Title: Response of pearl millet inbreds and hybrids to inoculation with Spirillum lipoferum
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Title: Response of pearl millet inbreds and hybrids to inoculation with Spirillum lipoferum
Physical Description: ix, 43 leaves : ; 28 cm.
Language: English
Creator: Bouton, Joseph Henry, 1948-
Copyright Date: 1977
 Subjects
Subject: Pearl millet   ( lcsh )
Nitrogen -- Fixation   ( lcsh )
Agronomy thesis Ph. D
Dissertations, Academic -- Agronomy -- UF
Genre: bibliography   ( marcgt )
non-fiction   ( marcgt )
 Notes
Statement of Responsibility: by Joseph Henry Bouton.
Thesis: Thesis--University of Florida.
Bibliography: Bibliography: leaves 39-42.
General Note: Typescript.
General Note: Vita.
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Bibliographic ID: UF00099257
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: alephbibnum - 000011452
oclc - 03403267
notis - AAB3953

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RESPONSE OF PEARL MILLET INBREDS AND HYBRIDS TO
INOCULATION WITH Spirillum iipoFerum






BY



JOSEPH HENRY BOUTON


A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY


UNIVERSITY OF FLORIDA
































Dedicated to the memory of my grandfather,

Basilio Fratesi; a farmer, a man.













ACKNOWLEDGEMENTS


The author wishes to express his sincere appreciation to Dr.

Dex L. Smith, chairman of the graduate coirmittee, for his guidance

throughout the course of this investigation as well as all areas

of the graduate program. Appreciation is extended to committee

members, Dr. S. C. Schank, Dr. R. J. Mans, Dr. J. R. Edwardson,

Dr. A. E. Dudeck, and Dr. D. H. Hubbell for their advice and assis-

tance. A special thanks is expressed to Dr. Ramon Littell for his

help in the statistical parts of this research and to Dr. Sherlie

West for his faith and support.

The author also wishes to acknowledge the following people whose

help made possible completion of this investigation: Loretta Tennant,

James Rarick, Doug Manning, Glen Weiser, Alice Kelly, Doug Baumer,

Bob Turnbull, Dr. Max Tyler, Mary Brown, Mary Leslie, Ken Cundiff,

Joe Rodrigues, and Dr. David Zuberer. The author will always

be indebted to Dr. Glenn Burton for his contribution of the plant

material used in this investigation.

Sincere appreciation is extended to the author's wife, Mary

Jeanne, and daughter, Melinda, for their love and understanding.













TABLE OF CONTENTS

Page


ACKNOWLEDGEMENTS ---------------------------------------- iii

LisT OF TABLES --------------------------------------------- v

ABSTRACT ---------------------------------------- vii

INTRODUCTION ----------------------------------- ----- 1

REVIEW OF LITERATURE ---------------------------------------- 2

Associative N2-Fixation --------------------------------- 2
Nitrogenase Enzyme and Assays of N2-Fixation ----------- 5
Taxonomy, Breeding and Cytogenetics in Pearl Millet ----- 9

MATERIALS AND METHODS --------------------------------------- 12

Standardized Procedures --------------------------------- 15
Screening Trials --------------------------------------- 17
Nitrogen Balance Study ---------------------------------- 20

RESULTS AND DISCUSSION -------------------------------------- 22

Screening Trials --------------------------------------- 22
Nitrogen Balance Study ---------------------------------- 31

CONCLUSIONS -------------------------------------------- 37

LITERATURE CITED ---------------------------------------- 39

BTOGORAPHICAL SKETCH ---------------------------------------- 43












LIST OF TABLES


Page

TALE 1 PEARL MILLET INBREDS AND HYBRIDS USED IN
SCREENING FOR RESPONSE TO Sp 13t INOCULUM
IN GREENHOUSE AND FIELD STUDIES -------------------- 13

TABLE 2 COMPOSITION OF NITROGEN FREE AND NITROGEN
CONTAINING MEDIA USED TO PRODUCE ALL LIQUID
BACTERIAL INOCULUM -------------------------------- 14

TABLE 3 DRY WEIGHT AND ACETYLENE REDUCTION RESPONSE OF
PEARL MILLET INBREDS TO INOCULATION WITH Sp 13t.
CONTROL TREATMENTS WERE AUTOCLAVED Sp 13t. PLANTS
WERE GROWN IN FLATS IN THE GREENHOUSE AND EFFECTS
OF TWO AUTOCLAVED SOIL TYPES (FIELD SOIL ANALYZED
AT 1.3% ORGANIC MATTER AND 0.05% NITROGEN AND
FIELD SOIL AMENDED WITH MANURE AND DECOMPOSED
PEAT TO ANALYZE AT 3.5% ORGANIC MATTER AND 0.10%
NITROGEN) WERE ALSO INVESTIGATED ------------------- 23

TABLE 4 DRY WEIGHT RESPONSE OF PEARL MILLET HYBRIDS TO
INOCULATION WITH Sp 13t. CONTROL TREATMENTS
WERE AUTOCLAVED Sp 13t. PLANTS WERE GROWN IN
THE GREENHOUSE IN POTS CONTAINING AUTOCLAVED
FIELD SOIL --------------------------------------- 24

TABLE 5 AGRONOMIC YIELD IN DRY MATTER, PERCENT NITROGEN,
AND TOTAL NITROGEN OF PEARL MILLET HYBRIDS FROM
THE FIELD SCREENING TRIALS ------------------------- 26

TABLE 6 AGRONOMIC YIELD IN DRY WEIGHT, PERCENT NITROGEN,
AND TOTAL NITROGEN OF PEARL MILLET INBREDS FROM
THE FIELD SCREENING TRIALS ------------------------- 27

TABLE 7 PERCENT HETEROSIS OF HYBRIDS OVER THEIR HIGHEST
YIELDING PARENT AS CALCULATED FROM THE CONTROL
(AUTOCLAVED INOCULUM) TREATMENT -------------------- 28

TABLE 8 ACETYLENE REDUCTION VALUES BY CORE AND WASHED
ROOT ASSAYS OF INBREDS AND HYBRIDS FROM THE
FIELD SCREENING TRIALS ----------------------------- 30

TABLE 9 DRY WEIGHT AND NITROGEN RESPONSE OF 23DA X 186
PLANTS TO INOCULATION WITH Sp 13t IN THE NITROGEN
BALANCE EXPERIMENT -------------------------------- 33













