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 Title Page
 Acknowledgement
 Table of Contents
 Abstract
 Introduction
 Review of literature
 Materials and methods
 Results and discussion
 Conclusions
 Bibliography
 Biographical sketch














Title: Evaluation of Panicum virgatum L. for nitrogen related agronomic characters /
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Title: Evaluation of Panicum virgatum L. for nitrogen related agronomic characters /
Alternate Title: Panicum virgatum
Physical Description: v, 54 leaves : ; 28 cm.
Language: English
Creator: Weiser, Glen C
Publication Date: 1980
Copyright Date: 1980
 Subjects
Subject: Forage plants -- Florida   ( lcsh )
Nitrogen -- Fixation   ( lcsh )
Switch grass
Agronomy thesis Ph. D
Dissertations, Academic -- Agronomy -- UF
Genre: bibliography   ( marcgt )
non-fiction   ( marcgt )
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Thesis: Thesis--University of Florida.
Bibliography: Bibliography: leaves 48-53.
General Note: Typescript.
General Note: Vita.
Statement of Responsibility: by Glen C. Weiser.
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Bibliographic ID: UF00099248
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 - 000014289
oclc - 06405972
notis - AAB7493

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Table of Contents
    Title Page
        Page i
    Acknowledgement
        Page ii
    Table of Contents
        Page iii
    Abstract
        Page iv
        Page v
    Introduction
        Page 1
        Page 2
    Review of literature
        Page 3
        Page 4
        Page 5
        Page 6
        Page 7
        Page 8
        Page 9
        Page 10
        Page 11
        Page 12
    Materials and methods
        Page 13
        Page 14
        Page 15
        Page 16
        Page 17
        Page 18
        Page 19
        Page 20
    Results and discussion
        Page 21
        Page 22
        Page 23
        Page 24
        Page 25
        Page 26
        Page 27
        Page 28
        Page 29
        Page 30
        Page 31
        Page 32
        Page 33
        Page 34
        Page 35
        Page 36
        Page 37
        Page 38
        Page 39
        Page 40
        Page 41
        Page 42
        Page 43
        Page 44
        Page 45
    Conclusions
        Page 46
        Page 47
    Bibliography
        Page 48
        Page 49
        Page 50
        Page 51
        Page 52
        Page 53
    Biographical sketch
        Page 54
        Page 55
        Page 56
        Page 57
Full Text













EVALUATION CF Panicum virgatum L.
FOR NITROGEN RELATED AGRONOMIC CHARACTERS


BY


GLEN C. WEISER


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















ACKNOWLEDGMENTS


I wish to sincerely thank Dr. Rex L. Smith, Chairman of the Graduate

Committee, for sharing his inimitable research genius, powers of observation

and valued friendship with me. The advice, guidance and friendship of

Drs. Stan C. Schank, Ken H. Quesenberry, 0. Charles Ruelke and

L. Curt Hannah are sincerely appreciated.

Salut6 to Doug Manning, Keith Parsons, Loretta Tennant, Alice Kelly.

Kathy Delate, Tony Gonzalez, Rex Glover, Peter Mansanow, Anthony Bouton,

Dale Bonnell, Elarige Lee, Verity Tennant, Ken Cundiff, Bob Green and

Austin Tyer.

Special thanks are due to Dr. John Cornell for statistical

consultations, Dr. Victor Green Jr. for his contribution of plant

materials and 3rs. Earl Horner, Gerald Mott, Murray Gaskins and

Steve Aibrecht for their inputs regarding research methodology and

interpretation.

I will always be indebted to Dr. Don Graetz, Candy Cantlin and

Don Mycoff for their help in soil and plant preparation; and to

Dr. R. H. Burris, Kathy Walsh, J. P. Houchins, Bill Sweet and Dan Arp

of the University of Wisconsin's Center for Nitrogen Fixation Studies

for their aid in mass spectrcmetric analyses.

The U. S. Agency for International Development contract ta-c-1376

provided partial financial support for this study.















TABLE OF CONTENTS

PAGE

ACKNOWLEDGMENTS ................. ................................. ii

ABSTRACT ................. .................. ...... ............. iv

INTRODUCTION ....................................... ............ 1

REVIEW OF LITERATURE ....................... ........... ....... 3

Nitrogen Fixation .................... ...... .......... 3
Nitrogen Uptake Efficiency .................... ............. 10
Switchgrass ................................................ 12

MATERIALS AND METHODS ............................ .............. 13

Plant Materials and Field Cultural Conditions ................. 13
Field Selection and Analysis of Acetylene Reduction
Activity (AR) ............................... .......... .. 15
Greenhouse Evaluations ............. ........... .. .. 17
Total N and 15N Determinations ............................... 19

RESULTS AND DISCUSSION ......................................... 21

Field Evaluations .................. ....................... 21
Greenhouse Evaluations .......... .. ............ ........... 31

CONCLUSIONS ...................................... ................ 46

LITERATURE CITED .......... ...................................... 48

BIOGRAPHICAL SKETCH .............................................. 54












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


EVALUATION OF Panicum virgatum L.
FOR NITROGEN RELATED AGRONOMIC CHARACTERS

By

Glen C. Weiser

March, 1980


Chairman: Rex L. Smrith
Major Department: Agronomy


Selected and non-selected half-sib progenies and their maternal

parents of switchgrass, Panicum virgatum L. were evaluated for nitrogen

related agronomic characters. The objectives were: 1) to evaluate

associative nitrogen fixation and nitrogen use efficiency in switchgrass:

2) to evaluate switchgrass as a suitable forage crop for the state of

Florida; and 3) to evaluate the potential for improvement through breeding.

In the field, over a two year period at two locations, significant

variations were observed for yield, percent nitrogen, nitrogen fertilizer

use efficiency and nitrogen fixation estimated by the total nitrogen

difference method. Responses were influenced by maternal line origin,

location, plant establishment and applied nitrogen fertilizer level.

Broad-sense heritabilities for the field measured parameters were generally

low, indicating that breeding progress will be slow. in the greenhouse.

four populations of switchgrass showed significant differences in yield,

nitrogen fertilizer use efficiency and nitrogen fixation estimated by

total nitrogen difference and by nitrogen-15 dilution. Responses were









influenced by maternal line origin, population of selection and plant

establishment.

Mean estimates of nitrogen fixation in the field by total nitrogen

difference and acetylene reduction cores were 20.8 and 6.5 kgN/ha,

respectively, and the two estimates were positively correlated (P> 0.001).

Mean estimates of nitrogen fixation in the greenhouse by total nitrogen

difference and nitrogen-15 dilution were 2.4 and 9.5 kgN/ha, respectively,

but in two combined harvests, the estimates were negatively correlated

(P = 0.02).

In all cases, %N in switchgrass was lower than in 'Pensacola'

bahiagrass which was included as a check species. Dry metter yields

were higher than in bahiagrass. This indicates that the ability of

switchgrass to dilute nitrogen is a factor that makes it a promising

forage and biomass producer, especially under low management and low

nitrogen input conditions.















