliums was 5.2.
3. Rock phosphate cannot be successfully substituted for
limestone.
4. The lime requirement of each soil type should be deter-
mined and the limestone application made sufficiently in advance
of seeding to permit the reduction of the soil acidity.
5. Approimately 5 to 10 times the amount of inoculum usually
recommended by commercial companies should be used for the
inoculation of clovers.
6. Recently processed cultures of bacteria isolated from
Florida-grown clovers are recommended.




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