Interaction of Rhizobium japonicum with soybean isolines carrying unique genes which affect nodulation at the Rj1 locus

interactionofrhi00payn ( .pdf )

INTERACTION OF RHIZOBIUM JAPONICUM WITH SOYBEAN ISOLINES
CARRYING UNIQUE GENES WHICH AFFECT NODULATION
AT THE RjI LOCUS





By

JOHN HOWARD PAYNE


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


UNIVERSITY OF FLORIDA


1985
















To Four Teachers


R. Guy Payne
Through his knowledge and enthusiasm he instilled
in me fascination for the natural world



Esther Ruth Collins
She introduced me to scientific study and always
encouraged enterprise and individuality



Robert G. Anderson
In socratic discourse he taught me the importance
of critical thinking



Steven G. Pueppke
He showed by his example the need for diligence
and integrity in scientific endeavor


















ACKNOWLEDGEMENTS


Dr. Steven Pueppke provided guidance as my committee

chairman. I thank him for his friendship and patience, and

for his consistent example of the finest in scholarly

research. Members of my committee, Drs. Raghavan

Charudattan, Ed Freeman, Bill Gurley, and George Bowes, have

always provided help when asked and have influenced the

direction of this work with their suggestions. I am

grateful for the friendship and advice of Drs. H. H. Luke

and Dave Mitchell. Mrs. Ulla Benny provided technical

assistance, including hours of her time assisting with the

tedious, repetitive procedures necessary for the study of

adsorption of bacteria to plant roots. Drs. R. Howard Berg,

III, and Greg Erdos provided instruction in the use of the

scanning electron microscope and made available the

facilities of the Biological Ultrastructure Laboratory. My

laboratory companions, Drs. Dan Kluepfel and Jill Winter,

Mr. Dave Heron, and others, have aided with discussions of

the research and have given helpful suggestions and

constructive criticism. To all of these I offer grateful

thanks.










My parents Rev. Richard and Mrs. Reva Payne, my

brother Mr. James Payne, my sisters Mrs. Rebecca McClanahan

and Mrs. Mary Jane Suppasansathorn and their families, and

my grandmother Mrs. Mable Speak, have provided abiding love

and constant encouragement. Their confidence in me and the

emotional support they gave were at times what kept me

going. I cannot thank them enough.

This research was supported in part by the National

Science Foundation through grant PCM 82-00110 to Dr.

Pueppke, and the University of Florida; each provided

research funds and contributed to my research assistantship.

Financial support also was provided by the Veterans

Administration.




















TABLE OF CONTENTS


Page

ACKNOWLEDGEMENTS...... ................................... i

ABSTRACT................................................* vii

CHAPTER ONE INTRODUCTION............................. 1

CHAPTERTWO REVIEW OF NODULATION SPECIFICITY AND MODE
OF INFECTION BY RHIZOBIUM JAPONICUM OF SOYBEAN
LINES WITH NODULATION RESTRICTIVE GENOTYPES......... 3

Introduction........ ..........................***** 3
"Cross-inoculation Groups" and Rhizobium Taxonomy.. 5
Genetics of Rhizobium Infection and Nodulation..... 8
Infection and Nodulation of Legumes............... 20
Phenotypic Nodulation Response of Soybean.......... 26
Nodulation Restrictive Soybean Genotypes........... 27
Perspective........................ ......... .....* 33

CHAPTER THREE EFFECT OF TEMPERATURE ON NODULATION OF
SOYBEAN ISOLINES DIFFERING AT THE Rjl LOCUS........ 35

Introduction.......................... ............. 35
Materials and Methods............................. 37
Results................... ....................... 39
Discussion.... ...................................... 53

CHAPTER FOUR ADSORPTION OF STRAINS OF RHIZOBIUM
JAPONICUM WITH DIFFERENTIAL NODULATING ABILITY TO
ROOTS OF SOYBEAN ISOLINES THAT DIFFER AT THE Ril
LOCUS............................ ................ .. 57

Introduction....................... .......- ........ 57
Materials and Methods.............. ............. 61
Results.................... ........................ 65
Discussion........................ ....... ........* 74










Page

CHAPTER FIVE INFECTION OF SOYBEAN ISOLINES DIFFERING AT
THE Ril LOCUS BY RHIZOBIUM JAPONICUM STRAINS WITH
DIFFERENTIAL NODULATING ABILITY..................... 77

Introduction ............................. ........... 77
Materials and Methods............................... 81
Results .............................. ............. .. 85
Discussion................................................. 110

CHAPTER SIX SUMMARY................................... 116

APPENDIX A PLASMIDS OF RHIZOBIUM JAPONICUM STRAINS THAT
NODULATE SOYBEAN ISOLINES DIFFERING AT THE Rj1
LOCUS .................................. ............. 120

APPENDIX B EVALUATION OF PROCEDURES REPORTED TO INDUCE
HIGH-FREQUENCY MUTATION OF STRAINS OF RHIZOBIUM
JAPONICUM............. ............................ 128

LITERATURE CITED................................................. 137

BIOGRAPHICAL SKETCH. ........ ............................ 151
















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


INTERACTION OF RHIZOBIUM JAPONICUM WITH SOYBEANISOLINES
CARRYING UNIQUE GENES WHICH AFFECT NODULATION
AT THE Rjl LOCUS

By

John Howard Payne

May 1985

Chairman: Steven G. Pueppke
Major Department: Plant Pathology

The soybean genotype rilril conditions the inability to

nodulate with most strains of Rhizobium japonicum. Certain

strains, termed overcoming strains, form a few nodules on

plants grown in hydroponic culture. The effect of

temperature on the number of nodules per plant and the

nodulation pattern was determined for the cultivar Clark

(R1lRil) and its isoline Clark-rj1 (rl1L1i). The

temperature treatments, 22 C, 27 C, and 32 C, had a

statistically significant effect on the number of nodules

for both genotypes. The percentage of Clark-jil plants

nodulated by overcoming strains was 44% at 22 C, 23% at 27

C, and 4% at 32 C for 120 plants tested. The nonovercoming

strain 110 did not nodulate Clark-rj1 at any temperature.

Ninety-eight percent of 270 Clark plants tested, including

all strains and treatments, were nodulated. Frequency plots











were generated for each isoline x strain combination for

each temperature. These indicated the number of nodules

produced at locations on the primary root relative to the

location of the root tip which was marked at the time of

inoculation. Plots for combinations of Clark with each

strain showed a peak near the root tip mark for plants grown

at 22 C or 27 C. The frequency plots for Clark plants grown

at 32 C were flattened and indicated a downward displacement

of nodulation on the primary root.

The adsorption of overcoming strain 94 and

nonovercoming strain 110 to roots of Clark and Clark-ril was

tested. After 2 hr inoculation, approximately 100 bacterial

cells were bound per plant, irrespective of strain. The

rank of isoline x strain combinations for the number of

bacteria bound to roots was opposite to that for nodule

number.

The roots of both plant types were examined by light

microscopy and scanning electron microscopy 10 d after

inoculation with strain 94 or 110. Curled root hairs with

infection threads were formed on Clark in response to either

bacterium, but were not observed on Clark-rj1. When Clark-

ril plants were inoculated at a high inoculum concentration,

perforation in the epidermis was apparent, suggesting a

potential infection pathway.


viii



















CHAPTER ONE
INTRODUCTION


The symbiosis of soybean and the bacterium Rhizobium

japonicum depends, even in early stages of its initiation,

on contributions from both the plant and the bacterium.

They interact in a coordinated, multi-step infection process

to form root nodules. The bacteria inhabit cells of the

nodule, where they fix atmospheric nitrogen, which is

unusable to the plant, to ammonium, a plant nutrient. The

rhizobia in turn derive sustenance from the plant in the

form of translocated nutrients. The series of events that

transpire during the infection process has been studied by

microscopy, by induced genetic change in the bacterium, and

by biochemical and microbiological examination. Some steps

leading to nodule formation have been described, but the

relative contributions of the plant and the bacterium to the

inception of a nodule are not well understood. The

literature that describes the infection process is reviewed

in Chapter Two. Studies are described in which genetic

changes are induced in Rhizobium and the effects of the

changes are correlated with the interaction phenotype with

the host. The contribution of studies of plants with

altered symbiotic phenotype also is described.






-2-


The soybean genotype, rJriil, conditions resistance of

soybean to most strains of R. japonicum. A few strains,

called the "overcoming strains," have the ability to

overcome the rji-resistance and form a few nodules on

EJilrl-plants. In this study the soybean cultivar Clark and

a line near-isogenic to it, Clark-rJi that carries the genes

rjl1fl, were used. Chapters Three through Five are reports
on aspects of the phenotypic expression of the symbiotic

interaction of soybean isolines and strains of R. japonicum.

Chapter Three describes the effect of temperature on the

differential nodulation response of the soybean isolines.

Chapter Four presents an evaluation of the role of bacterial

adsorption as a specific determinant of differential

nodulating ability. Chapter Five includes a report of the

interaction phenotype at the cellular level after

inoculation of seedlings with an overcoming or a

nonovercoming strain. Conclusions about some aspects of the

interaction of strains of R. japonicum with the soybean

isolines are summarized in Chapter Six. The appendices

include a short description of the plasmid complement of

strains of R. japonicum as determined by gel electrophoresis

and a report of an evaluation of procedures which had been

described to induce high-frequency mutations in symbiotic

functions.


















CHAPTER TWO
REVIEW OF NODULATION SPECIFICITY AND MODE OF INFECTION BY
RHIZOBIUM JAPONICUM OF SOYBEAN LINES WITH NODULATION
RESTRICTIVE GENOTYPES


Introduction

Soybean has had the largest increase in acreage for any

crop in American history. In 1930, 1 million acres produced

about 14 million bushels; in 1980 more than 70 million acres

produced over 2.25 billion bushels (Sundquist 1981). Over

the past 60 years there has been about one-quarter bushel

per acre increase in yield per year. This is due in great

measure to increased understanding through research on

soybean production. Included is the understanding and

manipulation of its symbiosis with root-nodule bacteria

(Hanson 1981, Weber 1981). Weber (1981) has estimated the

market value for the combined nitrogen produced in the

United States by the legume-Rhizobium symbiosis at $5

billion a year.

The value of the symbiosis and the impact of its

improvement are clear, but the exact nature of the

relationship of the bacterium to the plant and how best to

manipulate that relationship for greater productivityare

still unclear. The subtle interchange of signals between

plant and microsymbiont that leads to establishment and

maintenance of the complex partnership has largely remained











a mystery, in spite of considerable effort to unravel its

intricacies. More is known about the genetic and

physiological factors essential for the bacterium in this

relationship because of the greater ease with which these

elements can be manipulated in the relatively simpler

organism. Although a few genotypes are known to produce

qualitative changes in the phenotype of the interaction, our

knowledge of the role of the plant is limited mostly to

general observations of the effect of genetic constitution

on quantitative aspects of the symbiosis.

To replace the broad, nonanalytical descriptions of the

relationship, Vincent (1980) proposed terminology that

defines specific steps in the establishment of the

partnership. His terms are defined as specific phenotypes

meant to be applicable to most legume-Rhizobium symbioses,

although there is no provision for rhizobia that infect by

means other than through root hairs. The terms pertinent to

early infection processes will be used in this discussion,

and are as follows (derivation are indicated): Roc = Root

colonization, Roa = Root adhesion, Hac = Hair curling, and

Inf = Infection thread formation. Two common terms not

described by Vincent will be used for events representative

of the mature symbiosis. The term Nod (= Nodule formation)

will be used to describe the formation of macroscopic

nodules. The term Fix (= Fixation of nitrogen) will be

used, rather than Nif, to avoid confusion with bacterial

genetic nomenclature.










This review first examines the rhizobia and their

contribution to the infection of legumes. The taxonomy of

the Rhizobiaceae is discussed in light of its important role

in defining research directions and its implications for

better understanding of genetic data. Experimental genetic

manipulation of the rhizobia is included to provide a

background for discussion of the research presented in

subsequent chapters. The literature which describes the

phenotype of interaction of the bacterium and plant at the

cellular level is reviewed. Plant genotypes known to have

qualitative effects on the early stages of nodulation also

are described.



"Cross-inoculation Groups" and Rhizobium Taxonomy

The taxonomy of Rhizobium is based on the range of host

plants nodulated. Strains with similar host-range have been

given species status. This idea was formalized in the

landmark monograph of Fred, Baldwin, and McCoy in 1932. The

authors of this monograph expounded the concept of "the

cross-inoculation group." This amounted to an exhaustive

list of the legumes, species by species, which could be

nodulated by rhizobia that were isolated from other species

of legumes in that list but not by rhizobia from other

groups of legumes. Although there were clearly some

ambiguities, the concept of mutually exclusive inter-

nodulation within inoculation groups became an accepted

paradigm, and thus the basis of Rhizobium taxonomy. As a

result, a group of strains shared by an inoculation group











was given species status. The species recognized by this

criterion were I. Alfalfa group, R. meliloti; II. Clover

group, R. trifolii; III. Pea group, R. leguminosarum; IV.

Bean group, R. phaseoli; V. Lupine group, R. lupini; and VI.

Soybean group, R. japonicum. A seventh group, the cowpea

group (or cowpea miscellany) was not given species status

since the legumes that could be cross-inoculated were

numerous and taxonomically diverse. There also were

apparent subgroups within the cowpea group which did cross-

inoculate some subgroups but not others. Fred et al. (1932)

considered the cowpea group to be intermediate between the

soybean group and the lupine group, because many of the

rhizobia from the legume hosts in those groups formed

nodules with many of the hosts for the cowpea group.

Rhizobium strains fall naturally into two main

divisions on the basis of their physiology and morphology,

those which produce rapid growth on rich media and have

peritrichous flagella, and those which grow slowly on rich

media and have a polar or subpolar flagellum (Jordan and

Allen 1974). The fast-growing strains are generally those

included in R. leguminosarum, R. phaseoli, R. trifolii, and

R. meliloti. The slow-growing strains are usually those

included in R. lupini, R. jaSEoicum, and the cowpea

miscellany (Jordan and Allen 1974). Several strains from

the People's Republic of China nodulate soybean but are

similar in growth response and physiology to the fast-

growing species of Rhizobium (Keyser et al. 1982). These










strains were described taxonomically as R. japonicum and

noted parenthetically to be fast-growing strains (Keyser et

al. 1982, Jansen van Rensburg et al. 1983, Heron and Pueppke

1984). This taxonomic problem serves to underscore the

overall deficiencies of a taxonomy based on host range.