INTRODUCTION


Nitrogen is a vital element in the composition of plants and

animals. Even though our atmosphere contains 78% nitrogen, it exists

in the unavailable diatomic state. It is only after this diatomic

species has been chemically reduced that it is rendered available

for use in physiological systems. On a commercial scale, the reduction

of nitrogen for use as applied fertilizer requires high energy inputs.

With recent and justified concern over wise, efficient energy use,

the world must look towards biological nitrogen fixation as an asset

in food production.

Isolation of the diazotroph Azospirillum brasilense, from

the rhizospheres of several tropical Gramineae in Brazil, sparked

worldwide interest in the establishment of a highly efficient, asso-

ciative nitrogen-fixing system that would augment global nitrogen inputs

by the long and highly regarded legume-Rhizobia symbiotic systems.

Subsequent research in Brazil, the United States, Great Britian and

Australia repeatedly demonstrated yield increases due to inoculation

with strains of A. brasilense in many tropical and temperate grass

species. Confirmation of nitrogen fixation via acetylene reduction,
1N and Kjeldahl nitrogen difference methods has been accomplished in

ambient and axenic conditions; however, correlations to the observed

plant responses have not been adequate. Alternate hypotheses for

growtn stimulation, e.g., production of plant growth regulators by

bacterial inocula, have been proposed and demonstrated but these data









alone do not provide adequate explanations of observed responses.

Other existing and introduced soil diazotrophs, alone and in com-

binations, continue to be studied in associative systems involving

various grasses.

Advances in the understanding of associative biological nitrogen-

fixing systems remain elusive, despite intensive research efforts

of the past few years. Data relative to the magnitude and significance

of these systems are usually empirical and center around visual

observations of grass field crops and existing grassland ecosystems.

Gross nitrogen recoveries and efficiencies continue to be highly

encouraging; however, experimental data demonstrating nitrogen fixation

per se are, for the most part, inconclusive. It would seem that

either the individual systems themselves are highly variable and

unpredictable or that measurement techniques are inadequate, or both.

This study is based on empirical observations of established,

unfertilized high-yielding plots of switchgrass (Panicum virgatum L.)

growing at the Agronomy Farm, University of Florida. The objectives

are 1) to evaluate associative nitrogen fixation and nitrogen

efficiency in switchgrass; 2) to evaluate switchgrass as a suitable

forage crop for the state of Florida; and 3) to evaluate the potential

for improvement through breeding.















REVIEW OF LITERATURE


Nitrogen Fixation

Huge amounts of nitrogen required for the growth of plants

and animals are ultimately obtained from the atmosphere as chemically

fixed forms. Fixation catalyzed by lightning and ultraviolet

radiation may account for up to 0.5% of the global nitrogen require-

ment while man-made fertilizer nitrogen, requiring a large and costly

energy input, accounts for an additional 5%. Biologically fixed

nitrogen constitutes the remainder, estimated at 108 to 109 tons of

N per year (54).

Biological nitrogen fixation occurs in a variety of situations

under many different conditions. Plants belonging to the family

Leguminoseae have evolved efficient symbiotic systems that fix signi-

ficant amounts of nitrogen (27). These systems involve the formation

of nodules, small comoartments on the plant roots, that contain

bacteroids of Rhizobium spp. Associative nitrogen fixation involving

numerous bacteria of the family Enterobacteriaceae and grass crops

is regarded as one of the most promising areas in biological nitrogen

fixation research (45). Other documented biological nitrogen fixing

systems include Klebsiella pneumcniae living in the intestines of

humans on a high carbohydrate diet; various intestinal microflora in

some termite species; Actinomycetes in nodule-type structures in the

genera Ainus, Myrica, Ceanothus, Casuarina and Comotonia; and blue-green









bacteria growing as soil crusts, in aqueous solutions and in the

rhizoids of plants of the genus Azolla (16,30,66,75).


Measurements of Nitrogen Fixation

Three basic techniques are available to measure nitrogen

fixation. Total nitrogen present in the plant material can be measured

by the Kjeldahl method, and after the subtraction of applied nitrogen

sources, an estimate of nitrogen fixation may be obtained. Acetylene

reduction techniques use the alternative nitrogenase enzyme substrate

acetylene, which upon reduction produces ethylene that is measured by

gas chromatography, and an estimate of nitrogen fixation may be calcu-

lated. Nitrogen-15 enrichment and dilution techniques estimate

fixation by mass spectrometric measurements of 15N2 (gas) incorporation

in an enclosed system or the relative dilution of applied 15N

Fertilizer in plant tissue by fixed nitrogen. All three methods have

certain limitations and advantages.

Total nitrogen difference estimates by the Kjeldahl method (14)

require extremely precise sampling, sub-sampling and analytical

techniques. Serious errors may be introduced, especially in grass-

bacterial systems, where there exists a large amount of soil nitrogen

relative to fixed nitrogen, and the amounts of fixed nitrogen are on

the same order of magnitude as the experimental error. Nonetheless,

total nitrogen techniques have been widely used and provide gross

estimates of nitrogen fixation and overall nitrogen relationships.

Due to its relative rapidity, simplicity and sensitivity of 103

to 10 times that of 15N methods, the acetylene reduction technique is

presently used in mnst nitrogen fixation studies (33), Calculations




5




include the assumptions that a theoretical reduction conversion ratio

of 3C2H2 : 1N2 is maintained, non-interference by endogenous ethylene

evolution and microflora capable of metabolizing acetylene and

ethylene, and that the rates of acetylene reduction are linear over

time. The short periods of time, e.g., 1-24 hours, and the sometimes

destructive nature of the technique, i.e., the excavation of below-

ground plant parts to expose them to the assay, limit extrapolation to

intact systems over an entire growing season, although this assay may

also be done on nondisturbed systems.

Acetylene reduction techniques have been applied to various field

and laboratory conditions (38,60). Various reaction chambers are

utilized, such as plastic bags (59), soft-drink cans (13), serum-vials

(20) and soil cores (65). Problems with small plant samples,

initial periods of inactivity (lag) and non-linear reduction rates,

attributable in part to anaerobic nitrogen fixation, limit further

extrapolation to field conditions (39). Periods of preincubation may

alter the observed rates of nitrogen fixation. For example, Tjepkema

and Van Berkum (65) observed a 14-fold higher estimation of fixation

in preincubated vs. non-preincubated samples.

Nitrogen-15 enrichment and dilution techniques probably provide

the most reliable estimates of nitrogen fixation. The major limitations

are the high costs involved in reagents and in complex analyses. Such

studies also require stringent controls to measure background 15N

incorporation levels, soil N mineralization and denitrification

although such measurements are difficult to accomplish. The only

major assumption is non-discrimination of 15N and 14N by plants and

bacteria.









Enrichment experiments require that 15N2, in a known excess

concentration or 14N2, be introduced into a closed or limited access

vessel containing a nitrogen fixing system (22). The relative amount

of 15N is directly attributable to fixation; however, the elaborate

vessels needed, the high cost of 15N, which limits time of exposure

and number of samples, and the inability to provide natural field con-

ditions make extrapolations difficult. Enrichment experiments are

useful to calculate C2H2 conversion ratios for acetylene reduction

measurements.