Page
TABLE 10 NITROGEN BALANCE OF THE 23DA X 186 SOIL
SYSTEM IN THE NITROGEN BALANCE EXPERIMENT ---------- 34

TABLE 11 ACETYLENE REDUCTION VALUES ON TREATMENTS
IN THE NITROGEN BALANCE STUDY. SAMPLES
WERE TAKEN AT TERMINATION OF THE EXPERIMENT,
INCUBATED IN ARGON WITH 10% ACETYLENE,AND
SAMPLES ANALYZED BY GAS CHROMATOGRAPHY AT
17 AND 24 HOURS ------------------------------------ 36













Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fulfillment of
the Requirements for the Degree of Doctor of Philosophy



RESPONSE OF PEARL MILLET INBREDS AND HYBRIDS TO
INOCULATION WITH Spirillum lipoferum

By

Joseph Henry Bouton

August 1977

Chairman: Rex L. Smith
Major Department: Agronomy


Inoculation experiments were conducted on six hybrids and 15

inbreds (including the hybrid parents ) of pearl millet, Pennisetum

americanum (L.) K. Shun., using the N2-fixing bacterium, Spirillum

lipoferum Beijerinck (Sp 13t), as liquid inoculum. The objective of

this study was to investigate reconstitution of N2-fixation among

these inoculated plant genotypes by measuring enhancements of dry

weight, percent nitrogen, total nitrogen and acetylene reduction

(nitrogenase) activity.

One hybrid, Tift 23DA X Tift 186 ('Gahi 3'), gave significantly

(p = 0.05) higher dry weight yield in response to field inoculation.

Inoculated plots of 'Gahi 3' produced 32% more dry weight and 37%

more total plant nitrogen when compared to killed inoculum controls.







"o inbred was found to respond as vigorously as 'Gahi 3', but one,

Bil 3B, was enhanced 17% in dry weight by inoculation. Acetylene

reduction values were low (range 0 54 nmole/g dry root x hr) and

did not support yield effects. This assay was not used extensively

because of the damage it would cause plant agronomic yields. The

possibility exists that high levels of acetylene reduction were not

measured due to the limited sampling.

A nitrogen balance study was conducted in the greenhouse in

large ceramic containers on inoculated 'Gahi 3' plants to repeat

the yield differences observed in the field and to monitor inputs

of nitrogen into the soil-plant system. Increases of 3.2% in total

plant dry weight (p = 0.28) and 4.3% in total plant nitrogen (p =

0.21) were observed when compared to autoclaved inoculum controls.

No increase of nitrogen into the soil-plant system was found due to

inoculation. A sampling error of 3.48% was calculated and any

associative N2-fixation achieved could have beenwithin the limits of

this sampling error.

It is concluded that differences do exist among pearl millet

genotypes to respond to bacterial inoculum with increased agronomic

yield, but sensitive assays such as 15N2 incorporation must be used

to confirm N2-fixation in these responders. Further screening for

associative N 2-fixation achieved through inoculation should place

initial emphasis on well replicated experiments designed to show

increased nitrogenase activity by extensive sampling with acetylene









reduction or, if possible, 15N2 incorporation. Those genotypes which

are deemed responders should then be field tested individually for

increases in agronomic yield and acetylene reduction.




ChAirman'











INTRODUCTION


Biological nitrogen fixation (N2-fixation) by plants in associ-

ation with prokaryotic organisms is one of the most important natural

processes on the earth today. The legume-Rhizobium symbiosis is such

an association where N2-fixation is responsible for fulfilling the

plant's nitrogen requirement for growth and production. Inoculation

with Rhizobium bacteria to achieve N2-fixation is well known and

facilitates its agricultural use. Associative plant-microorganism

N2-fixing systems are found in agricultural soils. These groups

are believed to fix appreciable quantities of nitrogen worldwide.

An associative N2-fixing bacterium, Spirillum lipoferum Beijerinck,

was isolated from the roots of various tropical grasses and showed

potential for contributing nitrogen to this group of plants. Plant

genetic variation to associate with S. lipoferum was predicted (21).

Any plant variability offers excellent potential to enhance associative

N2-fixation by use of plant breeding.

The purpose of this study was to measure differences among inbreds

and hybrids of pearl millet, Pennisetum americanum (L.) K. Shum., to

reconstitute N2-fixing associations with Spirillum lipoferum, Spl3t,

i~oculum. Pearl millet is a diploid tropical forage and cereal grass

in which selling and hybridization is easily achieved. Variability

within this grass to associate with bacterial inoculum for increased

hiN-fixation is an important plant breeding consideration.










REVIEW OF LITERATURE


Associative N2-Fixation


Crop plants require a great deal of nitrogen for maximum growth

and production. Each hectare of land is surrounded by an atmosphere

containing over 80,000 MT of nitrogen yet none is directly useful to

plants. This atmospheric nitrogen must be reduced to usable plant

forms. Reducing nitrogen to synthetic fertilizer forms by the Haber-

Bosch process is wasteful of nonrenewable energy sources, but nitro-

gen produced by the process increased from 4.5 X 106 MT in 1957 to

36 x 106 MT in 1974. Biological N2-fixation uses renewable solar

energy, and though estimated to contribute 80 x 106 MT of nitrogen

per year, its contribution remained constant during the same time

(30). Efficient use of biological N2-fixation to supply nitrogen to

agricultural crops is needed as nonrenewable energy supplies dwindle

and become more expensive.

The legume-Rhizobium symbiosis is an efficient N2-fixing system.

But nitrogen contributed to agricultural land by associative N2-fixing

organisms is considerable. Estimates predict quantities of nitrogen

fixed by these systems to approach the 10.8 X 106 MT fixed by legumes

in the United States. Available energy limits N2-fixation by asso-

ciative systems in soils. Microorganisms which produce energy by

photosynthesis, or those which derive energy in association with plants

are more likely to fix significant amounts of nitrogen for agricultural

crops (44).









Nonleguminous plants, especially the cereal grains, constitute

the world's major food producing crops. Associative N2-fixation shows

promise for supplying nitrogen to these crops. Nitrogen-fixing systems

are now documented in rice (Cryza sativa L.), maize (Zea mays L.),

sugar cane (Saccharum officinarum L.), and several tropical pasture

grasses (18, 21, 24, 47). Rates of fixation in rice were reported

to be equal to some legumes in flooded paddies (24). The bacterium,

Azotobacter paspali, was discovered in close association with bahia-

grass, Paspalum notatum Flugge (20). Colonies of A. paspali were

found to establish in the root mucigel layer and N2-fixation rates

up to 90 kg N/ha/yr were predicted in this associative system (22).

A. paspali is found almost exclusively on broad leaved, pubescent

bahiagrass cultivars such as 'Batatais' (33).