Recently, the taxon R. fredii was proposed for the fast-

growing rhizobia that infect soybean (Scholla and Elkan

1984).

In 1964, Graham (1964) revised the taxonomy of the

Rhizobiaceae. He recognized previous criticism of the

cross-inoculation group concept (Wilson 1944), and based his

revision on a numerical taxonomy that compared 100

physiological characteristics. He proposed that R.

phaseoli, R. trifolii and R. leguminosarum be consolidated

into a single species R. leguminosarum. Agrobacterium

tumefaciens and A. radiobacter were to be included in the

genus Rhizobium as R. radiobacter. The fast-growing species

R. meliloti was retained. Graham also proposed that the

slow-growing strains be contained in a newly proposed genus

Phytomyxa. The taxonomic revision proposed by Graham was

not widely accepted, but some of the general features of his

system are included in recently proposed changes. The

International Subcommittee on Agrobacterium and Rhizobium

recently proposed that the slow-growing strains of Rhizobium

be transferred to a new genus Bradyrhizobium gen. nov.

(Jordan 1982). This taxon emphasizes the basic

physiological difference between the fast-growing and slow-

growing strains. Jordan (1984) included most of Graham's










proposed revisions in his recent description of the family

Rhizobiaceae. The proposed species are R. meliloti, R.

leguminosarum (with the biovars: trifolii, phaseoli, and

viceae) and R. loti, which includes strains that nodulate

Lotus spp. and related plants. The genus Bradyrhizobium

essentially represents the strains for which Graham proposed

the name Phytomyxa (Graham 1964). The genus Agrobacterium

was retained. This revision addresses many of the

deficiencies in the former taxonomic treatments of the

Rhizobiaceae. The new taxonomy should greatly facilitate

discussions of the comparative genetics of strains of

rhizobia, including the genetic basis of symbiotic

interaction. However, for this literature review I will

continue to use the nomenclature of Jordan and Allen (1974),

because all of the literature to be examined follows that

system. Nevertheless, the reader should consider the data

on the genetics of symbiosis and host range and the

comparisons in interaction phenotype in the framework of the

biological relationships suggested by the taxonomy described

by Jordan (1984).



Genetics of Rhizobium Infection and Nodulation

Much of what is known about the genetics of nodulation

has been developed with the four classical species of fast-

growing Rhizobium. The three allied species R.

leguminosarum, R. trifolii, and R. phaseoli will be

discussed together because similar procedures and genetic










probes have been used to study them. Both Ljunggren (1961)

and Beringer (1980) cite Krasilnikov (1941) as providing the

first evidence for transfer of nodulating ability from

strain to strain when he reported the transformation of

nonnodulating strains with culture filtrates from nodulating

strains. In 1961, Ljunggren reported the transformation of

the nonnodulating R. trifolii strain Bart A. The

transformed strain, Bart A'2, nodulated clover, had a smooth

colony morphology and produced a serological reaction

unrelated to that of Bart A and only partially related to

the transforming strain. However, one cannot rule out the

selection of a contaminant capable of nodulation.

The association of some of the symbiotic functions with

plasmids and the development of procedures for genetic

manipulation, including recombinant DNA techniques, have

greatly increased experimentation on the genetics of

nodulation. Plasmids were detected in various strains of

several of the Rhizobium spp. (Tshitenge et al. 1975, Nuti

et al. 1977). Higashi (1967) reported that R. phlaseoli

acquired the ablility to nodulate clover with the transfer

of an episomal factor from R. trifolii. Zurkowski et al.

(1973) used chemical agents to cure strains of R. trifolii

of plasmids and reported concomitant loss of ability to

nodulate clover. The introduction of the kanamycin-

resistance marker of the transposon Tn5 into R.

leguminosarum provided a means to both mutate and mark the

location of the mutation. This enabled genetic linkage

analysis and selection by DNA-DNA homology (Beringer et al.











1978). The presence of the Tn5 marker in the conjugative

plasmid, pRLlJI, enabled selection of transconjugants at a

high frequency (Johnston et al. 1978). The ability to

nodulate peas was restored to plasmid-cured strains of R.

leguminosarum by acquisition of pRLlJI. Ability to nodulate

peas was transferred with pRLlJI to strains of R. trifolii,

a strain of R. phaseoli, and a slow-growing strain of

Rhizobium from Cicer (chickpea). These strains retained

their ability to nodulate their normal hosts, but the number

of nodules per plant was somewhat reduced. In other

strains, nodulation ability co-transferred at greater than

95% with bacteriocin production, a natural marker for pRLlJI

(Brewin et al. 1980).

R. leguminosarum plasmids with genes for nodulation

exist in several incompatibility groups (Brewin et al.

1982). When conjugated into the same cell, the plasmids

either formed cointegrates or one of the plasmids was lost.

Three sizes of hybrid plasmids were formed after conjugation

of pJB5JI, which codes for pea nodulation and nitrogen

fixation genes, into strain T37 of R. trifolii. The size of

the cointegrate corresponded to a specific Nod and Fix

phenotype on pea or clover (Christensen and Schubert 1983).

Host-range specifying genes of R. leeuminosarum were

localized in a 10 kb fragment of pRLlJI by using sequences

adjacent to Tn5 insertion-induced nodulation mutants to

select cosmid clones with homology (Downie et al. 1983).

The insertion of the 10 kb DNA clone enabled a plasmid-cured










R. phaseoli strain to nodulate pea but not Phaseolus

vulgaris. The nodules produced on pea were normal appearing

and contained typical bacteroids.

At least some of the genes encoding enzymes for

nitrogen fixatation are on plasmids in fast-growing species.

Nuti et al. (1979) showed that cloned nitrogen fixation

(nif) genes from Klebsiella pneumoniae, when used as DNA

probes to Southern transfer blots of EcoRI-digested plasmid

DNA, hybridized to 1 or 2 unique restriction fragments from

several R. leguminosarum strains. Hooykaas et al. (1981)

reported that one particular plasmid in R. trifolii encoded

both nodulation functions and nif genes. This was

demonstrated by conjugating the plasmid, designated "Sym,"

into Nod- Fix- R. leguminosarum, which consequently became

Nod+ Fix+. The "Sym"-plasmid conjugated into a cured strain

of A. tumefaciens enabled it to form nodules on clover but

nitrogen fixation did not occur. Similar experiments with

"Sym" plasmids from other strains of R. trifolii and from

strains of R. leguminosarum yielded very similar results

(Hombrecher et al. 1981, Prakash et al. 1981, Hooykaas et

al. 1982). The restriction map of "Sym" plasmids from R.

leguminosarum, and DNA-DNA homology studies with the "Sym"

plasmid from R. trifolii and the Ti plasmid of A.

tumefaciens, show considerable conservation of sequences

(Prakash et al. 1982b). Some of these conserved regions

correspond to areas that are transcribed in bacteroids

isolated from nodule tissue, and to regions with homology to

nif probes (Prakash et al. 1982a). The use of the term










"Sym" for plasmids which code for some of the functions

necessary for symbiosis may serve to confuse the issue of

the role of chromosomal genes in symbiosis. The plasmid

genes have proven to be the most tractable, so have received

the greatest attention. Evidence for the necessity of an

appropriate chromosomal background for expression of the

plasmid is clear (Beringer 1982), and some genetic evidence

for the existence of specific chromosomal genes active in

symbiosis is developing (Noel et al. 1984). The actual

number of genes which are involved in coding for nodulation

specific functions is unknown (Long 1984).

Most of the products of the "symbiotic" genes are

unknown. Zurkowski (1980) correlated the presence of the R.

trifolii plasmid pWZ2 with the ability of strains to

specifically adsorb to clover roots. Cured strains did not

bind to the roots but a transconjugant did (Zurkowski and

Lorkiewicz 1979). When strains with the plasmid were assayed

for binding in the presence of 30 mM 2-deoxyglucose (a

hapten of the clover lectin) a reduction of adsorption was

observed. Dazzo and Hubbell (1975) described antigenic

differences between nodulating and nonnodulating strains of

R. trifolii; unfortunately, it is not clear that true

sibling strains were used. The possibility of multiple gene

differences make it difficult to evaluate the biological

significance of the correlation of antigenicity to

nodulating ability. Russa et al. (1982) correlated the

occurrence of plasmid pUCS202 with differences in






-13-


lipopolysaccharides of R. trifolii strains, an observation

corroborated by Raleigh and Signer (1982), who selected

nodulation-deficient R. phaseoli strains by enrichment of

populations for altered surfaces. This was done by

screening survivors refractory to infection by phage Fl.

These data point to a problem not often addressed in other

studies. Raleigh and Signer (1982) found considerable

genetic changes in strain physiology in addition to those

that had been selected, and it was difficult to identify

which of the changes led to the inability to nodulate. Many

of the studies reported here are based on the assumption

that the only genetic effect of plasmid loss is the

alteration of the component under study (i.e.,

lipopolysaccharide), and the component is correlated only to

the effect under study (i.e., nodulation), without careful

consideration of what other genetic systems may be disrupted

by loss of the plasmid. The plasmid may represent a

substantial amount of the potential genetic information of

the cell and many of the functions are, as yet, cryptic.

Simple correlations with assumptions as to cause and effect

in such complex systems with considerable potential for

pleiotropic effects are injudicious until specific genes and

gene products can be manipulated to test cause and effect

unequivocally.

Although much of the discussion has suggested the host-

specific nature of the genes encoded on the plasmids of

strains of these three species of Rhizobium, some genes are

clearly common to all of the strains. Djordjevic et al.











(1983) tested the effect of two self-transmissible "Sym"

plasmids, one (pJB5JI) from R. leguminosarum, the other

(pBRlAN) from R. trifolii, on the phenotype of various

strains of Rhizobium. When conjugated into a cured strain

of either R. lequminosarum or R. trifolii, the plasmid

conferred the expected Nod+ Fix+ phenotype, i.e. nodules

formed, on the host of the strain from which the plasmid was

derived. But either plasmid could restore the nodulating

phenotype on clover to two R. trifolli strains with Tn5-

induced "hair-curling" mutations. Neither plasmid restored

the wild type to nonmucoid mutant strains of R. trifolii,

regardless of whether the mutant was spontaneous or Tn5-

induced.

The study of the genetics of R. meliloti has been

somewhat slow to develop, but recently it has received more

study than the genetics of the other rhizobia. Bechet and

Guillaume (1978) provided the first evidence of very high

molecular weight plasmids in R. meliloti. These very large

plasmids ( > 300 megadaltons) were visualized and

characterized from several strains (Rosenberg et al. 1981).

Rosenberg et al.(1982) extended this observation by

demonstrating the presence of the very large plasmid in all

of the 27 strains that were examined from diverse origins.

Heat treatment-induced deletion mutants that were Nod- or

Fix- or both and lacked all or part of the very large

plasmid (BAnfalvi et al. 1981). The nif genes of R.

meliloti were cloned and selected by homology to nif






-15-


sequences from K. pneumoniae. These then were used to

demonstrate by hybridization analysis that most of the Nod-

mutants lacked at least 24 kb of DNA. The fragments all

included several of the nif structural genes as well as some

functions essential for nodulation (Bhnfalvi et al. 1981,

Ditta et al. 1980, Corbin et al. 1982). Transfer of

sequences from the very large plasmid of R. meliloti coding

for at least some of the nif genes and some nodulation genes

into A. tumefaciens or E. coli enabled the recipient

bacteria to form nodules or nodule-like structures on

alfalfa plants but not on clover (Hirsch et al. 1984,

Truchet et al. 1984). Even quite small ( < 8.7 kb )

fragments were active (Hirsh et al. 1985). When the "Sym"

plasmid from R. leguminosarum, which carries genes for

uptake hydrogenase activity, was transferred to R. meliloti,

the ability of the recipient to form nodules on alfalfa was

not impaired, but the uptake hydrogenase activity was

expressed very little, if at all (Bedmar et al. 1984).

Transposon mutagenesis has been used to study the

genetics of nodulation and nitrogen fixation in R. meliloti.

Mead et al. (1982) examined 6000 strains with presumptive

Tn5 insertions for auxotrophy and screened the prototrophs

on alfalfa plants. They detected 4 Nod- (0.07%) mutants, 46

Fix- (0.8%) mutants, and 20 (0.3%) auxotrophs, which

suggests that there are either very few genes coding

essential functions for nodulation or that Tn5 insertion is

not random. Long et al. (1982) described a system for

cloning nodulation genes by direct complementation of Nod-






-16-


mutants using members of an overlapping cosmid clone bank to

map the mutations. Using this system they found that genes

essential for nodulation in R. meliloti are located on the

very large plasmid within 30 kb of nifK (Long et al. 1982,

Zimmerman et al. 1983).

The transfer of RP4 and R68.45 factors into R. meliloti

enables the mobilization of the chromosome by conjugation

(Kowalczuk and Lorkiewicz 1979). This procedure has allowed

the development of linkage maps for chromosomal genes (Kiss

et al. 1980, Forrai et al. 1983), which are essentially

colinear with those of R. leguminosarum and R. trifolii

(Kondorosi and Johnston 1981, Beringer 1980). Of 13 Fix-

mutations mapped, 5 were localized to the chromosome and 8

were extrachromosomal. The chromosomally located Fix-

mutations were not clustered. None of the Nod- mutants

mapped to the chromosome (Forrai et al. 1983). There are

several reports of generalized transduction in R. meliloti

(Kowalski 1967, Sik et al. 1980, Finan et al. 1984, Martin

and Long 1984), but transduction has not been used

extensively in genetic studies of this bacterial species,

perhaps because of the limitation in size of DNA segments

which can be transferred (Beringer 1980).

Very few of the Nod- mutants have been characterized.

Hirsch et al. (1982) reported the infection phenotype of 4

mutants which had previously been derived by Tn5 mutagenesis

(Meade et al. 1982). Two of the Nod- mutants did not induce

root hair curling or penetrate host cells. The other two






-17-


induced root hair curling, and entered root epidermal cells

by some means other than infection thread formation. The

mode of entry of the bacteria into epidermal cells was not

determined, but it was clear that the process did not

maintain cell membrane integrity, because bacteria were

observed in cell cytoplasm.