Dilution experiments require the application of nitrogen fertilizer

containing a known excess of 1N, usually 1 to 5%. As the fertilizer

is taken up by the plant, any dilution of the original concentration

of "N may be attributable to the utilization of soil nitrogen and to

fixed, incorporated 14N from the air (31). After a series of calculations

(26,36) to derive "A" values, an estimate of nitrogen fixation is

obtained. These estimates usually correlate with total nitrogen

measurements. Williams et al. (73), using clover, found that Kjeldahl

methods consistently underestimated 15N dilution estimates by 40%.

Dilution experiments are done on a much larger scale than enrichment

experiments. Their advantages include a lower reagent cost, simple pot

containers (dilution experiments may also be done under field conditions)

and a long duration of exposure (20,21).


Legume-Rhizobium Symbioses

Most legumes, when in contact with the proper species and strains

of Rhizobium bacteria, form root nodules. Nodules enclose the bac-

teroids, specialized forms of nondividing bacteria, and provide an

environment for the reciprocal exchange of bacterial-fixed nitrogen









and plant-fixed carbon sources. One of the most important components

of the nodule is leghemoglobin, a plant product similar to human

hemoglobin, that binds 02 and reduces its concentration to low levels,

protecting rhizobia's oxygen sensitive nitrogenase, yet supplying

adequate quantities of 02 to maintain metabolic activities (15,23).

Well nodulated legumes fix agronomically important amounts of

nitrogen and are therefore extremely important in world agriculture.

Amounts of nitrogen fixed on a hectare basis range from 150 kg for soybean

(36) to 295 kg for clover and 59 kg for vetch (73). Nodulation and

nitrogen fixation are affected by many environmental and cultural con-

ditions. In alfalfa and trefoil, Barta (6) observed approximately a

50% decrease in nitrogen fixation when the plants were grown at 30 C

vs. 16 C. High levels of applied nitrogen and the location of application

within the root zone may severely affect nodulation and subsequent

nitrogen fixation. Molybdenum, a component of a nitrogenase co-factor,

is also important and may easily become limiting in the field (52).

Soil pH, p02, organic matter content and buffering capacity also affect

symbiotic nitrogen fixation (23,69).

Studies designed to assess plant breeding potential for nitrogen

fixation have been initiated with some major legumes. Wacek and Brill

(69) screened 45 soybean cultivars in 6 maturity groups for nitrogen

fixation. A broad range of relative acetylene reduction values were

obtained, representing a 20-fold difference between the cultivars.

Westermann and Kolar (72) also found a broad range of acetylene

reduction values in 18 field-grown common bean cultivars belonging to

several plant growth types and maturity groups. In 20 selfed and

hybrid progenies of alfalfa, significant variation in nodule number









and acetylene reduction and a significant positive correlation between

the two parameters have been observed (58). In a complete diallel cross

between 6 progenies selected for high and low acetylene reduction rates,

high X high crosses produced progenies with greater than twice the

acetylene reduction rates than low X low crosses. High X low crosses

produced progenies with intermediate rates. In another study (28),

individual plants within the alfalfa cultivar 'Mesilla' were evaluated

for nodulation, %N, total N and nitrogen fixation and positive

correlations between these traits were observed. Polycross progeny

from 15 plants selected for high trait levels showed a mean level of

acetylene reduction 82% over the mean level for the entire population.


Grass-Bacterial Associations

Because of their tremendous potential, associative nitrogen

fixing systems, involving various grasses and diazotrophs, have received

a great deal of recent attention. Most associative studies have

involved grass rhozosphere inoculation with previously isolated diazo-

trophs and the subsequent measurement of yield and nitrogen fixation by

total N, acetylene reduction and 15N methodology. Earlier studies have

involved bahiagrass-Azotobacter pasoali associations and a number of

forage and grain crops in association with Azospirillum brasilense and

A. lipoferum (formerly Spirillum lipoferum). Growth stimulation by

these bacteria is thought by some to be due, at least in part, to

bacterial plant growth regulator synthesis (5,17,63); however, many

nitrogen fixation studies per se have been and continue to be done. In

Brazil, Von Bulow and Dobereiner (68) screened corn genotypes in asso-

ciation with Azospirillum and indicated that sufficient variability

existed in nitrogenase activity to warrant plant breeding. Nitrogenase









activities equivalent to the fixation of 0 to 734 gN/ha/day in

Azospirillum inoculated corn have also been shown under greenhouse

conditions in Oregon, while field activities were much lower (4).

Schank et al. (55) also found nitrogenase activity differences in

breeding lines of 30 tropical forage grasses in Brazil. In Florida,

yield increases in some genotypes of Azospirillum inoculated pearl millet

(12,13,59), guineagrass (59), bahiagrass (7) and bermudagrass (3)

have been documented. Using fluorescent antibody labeling and conven-

tional microscopy, bacteria in such associations have been observed in

the root cortex and mucigel layer (56). Using electron microscopy,

Azospirillum cells have been observed intercellularly in field-grown

roots of pearl millet (46) and adsorbed to roots and root hairs and em-

bedded in the mucigel layer of axenically grown pearl millet and guineagrass

(67). Also using electron microscopy, the diazotroph Erwinia herbicola

has been observed embedded in the root cell walls of switchgrass (43).

In the rhizospheres of certain chromosome substitution lines of wheat,

diazotrophs have been isolated that were not found associated with the

chromosome donor or non-substituted lines (48). Other reports of

associative nitrogen fixing systems include stargrass (42), rice (39),

switchgrass (64) and Oryzopsis, Agropyron, Stipa and Aristida spp. in

xeric habitats (74).

Many factors influence associative nitrogen fixing systems, much

as those involved in legume symbioses. Field-applied nitrogen in amounts

greater than 22 to 40 kg/ha significantly reduce responses to diazo-

troph inoculation (34,59). In vitro studies with rice (44) also indicate

that nitrogen fixation is significantly inhibited by ammonium or

nitrate in concentrations exceeding 50 ppm. This inhibition is









markedly less in water saturated vs. aerobic media. Associative

systems may be temperature and light sensitive and may exhibit diurnal

variations (2), but such data are highly variable and do not solidly

support a positive conclusion. In addition to plant genetics, it is

also thought that carbon metabolism pathways are important. In

tropical associations, C4 grasses are hypothesized by Day et al. (25)

to have a competitive nitrogen fixation advantage over C3 species, but

no strong data to support such a hypothesis exist.

Other aoproaches to constitute new nitrogen fixing systems include

genetic engineering. Using a plasmid involved in tumor induction by

the crown gall bacterium, attempts to introduce nitrogen fixation

genes into higher plants are being undertaken (57). Direct attempts

to introduce nitrogen-fixing blue-green bacteria into corn and tobacco

protoplasts have been made (19); however, plant regeneration with the

incorporated bacteria is not yet possible. New, more precise approaches

to reconstitute natural associative systems are also currently underway.