Efficient N2-fixation in 'Transvala' digitgrass, Diiitaria

decumbens Stent., led to the isolation of strains of the bacterium,

Spirillum lipoferum, from the roots of this forage grass (21).

Spirillum strains were found to fix nitrogen in pure culture by

acetylene reduction, Kjeldahl total nitrogen, and 15N2 incorporation

(21, 39). Cultures showed optimum temperature for their N2-fixation

to range from 32 to 40 C with little fixation below 24 C or above

42 C (17). The bacterium reduced N2 only under microaerophylic

conditions and optimum p02 for acetylene reduction was between 0.006

and 0.02 atmospheres (41). Cell free extracts require both Mg2+ and

Sfor good nitrogenase activity (40). Spirillum lipoferum is a

co-ron soil organism and guineagrass, Panicum maximum Jacq., is

believed to be the most favored forage grass for isolating the organism

(23).









Differences for potential N2-fixation are found among cultivars

of Paspalum and Digitaria (21, 42). Screening maize for N2-fixing

ability by the acetylene reduction assay showed variability between

S1 lines of the rust resistant variety UR-1. The best lines exhibited

potential fixation of up to 2 kg N/ha/day and N2-fixing Spirillum

strains were isolated From these active S1 plants (47). This report

indicates genetic variation within maize to interact with Spirillum

for associative N2-fixation. Beneficial colonization of grass roots

by S. lipoferum as a criteria in plant breeding warrants investigations.

Inoculating legumes with Rhizobium bacteria facilitates its

experimental and agricultural use. Reconstitution of grass-Spirillu

N2-fixing associations through inoculation is therefore important.

When used as inoculum in the field, S. lipoferum (strain Sp 13t)

produced significantly higher yields of dry matter than did uninocu-

lated controls in field grown, lightly fertilized pearl millet and

guineagrass. Up to 42 and 39 kg N/ha were calculated by regression

analyses to be replaced by Sp 13t inoculum in these two respective

grasses (45). Work in Oregon with Spirillum inoculum (strain Sp 81)

or maize showed less than 4 g N/ha/day fixed. Yield and nitrogen

content were not enhanced, thus providing no evidence of appreciable

i2-fixation (1). Inoculation responses of enhanced dry matter and

tocal plant nitrogen in buffelgrass, Cenchrus ciliaris L., with Sp 13t

inoculum were not repeatable in the second year of testing (6).

Use of bacterial inoculants other than Rhizobium are reported

by Bron. (7) to be unsuccessful in achieving N2-fixation. Any

beneFiclal plant yield responses were thought to come from other









modes of action such as production of growth regulating substances,

suppression of plant pathogens, or mineralization of soil phosphates.

Culture supernatant of the N2-fixing bacterium Azotobacter paspali

was found to contain the plant growth regulators IAA, GAand cytokinin

in appreciable amounts (2). Inoculation studies on bahiagrass plants

grown in pots revealed no evidence of N2-fixation and increases in

plant yield were postulated to come from the growth regulating sub-

stances secreted by the bacteria. This study was conducted under

low light intensities. Achievement of N2-fixation under high light

conditions in the field was not discounted (8).


Nitrogenase Enzyme and Assays of N,-Fixation

Any study on biological N2-fixation requires a basic under-

standing of the nitrogenase enzyme and those assays which allow

measurement of the process. More intensive reviews are available

on the physiology and biochemistry of the process (4, 10, 48).

Nitrogenase catalyzes the reduction of atmospheric nitrogen

to ammonia and the reaction is shown in the following equation (43):

N2 + 6e + 12 ATP + 12 H20 ---- 2 NH3 + 12 ADP + 12 Pi + 4H+.

Thermodynamically the energy demanding step in N2-fixation is

the breakdown of the nitrogen-nitrogen triple bond in diatomic mole-

cular nitrogen. The bond energy required to reduce N--N to N=N is

126 kcal and is considered large for a biological system (32). Nitro-

genase is therefore a high energy electron acceptor. In the electron

donor system of the nodule bacteriods, NADPH which is generated by









glucose-6-phosphate dehydrogenase and the electron carriers, ferredoxin

and flavodoxin, will couple electrons with nitrogenase (49).

The reduction of N2 with H2 is exothermic at one atmosphere and

a free energy change of -17.5 kcal/N2 when 3 H are supplied has been

calculated for the system. The following reduction reactions show

the exothermic nature of N2 reduction with the exception of N2 to

the diimide. This tends to support the theory that the diimide is

not a free intermediate of the system (32).

Reduction Reaction H (kcal/mol at 25 C)

N2 + H2 --- N2H2 +44
N2H2 F H2 --- N2H4 -20

N2H4 + H2 --- 2NH3 -44

Nitrogen reduction to NH3 is therefore thermodynamically favored

and theoretically should need no input of energy or ATP. Energy is

shown to be essential to nitrogenase from ATP saturation curves and

must be present in any cell free preparation for completion of the

reaction (10). Since most nitrogenase reactions are shown as occurring

in two electron steps and expressed as electron pairs transferred;

2H+ + 2e --- H2

6H +N2 + 6e --- 2NiH3

this has led to speculation that ATP is utilized at the rate of 4.0

to 4.6 moles per 2e- (mole of H2 evolved or 1/3 N2 fixed). This

would give some 6.0 to 6.9 ATP molecules for each ammonium (NH4+)

ion as this ion is usually the form found in biological systems (5).

This need for available energy is thought to be the major limiting

factor in the development of associative N2-fixing systems (44).










Nitrogenase reactions utilizing substrate levels of ATP proceed

optimally when Mg2+ is supplied at a level which assumes the presence

of both an ATP:Mg2+ complex and free ATP. But there can be any number

of divalent cations used in the following order of importance: Mg,

Mn2, Co2+, Fe2+, and Ni2+ (10).

After N2 reduction, the ammonia produced is believed to be

assimilated by entering organic combination with keto acids produced

via the tricarboxylic acid cycle in one of two ways. With glutamate

dehydrogenase participating, the initial major product is glutamate,

followed by glutamine. This has been confirmed by 15N tracer studies

which showed these compounds to in fact be early labeled products (36).

Recently, another pathway of ammonia incorporation in free living

systems has been postulated via glutamine synthetase (36, 43). Gluta-

mine synthetase is believed to have the primary role of reacting with

the amino acid glutamate and NH3 produced from fixation to form the

amino acid glutamine (43).