Understanding of the genetics of the slow-growing

strains of R. japonicum has lagged behind that for the fast-

growing rhizobia, partly because of the truculence of the

slow-growing rhizobia to most of the techniques developed

for study of the fast-growing rhizobia. Maier and Brill

(1976) treated strain 61A76 with nitrosoguanidine and

screened 2500 colonies on Corsoy soybean. Five mutants were

selected on the basis of lack of nodule development or

altered nodule appearance (including reduced leghemoglobin).

Two of the five were reported not to form nodules but were

competent to fix nitrogen in vitro. Three of the five were

Fix-. It has since been reported that the two strains

reported to be Nod- are actually Nod+, but nodules are slow

to appear (Stacey et al. 1982). Maier and Brill (1978)

reported that two strains showed earlier nodulation greater

nitrogen fixation, and one of the strains produced more

nodules than the parent strain. It is not clear whether

these selected strains demonstrated an increase in symbiotic

efficiency through simple mutation or whether the changes

were partly due to selection for tolerance to the specific

growth conditions of the laboratory. Skogen-Hagenson and

Atherly (1983) used elevated temperature and either SDS or











ethidium bromide amendment of culture medium, procedures

designed to cure plasmids, to produce mutants in strains

USDA 74 and 61A76. They reported that none of the strains

showed altered plasmid content, but that more than 50 of 133

isolates were symbiotically altered. Of those tested none

was reported to be auxotrophic. Forty-four were Nod- and

ninewere Fix-. The exceptionally high ratios of symbiotic

mutants to auxotrophs and of Nod- to Fix- make these results

unique. If reproducible, the procedure should be very

useful for study of R. japonicum genetics.

The role of plasmids in R. japonicum is not well

understood. Gross et al. (1979) developed plasmid profiles

for a group of "extra-slow-growing" strains indigenous to

alkaline soils. Each strain had two to four plasmids, with

sizes ranging from 48 to 130 megadaltons. Plasmids of 91

and 118 megadaltons were common to all of the extra-slow-

growing strains, but no phenotype was correlated with a

particular plasmid. Plasmid content of several slow-growing

strains was examined (Haugland and Verma 1981, Masterson et

al. 1982). Strains were characterized as usually having one

plasmid, but some strains had two or none. When cloned K.

pneumoniae nif stuctural genes were used as a probe against

R. japonicum plasmid DNA, no hybridization was detected.

The nif genes with homology to cloned K. pneumoniae

genes, nifKDH, have been localized in R. japonicum strain

USDA 110 (Hennecke 1981, Fuhrmann and Hennecke 1982, Kaluza

et al. 1983, Fuhrmann and Hennecke 1983). Strain USDA 110







-19-


is a plasmid-less strain. In contrast to the fast-growing

strains of Rhizobium, where the nitrogenase structural genes

are clustered in one operon (nifKDH), in this strain nifDK

represents one operon and nifH is in another operon at least

12 kb away (Kaluza et al. 1983). Hadley et al. (1983)

reported that the nifKDH region of K. pneumoniae, when used

as a probe on blots of restricted DNA from 17 slow-growing

Rhizobium strains, hybridized to at least two EcoRI

fragments from each strain, suggesting that the separation

between the operons for nif structural genes is common among

a number of strains and not unique to USDA 110. Hahn and

Hennecke (1984) used a site-directed mutagenesis technique

in which Tn5 mutagenesis of the cloned R. japonicum nifDK

operon was carried out in E. coli. The operon was

transferred by conjugation to R. japonicum by suicide

vectors, and stable exconjugants were selected. Mutations

within nifD or nifK caused a Nod+ Fix- phenotype, whereas

Tn5 insertions in the immediate area to either side of nifDK

were Nod+ Fix+. This is evidence that, unlike R. meliloti,

the location of nodulation genes may not be closely linked

to nif structural genes in R. japonicum. Hom et al. (1984)

reported general mutagenesis of R. japonicum strain USDA 110

with Tn5 carried on a suicide plasmid. Of ten thousand

kanamycin resistant mutants, auxotrophs were detected at a

frequency of 0.5%. Two hundred mutants were screened on

plants and six Fix- mutants, but no Nod- mutants, were

detected.






-20-


These data suggest that the slow-growing strains have a

much different genome arrangement than the fast-growing

rhizobia. The strains included in R. japonicum have not

been shown to have the class of plasmids greater than 300

megadaltons which is nearly ubiquitous in the fast-growing

rhizobia. In fact, several strains of R. japonicum seem to

have no plasmids at all, and in at least one strain, the

genes which are commonly plasmid borne in the fast-growing

rhizobia have been mapped to the chromosome. The results of

the genetic studies of the fast-growing rhizobia, although

providing insight and guidance in the development of

suitable experimental approaches for study of R. japonicum

genetics, should not be indiscriminantly generalized to the

slow-growing strains without critical evaluation of their

broader applicability.



Infection and Nodulation of Legumes

The colonization of roots by rhizobia (Roc) is

generally believed to be a nonspecific phenomenon. At one

time it was assumed that a legume specifically enhanced the

growth of nodulating rhizobia, and that such enhancement was

one of the underlying causes of the observed specificity of

nodulation. The results of experiments measuring root

colonization have not.yielded clear-cut evidence for such a

specific plant effects on colonization. These studies have

been reviewed extensively by Fahraeus and Ljunggren (1968)

and Vest et al. (1973).






-21-


Debate continues as to the role of adsorption of

bacteria to roots (Roa) in the specific selection of strains

by the plant. The hypothesis that the control of bacterial

host range is mediated by the ability of the bacteria to

bind to plant surfaces is mostly supported by indirect

evidence. Support for this theory is principally in the form

of the correlation between the ability of a plant component

to bind in vitro to strains of rhizobia and the ability of

those strains to nodulate the plant. Such experiments have

given rise to the "lectin hypothesis" (reviewed by Dazzo and

Hubbell 1982). The hypothesis suggests that plant lectins

act as highly selective molecules on or near the surface of

roots, where they "recognize" potential microsymbionts by

attaching them to reactive sites on the root surface,

initiating infection. The ability of the lectin to

discriminate between even very closely related rhizobia is

thus believed to be the basis of the observed host range of

the rhizobia. Correlations of ability of the microsymbiont

to bind the host lectin are far from perfect. Even

correlations of 5 strains which bind lectin out of 7 strains

which nodulate have been cited as validating the lectin

hypothesis (Law and Strijdom 1984)! Rarely is an

explanation provided for the selective host range of those

strains which do not bind lectin in a host-specific manner

or those which bind the lectin in a hapten-reversible manner

yet do not infect the plant that produces the lectin.

Data are less conclusive in relating bacterial host

range to ability to adsorb to roots in experiments where






-22-


actual numbers of bound bacteria are determined.

Experiments designed to determine the amount of bacterial

binding are of three general types: i. measurement of

radioactivity of roots to which radiolabled bacteria have

been bound, ii. microscopic estimation of the number of

bacteria attached to roots, and iii. determination of the

number of bound bacteria by grinding root segments, plating

the grindate, and extrapolating the number of resulting

colonies to the number of bacteria bound to the root

segments. The relative advantages and drawbacks of these

experimental approaches were reviewed by Pueppke(1984a). A

comparison is made in the introduction to Chapter Four of

this dissertation of several of those studies as they relate

to bacterial host range determination.

The "Hac" phenotype refers to curling of root hairs by

infective strains of rhizobia. The curled root hairs are

often the ones that contain infection threads in soybean

(Ranga Rao and Keister 1978, Turgeon and Bauer 1982, Pueppke

1983). Callaham and Torrey (1981) demonstrated in white

clover that most infections arise at the inner curve of

strongly curled root hairs, but others occur in nearly

straight root hairs. Some have postulated the existence of

separate "curling factors," although no such factor is well

characterized, and experiments using crude preparations have

been ambiguous (Ervin and Hubbell 1985). Hubbell (1981) has

proposed a model of root hair curling that explains the

curling in terms of asymetric disruption of the elongating









root hair cell wall by bacterial enzymatic degradation,

suggesting that curling is an immediate consequence of the

incipient infection. This is corroborated in studies of

infection of alfalfa by A. tumefaciens containing cloned

genes from a region of the R. meliloti "Sym" plasmid shown

to have functions essential for nodulation (Hirsch et al.

1984). The strains which had a root hair curling phenotype

like the wild-type were those which formed infection

threads. The strains which produced various deformations of

root hairs, readily discriminated from the wild-type

phenotype, did not form infection threads. Sutton et al.

(1984) cloned genes from R. japonicum which cause distortion

of root hairs of Glycine soja. From the data they present

it is impossible to evaluate whether the root hair "curling"

is similar to that seen in root hairs with infections or is

some other type of distortion, although their

photomicrographs suggest the later.

The process of infection thread formation (Inf) was

examined in some detail in the small-seeded legumes using

techniques of light microscopy which allow direct

examination of living plants under nodulating conditions

(Fahraeus and Ljunggren 1968, Ljunggren 1969, Li and Hubbell

1969, Callaham and Torrey 1981). In those plants infection

thread formation is highly specific; strains which are

capable of nodulating a plant are found to form infection

threads; conversely, those strains which can form infection

threads, with rare exceptions, produce nodules.









The formation of infection threads in soybean was first

described by Bieberdorf in 1938. He noted that infection

leading to nodulation generally was by means of infection

threads in root hairs, though he stated that infection

directly through root epidermis occurred on occasion.

Direct penetration of soybean root epidermis has not been

corroborated, though infection by direct penetration or

through natural wounds leads to nodulation of some tropical

legumes (Allen and Allen 1940, Ranga Rao 1977, Chandler

1978, Chandler et al. 1982). Infection threads have been

described in soybean by Ranga Rao and Keister (1978),

Newcomb et al. (1979), Turgeon and Bauer (1982), Pueppke

(1983), and Heron and Pueppke (1984). Turgeon and Bauer (In

press) described root hair infection of soybean at the

ultrastructural level. Pueppke (1983) demonstrated that

when eight lines of soybean, four lines of wild soybean, and

one cowpea cultivar were inoculated separately with 18

Rhizobium strains, infection threads were formed in all

combinations which also formed nodules but were not formed

in any nonnodulating combination. The infection threads

almost exclusively were formed in root hairs which were

distal to the region with mature root hairs at the time of

infection.

The formation of nodules, the Nod phenotype, though not

an early infection event, is often used as a quick and

easily observed assay for mutations affecting infection.

Although this is understandable, particularly in large

screening experiments, too often the presence of nodules is











assumed, prima facie, to be evidence that all of a set of

specific infection events haveoccurred. Hirsch et al.

(1982, 1984) have demonstrated that nodules or

"pseudonodules" are produced on alfalfa by mutant strains of

R. meliloti which have altered infection phenotypes,

including Hac- and Inf-. In many studies the reason for the

relatively small number of Nod- mutants compared to Fix"

mutants may be that some mutants had altered infection

phenotypes but still produced nodules. Most or all of the

steps described above, including additional phenotypically

defined steps such as release of the bacteria from the

infection thread, are essential in legume-microsymbiont

combinations with only the root hair infection mechanism.

Symbiotically deficient nodules that are superficially

normal may form even though all of the steps do not occur.

It is not yet clear which of the infection steps are

necessary for the production of a macroscopic nodule.

An additional problem with using nodulation as a screen

for infection events is the assumption that infection

proceeds only by means of infection threads in root hairs.

For several tropical legumes nodulated by slow-growing

rhizobia this is not the case (Allen and Allen 1940, Ranga

Rao 1977, Chandler 1978, Chandler et al. 1982). In an

uncorroborated report Bieberdorf (1938) suggested that

soybean could likewise be infected by direct penetration of

the root epidermis in addition to infection threads in root

hairs. If two pathways were to exist for infection and they






-26-


depended on separate sets of genes in the bacterium,

screening for mutation in infection by nodule production

would cause many of the mutations to be missed due to

nodulation by the remaining pathway even when one pathway

was blocked.



Phenotypic Nodulation Response of Soybean

Variation in nodulation phenotypes of soybean cultivars

was noted early in the century by Vorhees (1915), who

reported that in plots which had been inoculated with either

of two commercial soybean inocula, the soybean variety

Haberlandt had no nodules while five other varieties were

well nodulated. Haberlandt had no nodules, even in plots

where it was interplanted with a cultivar which was well

nodulated. Vorhees concluded that different varieties of

soybean carried different levels of resistance to

association with symbiotic bacteria. In an addendum to

Vorhees' report, Morse (1915) notes that in subsequent years

he observed efficient nodulation of the variety Mammoth in

plots that did not support nodulation of the varieties Acme

and Tokio. Morse commented that in tests other than that

reported by Vorhees, Haberlandt nodulated as well as other

varieties, suggesting that Vorhees' data were indeed best

explained on the basis of strain-specific resistance rather

than basic incompatibility of the cultivar to nodulation.

In addition, Briscoe and Andrews (1938) observed that

differences between the nodulation responses of varieties of

soybeans were as great as the differences between cowpeas






-27-


and soybeans when reciprocal tests of their rhizobia were

made.

In a study covering three consecutive years (Caldwell

and Vest 1968, Vest et al. 1973), bacteria from nodules on

17 soybean genotypes were serotyped. The cultivar Lee was

used as a check variety. The population of strains

nodulating the cultivar Pickett was not significantly

different from the backcross parent Lee, but many of the

soybean lines less closely related were significantly

different when the serogroups of strains which formed

nodules were compared. This was true even for varieties

grown side-by-side. The cultivar Peking was included in

this study. Even though it was planted in a field known to

have a natural population of mixed strains of R. japonicum

of which strain 110 was a major component, fewer than 1% of

the nodules on Peking contained strain 110. And yet, when

Peking was inoculated with strain 110 in pots of sterile

soil, the plants were nodulated very efficiently. Of course,

competition between the rhizobia may account for some of

these differences.


Nodulation Restrictive Soybean Genotypes

In 1954, Williams and Lynch reported the inheritance of

a nonnodulating character in soybean. The trait was found

in 1947 among breeding selections from a cross between

Lincoln and a line selected from a Lincoln X Richland cross.

Resistance to nodulation segregated as a single recessive

character, with the homozygous recessive plant expressing






-23-


the resistance to nodulation. Selfs of both parents and

test crosses between the parents all gave the normal

nodulating phenotype, indicating that the characteristic

apparently had arisen as a mutant. Williams and Lynch

(1954) called the gene no and its dominant allele No.

Caldwell (1966) renamed these genes rjl and Ril to conform

with general soybean genetic nomenclature.