Gilmour et al. (34), using diazotrophs isolated from native grass

rhizospheres, have devised axenic and natural systems to test the patterns

of root-bacterial associations. Plant-bacterial combinations that show

a close association have been placed in the greenhouse and the field,

and significant yield and nitrogenase activity increases have been

observed.


Nitrogen Uptake Efficiency

Nitrogen uptake efficiency, the ability of a plant to recover

applied, soil and fixed nitrogen, is a very important agronomic and

plant breeding consideration. Reported efficiencies generally vary

with species and genotype and range from 10 to 70%. In addition to species









and genotype differences (53), it has been documented that C4 grasses

have higher nitrogen efficiencies than C3 grasses (18). An evolutionary

advantage of C4 over C3 grasses has been postulated, based on relative

recovery rates of various species. It is assumed in this hypothesis

that C4 photosynthesis evolved in tropical regions with soils low in

nitrogen, and that nitrogen use efficiencies and this photosynthetic

pathway are therefore linked. High nitrogen efficiencies in tropical

regions with sandy soils and high rainfall, where typically less

than 50% of applied nitrogen is actually recovered, are important

plant traits (8). Also, in temperate regions with heavier soils that

bind and immobilize applied nitrogen, high nitrogen uptake efficiencies

are valuable (37,41). High nitrogen efficiencies are not always bene-

ficial, however, and may result in toxic compound conditions under

some conditions (35).

Time, rate of application and the nitrogen source used oftentimes

influence efficient fertilizer use. Blue (9,10), using bahiagrass,

observed low (40-50%) recoveries during the first four years of

pasture establishment with recoveries of 60-70% after the fifth year.

High recoveries were generally associated with higher rates of apoli-

cation, when applied during periods most favorable for plant growth.

Efficiencies related to nitrogen source were noted, with the uptake of

ureaform nitrogen markedly less than that of urea, calcium nitrate,

ammonium nitrate and ammonium sulfate. Over a 25 year period with these

experiments, soil nitrogen was increased by 800 kg/ha, indicating that

nitrogen efficiency is related to nitrogen recycling and soil improvement

(11).










Switchgrass

Switchgrass, Panicum virgatum L., is a member of the Virgata

section of the Panicum subgenus Eupanicum. It is a large, bunch-type

grass found in prairies, open ground, open woods and brackish marshes

from Nova Scotia to Central America and west to North Dakota, Wycming,

Nevada and Arizona (40). Grown extensively in the midwestern United

States for many years, released synthetic cultivars include 'Caddo,'

'Pathfinder,' 'Nebraska 28' and 'Blackwell' (1). Switchgrass pastures

perform well in comparison to other native, adapted grasses and provide

high yields of good quality forage with good stand persistence (71).

Switchgrass strains respond differently to soil types and cultural

practices and respond well to nitrogen fertilization (48). In Nebraska,

switchgrass yields were increased by approximately 75% with the appli-

cation of 35 kgN/ha/year over a two year period (70). Percent N in

switchgrass forage varies with strain, location of growth and nitrogen

fertilization. Newell (50) observed 0.95%N in switchgrass forage

compared to 0.85%N in bluestem forage from the same study; and McMurphy

ea al. (47) found switchgrass to be intermediate in %N when compared to

bluestem, indiangrass and lovegrass. Heritabilities and expected gains

from selection are usually high for collected genotypes of switchgrass

with regard to yield, quality, disease resistance and desirable

morphological traits, making breeding and selection profitable in

many instances (29,49,51).














MATERIALS AND METHODS


Plant Materials and Field Cultural Conditions

Six accessions of switchgrass were used as the base population for

this study (Table 1). Large, field-grown plants, one of each genotype,

were split into 12 uniform clones and established on 0.6 m centers in a

completely randomized polycross field block in June, 1977. In October 1977,

mature spikelets were harvested and equal amounts of seed from each maternal

plant were mixed and pooled into 6 lots. These half-sib progenies were

established in flats of non-amended field soil in the greenhouse. Randomly

chosen seedlings were placed individually in cell-pack flats containing

non-amended field soil, lightly fertilized with the equivalent of 400 kg/ha

6-6-6 organic fertilizer and allowed to become well established.

The ramets were planted in the field at Gainesville and hague, Florida,

in May 1978. The Gainesville location is well-drained Gainesville sand,

loamy, hyperthermic Typic Quartzipsamments, pH 4.5-6.0, with an analyzed

soil N content of 0.046%. The Hague location is Sparr sand, loamy,

siliceous, hyperthermic Grossarenic Paleudalts, pH 4.5-6.5, with an analyzed

soil N content of 0.056%, and is susceptable to partial flooding after a

heavy rain. Percent N in each soil type did not change over the duration

of the experiment. These locations were chosen because of their diversity

in soil type and moisture relationships to adequately test the genotypes.

Prior to planting, all field locations were sprayed with 5 1/ha of

glyphosate herbicide, plowed 10 days later, fertilized with the

equivalent of 1,000 kg/ha of 0-10-20 fertilizer with fritted






















Table 1. Plant Materials Center accession numbers of the Switchgrass
(Panicum virgatum L.) parental clone genotypes and their
locations of collection.


Accession number*

F-687

F-1666


F-1668

F-1716

F-4685

F-3115


Location of collection

Stuart, FL

West Palm Beach, FL


Ft. Pierce, FL

Arcadia, FL

George West, TX

Miami, FL


*
All accessions were obtained courtesy
U.S. Soil Conservation Service Plant
FL.


of R. D. Roush, Manager,
Materials Center, Brooksville,









trace elements (FTE 503; 5 B: 5 Cu: 29 Fe: 12 Mn: 0.3 Mo: 11 Zn,

in g/lOOkg) and lightly disced.

The field trials consisted of 3 fertilizer N levels applied to

6 polycross half-sib lines of switchgrass, and a bahiagrass check

species (Paspalum notatum Flugge cv 'Pensacola'). The total number of

switchgrass plants evaluated rias 1,260. Fertilizer N levels were the

equivalent of 10, 50 and 90 kg/ha elemental N applied by hand as

NH4NO3. Bahiagrass was obtained as 7.5 cm plugs from a well-established

sod. Plots were 4 m long and 1 m wide and consisted of 7 ramets per

plot on 0.6 m centers with 5 completely randomized block replications

at the 2 locations over a 2 year period. Plots were separated by

2 m and during 1978, a border row of a sorghum-sudangrass hybrid was

included between each plot. Border rows were omitted in 1979. Plots

were irrigated as needed during early establishment in 1978. Weeds

were controlled by mowing and hand-pulling. Forage was harvested in

October of both years with a flail-type plot harvester for yield and

nitrogen analyses. October was chosen as a harvest date in order to

obtain mature seeds from the plants for further studies.


Field Selection and Analysis of
Acetylene Reduction Activity (AR)

During the 1978 growing season, each switchgrass plant was rated

several times on the basis of vigor, decumbance, lateness of flowering

and color. Vigor was scored on 5 levels (1,3,5,7,9). Decumbance, a

desirable trait for a bunch-type forage grass, was scored on 2 levels

(0 for completely upright types and 1 for any degree of spreading).