Inhibitors of nitrogenase include carbon monoxide, hydrogen,

oxygen, and ammonia (32). Nitrogen fixation is an anaerobic process

with oxygen inhibiting the activity of nitrogenase (32, 36). Natural

systems to protect the enzyme from this element have been evolved. The

nodulating bacteriods are surrounded by leghemoglobin to give the

enzyme protection from 02. In free living Azotobacter, 02 is consumed

via cytochrome oxidase system bound to its membrane. But 02 sensitivity

would be another limiting factor in development of associative N2-fixing

sy-tems (44).









Three methods of assaying N2-fixation are used: Kjeldahl total

nitrogen, incorporation of 15N,and acetylene reduction. The Kjeldahl

method for total nitrogen determination is slow, destroys the plant

and fixed nitrogen is not distinguishable from that obtained from

other sources (29). Even with these drawbacks, total nitrogen is

a good method for confirming the results of other indirect N2-fixing

assays such as acetylene reduction (27). Alone, the method is satis-

factory if done with extensive replications and statistical analyses

to show total nitrogen gain in a system (11, 19). A nitrogen increase

of 1% needs to take place before differences can be detected (11).

The isotope, 15N, provides a more direct assay method and any

increase from the gaseous form (15N2) in the plant gives positive

evidence of N2-fixation (11, 29). It is also not subject to the

sampling problems of the Kjeldahl total nitrogen procedure. Accumu-

lation of 0.015 atmo % excess of 15N would require only 0.025% increase

in total plant nitrogen, thus it would be 100 times more sensitive

than the total nitrogen assay (11). But, 15N is expensive and a mass

spectrometer is required in the analysis (27).

An important and extensively used assay method is acetylene re-

duction (3, 19, 27, 29). It is based on the fact that nitrogenase

catalyzes the reduction of acetylene to ethylene (C2H2 to C2H4) as

well as the reduction of N2 to NH3 (34). In its simplest form the

assay is conducted on the desired plant material or microbial culture

in a sealed chamber which has a port for exchanging gases. Acetylene

is introduced into the chamber in known concentrations with an inert









gas such as argon and incubated for specific periods of time. A

sample is taken and gas fractions separated through a gas chromato-

graph to measure amounts of ethylene reduced (29).

Acetylene reduction is very useful for investigations into the

biochemistry and physiology of nitrogenase and N2-fixation, as well

as defining N2-fixing systems of soil and plants. Although sensitive

to the nanomole range, this assay must be supplemented by other

nitrogen determining methods as the 3:1 theoretical conversion ratio

(ethylene:nitrogen) is not consistent for all systems (31).


Taxonomy, Breeding,and Cytogenetics in Pearl Millet

Pearl millet dry matter yields were significantly enhanced with

Spirillum lipoferum (Sp 13t) inoculation (45). This enhanced response

and the ease of manipulating this grass genetically led to its selec-

tion for use in this investigation. A review of its taxonomic,

breeding,and cytogenetic characteristics is nowv presented.

Pearl millet is an annual tropical grass found to be very

resistant to low soil moisture situations. It is thought to have

originated in tropical Africa and is cultivated as a food grain in

Africa and India. It is used in the Southern coastal plain of the

United States for temporary summer grazing (35). The scientific

name of pearl millet has changed over the past two decades and the

literature bears such names as Pennisetum glaucum, P. typhoideum,

P. typhoides (Burm) and presently P. americanum (L.) K. Shum (15,35).

At maturity, plants of pearl millet range from 1.5 to 3 meters

in height and have flat leaf blades which reach to 1 meter in length.










Panicles of this grass are stiff and cylindrical from 20 to 45 cm

in length and are similar in size and shape to common cattail growing

in marsh areas. Panicles possess short-pedicled, two-flowered spikelets

with the lower floret imperfect and usually staminate. The upper

floret is fertile and perfect with the caryopsis surrounded by a

bristled involucre (35).

Pearl millet is highly cross-pollinated with its stigmas being

exerted several days before the anthers. This flowering characteristic

facilitates making hybrids in a plant breeding program. Heterosis

has resulted in increased forage yields of up to 71% when hybrids

are compared to their inbred parents. But even with its cross-

pollinated character, selfed seed is easily obtained by bagging (15,

35).

Pearl millet is generally a diploid with a basic chromosome

number of 7. Its chromosome pairing at metaphase I was studied

and found to give 59.3% closed bivalents, 34% open bivalents and

6.8% univalents. Autotetraploids were induced and found to be less

fertile than diploids. Autotriploids are produced by crossing

tetraploids to diploids. Autotriploids will occur spontaneously

from fertilization of unreduced female gametes (15).

Interspecific hybrids can be produced with pearl millet. It

is generally easier to obtain these hybrids with those species having

the same basic chromosome number. Pennisetum purpureum (4x=23) has

been hybridized successfully with pearl millet (15).










Burton (12) found plants from pearl millet crosses involving in-

bred 556 x inbred 23 failed to shed pollen or set selfed seed. Seed

were set if dusted with pollen from inbred 23. Plants from the back-

cross, (556 X 23) X 23, shed no pollen or selfed seed yet when polli-

nated with other lines readily set seed. This was the first recorded

case of cytoplasmic male sterility (CMS) in pearl millet. Further

work resulted in the release of the male sterile inbred lines 23A

and 23DA and their maintainers 23B and 23DB (14). Several other

sources of CMS lines have also been located (16). This characteristic

has made possible commercial production of hybrid pearl millet seed.

Spontaneous mutations from male sterile to male fertile plants

were observed and thought be be both cytoplasmic and genetic in

nature (13). Subsequent studies on four different male sterile

sources indicated the mutations to be cytoplasmic with mutation

rates of 0.15 to 1.02/100 seedheads greatly influenced by the plant's

genotype (16).









MATERIALS AND METHODS


Fifteen inbreds and five hybrids of pearl millet were obtained

(courtesy Dr. Glenn Burton, USDA-ARS, Tifton, GA) for use in screening

for inoculation response. The sixth hybrid (23 DA x PMB004, courtesy

Jimmy Barber, North American Plant Breeders, Hutchinson, Kansas) was

the female of the three-way cross shown to respond to Spirillum

inoculation (45). All genotypes are shown in Table 1. The A lines

are cytoplasmic male sterile and B lines are maintainers for them.

D lines carry the dwarf gene. Any commercial hybrid is listed in

parenthesis by its varietal name.

Inoculum was Spirillum lipoferum, strain Sp 13t (courtesy Dr.