Genotypes conditioning resistance to Rhizobium

infection have been characterized in several legumes, other

than soybean. The best characterized phenotypic expression

of such a genotype is in red clover (Nutman 1949).

Resistance is expressed as lack of any infection threads,

although some root hair deformation occurs upon inoculation

with R. trifolii. The resistant genotype is described as

homozygous recessive, rr. A cytoplasmic factor, p,

interacts with rr and is inherited in a complex manner. A

genotype resulting in a Fix- phenotype is designated ii.

The gene i segregates independently of r (Nutman 1949).

Two independently segregating genes affect symbiosis in

field peas. The genotype sym2sym2 conditions resistance to

nodulation, and sym3sym3 prevents nitrogen fixation (Holl

1975). A Fix- phenotype is produced in crimson clover (T.

incarnatum), irrespective of strain, by the single recessive

gene pair rtlrtl, with possible modifiers (Smith and Knight

1983). The genetic constitution of phenotypically Nod-

peanut (Arachis hypojaea) requires the independently

segregating double recessive gene complement, n1 il2n2










(Nigam et al. 1980). None of these nodulation resistant

genotypes has been characterized at the cellular or

biochemical level. It is interesting to note that even

peanut, which is infected through natural wounds (Allen and

Allen 1940, Chandler 1978), often considered a passive mode

of infection, expresses resistance in the recessive

genotype.

Several soybean genes condition strain-specific Fix-

phenotypes. In each case the Fix- phenotype is conditioned

by the dominant allele. The Rj2 allele conditions against

strains of the 122 and cl serogroups (Caldwell 1966), the

Ri3 allele conditions against strain USDA 33 (Vest 1970),
and the Ri4 allele conditions against strain USDA 61 (Ham et

al. 1971). One or both of the alleles R_2 or Rj4 are

present in 30% of the plant introduction lines, but have

generally been selected against in breeding programs and are

present in only a few named cultivars (Devine and Breithaupt

1980c, 1981, Devine 1984a). When plants carrying the

dominant allele are inoculated with the restricted strains,

they produce small nodules or nodule-like proliferations on

the roots. Pueppke (1983) showed that the cultivar Hardee,

which carries the genotype RJi2R3, developed infection

threads which appeared to be normal with strain USDA 138 (cl

serogroup), so the block in nodule function appears to occur

late in development. Although Ri2j, 13, and Rj4 are often

described with rjl as a group of nodulationn restrictive

genes," clear differences are apparent. Whereas the rjlrj1

genotype conditions against most strains of R. japonicum











with no nodules or nodule-like structures formed by

restricted strains; the other nodulationn restrictive genes"

are restrictive only to a few strains or specific serotypes,

and nodules or nodule-like proliferations are formed. Rj2,

Rj3, and RJ4 restrict symbiotic effectiveness not nodulation

so the term nodulationn restrictive genes" is a misnomer for

them.

The ri41il-soybean was originally described as

nonnodulating (Williams and Lynch 1954). Clark (1957) found

that a few nodules were formed by a few strains when plants

were grown hydroponically using sand as the support medium,

but the plants were incapable of being nodulated in soil.

The typical nodulation response in sand culture was about

one nodule per plant. Nodulation response was reduced for

plants grown in sand amended with soil, and no nodules were

formed when the sand was amended with 10% bentonite clay.

Isolines of soybean differing at the Rj1 locus were

found in one study of root colonization to harbor

approximately equal numbers of rhizobia (Clark 1957). Elkan

(1962) later reported larger numbers of rhizobia in

rhizospheres of rjlrjl-soybean than in its isoline for

approximately the first 40 d of plant growth in the field.

Clark (1957) reported no differences in the kinds or amounts

of amino acids in the two isolines, but Hubbell and Elkan

(1967b) noted that roots of uninoculated Ril-plants

contained larger amounts of protein and reducing sugars and

smaller amounts of free amino acids than did uninoculated






-31-


rJlrJl-plants. The biological significance of this latter
finding is not apparent in light of Elkan's (1962) earlier

data on root colonization, which certainly suggests no basic

growth inhibition of rhizobia. Elkan (1961) suggested that

the rijilr-soybean produced a nodulation-inhibiting

excretion capable of a highly significant reduction of

nodulation on the Rjl-genotype. The amount of nitrate added

to culture medium for container-grown plants, however, was

sufficient to have a potential effect on nodule number.

Eskew and Schrader (1977) reexamined the putative

nodulation-inhibiting excretion from rjl-soybean using

modifications of Elkan's (1961) experimental design. They

found no statistically significant reduction in nodule

number due to co-cultivation of nodulating plants with rjl-

isolines, but a strong inhibitory effect of nitrate was

noted.

Hubbell and Elkan (1967a) compared thephysiological

characteristics of strains of R. japonicum with differential

abilities to nodulate isogenic lines of soybean differing at

the Rjl locus. High measurable indoleacetic acid formation,

low indoleacetic acid destruction, formation of large

amounts of capsular material, failure to metabolize nitrate,

and failure to reduce tr.iphenyl tetrazolium chloride were

properties associated with ability to nodulate both normal

and mutant soybean. Stains with the opposite properties

generally were able to nodulate only the normal soybean. A

mode of infection could not be educed from correlations










between physiological characteristics and nodulation

phenotype.

Devine and Weber (1977) observed that many R. japonicum

strains capable of overcoming ril-conditioned resistance to

nodulation produced a previously reported soybean foliar

chlorosis (Erdman et al. 1956, Johnson and Means 1960). The

chlorosis symptoms were ascribed to the formation of

rhizobitoxine, 2-amino-3-hydroxypropoxyvinylglycine by the

bacteria (Owens and Wright 1965, Owens 1969, Giovanelli et

al. 1971, Owens et al. 1972). Devine and Weber (1977)

suggested that the production of rhizobitoxine might enable

the infection of rilril-soybean by overcoming strains. This

was examined indirectly by Devine and Breithaupt (1980b),

who tested the effect of three temperature regimes on

nodulation and chlorosis. The effects were opposite, in

that the chlorosis symptoms were greatest at the highest

temperature (32 C), but the most nodules were formed at the

low and intermediate temperatures (21 C and 27 C). No

evidence was found for a diffusible compound capable of

endowing the rjl-incompatible strains with ability to

nodulate rjillj-soybean (Devine et al. 1981). The ethoxy

analog of rhizobitoxine, when added to bacteria and used to

inoculate soybeans in Leonard jars, did not modify the

nodulating ability of strains of R. japonicum on rjlrjl-

soybean (Devine and Breithaupt 1980a). Devine (1984a)

concludes from these data that rhizobitoxine probably has no

enabling role in infection. Devine suggests that the

rhizobitoxine is only correlated with the rJilr1-overcoming











strains due to fixation of the separate genetic factors in

the same population by "random drift."

Devine et al. (1980) determined that the _ll1-

resistance was not a basic incompatibility with the

nonnodulating stains. When the strains capable of

nodulation and those which were not were mixed and used as

inoculum, 32% of the resulting nodules on rllr_1i-soybean

contained both strains, 36% contained only the usually

nonnodulating strain, and 32% contained only the usually

nodulating stain.

The mode of infection of rilj l-soybean has not been

determined. Nutman (1981) notes that the rr phenotype of

red clover conditions inability of the plant to form

infection threads; and, since the soybean resistance to

nodulation is likewise a recessive trait, it seems likely to

condition a similar block early in infection. Devine

(1984a) notes that no nodules or nodule-like proliferations

are formed on roots inoculated with incompatible strains,

and likewise suggests that the block is early in infection.

Tanner and Anderson (1963) examined soybean roots for

infection in root hairs but unfortunately were unable to

find infection threads in either the 1ilrjl-soybean or the

normally nodulating line.



Perspective

The challenge remains to find the point at which the

ril-plant blocks infection and to elucidate the pathway of






-34-


infection for those strains which can overcome the rilrjl-

resistance to nodulation. These problems are the focus for

the studies reported in this dissertation. The function of

temperature on nodulation number and pattern was studied to

find the conditions under which infections were most likely

to be observed in the rjillj-soybean. The hypothesis that

bacterial adsorption has a role in determining differential

nodulation ablilty of strains between normally nodulating

and restrictive lines of soybean was tested. The plasmid

content of strains was determined and attempts were made to

alter the genetic complement of strains, in search of clues

to the genetic basis for infection. Finally, roots were

examined using light and scanning electron microscopy to

determine the phenotype of infection at the cellular level.


















CHAPTER THREE
EFFECT OF TEMPERATURE ON NODULATION OF SOYBEAN ISOLINES
DIFFERING AT THE Ril LOCUS


Introduction

The nodulation restrictive genotype of soybean, rjilrl,

identified as a spontaneous mutant in a soybean breeding

program, was reported by Williams and Lynch in 1954.

Initial work determined that one genetic locus is involved

in conditioning the restrictive phenotype and that the

homozygous recessive genotype is required for expression of

the trait. The alleles originally were named no and NO for

nonnodulating, but since have been redesignated Kil and Rj1

to conform to currently accepted terminology for soybean

genetics (Caldwell 1966).

It once was believed that the nodulation restrictive

plants are unable to be nodulated (Williams and Lynch 1954,

Caldwell 1966). Now it is clear that although most strains

of Rhizobium japonicum are unable to nodulate these plants,

several strains produce a small number of nodules on plants

grown in hydroponic culture (Clark 1957). These strains are

called the "overcoming" strains because they overcome the

plant resistance. Devine and Breithaupt (1980b) reported a

temperature effect on nodulation of the soybean cultivar

Clark and its nodulation-restrictive isoline Clark-ril by


-35-









two overcoming strains. The trends in nodulation response

of both isolines to temperature are similar. The two

bacterial strains have different temperature optima for

nodulation with the greatest number of nodules per plant

formed at 27 C and 21 C, respectively, for the two strains.

Fewer nodules were formed at 32 C for both strains (Devine

and Breithaupt 1980b).

Bhuvaneswari and colleagues (Bhuvaneswari 1981,

Bhuvaneswari et al. 1980, 1981) developed a model for

nodulation of soybean. The model predicts that most nodules

will be clustered near the point that represents the

position of the root tip at the time of inoculation. The

model is supported by a correlation between nodule

distribution and the position of areas that had immature

root hairs or had not yet developed root hairs at the time

of inoculation. This developmental model of nodulation is

extended by the observations of Pueppke (1983) and Calvert

et al. (1984), who demonstrated that the formation of

infection threads is the developmentally restricted event in

soybean and two other legumes. In accordance with this

model, no nodules are expected to form above the zone of

developing root hairs on the primary root (Bhuvaneswari et

al. 1980).

The objectives of my study were to i. find the

temperature optima for overcoming strains, ii. test the

appropriateness to rjflrl-soybean of the Bhuvaneswari model

of transient susceptibility of root cells to infection

leading to nodulation, and iii. extend the study (Devine and







-37-


Breithaupt 1980b) of the effect of temperature on nodulation

of Clark and Clark-rji isolines to additional overcoming

strains.



Materials and Methods

The bacteria all were obtained from the U. S.

Department of Agriculture, Nitrogen Fixation and Soybean

Genetics Laboratory, Beltsville, MD, courtesy of H. H.

Keyser, D. F. Weber, and R. Griffin. All bacteria are

USDA strains of R. aponicum. The overcoming strains used

were 61, 84, 94, and 119. The nonovercoming strain 110 was

used as a control in all experiments. The bacteria were

maintained at 4 C on yeast extract-mannitol agar slants

(Vincent 1970).

Seeds of Glycine max (L.) Merr. cultivar Clark-L1

(RjilRj) and the nodulation restrictive isoline of Clark-Ll,

L63-1889 (rjlil), were obtained from R. L. Bernard, USDA

Regional Soybean Laboratory, University of Illinois, Urbana,

and D. A. Phillips, Agronomy and Range Science Department,

University of California, Davis. The isolines are

designated Clark and Clark-ril according to the nomenclature

of Devine and Breithaupt (1980b). Seeds were surface

disinfested by soaking in 50% ethanol for 2 min with

agitation, rinsing in deionized water, and then shaking in

0.5% aqueous sodium hypochlorite for 2 min. Seeds were

washed for 20 min in running deionized water and were

germinated in the dark on water agar plates for 4 to 5 d at









22 C, 27 C, or 32 C, depending on the temperature to be used

for nodulation experiments.

Inoculum was produced from 50 ml log-phase cultures

grown in liquid gluconate-mannitol medium (Bhuvaneswari et

al. 1977) at 28 C with rotory shaking at 120 rpm. Bacterial

suspensions were centrifuged at 7500 x g for 10 min and the

bacteria were resuspended in sterile nitrogen-free Jensen's

plant-growth solution (Vincent 1970). Cell concentration

was adjusted turbidimetrically to 5 x 108 cells/ml.

Seedlings with roots approximately 4 cm long were inoculated

by dipping the roots for 10 min in the bacterial suspension.

Inoculated seedlings were placed, 2 per pouch, in

autbclaved plastic growth pouches (Northrup King Seed Co,

Minneapolis, MN) containing 15 ml of Jensen's solution. The

surface of the growth pouch was marked at the location of

the primary root tip of each plant (Bhuvaneswari et al.

1980b). This mark was designated the root tip mark (RTM).

Plants were grown for 30 d at continuous temperatures

of 22, 27, or 32 C with a 12 hr light/dark cycle in a

Conviron E-15 growth chamber with 900 uE/m2/sec (400-700 nm)

irradiance at canopy height. Each temperature experiment

was repeated 3 times with 6 plants of Clark and 10 plants of

Clark-ril tested for each R. japonicum strain in each

experiment. Appropriate control plants sham-inoculated with

sterile Jensen's solution were included in each experiment.

Plants were watered as needed with deionized water.

The distance (to the nearest mm) from the RTM to the

root crown and from the RTM to each nodule on the primary






-39-


root was measured on each plant at harvest. The number of

nodules on secondary roots was counted. Data were analyzed

by analysis of variance using the general linear models

(GLM) procedure of the Statistical Analysis System (SAS

Institute Inc., Cary, NC). Computing was done utilizing the

facilities of the Northeast Regional Data Center of the

State University System of Florida.



Results

The mean number of nodules per plant on Clark ranged

from 2 to 8 at 32 C, up to 15 to 19 at 22 C (Figure 3.1,

Table 3.1). On Clark-ril inoculated with overcoming

strains, the mean number of nodules ranged from 0 to about

0.2 nodules per plant at 32 C, to from 0.7 to 2.2 nodules

per plant at 22 C (Table 3.2, Figure 3.1). Nodules were not

formed on Clark-rjl inoculated with the nonovercoming strain

110 or on plants sham-inoculated with plant-growth medium.