Lateness of flowering, also a desirable trait, was scored on 2 levels










(0 for plants flowering before August 15 and 1 for those that flowered

afterwards). Color, thought to be a neutral trait, was scored as

either blueish or completely green. Broad-sense heritabilities for

the numerically scored traits were calculated. The most highly rated

plant in each plot, representing the best 1 out of 7 or the top 14%,

was used to generate further plant material, and the most highly rated

plants in plots of 2 replications at each location were sampled for AR

in 1978. In 1979, the most highly rated plants in plots of all 5

replications were sampled.

The AR sampling procedure involved taking soil-root cores in

tubes, consisting of a 7.5 cm diameter piece of steel electrical conduit,

36 cm long. One end of the core tube had either a welded steel top

or a No. 13 rubber stopper with a sampling tube for the insertion of

a rubber septum. The opposite end of the tube was sharpened. At

sampling time, the cores were taken by gently pressing the tubes into

the root zone of each plant to a depth of 18 cn, and carefully removing

and sealing with a Jim-Cap secured with a steel hose clamp. Each core

was then freely flushed with argon for 1 min through the top sampling

tube and sealed with the septum. Acetylene was added through the septum

to an approximate concentration of 10% (v/v). Cores were incubated

at 30Cin a growth chamber. Internal atmospheres were monitored for

ethylene evolution by gas chromatography at various incubation times.

A lag time of approximately 5 hours was commonly observed, and the

rates of acetylene reduction were linear after the lag and up to 24 hours.

A core consisting of bare soil was included after every 21 plant samples,

and always exhibited zero or near zero rates of acetylene reduction.

Extrapolations of N fixed were all based on 24 hour readings and included









the assumptions of a theoretical ratio of 3.0 moles ethylene per mole

N2, 18 hours of activity per day and 22 x 106 cores per ha.


Greenhouse Evaluations

Four populations of switchgrass selected under various selection

intensities (as described in Table 2) and bahiagrass plugs were esta-

blished with 5 replications in 15 cm plastic pots, and randomly placed

in the greenhouse in March, 1979. Populations 1, 2 and 3 were derived

as previously discussed. Population 4 consisted of the best 1 out of

100 seedlings generated from field-grown seed from plants of population

3. The plants were outcrossed, but not in a true polycross layout, and

some inbreeding is therefore expected in population 4. A polycross

block was not established in order to obtain a maximum number of

populations without extending the work an additional season.

All pots contained uniform amounts of screened, non-amended field

soil, later fertilized with the equivalent of 1,000 kg/ha of 0-10-20

with FTE 503 and 10 kg/ha of N. Soil N content was analyzed before

fertilization at 0.050% and did not chance over the duration of the

experiment. Nitrogen was applied in aqueous solution as (NH4)2S04 with

a 3.00 atom % excess of 15N. Homogenate of excavated field-grown roots

from plots exhibiting the highest levels of AR was added to the pots

at the time of fertilization to insure the presence of diazotrophic

bacteria. During the course of the experiment, any leachate was returned

to the pots. Verdure was harvested in July, 1979, N fertilizer and

fresh root homogenate reapplied, and the regrowth harvested in October.

The material from each harvest was dried and weighed for yield, pooled

over the 5 replications, and analyzed for total N and 15N content.


























Table 2. Description
experiments


Population


of the populations used in the greenhouse
and their selection intensities.


Description


Selection
Intensity


Original parental clones
(see Table 1).

Randomly selected poly-
cross progeny from
population 1.

Field selected individuals
from population 2.

Greenhouse selected out-
crossed progeny from
population 3.









Total N and 15N Determinations

For total nitrogen, forage samples from field plots and the pooled

samples from the greenhouse pots were dried at 60 Cand ground through a

Wiley mill to pass a 1 mm mesh stainless steel screen. Sub-samples of

the mixed, ground material, weighing 0.1 g, were placed in test tubes

containing 2-3 boiling chips and 3.2 g of a K2SO4 : CuSO4 (9:1 w/w)

catalyst. After the addition of 10 ml conc. H2S04 and 2 ml 30% H202,

the samples were digested on an aluminum block at 360C, to give a

solution boiling temperature of approximately 342C, for 3 hours. The

digestates were diluted to 75 ml with deionized water and analyzed by

colorimeter autoanalyzer (32). This N content data were used to calculate

gross fertilizer use efficiency by the equation:




[(%N) (ory matter yield) 100-1 (rate of N applied)-1].


Such N fertilizer use efficiencies do not discriminate between soil,

applied and fixed N and are therefore only an overall estimate of this

parameter.

Total nitrogen in field and greenhouse soils was determined by

digesting 1 g of screened soil in the manner above. After the addition

of 15 ml of 10 M NaOH, 30 ml of the digestate was steam distilled into

5 ml 0.1 M H303 and 2 drops of mixed indicator (2 parts 0.2% methyl

red and 1 part 0.2% methylene blue in ethanol) and titrated to end-point

with 0.0065 N H2SO4 (14).

Nitrogen-15 determinations were made on 0.2 g of dried, ground

plant material. Samples were placed in test tubes containing 1-3

boiling chips and 1.5 g K2S04. After the addition of 1.5 ml mercuric









sulfate solution (12H2S04 : 88H20 : lOHgO v/v/w) and 3 ml of conc. H2SO4,

the samples were digested on aluminum block at 360C for 3 hours and then

diluted with 25 ml deionized H20. The digestates were neutralized with

15 ml 10 M NaOH and steam distilled into 10 ml of 0.01 N H2SO. The

distillates were condensed to ca. 0.5 ml and dried on 1 x 11 cm filter

paper strips. Ammonium on the strips was converted to N2 gas with

1.5 ml of alkaline hypobromite (8Br : 40 13 N NaOH : 30H20 v/v/v) and

analyzed for 15N atom% excess with a Consolidated-Nier isotope ratio

mass spectrometer (20,21,22).

Nitrogen-15 atom % excess and 1N "A" values were used in the

analyses (31,36,73). "A" values were calculated with the equation:


[(total N fertilizer N) (fertilizer N rate of applied N)-]


Estimated N fixed was calculated by the equation:

[("A" for the fixing plant "A" for the control) (fertilizer N)

(rate of applied N)-1]

Since a suitable control is not available in studies involving

associative nitrogen fixation, the control values used in the above

equation were assumed to be zero. The implications of this assumption

will be discussed. Additional calculations of N fertilizer use effi-

ciency were made using the 15N data with the equation:


[(total plant N) (atom % 15N) (amount of 15N applied)-]


Such N fertilizer use efficiencies discriminate between applied and

other forms of N and are a more accurate assessment of actual fertilizer

uptake.














RESULTS AND DISCUSSION


Field Evaluations

A summary of the analysis of variance for yield, %N, fertilizer

use efficiency (calculated by total N) and estimates of

nitrogen fixation by total N difference and acetylene reduction,

combined over both locations and both years, is presented in Table 3.