Johanna Dobereiner, Empresa Brazileira de Pesquisa Agropecuaria,

Rio de Janeiro, Brazil) grown in sealed 12 1 glass containers with

aeration in either nitrogen free or nitrogen containing liquid medium

(Table 2). To standardize the Sp 13t inoculum applied in different

experiments, cells were first cultured in rich peptone medium for 24

hours, centrifuged and supernatant removed. Peptone medium was 15%

glycerol substituted for the water was added to an equal volume of

cells and sedimented cells resuspended. This heavy suspension (1011

cells) was placed in 1 ml aliquots in Cooke pro-vials and stored

under liquid nitrogen (courtesy Dr. Max E. Tyler, Department of

Microbiology, University of Florida, Gainesville, FL).

All fertilizer was applied at a calibrated per hectare rate of

60 kog (as ';' "^ ), 7.9 kg P, 30.1 kg K and 5.5 kg of fritted trace











TABLE 1


PEARL MILLET INBREDS AND HYBRIDS USED IN SCREENING FOR
RESPONSE TO Sp 13t INOCULUM IN GREENHOUSE AND FIELD STUDIES


Tift 23D
Tift 23D
Tift 23A
Tift 23B
Tift 239






Tift
Tift
Tift
Tift
Tift
Tift


!A
IB



A


INBREDS

Tift 186
Tift 239B
Tift 383
Tiftlate
Tift 23SB


Tift 131
Tift 123
Tift 18DB
Tift 13
Bil 3B


HYBRIDS
23DA x Tiflate
23DA x Tift 18DB
23DA x Tift 186 (Gahi 3)
23DA x Tift 383 (Tifleaf)
23DA x PMBO04
23A x Bil 3B












TABLE 2


COMPOSITION OF NITROGEN FREE AND NITROGEN CONTAINING
MEDIA USED TO PRODUCE ALL LIQUID BACTERIAL INOCULUM


D-L Malic Acid

NaC1

MgSO4 7 H20

CaC12

FeC13

K2HPO4

KH2PO4

NaMoO4 2H20

Yeast extract

NHr4 Cl

Tryptocase(l'6 N)


N Free*
g/liter

5

0.1

0.2

0.02

0.01

0.1

0.4

0.002


N containing
g/liter

5

0.1

0.2

0.02

0.01

6

4

0.002

0.05

1

5


*From Dobereiner and Day (21)









elements (5 g B, 5 g Cu, 29 g Fe, 12 g Mn,0.3 g MO,and 11 g Zn).

Fertilizer rates and Sp 13t were selected on the basis of previous

success of enhancing growth in pearl millet with these two parameters

as variables (45).

Soil used in greenhouse and field studies was an Arredondo loamy

fine sane, pH 6.0 co 6.5. Any soil autoclaving was achieved at .844

kg/cm2 steam pressure for five hours then repeated 24 hours later.


Standardized Procedures

Throughout this investigation, increases in plant dry weight,

percent nitrogen, total nitrogen,and acetylene reduction were used as

predictors of N2-fixation achieved through Sp 13t inoculation. Data

were statistically analyzed by analysis of variance and comparisons

between means made by LSD. Mention of a trademark or proprietary

product does not constitute or guarantee warranty of the product,

and does not imply its approval to the exclusion of other products

that may be suitable.

Dry weight measurements were determined on soil and plant tissue

dried at 60 C for 48 hours in a forced air drier. Dried material was

then prepared for nitrogen analyses. Soil was screened four times

through a 2 mm screen and plant tissue was ground in a 1 mm mesh Wylie

mill and/or a 1 mm mesh disintegrator (Christy Norris LTD, Chelmsford,

England). Material was stored in sealed, plastic bags in a freezer

(-12 C) until ready for use.

All nitrogen determinations were conducted by first performing a

block digestion (25) on 2 g of the screened soil or .1 g of the ground










plant tissue. This digestion was conducted in 10 ml of concentrated

H2S04, 2 ml of concentrated H202, 3.5 gn of catalyst-salt mixture

(90% K2504 : 10% CuSO4),and 2 to 3 boiling chips for 1.5 hours on an

aluminum block heated to 400 C (25). The digestate was then diluted

to 75 ml with distilled water and analyzed by a Technicon nitrogen

autoanalyzer for nitrogen concentration.

Acetylene reduction assays were conducted on root-soil cores or

preincubated, excised washed roots. Preincubation denotes that time

before acetylene is introduced into the chamber containing the roots.

Underestimations of acetylene reduction in freshly washed roots are

reported due to a lag period in nitrogenese activity. Preincubation

of washed roots is conducted to overcome this problem (27). But

preincubation is believed to overestimate predictions of quantities

of nitrogen fixed possibly due to increases in microbial populations

(1). The core method was used to overcome the preincubation problems

and was thought to offer least disturbances of root-bacteria systems.

Washed roots were sealed in plastic bags, then preincubated over-

night in an atmosphere of 5% 02 95% N2 at 30 C. Plastic bags con-

tained a rubber septum taped on its side to allow exchanging of gases.

After preincubation, the atmosphere was replaced with a fresh 02 NI2

mixture and 10% acetylene added. After three hours incubation at 30 C,

samples (11 cc) were taken, placed in 8 cc evacuated tubes,and subse-

quently analyzed by gas chromatography. Details of chromatographic

analysis have previously been reported (27).










Core samples were prepared as follows: The top of pearl millet

plants were cut away at ground level. A soil core containing the

roots were removed by inserting a metal collar (made by removing both

ends of a soft drink can and sharpening the bottom rim) over the plant

stubble, forcing the collar into the soil, and then removing the soil

core along with the metal collar. The intact soil core with metal

collars was placed in glass jars with a void volume of 473 ml. Glass

jars were sealed with metal lids and threaded rims (Mason jars). Metal

lids contained a sealed rubber septum for gas exchange. The atmosphere

was replaced with gas containing partial pressures of 0.86 atm N2,

0.05 atm 02 and 0.09 atm acetylene. After a four-hour incubation at

30 C, samples were taken and analyzed as described in the washed root

procedure.


Screening Trials

Greenhouse screenings were conducted to supplement field screening

trials. It was believed that the field trials represented the best

way to screen for inoculation response since a plant achieves maximum

growth and development in the field. But field experiments require

much time and space to conduct, whereas planting in small greenhouse

containers allows screening of many plants anytime during the year.

Also, the greenhouse screening studies permit testing the effects of

added organic matter and nitrogen to the system. Genotype responses

in the greenhouse flat and pot studies were compared to their field

results.