Analysis of variance was conducted using the number of

nodules per plant as the dependent variable. In a

preliminary test, the responses of Clark and Clark-ril were

demonstrated to be significantly different. All subsequent

analysis thus was conducted separately for the two genotypes

to avoid heterogeneity of variance. Temperature

significantly (p < 0.01) influenced nodule number for each

plant genotype. F-values for strain, replication, and the

interactions of each of the independent variables were
























Figure 3.1. The mean number of nodules formed per
plant at three temperatures with five strains of
Rhizobium japonicuTr Plants were dip inoculated in
suspensions (5 x 10 cells/ml) of one of the strains
indicated, placed in plastic growth pouches, and grown
at constant temperature for 30 d. For each strain at
each temperature the experiment was replicated three
times with six plants per treatment for Clark and ten
plants per treatment for Clark-rjI. Treatments with
Clark are indicated by the solid line, Clark-rjI with
the broken line. 0= strain 61, U = strain 84, 6 =
strain 94, 0= strain 110, and A = strain 119.








20-

19-

18-

17-

16-

15- 0

14-

13 -

12 -

'11 -

-10- a


S 8 --






a- a,
5-

14-

3-

2-




22 27 32
TEMPERATURE ICI



















Table 3.1. The effect of temperature on the nodulation of
Clark soybean by Rhizobium japonicum

Temperature

Strain 22 C 27 C 32 C


15 6a


16 1 7


18 5


16 9


19 7


12 4


10 3


15 6


12 5


15- 6


8 3


7 3


2 2


7 3


6 3


a Mean number of nodules per plant for 3 replications with
6 plants per replication standard deviation.






















Table 3.2. Effect of temperature on the nodulation of Clark-
rJl Rhizobium japonicum
---------------------------------------------------
Temperature
Straina 22 C 27 C 32 C
Straina 22 C 27 C 32 C
---------------------------------------------------


2.2 2.0b


0.8 1.4


0.9 1.4


1.0 1.2


0.9 1.7


0.1 0.4


0.1 0.3


0.2 0.7


0.7 1.1


--------------------------------------------------

a Strain 110 is a nonovercoming control. All others are
overcoming strains.

b Mean number per plant for 3 replications with 10 plants
per replication standard deviation.






-44-


not significant. A dramatic indication of the effect of

temperature on nodulation of Clark-irj is provided when the

data are expressed as the percentage of plants developing at

least one nodule per plant after inoculation with an

overcoming strain (Table 3.3). At each temperature a total

of 120 plants was inoculated with one of the four overcoming

strains; of those, 4% of the plants were nodulated at 32 C,

23% at 27 C and 44% at 22 C.

The ratio of primary to secondary nodules ranged from

1:0.6 to 1:0.9 on Clark. On Clark-rjI the ratios were 1:9

at both 32 C and 27 C, and 1:4 at 22 C.

Histograms were developed for each interaction of

bacterial strain x isoline for each temperature as described

for various other legume x microsymbiont combinations

(Bhuvaneswari 1981, Bhuvaneswari et al. 1980, 1981,

Halverson and Stacey 1984, Heron and Pueppke 1984). These

are shown in Figures 3.2 and 3.3. In most interactions with

overcoming strains, nodules formed well above what has been

considered the zone of infectibility, defined as that area

which has only emerging root hairs or no root hairs at the

time of inoculation. This type of anomolous nodulation is

seen at 22 C and 27 C for combinations of Clark with the

strains 61, 84, and 94, each an overcoming strain. A

pattern of nodulation similar to those described as fitting

the nodulation model of Bhuvaneswari (1981) is seen in the

combination of Clark with strain 110, a nonovercoming

strain, at 22 C and 27 C. From the nodule profiles






-45-


(Figure 3.2), it can be seen that the pattern of nodulation

at 32 C of Clark by all of the tested bacterial strains

produced flattened peak and a population of nodules

displaced downward with respect to the profiles observed at

22 C and 27 C. This downward displacement is clearly

evident when the data are expressed as the mean distance of

all primary root nodules from the RTM (Table 3.4). The

nodule profiles of the overcoming strains on Clark-ril

showed sparse nodulation down the length of the root from

just above the RTM.



Discussion

The soybean cultivar Clark and its isoline, Clark-ril,

were used by Devine and Breithaupt (1980b) to study the

effect of temperature on nodulation. They tested the two

overcoming strains USDA 61 (used in this study) and 76.

Strain 76 produced the most nodules on Clark-ril at 27 C

with few nodules formed at 21 C or 32 C. The combination of

Clark-rjl with strain 61 developed the most nodules at 21 C

(7.5), with 6.4 and 3.1 nodules at 27 C and 32 C,

respectively. In my study the slope of the regression of a

plot--number of nodules versus temperature--was similar to

that observed by Devine and Breithaupt (1980b), but the

absolute numbers of nodules per plant were lower (Figure

3.1). All of the overcoming strains that I tested responded

similarly to strain 61 in the previous study, except strain

84 which had slightly fewer nodules at 22 C than at 27 C.






-46-


Table 3.3. The percentage of Clark-rj1 soybean plants
nodulated by overcoming strains of Rhizobium japonicum
at three temperatures

Temperature

22 C 27 C 32 C

Strain pri sec anya pri sec any pri sec any


61 23 73 77


84 3 30 30


94 13 27 40


10 50 53


7 30 30


0 10 10


3 3 7


0 10 10


0 0 0


3 27 30


0 0 0 0 0 0


Totalb 11 39 44


4 23 23


1 3 4


a Percentage of 30 plants nodulated (3 replications x 10
plants) at the locations indicated. Pri = percentage of
plants with at least one nodule on the primary root.
Sec = percentage of plants with at least one nodule on
the secondary roots. Any = percentage of plants with
at least one nodule.

b Percentage of all plants in each temperature experiment
with at least one nodule (120 plants per temperature).



















Figure 3.2. Frequency histogram of nodule distribution on the primary root of Clark
soybean. Plants were grown 30 d in plastic growth pouches at 22, 27, or 32 C after
dip inoculation with one of the five strains of Rhizobium japonicum indicated. Each
nodule on the primary root was measured to the nearest millimeter from the RTM (root
tip mark, placed on the growth pouch at the time of inoculation). The frequency
diagrams represent the plants from three replications at each temperature with six
plants per treatment.









STRAIN 61
22 C 27 C 32 C


STRAIN 84
22 C 27 C 32 C


40

20

RTM

20

40

60

80

100

120

140


STRAIN 94


STRAIN 94
22 C 27 C 32 C





-


r[











STRAIN I 10

22 C 27 C 32 C


-= ONE
NODULE

Figure 3.2 Continued


STRAIN 119

22 C 27 C 32 C


40

20

RTM

20

40

60

80

100

120

140




















Figure 3.3. Frequency histogram of nodule distribution on the primary root of
Clark-rjl soybean. Plants were grown 30 d in plastic growth pouches at 22, 27, or
32 C after dip inoculation with one of the four strains of Rhizobium j~iponicum
indicated, or the control strain 110. Each nodule on the primary root was measured
to the nearest millimeter from the RTM (root tip mark, placed on the growth pouch at
the time of inoculation). The frequency diagrams represent the plants from three
replications at each temperature with ten plants per treatment. No nodules were
observed on plants inoculated with strain 110 (not shown) at any temperature.








-51-






3 I 3



-I
I0
-I






IC,















cm
10.
IC











CI

















nc
u


















I
.c-
IN


















C.,
-I o
CM






0 0--0--0 0-0-0-0- -









Ca I- C,' o = UC h DQ C






-52-


Table 3.4. Mean distance of primary root nodules on Clark
soybean from the root tip mark at time of inoculation

Temperature

Strain 22 C 27 C 32 C


16 2a


16 2


11 2


16 1


61 5


56 4


13 2


16 2


7 2


14 1


9 1


53 7


43 -+ 5


a Mean nodule distance in millimeters for 18 plants (3
replications x 6 plants) standard error of the mean.
Measurements for nodules above the RTM were given a
negative value.

bOnly one nodule produced on the primary root of a plant
in 3 replications of this treatment.






-53-


At 22 C only the combination of the nonovercoming strain 110

and Clark-ril failed to produce nodules on at least some of

the plants. At 27 C, Clark-rj1 failed to form nodules with

strain 119 as well as with strain 110, and at 32 C no

nodules were formed by strains 94 and 119, as well as 110.

The effect of temperature on nodulation of both Clark and

Clark-rj1 had the same trend (Figure 3.1), although the

number of nodules per plant was much different for the two

plant types. Nodulation of nonovercoming strain 110 on

Clark was affected by temperature in a manner similar to the

overcoming strains, but strain 110 did not nodulate the

Clark-ril isoline at any temperature.

Reports have been made on the effect of temperature on

numbers of soybean nodules per plant both in the greenhouse

and field, as well as under controlled conditions (Devine

and Breithaupt 1980b, Munevar and Wollum 1982, Weber and

Miller 1972), but this study is the first to examine the

effect of temperature on the pattern of nodulation. The

pattern of nodulation of strain 110 on Clark soybean at 27 C

and 22 C was similar to the pattern reported by others for

compatible interactions and follows that predicted by the

model of transient susceptibility to nodulation (Bhuvaneswari

1981, Bhuvaneswari et al. 1980, 1981). The general shape of

the profiles for the overcoming strains on Clark is similar

to strain 110 on Clark, suggesting that at least most of the

nodules that arise are the result of infections constrained

by developmental processes to areas of susceptibility, as






-54-


defined by the region of the root on which the root hairs

are immature or are not yet formed at the time of

inoculation. Overcoming strains 61, 84, and 94 produced some

nodules well above that region known to be infectible by the

model of infection. The presence of these nodules can be

explained in two ways. First, immature root hairs may exist

in this region and remain infectible after the surrounding

root hairs have matured. Alternatively, these overcoming

strains may have an additional infection sequence that is

not restricted to areas with developing root hairs. If the

first is true, one would expect that strain 110 and other

nonovercoming strains also would produce nodules in this

area, at least occasionally. Since nodules are produced in

this area only by overcoming strains, an alternative

infection mechanism is suggested.

The pattern of nodulation at 32 C of Clark soybean with

the five strains of R. japonicum gave a more flattened curve

which was displaced downward relative to those at the lower

temperatures. Nodulation generally was lower on the primary

root, and some nodules were very far below the RTM. The

pattern of nodulation obtained at this temperature looks

much like that reported (Halverson and Stacey 1984, Heron

and Pueppke 1984) for interactions with fewer nodules

relative to other interacti-ons tested in those studies.

Heron and Pueppke (1984) reported a similar pattern on the

soybean cultivar Vicoja inoculated with the fast growing R.

japonicum strain 191. Halverson and Stacey (1984) reported

that the delayed nodulating mutant strain HS111 produced a







-55-


similar scattered and downwardly displaced pattern on Essex

soybean as compared to the pattern obtained with strain 110.

An explanation for the striking similarity in the

nodulation pattern for all of these interactions is that the

they are merely diagnostic for any interaction in which the

initial number of successful infections is reduced and plant

regulation of additional nodulation is not triggered (Pierce

and Bauer 1983). That is, the similarities of the patterns

may be coincidental. But the surprising similarity of those

nodulation patterns to the ones in this study resulting from

restrictive temperature, brings up the question as to what

effect temperature might have on those interactions. The

soybean cultivar Vicoja was developed at the Universidade

Federal de Vicosa, Vicosa, Minas Gerais, Brazil,

specifically for local Brazilian conditions, including high

temperature. Thus the observed inefficiency of nodulation

is perhaps due to assay temperatures below those to which

Vicoja is adapted. Similarly, the possibility that HS111 is

a temperature sensitive mutant that has been tested only at

restrictive temperatures cannot be ruled out.

The nodulation of Clark-r.E is so sparse that

interpretation of the nodule profile is difficult. There is

certainly no clear peak in the histogram near the point

corresponding to the RTM. Whether such a peak would become

evident if much greater numbers of plants were examined is

uncertain, but seems unlikely; the pattern appears to be

scattered and random. Although these data are insufficient






-56-


to either validate or invalidate the application of the

model of Bhuvaneswari (1981) to Clark-riI, these data show

only limited correspondence with the expected pattern of

nodulation predicted by the Bhuvaneswari model. The

scattered and sparse nodulation and the reduced correlation

of nodulation with the region near the RTM are likely to

make the elucidation of early infection events in the

nodulation restrictive rj1 rj-soybean that much more

challenging.


















CHAPTER FOUR
ADSORPTION OF STRAINS OF RHIZOBIUM JAPONICUM WITH
DIFFERENTIAL NODULATING ABILITY TO ROOTS OF SOYBEAN ISOLINES
THAT DIFFER AT THE Rji LOCUS


Introduction

The ability of rhizobia to bind to roots of their

legume hosts has long been assumed to have a principal role

in the specificity of the infection process and is believed

to be a major determinant of host range (Bhuvaneswari 1981,

Dazzo 1981). These assumptions often are based on the

correlation between binding of some component of a plant to

a microbe in vitro, and the ability of that microbe to

infect the plant (Dazzo and Hubbell 1975, Robertson et al.

1981). These and other indirect tests have been interpreted

as evidence for a direct role of binding in determining host

range of microsymbionts. The tests have included studies of

cross-reacting antigens on plant and bacterial surfaces

(Bishop et al. 1977, Dazzo and Hubbell 1975), the

determination of number of nodules formed after various

substances were added to plant roots with inoculum

(Halverson and Stacey 1984), and tests for lectin binding to

bacteria and the search for those lectins on or in plant

roots (Bohlool and Schmidt 1974, Bhuvaneswari et al. 1977,

Dazzo et al. 1978, Stacey et al. 1980, Gade et al. 1981, Law

et al. 1982, Law and Strijdom 1984). Although these studies


-57-






-58-


have not always addressed the subject of bacterial binding

to plant roots, they have been interpreted in terms of the

role of adsorption. The underlying assumptions are i. that

the examined process is a necessary antecedent to

adsorption, or ii. that a tested factor has a direct

intermediary role in adsortion. Unfortunately, these

assuptions have not been tested.