Main effects, i.e. location, fertilizer, line and year were significant

for yield, fertilizer use efficiency and the estimate of nitrogen

fixation by total N difference. Location, line and year significantly

influenced %N but nitrogen fertilizer rate did not. Estimated nitrogen

fixation by acetylene reduction showed no influence by main effects and

only the line X year interaction was significant. Two-factor inter-

actions showed mixed effects for the other parameters. The location X

line (environment X genotype) interaction is an extremely important one

and was significant for yield, %N and fertilizer use efficiency,

indicating the need for multiple test sites for switchgrass in further

evaluations. Both estimates of nitrogen fixation failed to show

significant location X line interactions, however, and these parameters

may be assumed to be relatively constant. Although both locations

responded the same in both years, yield and %N were not consistent

over locations and fertilizers, indicating a need for specialized

fertilizer requirements due to different soil types. Significance in

the year X fertilizer and year X line interactions indicate that plant










Table 3. Summary of the analysis of variance for yield, %N, N fertilizer
use efficiency (FEFF) and estimates of nitrogen fixation by
total N methods (NF TN) and acetylene reduction (NF AR) of
main effects and two-factor interactions in the field
experiments.


d.f. YIELD %N FEFF


NF TN NF AR


1 ** ** ** NS

2 ** NS ** ** NS

6 ** ** ** ** NS

1 ** ** ** ** NS


LOCATION X FERTILIZER

LOCATION X LINE

FERTILIZER X LINE

LOCATION X YEAR

FERTILIZER X YEAR


LINE X YEAR


NS ** NS NS


** ** ** NS NS


12 ** **

1 NS NS *


** NS


** NS NS


6 ** ** **


*,** are significant at the
NS, not significant.


probability, respectively.


SOURCE


LOCATION

FERTILIZER


5% and 1% levels of









establishment over the two years is important in yield and N fertilizer

response. Percent N and nitrogen fixation are also significantly

influenced by plant establishment.

Half-sib switchgrass lines and bahiagrass varied significantly with

regards to yield, %N, N fertilizer use efficiency and nitrogen fixation

estimated by total N difference (Table 4). All switchgrass yielded

significantly more dry matter than bahiagrass, but the latter was higher

in %N, indicating the ability of switchgrass to dilute nitrogen. Nitrogen

dilution, as defined by Terman and Allen (61) refers to a relatively high

concentration of N in young plants that becomes lower with increasing dry

matter production and age. This type of nitrogen dilution should not be con-

fused with 15N dilution estimates of nitrogen fixation. Nitrogen dilution

is particularly noticeable in pot experiments having a finite volume of soil

for root development and occurs more rapidly under pot vs. field conditions.

Dilution has been observed in corn (60,61) and Italian ryegrass (24). The

present data show nitrogen dilution with reference to a species difference

between switchgrass and bahiagrass in the field, in addition to limited

half-sib line dilution differences within switchgrass. This ability,

coupled with the high fertilizer use efficiency of switchgrass (Table 4),

may be why this species does so well on poor soils with little or no nitrogen

input. Switchgrass was also higher than bahiagrass in nitrogen fixation

estimated by total N difference and showed a trend towards higher acetylene

reduction.

Applied fertilizer nitrogen had a significant positive influence on

crop yield, a negative influence on fertilizer use efficiency and no

influence on %N (Table 5). Nitrogen fixation estimates were also

negatively affected by fertilizer nitrogen in agreement with other

observations with grasses (59) and legumes (52). This effect was











Table 4. Mean Yield,%N, N fertilizer use efficiency (FEFF) and estimates
of nitrogen fixation by total N methods (NF TN) and acetylene
reduction (NF AR) by lines in the field experiments.


LINE YIELD %N FEFF NF TN NF AR
kg/ha kg/ha kg/ha

F-3115 7954a* 0.84c 255a 28.0ab 3.6a

F-4685 7381b 0.88bc 245ab 26.9ab 3.7a

F-1116 6543c 0.89b 223bc 34.9a 23.2a

F-1666 5869d 0.88bc 198c 16.9b 4.4a

F-1668 5776d 0.92ab 224bc 19.lab 3.9a

F-687 5731d 0.92ab 209c 18.6ab 3.9a

Bahiagrass 945e 0.93a 35d 0.9c 2.7a


*


Means followed by different letters
5% level by Duncan's Multiple Range


are significantly different at the
Test.












Table 5. Mean Yield, .%, N fertilizer use efficiency (FEFF) and estimates
of nitrogen fixation by total N methods (NF TN) and acetylene
reduction (NF AR) by fertilizer treatment in the field
experiments.


FERTILIZER YIELD %N FEFF NF TN NF AR
kgN/ha kg/ha __ kg/ha kg/ha

90 6434a* 0.88a 61c 5.2c 3.6a

50 5828b 0.91a 104b 17.ib 3.7a

10 4965c 0.90a 429a 39.9a 12.3a


*
Means followed by different letters are significantly different at the
5% level by Duncan:s Multiple Range Test.









significant for each increment of applied N when estimated by total N

difference but showed only a trend in the acetylene reduction measure-

ments.

All measured parameters, excluding the nitrogen fixation estimate

by acetylene reduction, showed significant effects due to year and

location. The significantly higher values obtained in 1979 (Table 6)

probably relate most importantly to plant establishment. Plants grown at

the Gainesville location were higher in yield, fertilizer use efficiency

and nitrogen fixation by total N difference (Table 7). Since the Hague

location represents a poorly drained site, the sometimes waterlogged soil

seemed to account for decreases in yield, and inhibited root development

that would decrease plant uptake of N and other nutrients, including water.

Percent N, however, was significantly higher at Hague, possibly due to a

higher overall soil N content.

In the correlation analysis, yield and percent N showed a significant

negative correlation while yield ana fertilizer use efficiency and nitrogen

fixation estimated by total N difference were positively correlated, as

was the latter with %N (Table 8). Positive correlation (p> 0.001) exists

between the two nitrogen fixation estimates. Although the relative mag-

nitude of the values differs greatly (Tables 5, 6, 7, 8), their correlation

indicates that both are applicable to these systems and may be used

concurrently or alone.

Broad-sense heritabilities (Table 9) were low for most of the

parameters measured in the field. Only decumbance and lateness of

flowering exhibited relatively high heritabilities that were stable over

locations. The surprisingly low values for yield at Hague and the

combined locations illustrates the effects of inhibited plant growth














Table 6. Mean Yield, N, N fertilizer use efficiency (FEFF) and estimates
of nitrogen fixation by total N methods (NF TN) and acetylene
reduction (NF AR) by year in the field experiments.


YEAR YIELD %N FEFF NF TN NF AR
_ha kg/ha kg/ha kg/ha

1979 8297a* 0.92a 293a 32.5a 4.0a

1978 3187b 0.87b 103b 9.1b 12.8a



*
Means followed by different letters are significantly different at the
5% level by Duncan's Multiple Range Test.