Greenhouse Screening

Two types of greenhouse screening experiments were conducted: one

in flats, the other in pots. In the experiments conducted in flats,

Tift inbreds 123, 13, 23 SB and 131 were used. Seed of each inbred

were planted in rows at a rate of .5 g/m of row in the 54 cm x 38 cm

x 6 cm flats. Each flat contained either autoclaved field soil analyzed

at 1.3' organic matter and 0.05% nitrogen or an autoclaved field soil

amended with manure and decomposed organic matter to analyze at 3.5%

organic matter and 0.10% nitrogen. All flats were given standard

fertilizer rates. Seven days after planting, 30 ml of Sp 13t inoculum,

grown under standard conditions in nitrogen free medium to approximately

107 cells/ml, were drenched onto each row within each flat. Control

flats received the same rate of autoclaved inoculum in the same manner.

Treatments were thus a factorial combination, inbred X soil type X

inoculum, at one flat each. Plant tops were harvested for dry matter

28 days after inoculation. A washed root acetylene reduction assay

was also conducted at harvest on plants grown under each treatment.

The experiment was repeated one month later but with two flats per

treatment.

In the pot experiments, four half-sib hybrids, 23DA X PMB004,

23DA X 333, 23DA X 186,and 23DA X 18DB, and the maintainer line 23DB

were germinated in cell packs. Four uniform seedlings were then trans-

planted into 25 cm plastic pots containing autoclaved field soil from

the nursery area where the field screenings were conducted. Pots con-

tainiing the plants were given standardized fertilizer rates. Three










days later, 25 ml treatments of Sp 13t inoculum or autoclaved inoculum,

grown under standard conditions in nitrogen free media to approximately

107 cells/mi, were injected by a needle and syringe into the root zone

of respective plants. Treatments were thus a factorial combination,

genotype X inoculum at three replications each. After a 21-day re-

growth, plants were harvested a second time for dry matter.


Field Screening

All inbreds and hybrids were tested in a field production nursery

for enhanced plant dry matter, nitrogen,and acetylene reduction to

Sp 13t inoculation. The experiment was conducted in split-plot design

with genotypes as a six row main plot, replicated three times. All

rows were 2.7 meters long. Subplots of Sp 13t inoculum or autoclaved

inoculum were drenched onto the second or fifth row with watering cans

then lightly irrigated to wash the bacteria into the soil. Inoculum

was grown under standard conditions in nitrogen containing medium and

applied at the rate of 4 X 107 cells/cm of row. Inoculum treatments

were reapplied two weeks later with a small push plow rigged with a

CO constant delivery sprayer. This device injected the liquid into

the plant's root zone through a nozzle behind the furrow opener. In-

jection was used to avoid the need to irrigate as with the drench

method. Also, this sprayer delivered a constant supply of liquid

over a given area at a pressure of 3.57 kg/cm2

Seed of each genotype were planted at a rate of .5 g/n of row

with a belt planter. Standard fertilizer and inoculum treatments










were applied after seedling emergence. General maintenance of hand

weed control, irrigation,and insect pest control with Lanate were

used as needed. At anthesis (70 to 80 days) plants were sampled for

acetylene reduction by both core and excised washed root assays.

Sampling at full anthesis was reported to be the best time to sample

for highest activity in other grass Spirillum systems (47). Four

random plants per treatment row were taken for percent nitrogen deter-

minations. The complete row was harvested with a Carter flail har-

vester and total wet forage weighed. A subsample of the forage was

taken for dry weight calculations.


Nitrogen Balance Study

Hybrid 23DA X 186, that responded to Sp 13t inoculation with in-

creased yield in the field screening trials was subjected to a nitrogen

balance study. Inputs of nitrogen due to inoculations into the plant -

soil system were monitored.

Glazed, ceramic containers, 23 cm in diameter and 30.5 cm deep,

with a drain in the bottom for catching leachable liquid were used

in this study. Nonautoclaved bulk-mixed soil from the field nursery

area was weighed for each container and a sample taken with a 2.5 cm

probe for soil dry weight determination. Standard fertilizer rates

were added to the soil and mixed in a clean cement mixer for 15 minutes

per container. Two uniform seedlings of 23DA X 186 were transferred

into each container and 100 ml of inoculum or autoclaved inoculum were

injected into each container with needle and syringe. Inoculum was

grown under standard conditions to 108 cells/ml in nitrogen containing









medium. After 24 hours, five soil samples were taken from each con-

tainer with a 2.5 cm probe through the soil profile to give the initial

nitrogen determinations for each container. The experiment thus con-

sisted of two treatments, inoculum and autoclaved inoculum, as pairs

within each of 18 blocks. Throughout the study, each pair was rotated

weekly within its block to overcome border effects. Light was supple-

mented from incandescent and flourescent sources at the end of the

day to achieve a 13.5 hour photoperiod. Plants were hand watered once

a day to completely wet soil profile. Any leachable liquid was col-

lected and returned to its respective container.

After 42 days growth, five soil samples were taken as before.

Plants were then removed from their containers and washed free of

remaining soil. A small sample of roots was excised for a washed

root acetylene reduction assay. This washed root assay differed

from the one previously described in that no preincubation was used,

argon was substituted for the 95% N2 5% 02 air mixture and samples

were taken directly for chromatography at 17 and 24 hours. Soil and

plant tissue was dried and prepared for nitrogen analysis.













RESULTS AND DISCUSSION


Screening Trials

Greenhouse Screening

Results of the experiment conducted in flats are presented in

Table 3. Data from the two studies which were repeated over time

were composite as replications. This was done to give a truer

representation of inoculation response as the number of replications

at each time were limited. Trends of increasing dry weight are found

within each soil type. Acetylene reduction activity was similar

within amended field soil but decreased in the nonamended field soil.

Data for the experiment conducted in pots are presented in Table

4. In harvest 1, inoculum significantly (LSD = 0.35, p = 0.05)

enhanced overall dry weight (data pooled across all hybrids). This

trend was significantly reversed (LSD = 0.56, p = 0.05) in favor of

autoclaved inoculum on regrowth to havest 2.

Limited replications and possible contamination of control treat-

nmnts probably contributed to the erratic greenhouse screening data.

These problems resulted in measuring no differences among inbreds

and hybrids for response to Sp 13t inoculum. All statistically signi-

ficant inoculum responses occurred in overall means for each treatment

as these data have more replications (pooled across all genotypes).


