Measurement of bacterial binding per se has been

reported with mixed results in terms of its perceived role

in host-range determination. Broughton et al. (1980) tested

binding of 35S-radioisotope-labelled strains of R.

leguminosarum to roots of Pisum sativum. Their results are

difficult to evaluate due to the very low specific activity

of labelling attained and the high variability between

experiments. They concluded that ability to adsorb to plant

roots was not a determining factor in the differential

ability of the bacteria tested to nodulate cultivars of pea.

This conclusion was corroborated by microscopic examination

of inoculated pea roots (Broughton et al. 1982). Chen and

Phillips (1976) used 32P-radioisotope-labelling of several

rhizobia to test adsorption to severed root segments in

vitro. Considerable radioactivity was taken up by the root

segment tissues during incubation with the radioisotope-

labelled bacteria. This fact and the artificiality of their

assay conditions confound the conclusions. Their data tend

to discredit the role of adsorption in host range










determination, because binding between their bacterial

strains and plant roots was rather nonspecific.

Light microscopic examination of binding, either with

transmitted light or fluorescence labelling techniques, has

been used as an assay for adherence. From such studies

qualitative rather than quantitative data are generally

reported. Dazzo and Brill (1979) reported specific

adherence of R. trifolii strain 0403 to root hairs of

Trifolium repens. A strain of Azotobacter vinelandii that

had been transformed with R. trifolii DNA and selected for

antigenic cross-reactivity with T. repens also bound. They

reported that an A. vinelandii revertant "did not adhere."

Chen and Phillips (1976) reported that no differences were

apparent between the binding of fluoresescent-labelled R.

leguminosarum to roots of pea, which it normally nodulates,

and the roots of several legumes which it does not nodulate.

Conversely, a direct role in adsorption has been

inferred by Stacey et al. (1980) from their light

microscopic and scanning electron microscopic examination of

the binding of rhizobia to soybean (Glycine max) and wild

soybean (G. soja). Unfortunately, they tested various

haptens of lectins for their effect on binding but not on

nodulation. The assays involved adsorption of rhizobia to

the elongated root hairs, which are not believed to be

normally infected in soybean (Bhuvaneswari 1981, Pueppke

1983, 1984a). Using similar binding assays in another

study, Stacey et al. (1982) reported that, of a number of









mutant R. japonicum strains with genetic lesions affecting

nodulation, only two failed to bind to soybean root hairs.

Two studies have measured bacterial binding to roots of

soybean directly (Law et al. 1982, Pueppke 1984b). Law et

al. (1982) determined the number of cells of mutant isolates

which bound to 1 cm segments of excised soybean root. The

root pieces were incubated for 1 hr in a dilute bacterial

suspension, gently washed, ground, diluted, and plated.

Between 1000 and 2300 bacteria bound per root segment.

Unfortunately, nodulation cannot be compared to binding

using inoculation conditions similar to those used in this

assay since the binding assay uses severed roots. The loss

of plant sap from the cut ends may also affect the number of

bacteria bound. These problems were answered in the

procedure devised by Pueppke (1984b), in which intact

seedlings were suspended with their roots dangling in

bacterial suspensions. After timed incubation the plants

were removed and rinsed vigorously. A root segment was

removed, ground, and plated for determination of colony

forming units. Adsorption to seedling roots of soybean and

cowpea by one fast-growing and four slow-growing rhizobia

was independent of plant species and of the ability of the

strains to nodulate these hosts. This procedure (Pueppke

1984b), although more cumbersome than the previous assay

(Law et al. 1982), allows examination of root adsorption in

a system similar to a commonly used inoculation protocol.

The effect of the binding and rinsing conditions on






-6 -


nodulation can be tested by creating seedlings and then

transferring them to plastic g-owth pouches.

The objective of my research was to compare the binding

of rhizobia to soybean roots, with the known abilities of

the Rhizobium strains to nodulate specific genotypes of

soybean (Devine 1984a). The study used the procedures

developed Pueppke (1984b) to enable direct assay of

bacterial binding to living plants. The assay conditions

were similar to the customary inoculation procedure used for

nodulation studies. Near-isogenic lines of soybean that

have differential ability to form nodules with the R.

japonicum strains were utilized. Two temperatures known to

affect the number of nodules formed on these isolines

(Devine and Breithaupt 1980b, Chapter Three) were tested.



Materials and Methods

The USDA strains 94 and 110 of Rhizobium japonicum were

obtained from the USDA Nitrogen Fixation and Soybean

Genetics Laboratory, Beltsville, MD, courtesy of H. H.

Keyser, D. F. Weber, and R. Griffin. They were maintained

on yeast extract mannitol (YEM) agar (Vincent 1970) slants

at 4 C. Cultures for adsorption studies were grown at 28 C

with shaking in liquid defined gluconate-mannitol medium

(Bhuvaneswari et al. 1977). Bacterial concentration was

estimated turbidimetrically. Bacteria were pelleted by

centrifugation at 7500 x _, washed once in sterile filtered

nitrogen-free Jensen's plant growth solution (Vincent 1970),

repelleted, and resuspended in Jensen's solution at










approximating 1 x 104 bacteria/ml. Aliquots were plated on

YEM agar for determination of colony forming units to

quantify viable bacteria in the inoculum.

Seeds of Glycine max (L.) Merr. cultivar Clark-Ll

(RJlRJl) and its isoline, L63-1889 (.illil), were obtained
from R. L. Bernard, USDA Regional Soybean Laboratory,

University of Illinois, Urbana, and D. A. Phillips,

Department of Agronomy and Range Science, University of

California, Davis. In this study the terminology of Devine

and Breithaupt (1980b) is followed; the term Clark is used

for the parent cultivar Clark-Ll, and Clark-rj1 designates

the isoline L63-1889 carrying the nodulation-restrictive

genes. The nodulation-restrictive genotype conditions

resistance to nodulation by most strains of R. japonicum,

including strain 110. Strain 94 is one of the "overcoming"

strains which overcome rJi-resistance and form a few

nodules. Clark is nodulated abundantly by both strains.

Seeds were surface disinfested by soaking them in 50

percent ethanol for 2 min, rinsing, shaking in 0.5% aqueous

sodium hypochlorite for 2 min, and rinsing in running

deionized water for 20 min. Seeds were germinated on water

agar for 4 d at 28 C in the dark. Seedlings lacking

bacterial or fungal growth were placed, three per pouch, in

autoclaved plastic growth pouches (Northrup King Seed Co,

Minneapolis, MN) containing 15 ml of Jensen's solution.

Pouches were covered with plastic sleeves to maintain

sterility. Seedlings were placed under fluorescent lights










with a 12 hr light/dark cycle at 450 uE/m^2/sec irradiance

for 1 d to allow root elongation.

The adsorption assays were completed as previously

reported (Pueppke 1984b). All assays were done in a laminar

flow hood under aseptic conditions. Bacterial inoculum (25

ml) was placed in each of sixteen 100 x 25 mm sterile test

tubes. Two bent paper clips were hooked on each test tube

rim, each to support a seedling. Seedlings were suspended

with the roots immersed in the bacterial suspension. After

30, 60, 90, and 120 min, four sets of two seedlings were

harvested. The roots of each seedling were washed

vigorously with 25 ml of sterile filtered Jensen's solution

delivered from a Brinkman Dispensette. The solution was

delivered with the maximum possible stream that would still

run down around the root when the seedling was held

intersecting the stream. Each root was severed 2 cm from

the root tip. The distal segments of the two roots from

each tube were ground together in a ground-glass tissue

grinder in 1 ml of sterile Jensen's solution. The resulting

pulp was appropriately diluted and five 0.1 ml aliquots were

plated on YEM agar plates. The plates were incubated in the

dark at 28 C, and the colonies were counted after 7 to 10 d.

In each experiment, for each plant type at each time, there

were four replications represented by four tubes. Each

experiment was repeated at least five times for each strain

x plant combination.

The assays described above were completed at an ambient

air temperature of approximately 27 C. Additional assays











were completed for the strain 94 x Clark-rjl combination at

22 C. All procedures were completed as described, except

that the test tubes with inoculum were placed in a water

bath and equilibrated to 22 C for 30 min before seedlings

were added. Temperature was monitored carefully during the

assay to maintain 22 0.5 C.

Data were collected as the mean number of colonies

formed on five replicate plates spread with suspensions

resulting from grinding each set of root segments. The data

were normalized to correct for experiment-to-experiment

variation in actual inoculum density. This was accomplished

by dividing treatment means by a ratio representing the

turbidimetrically estimated inoculum (1 x 104) divided by

the actual colony forming units in the inoculum. Normalized

data are expressed as the number of bacteria adsorbed per

plant.

Two kinds of control experiments were conducted. Known

amounts of bacteria were ground with root tissue to

determine the effect of grinding and of plant tissue

constituents on numbers of bacteria producing colonies on

YEM. The other control tested the effect of adsorption

assay conditions on nodulation. Seedlings that had been

incubated with bacteria were washed as described above and

then directly placed into prepared growth pouches and

maintained in a Conviron E-15 growth chamber at 22 C for 20

d with 900 uE/m2/sec irradiance (400-700 nm) with a cycle 12

hr light and 12 hr dark.












Results

In the adsorption assay, both bacterial strains bound

to Clark soybean roots in roughly similar numbers with a

near linear increase with time. Approximately 100 bacteria

were bound per plant at 2 hr (Figure 4.1). The capacity of

Clark-rjl to adsorb overcoming strain 94 was very similar to

that of Clark. Somewhat surprising was the binding of

greater numbers of bacteria in the nonnodulating combination

of Clark-rjl x strain 110. In this combination, the mean

number of bacteria bound per plant was nearly 100 at 30 min,

about twice the number bound in the Clark x 110 combination.

A reduction in assay temperature from 27 to 22 C

markedly decreased the binding of strain 94 to the isoline

Clark-ril (Figure 4.2). After 1 hr the number bound at 27 C

was 59 4 ( SE), but at 22 C was 15 1, about a 75%

reduction. After 2 hr, the number bound at 27 C was 105 8

but for 22 C was 21 2, an 80% reduction. The effect of

these temperatures on adsorption was opposite to their

effect on nodulation. At 22 C this plant-strain combination

had a greater number of nodules, a greater percentage of the

plants with nodules, and a greater number of nodules on the

primary root than at 27 C (Tables 4.1 and 4.2 [data from

Chapter Three]).

Controls in which inoculum with known numbers of

bacteria was ground with either type of root tissue produced

colony counts which were not significantly different from






-66-


each other or from those plated directly from the inoculum.

Controls to test the suitability of the conditions of the

adsorption assay for nodulation were examined 20 d after

placing seedlings in growth pouches. The seedling had been

incubated in inoculum for 120 min and washed in parallel

treatments to seedlings in adsorption assays. The mean

number of nodules per plant for each combination is

presented in Table 4.3. Only the combination of strain 110

x Clark-rji and the seedlings incubated in sterile Jensen's

solution without bacteria failed to form nodules. The

following number of plants were nodulated in the other

combinations: Clark-ril x strain 94, 1 of 8; Clark x strain

110, 8 of 8; and Clark x strain 94, 8 of 8. Thus, it is

clear that adsorption was assayed under conditions which

were conducive to infection leading to nodulation.



Discussion

One objective of this study was to determine whether

the nodulation restrictive genotype, Clark-rj1, reduces the

ability of the plants to adsorb nonovercoming strains of

Rhizobium, thus influencing bacterial host range. This is

not the case, because after 2 hr Clark-rJl adsorbed more of

either R. japonicum strain tested that did Clark. In fact,

the ranking of plant type x strain combinations based on

numbers of nodules is precisely opposite to their ranking by

numbers of bacteria adsorbed at 2 hr. Although no

biological significance is apparent, this inverse



























Figure 4.1. Adsorption of cells of Rhizobium japonicum
to soybean roots. The experiments were completed at 27
C. Each point represents the mean from five
experiments with four pairs of plants tested at each
time for each plant-strain combination at each
replication of the experiment. The roots of soybean
seedlings were incubated in bacterial suspensions (1 x
10 cells/ml) for the times indicated and rinsed
vigorously. The terminal 2 cm of the primary roots
were excised, ground and plated for determination of
colony forming units. O = Clark x strain 110, U =
Clark x strain 94, O = Clark-ril x strain 110, and
* = Clark-rji x strain 94.Bars represent the standard
error of the mean.





-68-


0 30 60 90
MINUTES


150





I-

t--oo
.100








" 50
I-


120




























Figure 4.2. Adsorption of cells of Rhizobium japonicum
strain 94 to roots of Clark-rj1 soybean at two
temperatures. Each point represents the mean of four
pairs of plants tested at each time for each
temperature from each of five replications of the
experiment. The roots of seedlings were incubated in
bacterial suspensions (1 x 10 cells/ml) equilibrated
at 22 C or 27 C for the times indicated and rinsed
vigorously. The terminal 2 cm of the primary roots
were excised, ground and plated for determination of
colony forming units. V = 22 C, and 0 = 27 C (The 27
C curve is duplicated from Figure 3.2.). Bars
represent the standard error of the mean.






























30 60 90
MINUTES


150






100






50


120





















Table 4.1. Percentage of plants nodulated at 27 C and 22 C

Primary Secondary Any Root

Combination 27 C 22 C 27 C 22 C 27 C 22 C


Clark x 94


Clark x 110


Clark-rjl x 94


Clark-ril x 110


89a 100 89 100 100 100


89 100 50 89 100 100


0 13 10 27 10 40


0 0 0 0 0 0


a Data in table from Chapter Three. Percentage of plants
nodulated at each indicated location. Data are for all
plants from 3 replications at each temperature with 6
to 10 plants per replication.





















Table 4.2. Mean number of nodules per plant at 27 C and 22 C

Primary Secondary Any Root

Combination 27 C 22 C 27 C 22 C 27 C 22 C


Clark x 94


Clark x 110


Clark-ril x 94


Clark-rjl x 110


8.8a 7.9


8.3 8.9


5.8 10.2 14.6 18.1


3.6 6.8 11.9 15.7


0 0.3 0.1 0.5 0.1 0.9


0 0 0 0 0 0


a Data in table from Chapter Three. Mean number of
nodules per plant on primary roots, secondary roots, or
on any root of the plant as indicated. Each value
represents the average for 3 replications at each
temperature with 6 to 10 plants per replication.