Table 7. Mean Yield,%N, N fertilizer use efficiency (FEFF) and estimates
of nitrogen fixation by total N methods (NF TN) and acetylene
reduction (NF AR) by location in the field experiments.


LOCATION YIELD %N FEFF NF TN NF AR
kg/ha kgha kg/ha

GAINESVILLE 7510a* 0.85b 243a 25.3a 2.4a

HAGUE 3976b 0.95a 153b 16.3b 10.6a


*
Means followed by different letters are significantly different at the
5% level by Duncan's Multiple Range Test.












Table 8. Pearson correlation coefficients (and their probabilities)
for yield, %N,Nfertilizer use efficiency (FEFF) and estimates
of nitrogen fixation by total N methods (NF TN) and acetylene
reduction (NF AR) in the field experiments.


%N FEFF NF TN NF AR

YIELD -0.176 0.409 0.335 -0.069
(>0.001) (>0.001) (>0.00l) (0.267)

%N 0.068 0.112 0.041
(0.163) (0.022) (0.419)

FEFF 0.455 -0.047
(>0.001) (0.419)

NFKJ 0.835
(>0.001)












Table 9. Broad-sense heritabilities, in percent, for yield, vigor,
decumbance, lateness of flowering, %N, N fertilizer use
efficiency by total N difference (FEFF TN) and estimates
of nitrogen fixation by total N difference (NF TN) and by
acetylene reduction (NF AR) in the field experiments at
Gainesville and Hague and the combined locations.


GAINESVILLE


YIELD

VIGOR


DECUMBANCE


LATENESS OF FLOWERING


S COMBINED
HAGUE LOCATIONS
LOCATIONS


22.2

44.6

70.1

56.8


6.8

3.9

68.2

65.3

5.9

2.9

5.1


8.5 6.1


FEFF TN


NF TN

NF AR










within a location and a very large location variance on heritability

estimates. It is evident from these data that different switchgrass

lines, in terms of yield, are suited to different locations, and that

breeding progress would be slower under Hague conditions. This same

sort of situation exists for %N. Heritabilities for fertilizer use

efficiency and the estimates of nitrogen fixation are very low, as

may be expected, and indicate that any breeding progress for these traits

would be slow.


Greenhouse Evaluations

Mean yields, %N, fertilizer use efficiencies and estimates of

nitrogen fixation by 15N and Kjeldahl N methods for the switchgrass lines

and bahiagrass in the first, second and combined harvests are presented

in Tables 10, 11 and 12, respectively. The plants were at a pre-flowering

stage of maturity when harvested. Although ample time for flowering was

allowed (120 days for harvest 1 and 92 days for harvest 2) a delay was

brought about by the greenhouse environmental conditions and time of

year. Significant variations in yield and consistent yield ranking by

lines were noted throughout the experiment. As in the field results,

bahiagrass yielded less than switchgrass, but not as markedly. In

addition, switchgrass line rankings were different between the greenhouse

and the field. Percent N in switchgrass was significantly lower than

that of bahiagrass, indicating that switchgrass is able to dilute nitrogen

to a great extent. This, again, is one reason why switchgrass grows well

under a low nitrogen input and makes the species an excellent biomass

producer on poor soils. The extremely low %N values observed for pot-

grown plants is in agreement with other published reports (24,61,62)

and agrees with the species differences shown in the field.





















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Fertilizer N use efficiencies estimated by 15N uptake were low

throughout the experiment and no line differences were observed. This

measurement includes only fertilizer N uptake, and is not confounded

with fixed or soil N. Nitrogen efficiencies estimated by total N

difference were higher than 1N uptake values, but are confounded with

applied, fixed and soil N, which are not separable by the total N method.

It is assumed that the N uptake values are most accurate; however, it

seems unrealistic that less than 25% of the applied N was actually

recovered in the verdure, especially since leaching was avoided. It

may be possible that applied N was incorporated into stable, non-available

soil N forms and a turnover of non-labeled soil N was released for

plant uptake. Further studies would be needed to support this hypothesis.

Estimates of nitrogen fixation were also low; however, in the second

and combined harvests, nitrogen fixation (by 15N dilution) was signifi-

cantly lower in bahiagrass than in switchgrass, indicating an important

advantage of switchgrass over bahiagrass under nitrogen limited conditions.

This result is in agreement with field estimates of nitrogen fixation

in the two species. No overall differences existed between switchgrass lines

for %N, fertilizer use efficiency or nitrogen fixation. This may be

indicative of a species trait or a starting population of plants with

low genetic variance.

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are obtained when switchgrass lines are pooled over the four populations.

In the first harvest (Table 13), significant differences due to

population were found for yield, fertilizer use efficiency and nitrogen

fixation. Since selection procedures were visual, and included yield

as one criterion, increases in yield are expected, and populations 3 and


















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4 were significantly higher than the unselected population 2 in the

first and combined harvests (Table 14), although the selected popula-

tions did not exceed population 1. This may be due to a highly selected

population 1, which is reasonable to assume since a plant collector

would intuitively select the visually best plants within a location.

In addition to yield, fertilizer efficiency by 15N uptake in

population 1 was higher than population 3, although populations 2, 3

and 4 did not differ indicating no selection improvement for this trait.

The most exciting observations are in the estimates of nitrogen fixation

by 15N dilution. In the first harvest, population 3 was significantly

higher than population 1 for nitrogen fixation, and in the combined

harvests, population 4 significantly exceeded population 1. In the

second harvest (Table 15), nitrogen fixation was also highest in

population 4 and significantly exceeded population 3, but was not

different from populations 1 or 2. The results suggest that selection

for nitrogen fixation may be possible but will require further refinement

of the measurement techniques and understanding of the cultural conditions

(e.g., plant establishment, growth environment, time of year) that affect

genetic expression of associative nitrogen fixing abilities. For example,

harvest time played an important role in all traits measured (Table 16).

Yield and estimated nitrogen fixation by the total N method were signi-

ficantly higher in harvest 1, due to better plant growth, whereas growth

was restricted by the pots in harvest 2. While total N difference

calculations did not agree, estimated nitrogen fixation by the more

sensitive and reliable 15N dilution method was higher in harvest 2 and

illustrates the importance of time of year and plant establishment on

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in harvest 2 also indicates the effect of plant establishment on

these parameters and includes the utilization of soil nitrogen during

the first growth period.

Correlation analyses for the six parameters in harvests 1, 2 and

the combined harvests are presented in Tables 17, 18 and 19, respectively.

In the combined harvests, yield and %N were negatively correlated,

indicating nitrogen dilution. Yield and fertilizer use efficiency by

15N uptake, while positively correlated in harvest 2, showed an overall

negative correlation, and fertilizer efficiency was positively related

to %N in the combined harvests. Nitrogen fixation by 15N dilution was,

over both harvests, negatively correlated to yield and positively so

to %N, indicating that these factors may be of value in initial selection

and screening procedures in a breeding program. Yield may be sacrificed

to support associative diazotrophs, as previously hypothesized by

Brill (15). In contrast, nitrogen fixation estimated by the total N

method was positively correlated to yield in the first and combined

harvests indicating again the need for technique refinement. This and

the non-agreement and overall negative correlation of total N and 15

dilution estimates indicate the relative insensitivity of the Kjeldahl

method. The importance of such measurements lies in an overall esti-

mation of gross nitrogen relationships.