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Field Studies

Agronomic dry weight, percent nitrogen,and total nitrogen for

the hybrids are presented in Table 5. The 23DA X 186 ('Gahi 3')

hybrid gave a significant response in dry weight (LSD = 2372, p = 0.05)

to Sp 13t inoculation when compared to its autoclaved control. In

this hybrid, the autoclaved control yielded approximately 13 MT/ha

over 80 days with inoculation enhancing dry weight to approximately

17 EK/ha for a 32% increase. A 37% increase in total plant nitrogen

was brought about by inoculation in this hybrid. Other hybrids did

not respond significantly to inoculation for any agronomic parameter.

Inbred dry weight, percent nitrogen,and total nitrogen data are shown

in Table 6. It is noted that enhanced responses in dry weight of 1192

and 1151 kg/ha are seen in inbreds Bil 38 and Tiflate respectively.

This would indicate trends that warrant further testing.

The hybrid, 23DA X 186 ('Gahi 3'), responded in dry weight to

inoculation while its parents showed no response. If the degree of

heterosis of hybrids over their highest yielding parent is examined

in Table 7, 'Gahi 3' exhibits a low degree of heterosis compared to

other hybrids in the study. It appears from these results that the

degree of hybrid vigor or heterosis would not be a good predictor of

plant response to inoculation, but rather the specific plant genetic

might be. This is especially interesting when compared to work in

what by Neal and Larson (37) where an asymbiotic bacillus possessing

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TABLE 7

PERCENT HETEROSIS OF HYBRIDS OVER THEIR HIGHEST YIELDING PARENT
AS CALCULATED FROM THE CONTROL (AUTOCLAVED INOCULUM) TREATMENT





Hybrid % Heterosis

23DA X 18DB 42.5

23DA X Tiflate 31.8

230A X 383 50.7

23DA x 186 15.4

23DA X Bil 3B 8.6









They suggested that the genetics of the wheat plant might play a large

role in altering establishment of such bacilli in its root environment.

Acetylene reduction values in both inbreds and hybrids were low

(range 0-54 nmole/g dry root x hr) and did not support the yield para-

meters (Table 8). Plots were not sampled extensively for acetylene

reduction because of the damage it would cause agronomic yields.

Acetylene reduction only measures nitrogenase activity during the

time of the assay and the active time of the enzyme might have been

missed. The possibility therefore exists that N2-fixation was not

measured due to the limited samplings (once for each assay at harvest).

Nitrogen content (percent nitrogen) was not increased by inocu-

lation. Normally one expects nitrogen content to increase with better

nitrogen nutrition of the plant, but percent nitrogen has been reported

to drop in some tropical forage grasses when nitrogen fertilization

was increased to 200 kg N/ha (46). Total nitrogen per unit of area

might therefore be a better predictor of added nitrogen to a grass

especially in the range of nitrogen inputs of any associative N2-

fixing system. But total nitrogen cannot be the only predictor of

N2-fixation because it will increase proportional to dry weight.

Other mechanisms besides IN2-fixation will cause a plant to accumulate

dry matter (7).

The experimental design of confining inoculum and autoclaved

inoculum treatments to paired subplots was thought to reduce plot

to plot variation and allow use of less replication For better land

utilization. A significant interaction was then defined as differences










TABLE 8

ACETYLENE REDUCTION VALUES BY CORE AND WASHED ROOT ASSAYS OF
INBREDS AND HYBRIDS FROM THE FIELD SCREENING TRIALS


Inbreds

Tift 23DA
Tift 23DB
Tift 23A
Tift 23D
Tift 239A
Tift 239B
Tift 186
Tift 383
Tiflate
Tift 23SB
Bil 3B
Tift 13
Tift 131
Tift 123
Tift 18DB


Core
Acetylene Redcution
T(nmole/g dry root x hr.)
Auto


Inoc.

2.0
6.3
7.0
8.0
0
0
0.3
1.6
0
2.0
0
0
7.6
6.7
0


Hybrids

23DA X 18DB
23DA X Tiflate
23DA X 383
23DA X 186
23DA X PMB004
23A X Bil 3B


6.0
0
25.3
2.0
8.3
15.0


Diff.

- 1.0
+ 5.3
+ 2.4
+ 6.7
0
0
- 1.3
- 4.7
0
- 1.3
0
0
- 5.4
+ 4.1
0


- 4.0
0
+ 1.7
- 1.0
+15.4
+19.3


Washed Root
Acetylene Reduction
(nmole/g dry root x hr.)
Auto


Inoc.

13.0
0
3.5
27.5
9.0
11.0
10.0
4.5
15.5
12.5
6.0
8.5
10.5
11.5
9.5


4.5
5.5
14.0
0
31.5
14.0


Inoc.

9.0
10.5
0
14.5
10.5
6.0
11.0
1.0
7.0
3.0
0
11.5
7.5
3.5
10.0


1.5
2.0
7.5
0
12.0
8.0


Diff.

+ 4.0
-10.5
+ 3.5
+13.0
- 1.5
+ 5.0
- 1.0
+ 3.5
+ 8.5
+ 9.5
6.0
- 3.0
+ 3.0
+ 8.0
- 0.5


+ 3.0
+ 3.5
+ 6.5
0
+19.5
+ 6.0


--









among genotypes to respond to inoculation. If the effect in any one

genotype is small but consistent, it could be masked by the random

variation created by many genotypes not responding in the population.

Another method of locating one or two responding genotypes would be

the use of outlier tests such as the Grubbs Test (28). Outlier tests

allow screening for those outling values which are not predicted by

the interaction term. In the inbred population, it was found that a

difference in dry weight of 1736 kg/ha was needed for any genotype

to be an outlier (p = 0.05). Even with this test, no response is

observed with Bil 3B (1192 kg/ha) or Tiflate (1151 kg/ha). But

1736 kg/ha represents a 25% increase in Bil 3B and only a 12% increase

in Tiflate. This is a discrepancy in this experiment to measure

individual responding genotypes. Screening within a population with

wide yield variability might not be a valid approach when inoculation

response emphasizes enhancement of the same yield parameters.

Results of the field and greenhouse studies indicate that a

schedule to screen for associative N2-fixation should place initial

emphasis on experiments designed to show increased nitrogenase activity

achieved through inoculation. Well replicated genotypes could be

screened each cycle of selection by extensive sampling with acetylene

reduction or if possible, 15N2. Responders should then be field tested

individually for increases in agronomic yield and acetylene reduction.


Nitrogen Balance Study

Though the 32' yield increase by inoculated 'Gahi 3' is important

agronomically, data did not show adequate N'2-fixation to explain this










increase. There are also reports that Sp 13t is in a group of Spirillum

lipoferum strains which show potential for dinitrification (38). This

nitrogen balance study was conducted to monitor increases of nitrogen

in inoculated 'Gahi 3' plants and to verify its field growth responses.