-73-


Table 4.3. Nodulation of plants inoculated with Rhizobium
japonicum under the conditions of the adsorption assay

Bacterial strain
Soybean------ --------- ------
isoline 94 110
- - - - - - - - - - - - - - - - - - - -


Clark


8.3 5.7a



0.1 0.4


Clark-rji


6.5 2.1


a Seedlings were inoculated by dipping the roots in a
bacterial suspension containing approximately 104
cells/ml for 2 hr. The roots were rinsed vigorously.
Plants were grown for 20 d in plastic growth pouches
(see Materials and Methods for detailed description of
procedures).











relationship underscores the rejection of the hypothesis

that host range is primarily dependent on ability of

rhizobia to adsorb to host roots, at least for the

differential nodulation of Clark and its isoline, Clark-ril.

Clark (1957) reported that similar numbers of rhizobia

were recovered from the roots of plants carrying the rJlr11

genotype and plants that carried a nonrestrictive genotype

whether they were grown in a greenhouse or in the field.

Elkan (1962) demonstrated that Clark-ril actually maintained

substantially higher populations of rhizobia in rhizosphere

soil in the field than did Clark for 45 out of the first 60

d of plant growth. These observations, in conjunction with

the results of the present study, suggest that the limiting

step in nodulation of these plants occurs post-adsorption.

Pueppke (1984b) showed that adsorption of R. japonicum

strain 138 to roots of the soybean cultivar Hardee is

temperature sensitive; when that combination was subjected

to assay temperatures of 4 C, 27 C, and 37 C, the optimum

binding temperature was 27 C. The number of bacteria bound

per plant after co-incubation for 1 hr was reduced

approximately 90% at 4 C and approximately 65% at 37 C,

compared to the number bound at 27 C. In the present study,

the binding of strain 94 to roots of Clark-ril similarly was

temperature-sensitive. The reduction in the number of

bacteria bound per plant in 1 hr with a drop in assay

temperature from 27 C to 22 C was about 75%. The large

reduction in adsorption with only a 5 degree temperature





-75-


difference suggests that this combination is either more

highly temperature sensitive than the combination used in

the previous study (1984b), or that perhaps the curve of

temperature sensitivity for both combinations is very steep

on either side of an optimum temperature. The shape of the

response curve has not yet been determined, and would

require testing adsorption at considerably more temperatures

than have been used as treatments in either report.

For the combination of Clark-ril with strain 94, the

response of adsorption to temperature is opposite to that of

nodulation to temperature for the same (Tables 4.1 and 4.2

[data from Chapter Three], Devine and Breithaupt 1980b).

Although 40% of the plants were nodulated at 22 C, only 10%

were nodulated at 27 C. The number of bacteria bound per

plant after 2 hr at 22 C was 21 2. At 27 C 105 8

bacteria were bound, a five-fold increase. Clearly the

temperature effect on nodulation of these plants is

independent of its effect on binding.

The following conclusions are drawn from the data

presented in this report: i. Compared to 27 C, 22 C favors

nodulation in the Clark-ril x strain 94 combination,

whereas, the effect of temperature on adsorption is

precisely the opposite. ii. There is not a qualitative

difference between the adsorption of strains 94 and 110 to

Clark and Clark-ril soybean roots. iii. The rates of

adsorption in these combinations are similar to the rates

reported for other strain x soybean cultivar combinations





-76-


(Pueppke 1984b). iv. Under these experimental conditions,

there is no correlation between the number of rhizobia bound

to roots and the extent of nodulation of Clark or Clark-rji

soybean.

















CHAPTER FIVE
INFECTION OF SOYBEAN ISOLINES DIFFERING AT THE RJ_ LOCUS
BY RHIZOBIUM JAPONICUM STRAINS WITH DIFFERENTIAL
NODULATING ABILITY



Introduction

The nodulation-restrictive genotype of soybean, rJilrJI

originated as a field mutant in a soybean breeding program

(Williams and Lynch 1954). The phenotype was first

characterized as nonnodulating with Rhizobium japonicum

(Williams and Lynch 1954), but Clark (1957) reported that a

few strains, called overcoming strains, form a few nodules

on plants growing in sand or vermiculite but not soil.

Several investigators have sought to identify the factor

that differentiates rilrjl-plants from those that nodulate

normally. The amino acid content of both plant types has

been compared and found to be similar (Clark 1957, Hubbell

and Elkan 1967b). The restrictive step is not the inability

of the jilrjl-plants to support rhizobia in the rhizosphere;

such plants support populations equal to (Clark 1957) or

greater than (Elkan 1962) those supported by a near-isogenic

but normally nodulating soybean line. Devine and Weber

(1977) determined that successfully nodulated rJlrjl plants

were capable of fixing nitrogen. From these studies, Devine

(1984a) inferred that the incompatibility conditioned by the





-78-


illjll genotype is not a general antagonism to Rhizobium

metabolism and function, but that the answer to

incompatibility is likely to be found in early stages of the

infection process.

A number of investigators have attempted to elucidate

the bacterial property that enables a few strains to

overcome the plant's resistance and produce some nodules.

Hubbell and Elkan (1967a) reported that several

physiological characteristics of R. japonicum strains were

correlated with the ability to nodulate jilrJl-soybean, but

none of the factors was implicated in infection. Devine and

Weber (1977) noted that the ability of bacteria to nodulate

the rilrjl genotype was highly correlated with the

production of a bacterial metabolite that induces chlorosis

in several soybean lines. The bacterial product was latter

shown to be rhizobitoxine (2-amino-3-hydroxypropoxy-

vinylglycine) (Owens et al. 1972). They postulated that

this chlorosis-causing agent had an enabling role in

infection of rjlril-soybean. Devine and Breithaupt (1980b)

showed that processes leading to nodulation and to the

expression of chlorosis had different temperature optima.

When added to a Rhizobium inoculum, an analog of

rhizobitoxine did not enhance nodulation of the restrictive

soybean (Devine and Breithaupt 1980a). No diffusible

compound was detected in tests for a factor to endow rJ1-

incompatible strains with the ability to nodulate the

rJilrl-soybean (Devine et al. 1981). Devine concluded that

the correlation of rhizobitoxine-induced chlorosis and





-79-


ability to form nodules on the restrictive soybean is likely

incidental and "not the result of an intrinsic physiological

relationship" (Devine 1984a [p 150]).

Infection of the small-seeded temperate legumes has

been studied in the most detail due to the development of

the Fahraeus slide technique enabling study of the root

surface of living plants with the light microscope (Nutman

1981). These plants, which are are nodulated by fast-

growing rhizobia, are believed to be infected exclusively

through infection threads in root hairs (Fahraeus 1957,

Nutman 1959, Ljunggren 1969, Callaham and Torrey 1981).

Some tropical legumes, such as peanut (Arachis hypogaea L.)

and several species of Stylosanthes, are infected through

natural wounds caused by emergence of lateral roots, with no

infection thread formation in root hairs (Allen and Allen

1940, Ranga Rao 1977, Chandler 1978, Chandler et al. 1982).

In the tropical genus Lotus, one species (L. corniculatus

L.) was reported to be infected only by means of infection

threads in root hairs, but in another (L. hispidus Desf.)

most nodules originated by bacterial penetration directly

through the epidermis, and infected root hairs were rare

(Ranga Rao 1977).

In 1938, Bieberdorf described the infection process in

soybean in considerable detail. He was the first to note

that infections usually progress via infection threads in

soybean, but he states that rhizobia also infect directly

through root epidermal cells. Apparently, corroboration of





-80-


the initial report of root hair infection of soybean was not

made until fifty years later (Ranga Rao and Keister 1978)

and direct infection processes have not been corroborated.

Several studies of nodule structure have included some

details of early infection (Cabezas de Herrera and Fernandez

1982, Goodchild and Bergersen 1966, Newcomb et al. 1979).

Pueppke (1983) recently examined soybean in nodulating and

nonnodulating combinations with eighteen strains of rhizobia

for signs of early infection. He showed that infection

threads were formed exclusively in nodulating combinations

and that infection threads were restricted to locations

distal to the region of the root on which root hairs were

fully elongated at the time of inoculation. A thorough

description of root hair infection at the ultrastructural

level was recently reported by Turgeon and Bauer (Turgeon

and Bauer 1982, In press).

Near-isogenic lines of soybean were examined in this

study to determine whether the difference between the

phenotypes of normally nodulating soybean and nodulation-

restrictive (llrJil) soybean were expressed at the level of

infection. Both Nutman (1981) and Devine (1984a) have

suggested that the rjlrjl-soybean might be infected

exclusively by means other than infection threads in root

hairs. The mode of infection of Jlrj4l-soybean has not

previously been reported.







-81-


Materials and Methods

The bacteria utilized were R. japonicum strains 94 and

110 obtained from the U. S. Department of Agriculture

Nitrogen Fixation and Soybean Genetics Laboratory,

Beltsville, MD, courtesy of H. H. Keyser, D. F. Weber,

and R. Griffin. Strain 94 is an overcoming strain that

forms a few nodules on the nodulation-restrictive isoline,

but forms abundant nodules on Clark (Chapter Three). Strain

110 nodulates Clark abundantly but does not nodulate the

restrictive isoline (Chapter Three). Rhizobia were

maintained on yeast extract-mannitol (YEM) agar (Vincent

1970) slants at 4 C, and stock cultures were transferred

about every three months. Bacteria for inoculation were

grown to mid-log phase at 28 C with shaking in 50 ml liquid

defined gluconate-mannitol medium (Bhuvaneswari et al.

1977). Cell number was estimated turbidimetrically.

Cultures then were centrifuged at 7500 x for 10 min and

bacteria were resuspended at a concentration of

approximately 5 x 108 cells/ml (except where noted) in

sterile Jensen's solution (Vincent 1970).

The plants were two near-isogenic lines of soybean

Glycine max (L.) Merr., the cultivar 'Clark-Ll' (RilRil) and

its isoline L63-1889, which carries the nodulation-

restriction genotype, rJlrJl. The isolines will be referred

to as Clark and Clark-ril, respectively, following the

terminology of Devine and Breithaupt (1980b). Seeds were

obtained from R. L. Bernard, USDA Regional Soybean

Laboratory, University of Illinois, Urbana, and D. A.











Phillips, Department of Agronomy and Range Science,

University of California, Davis. Seeds were soaked in 50%

aqueous ethanol for 2 min with agitation, rinsed with water,

soaked in 0.5% aqueous sodium hypochlorite for 2 min with

agitation, and rinsed for 20 min under running deionized

water. Surface disinfested seeds were germinated at 22 C in

the dark for 5 d on water agar plates.

Inoculation and seedling transfer were completed in a

laminar flow hood using procedures designed to maintain

sterility. Germinated seedlings were carefully selected for

lack of any sign of contaminating microorganisms and for

uniform size (ca. 4 cm, excluding cotyledons). Seedlings

were inoculated by immersing the roots in the bacterial

suspension for 10 min. Seedlings were placed, 2 per pouch,

in plastic growth pouches (Northrup King Seed, Co.,

Minneapolis, MN). Growth pouches had been prepared by

adding 15 ml nitrogen-free Jensen's plant growth solution to

each and autoclaving. Control plants were sham-inoculated

with sterile Jensen's solution only. A mark, called the

root tip mark (RTM), was made on the growth-pouch surface at

the position of the primary root tip. Except where noted,

the tops of growth pouches were covered with plastic sleeves

for several days to prevent contamination. Plants were

grown at 22 C in a Conviron E-15 growth chamber at an

irradiance of 900 uE/m2/sec (400-700 nm) with 12 hr light,

12 hr dark.










Root surfaces were examined by light microscopy for

curled root hairs of the type referred to previously as

"question mark shaped" (Pueppke 1983), and for the presence

of infection threads. Plants used in light microscopy

experiments were grown essentially as stated above, except

that seeds occasionally were germinated and plants grown at

other temperatures and light intensities. Growth pouches

were not covered during plant growth. Plants were sampled

for light microscopy as previously described (Pueppke 1983)

by stripping away very thin strips of tissue from root

surfaces for several centimeters on either side of a point

corresponding to the RTM. Two to four strips were made for

each plant. These collectively included almost all of the

root surface in the region of the RTM. Root surfaces of 18

Clark plants inoculated with strains 94 or 110, and 27

plants of Clark-rji inoculated with strain 94 were examined.

These strips were mounted in phosphate-buffered saline

(Bhuvaneswari et al. 1977) with or without prior staining

with toluidine blue 0 and were examined using bright-field

or interference-contrast optics (Hoffman modulation). The

entire strips were scanned for short, curled root hairs and

infection threads. Light microscopy also was used to

examine the surface of nodules for residual curled root

hairs and infection threads. Plants were examined for very

young developing nodules. The nodules were removed from the

plants and a thin layer of the nodule surface distal to the

plant was sliced away with a sharp scalpel. These thin






-84-


disks of tissue were mounted and examined as described for

root surface strips.

Samples for scanning electron microscopy (SEM) were

obtained from 10 day-old plants by severing the primary root

with a scalpel at the RTM and at 10 mm above the RTM.

Secondary roots attached to these sections were severed at a

line parallel to the primary root, at 5 mm perpendicular

from the primary root. The root sections immediately were

placed into a fixing solution consisting of 4%

glutaraldehyde (Eastman Kodak Co.) in 50 mM sodium

cacodylate buffer (pH 7.4). After fixation overnight at 4

C, samples were rinsed with distilled water and post-fixed

overnight at 4 C in 2% aqueous osmium tetroxide. Sections

were washed in 5 changes of distilled water and dehydrated

progressively in a graded water-ethanol series with never

more than a 10% increase in ethanol concentration per step

or less than 15 min per step. A faster dehydration or

steeper series caused distortion. The samples were

transferred in anhydrous ethanol to a critical point dryer

(Balzers Model H) and dried from liquid carbon dioxide.

Dried samples were attached to Cambridge mounts (Ernest F.

Fullam, Inc.) with double-sided tape, sputter-coated with

ionized gold, and examined with a Hitachi S-450 scanning

electron microscope at 20 kV.

Two or more groups of 5 plants were sampled and fixed

separately for each combination of Clark x strain 110, Clark

x strain 94, and Clark-rjl x strain 94. Additional plants

were examined for some combinations. Two uninoculated Clark







-85-


control plants and two of the Clark-ril x strain 94

combination that had been inoculated at 1 x 1010 cells/ml

were examined. Root sections were examined using SEM at low

magnification (50x) and photomicrographs were made of the

entire root segment to enable the mapping of higher

magnification photomicrographs for interpretation in

context. Nodule surfaces and root surfaces were examined.