In the combined greenhouse harvests (Table 14), mean estimates of

nitrogen fixation by the total N method were 75% lower than 1N dilution

estimates. This is in contrast to the 40% underestimates observed by

Williams et al. (73) using legume systems. In "A value" calculations

(see Materials and Methods section) involving legume systems, either a

grassor non-nodulating legume is used as a control, and includes N











Table 17. Pearson correlation coefficients (and their probabilities)
for Yield, %N, N fertilizer use efficiencies by 15N uptake
(FEFF 15N), N fertilizer use efficiencies by total N
difference (FEFF TN) and estimates of nitrogen fixation by
15N dilution (NF 15N) and total N difference (NF TN) in the
first greenhouse harvest.


%N

YIELD -0.033
(0.879)


FEFF TN

0.936
(>0.001)


0.304 0.049
(0.149) (0.820)

0.478 -0.559
(0.018) (0.005)

-0.169
(0.429)


FEFF 15N

0.392
(0.058)

0.332
(0.113)


NF 15N

-0.214
(0.316)


FEFF 15 N


FEFF KJ


NF 15N


NF TN

0.851
(>0.001)

0.184
(0.390)

0.358
(0.086)

0.884
(>0.001)

-0.094
(0.663)











Table 18. Pearson correlation coefficients (and their probabilities)
for Yield, %N, N fertilizer use efficiences by 15N uptake
(FEFF 15N), N fertilizer use efficiencies by total N
difference (FEFF TN) and estimates of nitrogen fixation
by 15N dilution (NF 15N) and total N difference (NF TN) in
the second greenhouse harvest.


YIELD -0.332
(0.113)


FEFF 15N

0.467
(0.021)

0.040
(0.852)


FEFF 15N


FEFF KJ


FEFF TN NF 15N

0.004 -0.327
(0.987) (0.119)


0.214
(0.315)


-0.060
(0.781)


0.089 -0.734
(0.678) (>0.001)


-0.165
(0.440)


NF 15N


NF TN

0.051
(0.811)

0.425
(0.039)

-0.015
(0.947)

-0.062
(0.773)

0.123
(0.567)












Table 19. Pearson correlation coefficients (and their probabilities)
for Yield, %N, N fertilizer use efficiencies by 15N uptake
(FEFF 15N), N fertilizer use efficiencies by total N
difference (FEFF TN) and estimates of nitrogen fixation by
15N dilution (NF 15N) and total N difference (NF TN) in the
combined greenhouse harvests.


%N FEFF 15N FEFF TN NF 15 NF TN

YIELD -0.663 -0.446 0.926 -0.628 0.762
(>0.001) (0.002) (>0.001) (>0.001) (>0.001)

%N 0.676 -0.426 0.546 -0.218
(>0.001) (0.003) (>0.001) (0.134)

FEFF 15N -0.306 0.249 -0.179
(0.035) (0.091) (0.225)

FEFF KJ -0.529 0.819
(>0.001) (>0.001)

NF 15N -0.529
(>0.001)









inputs by associative and free-living nitrogen fixation in addition

to the contribution by soil N. In this study, no suitable control was

available and the 15N dilution estimates are, therefore, absolute ones.

Originally, bahiagrass was to be used as a control in these calculations

based on prior observations of low acetylene reduction in this species (7);

however, some bahiagrass "A" values were higher than those of some

switchgrasses. Also, the relative contribution of soil and fixed N in

the 15N nitrogen fixation estimates is not known.

It would also be desirable to have acetylene reduction data for

the greenhouse experiment. Attempts to obtain such data were made in

large plexiglass chambers designed to accommodate a 15 cm pot. Nearly

300 such measurmeents were made; however, the data were rendered invalid

by a blue-green bacterial bloom during the first growth period and a

general non-response in the second, possibly due to the root-bound nature

of the pots. The latter observation was not, however, consistent

with 15N dilution data (Table 16) which indicate more nitrogen fixation

in harvest 2 than in harvest 1, when the plants were better established.

Since the blue-green bacterial crusts were scraped from the soil surface

and discarded, their fixed nitrogen may have also been removed, a

speculation consistent with these data.















CONCLUSIONS


Combined field and greenhouse studies with switchgrass breeding

lines and Pensacola bahiagrass showed differences in yield, %N,

fertilizer use efficiency and nitrogen fixation measured by three

methods: total nitrogen difference, acetylene reduction and 15N

dilution. In the field, nitrogen fixation estimates by the total N

method and acetylene reduction showed a significant positive correlation,

but in the greenhouse, total N methods were negatively correlated to

15N dilution estimates, indicating a great need for further refinement

of these techniques. Mean estimates of the amount of nitrogen fixed

by the total N method were 20.8 and 2.4 kgN/ha in the field and green-

house, respectively. Acetylene reduction estimated a mean of 6.5 kgN/ha

fixed in the field, while 15N dilution in the greenhouse estimated

9.5 kgN/ha. The results verify that an agronomically significant amount

of nitrogen is being fixed by indigenous diazotrophs in the rhizospheres

of these forage grasses.

From a plant breeding standpoint, improvement of switchgrass for

yield and fertilizer use efficiency seems possible. Improvement for

associative nitrogen fixation also appears possible, but the results of

a breeding and selection program will be dependent, in part, on the type

of measurements made and the environmental and cultural conditions.

Broad-sense heritabilities for these parameters were generally low,

indicating that such breeding progress will be slow. Overall results




47





indicate the need for further collection of a broad-based germplasm

switchgrass population.

Switchgrass yields well on sandy soils under low nitrogen inputs

and also responds to applied fertilizer nitrogen. In addition, the lines

evaluated have the ability to dilute nitrogen to a great extent. It

is concluded that switchgrass is a promising new forage and biomass

producer for Florida, especially in present times due to increasing

nitrogen fertilizer costs and decreasing availability.














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BIOGRAPHICAL SKETCH


Glen C. Weiser was born in Salt Lake City, Utah, where he graduated

from Highland High School in 1970. After attending Utah State University

and the University of Utah, he received the degree of Bachelor of Science

from the University of Idaho in 1974. He received the Master of Science

degree in crop physiology in 1976, and began a program of study leading

towards the degree of Doctor of Philosophy in plant breeding and genetics

at the University of Florida in 1977.

The author is a member of the American Society of Agronomy, the

Crop Science Society of America, the Botanical Society of America and is

an honorary member of Gamma Sigma Delta.










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
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.



0. Charles Ruelke
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.



L. Curtis Hannah
Associate Professor of
Vegetable Crops









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.



Kenneth H. Quesenberry
Assistant Professor of Agronomy




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.

March 1980


Dean college of Agricuj re




Dean, Graduate School




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