The effects of Sp 13t inoculum on 'Gahi 3' plants are seen in

Table 9. Increases from .5 to 4.7% are found in the various plant

parameters due to inoculation. Only total plant dry weight (p = 0.28)

and total plant nitrogen (p = 0.21) showed any noticeable statistical

response. This experiment was conducted for a 42 day growth period,

while in the field screening trials plants were grown for 80 days.

At termination of this nitrogen balance study plants were yellow and

seemed to be uniformly deficient in nitrogen, but possibly a longer

growth period was needed to duplicate the statistically significant

(p = 0.05) field yield enhancements.

The nitrogen balance for the soil-plant system is presented in

Table 10. Inoculation with Sp 13t did not increase the nitrogen

content for the total system. It is interesting that an overall

experimental nitrogen increase of 0.2628 g (combining all treatments)

is seen. This amount of nitrogen represents 3.48% of the initial

soil nitrogen and might be viewed as the sampling error within this

nitrogen balance study. From previous work with pearl millet, it

was predicted that Sp 13t inoculum could replace 39 kg N/ha if 60

kg h/ha was added as fertilizer and plants achieved a 22% increase

in yield (45). This soil was found to have about 0.055% nitrogen;and










33



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if one assumes that an acre furrow slice of soil weighs 2,000,000

Ibs. (9), then there would be 1171 kg of nitrogen after fertilizer

addition. Therefore the predicted increase of 39 kg N/ha would be

about 3.33% of this total. It does appear that the amount of nitro-

gen that could be fixad was within the sampling error of the experi-

ment. This supports the predictions of total nitrogen determinations

not being sensitive enough to show small levels of N2-fixation (11).

Also, the low levels of plant yield possibly negated finding a nitro-

gen increase for the system. As there was no overall decrease in

nitrogen, dinitrification resulting in loss of nitrogen from the

system was not measured. More sensitive assay such as 15N should

be used to prove whether N2-fixation is taking place in the 'Gahi 3'

Sp 13t system.

Plant root acetylene reduction values are presented in Table 11.

Inoculation with live Sp 13t cells enhanced acetylene reduction by

21 nmoles/g dry root x hr and are of some statistical value when

compared to autoclaved controls (p = 0.21). This level of increased

nitrogenase activity attained with the hybrid is encouraging, but, as

stated previously, limited sampling does not allow predicting N2-

fixation for the whole term of the experiment.











TABLE 11

ACETYLENE REDUCTION VALUES ON TREATMENTS IN THE NITROGEN
BALANCE STUDY. SAMPLES WERE TAKEN AT TERMINATION OF THE
EXPERIMENT, INCUBATED IN ARGON WITH 10% ACETYLENE,AND
SAMPLES ANALYZED BY GAS CHROMATOGRAPHY AT 17 AND 24 HOURS


17 Hours 24 Hours
Treatment (nmole/g dry root x hr) (nmole/g dry root x hr)


Live Sp 13t 84.89 108.94

Autoclaved Sp 13t 72.78 87.11












CONCLUSIONS


Differences among pearl millet inbreds and hybrids to respond to

Sp 13t liquid inoculation were found. A hybrid, 23DA X 186 ('Gahi 3'),

responded in the field with a 32% increase in dry weight and a 37%

increase in total nitrogen while its parents showed no response.

Heterosis is not thought to be the sole cause of this response. An

inbred, Bil 3B, was enhanced 17% in dry weight by Sp 13t inoculation

and has potential for further study.

All screening studies reported in this investigation placed

emphasis on increased plant dry weight, percent nitrogen, total nitro-

gen,and acetylene reduction achieved through inoculation. Of these

acetylene reduction represents the most sensitive assay for indicating

increased nitrogenase activity. In a population with wide yield

variation it is difficult to predict N2-fixation on any one genotype

when response is measured by further yield enhancements. Screening

pearl millet for associative N2-fixation achieved through inoculation

should place initial emphasis on well replicated experiments designed

to show increased nitrogenase activity by extensive sampling with

acetylene reduction or, if possible, 'iN2. Those genotypes which are

deemed responders should then be field tested individually for increases

in agronomic yield and acetylene reduction.

Liquid inoculation of 'Gahi 3' did result in very substantial

increases in dry weight and nitrogen. A nitrogen balance study with


37






38


this hybrid did not indicate that the growth enhancements were based on

significant amounts of nitrogen added into the plant-soil system. It

is possible that any N2-fixation achieved through inoculation might

have been within the limits of sampling error for the nitrogen balance

system. Sensitive assays such as 15N2 are needed to confirm recon-

stitution of N2-fixation by Sp 13t inoculation in the 'Gahi 3' hybrid.










LITERATURE CITED


1. Barber, L. E., J. D, Tjepkema, S. A. Russell and H. J. Evans. 1974.
Acetylene reduction (nitrogen fixation associated with corn
inoculated with Spirillum. Appl. Environ. Microbiol. 32:108-
113.

2. Barea, J. M. and M. E. Brown. 1974. Effects on plant growth pro-
duced by Azotobacter paspali related to synthesis of plant
growth regulating substances. J. Appl. Bacteriol. 37:583-593.

3. Bergersen, F. J. 1970. The quantitative relationship between
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BIOGRAPHICAL SKETCH


Joseph Henry Bouton was born on March 24, 1948,in Greenville,

Nississippi,and graduated from Leland High School in Leland,

Mississippi,in May 1966. He received the degrees of Bachelor of

Science in Education and Bachelor of Science in Agronomy from

Mississippi State University in June 1970 and June 1972, respectively.

He entered the graduate school of the University of Florida

and completed the degree of Master of Science in agriculture with

a major in ornamental horticulture. In September 1974, he started

a program of study and research at the University of Florida leading

to the degree of Doctor of Philosophy with a major in agronomy and

a minor in biochemistry.

He is married to the former Mary Jeanne Carr and they have a

daughter, Melinda.










I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.



Rex L. Smith, Chairman
Associate Professor of Agronomy

I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.



Stanley C. Schank
Professor of Agronomy


I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.



rochn R. Edwardson
Professor of Agronomy


I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.



/Albert E. Dudeck
Associate Professor of Ornamental
Horticulture











I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.



Rusty an
S rofe sor Io Biochemistry


I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.



David H. Hubbell
Associate Professor of Soil
Science



This dissertation was submitted to the Graduate Faculty of the College
of Agriculture and to the Graduate Council, and was accepted as partial
fulfillment of the requirements for the degree of Doctor of Philosophy.



Dean, College of Agriculture


Dean, Graduate School




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