All samples were scanned at 150x and 250x for curled root

hairs of the type previously identified by light microscopy

to harbor infection threads.



Results

Clark soybean inoculated with either of the fully

compatible strains 110 or 94 at 5 x 108 cells/ml had short,

tightly curled root hairs (Figure 5.1), many containing

infection threads. The tightly curled root hairs with

infection threads sometimes occurred in clusters. A number

of root surface strips were examined from 27 plants for the

combination Clark-ril x strain 94, which was previously

demonstrated to produce a few nodules (Chapter Three). No

short, tightly curled root hairs similar to those seen in

the fully compatible combinations were found and no

infection threads were ever observed.

Light microscopy was used to examine nodule surfaces

for residual root hairs with infection threads. Thirteen of

twenty sections of nodule surface tissue examined for the





























Figure 5.1. Curled root hair on Clark soybean 10 days
after inoculation with Rhizobium japonicum strain 110.
Phase contrast optics.





-87-


I )I' i
r
LFi ~


i ~~*F"F
'"


\\










Clark x strain 94 combination had curled root hairs, and in

9 the infection thread(s) was still visible. None of twelve

nodules examined similarly from the Clark-rjl x 94

combination had curled root hairs or visible infection

threads.

Primary root segments extending from the RTM to 10 mm

above the RTM were examined using SEM. Uninoculated plants

developed long root hairs that were free of detectable

bacteria (Figure 5.2). No short, curled root hairs were

apparent except where it was obvious that the root had come

in contact with a solid surface such as the plastic of the

growth pouch. In these areas the individual deformed root

hairs were easily discriminated from root hairs

characteristic of inoculated roots of compatible

combinations. Root segments from Clark soybean inoculated

with 5 x 108 cells/ml of fully compatible strains of R.

japonicum were examined using SEM. The root segments from

Clark soybean inoculated with strain 110 had clusters of

short curled root hairs similar in appearance to the root

hairs shown by light microscopy to contain infection threads

(Figure 5.3). Clark inoculated with strain 94 produced

short curled root hairs very similar to those seen in the

the combination Clark x strain 110 (Figures 5.4 and 5.5).

There were differences in the general appearance of root

segments from these two combinations. The root surfaces of

Clark were smooth (almost waxy appearing in SEM micrographs)

























Figure 5.2. Clark soybean root hairs on a control
plant sampled and fixed 10 days after sham inoculation
with plant growth solution. The area sampled was the
10 cm of the primary root immediately above the point
representing the root tip at the initiation of the
experiment.






-90-


V l(


'1


I j,


br
k* -
_^^^^ -
'r*\^ c


'~nE



























Figure 5.3. Clark soybean root hairs on plants sampled
10 days after inoculation with Rhizobium japonicum
strain 110. Clusters of tightly curled root hairs
characteristic of this plant-strain combination are
marked by arrows.







-92-


-. -J" ^&l^-^ '''^^t^f.^ ^B
-'3 *
-7-- p a A -


Vo"ff s. Is At i .f
S.,Vow.-
aM g" w
A..k i -a b i


...'.
"- ** '


i L-_z

xml version 1.0 encoding UTF-8
REPORT xmlns http:www.fcla.edudlsmddaitss xmlns:xsi http:www.w3.org2001XMLSchema-instance xsi:schemaLocation http:www.fcla.edudlsmddaitssdaitssReport.xsd
INGEST IEID EGUYKTBHP_JC3IQX INGEST_TIME 2017-08-14T21:19:37Z PACKAGE UF00099339_00001
AGREEMENT_INFO ACCOUNT UF PROJECT UFDC
FILES



PAGE 1

INTERACTION OF RHIZOBIUM JAPONICUM WITH SOYBEAN ISOLINES CARRYING UNIQUE GENES WHICH AFFECT NODULATION AT THE RJ! LOCUS By JOHN HOWARD PAYNE A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 1985

PAGE 2

To Four Teachers R. Guy Payne Through his knowledge and enthusiasm he instilled in me fascination for the natural world Esther Ruth Collins She introduced me to scientific study and always encouraged enterprise and individuality Robert G. Anderson In socratic discourse he taught me the importance of critical thinking Steven G. Pueppke He showed by his example the need for diligence and integrity in scientific endeavor

PAGE 3

ACKNOWLEDGEMENTS Dr. Steven Pueppke provided guidance as my committee chairman. I thank him for his friendship and patience, and for his consistent example of the finest in scholarly research. Members of my committee, Drs. Raghavan Charudattan, Ed Freeman, Bill Gurley, and George Bowes, have always provided help when asked and have influenced the direction of this work with their suggestions. I am grateful for the friendship and advice of Drs. H. H. Luke and Dave Mitchell. Mrs. U 1 1 a Benny provided technical assistance, including hours of her time assisting with the tedious, repetitive procedures necessary for the study of adsorption of bacteria to plant roots. Drs. R. Howard Berg, III, and Greg Erdos provided instruction in the use of the scanning electron microscope and made available the facilities of the Biological Ul trastructure Laboratory. My laboratory companions, Drs. Dan Kluepfel and Jill Winter, Mr. Dave Heron, and others, have aided with discussions of the research and have given helpful suggestions and constructive criticism. To all of these I offer grateful thanks .

PAGE 4

My parents Rev. Richard and Mrs. Reva Payne, my brother Mr. James Payne, my sisters Mrs. Rebecca McClanahan and Mrs. Mary Jane Suppasansathorn and their families, and my grandmother Mrs. Mable Speak, have provided abiding love and constant encouragement. Their confidence in me and the emotional support they gave were at times what kept me going. I cannot thank them enough. This research was supported in part by the National Science Foundation through grant PCM 82-00110 to Dr. Pueppke, and the University of Florida; each provided research funds and contributed to my research assistantship. Financial support also was provided by the Veterans Administration .

PAGE 5

TABLE OF CONTENTS Page ACKNOWLEDGEMENTS iii ABSTRACT vii CHAPTER ONE INTRODUCTION 1 CHAPTERTWO REVIEW OF NODULATION SPECIFICITY AND MODE OF INFECTION BY RHIZOBIU M JAPONICU M OF SOYBEAN LINES WITH NODULATION RESTRICTIVE GENOTYPES 3 Introduction 3 "Cross-inoculation Groups" and Rhizobium Taxonomy.. 5 Genetics of Rhizobium Infection and Nodulation 8 Infection and Nodulation of Legumes 20 Phenotypic Nodulation Response of Soybean 26 Nodulation Restrictive Soybean Genotypes 27 Perspective 33 CHAPTER THREE EFFECT OF TEMPERATURE ON NODULATION OF SOYBEAN ISOLINES DIFFERING AT THE R_2]_ LOCUS 35 Introduction 35 Materials and Methods 37 Results 39 Discussion 53 CHAPTER FOUR ADSORPTION OF STRAINS OF RHI10_B_I_U_M_ JAPONICUM WITH DIFFERENTIAL NODULATING ABILITY TO ROOTS OF SOYBEAN ISOLINES THAT DIFFER AT THE Rj 1 LOCUS 57 Introduction 57 Materials and Methods 61 Results 6 5 Discussion 74

PAGE 6

Page CHAPTER FIVE INFECTION OF SOYBEAN ISOLINES DIFFERING AT THE Rj 1 LOCUS BY RHIZOBIU M JAPONICU M STRAINS WITH DIFFERENTIAL NODULATING ABILITY 77 Introduction 77 Materials and Methods 81 Results 85 Discussion HO CHAPTER SIX SUMMARY 116 APPENDIX A PLASMIDS OF RHIZOBIUM JAPONICUM STRAINS THAT NODULATE SOYBEAN ISOLINES DIFFERING AT THE Rj ]^ LOCUS 120 APPENDIX B EVALUATION OF PROCEDURES REPORTED TO INDUCE HIGH-FREQUENCY MUTATION OF STRAINS OF RHIZOBIU M JAPONICUM 12 8 LITERATURE CITED 137 BIOGRAPHICAL SKETCH 151

PAGE 7

Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy INTERACTION OF RHIZOBIU M JAPONICUM WITH SOYBEAN ISOLINES CARRYING UNIQUE GENES WHICH AFFECT MODULATION AT THE Rjj LOCUS By John Howard Payne May 1985 Chairman: Steven G. Pueppke Major Department: Plant Pathology The soybean genotype r j 1 r j-^ conditions the inability to nodulate with most strains of Rhizobium japonicum . Certain strains, termed overcoming strains, form a few nodules on plants grown in hydroponic culture. The effect of temperature on the number of nodules per plant and the nodulation pattern was determined for the cultivar Clark (R_ilR_il) and its isoline Clark-rJ! (LllLll^ ' The temperature treatments, 22 C, 27 C, and 32 C, had a statistically significant effect on the number of nodules for both genotypes. The percentage of Clark-rj^ plants nodulated by overcoming strains was 44% at 22 C, 23% at 27 C, and 4% at 32 C for 120 plants tested. The nonovercoming strain 110 did not nodulate Clark-rJ! at any temperature. Ninety-eight percent of 270 Clark plants tested, including all strains and treatments, were nodulated. Frequency plots

PAGE 8

were generated for each isoline x strain combination for each temperature. These indicated the number of nodules produced at locations on the primary root relative to the location of the root tip which was marked at the time of inoculation. Plots for combinations of Clark with each strain showed a peak near the root tip mark for plants grown at 22 C or 27 C. The frequency plots for Clark plants grown at 32 C were flattened and indicated a downward displacement of nodulation on the primary root. The adsorption of overcoming strain 94 and nonovercoming strain 110 to roots of Clark and Clark-rj^ was tested. After 2 hr inoculation, approximately 100 bacterial cells were bound per plant, irrespective of strain. The rank of isoline x strain combinations for the number of bacteria bound to roots was opposite to that for nodule number . The roots of both plant types were examined by light microscopy and scanning electron microscopy 10 d after inoculation with strain 94 or 110. Curled root hairs with infection threads were formed on Clark in response to either bacterium, but were not observed on Clark-rj^. When Clarkr j -i plants were inoculated at a high inoculum concentration, perforation in the epidermis was apparent, suggesting a potential infection pathway.

PAGE 9

CHAPTER ONE INTRODUCTION The symbiosis of soybean and the bacterium Rhizobium japonicum depends, even in early stages of its initiation, on contributions from both the plant and the bacterium. They interact in a coordinated, multi-step infection process to form root nodules. The bacteria inhabit cells of the nodule, where they fix atmospheric nitrogen, which is unusable to the plant, to ammonium, a plant nutrient. The rhizobia in turn derive sustenance from the plant in the form of translocated nutrients. The series of events that transpire during the infection process has been studied by microscopy, by induced genetic change in the bacterium, and by biochemical and microbiological examination. Some steps leading to nodule formation have been described, but the relative contributions of the plant and the bacterium to the inception of a nodule are not well understood. The literature that describes the infection process is reviewed in Chapter Two. Studies are described in which genetic changes are induced in Rhi zobium and the effects of the changes are correlated with the interaction phenotype with the host. The contribution of studies of plants with altered symbiotic phenotype also is described. -1-

PAGE 10

-2The soybean genotype, r j^r j-i , conditions resistance of soybean to most strains of R^_ japonicum . A few strains, called the "overcoming strains," have the ability to overcome the rj i -res is tance and form a few nodules on r_2 1 rj 1 -plants. In this study the soybean cultivar Clark and a line near-isogenic to it, Clark-rjj that carries the genes rj-, rji i were used. Chapters Three through Five are reports on aspects of the phenotypic expression of the symbiotic interaction of soybean isolines and strains of R^_ japonicum . Chapter Three describes the effect of temperature on the differential nodulation response of the soybean isolines. Chapter Four presents an evaluation of the role of bacterial adsorption as a specific determinant of differential nodulating ability. Chapter Five includes a report of the interaction phenotype at the cellular level after inoculation of seedlings with an overcoming or a nonovercoming strain. Conclusions about some aspects of the interaction of strains of R^_ j apon icum with the soybean isolines are summarized in Chapter Six. The appendices include a short description of the plasmid complement of strains of R^ japonicum as determined by gel electrophoresis and a report of an evaluation of procedures which had been described to induce high-frequency mutations in symbiotic functions.

PAGE 11

CHAPTER TWO REVIEW OF NODULATION SPECIFICITY AND MODE OF INFECTION BY R HIZOBIU M JAPONICU M OF SOYBEAN LINES WITH NODULATION RESTRICTIVE GENOTYPES Introduction Soybean has had the largest increase in acreage for any crop in American history. In 1930, 1 million acres produced about 14 million bushels; in 1980 more than 70 million acres produced over 2.25 billion bushels (Sundquist 1981). Over the past 60 years there has been about one-quarter bushel per acre increase in yield per year. This is due in great measure to increased understanding through research on soybean production. Included is the understanding and manipulation of its symbiosis with root-nodule bacteria (Hanson 1981, Weber 1981). Weber (1981) has estimated the market value for the combined nitrogen produced in the United States by the legumeRhizobium symbiosis at $5 billion a year. The value of the symbiosis and the impact of its improvement are clear, but the exact nature of the relationship of the bacterium -to the plant and how best to manipulate that relationship for greater pr oducti v i tyare still unclear. The subtle interchange of signals between plant and mi cr osymbi ont that leads to establishment and maintenance of the complex partnership has largely remained -3-

PAGE 12

-4a mystery, in spite of considerable effort to unravel its intricacies. More is known about the genetic and physiological factors essential for the bacterium in this relationship because of the greater ease with which these elements can be manipulated in the relatively simpler organism. Although a few genotypes are known to produce qualitative changes in the phenotype of the interaction, our knowledge of the role of the plant is limited mostly to general observations of the effect of genetic constitution on quantitative aspects of the symbiosis. To replace the broad, nonanalytical descriptions of the relationship, Vincent (1980) proposed terminology that defines specific steps in the establishment of the partnership. His terms are defined as specific phenotypes meant to be applicable to most legumeRhizobium symbioses, although there is no provision for rhizobia that infect by means other than through root hairs. The terms pertinent to early infection processes will be used in this discussion, and are as follows (derivation are indicated): Roc = Root colonization, Roa = Root adhesion, Hac = Hair curling, and Inf = Inf ection thread formation. Two common terms not described by Vincent will be used for events representative of the mature symbiosis. The term Nod (= Nod ule formation) will be used to describe the formation of macroscopic nodules. The term Fix (= Fix ation of nitrogen) will be used, rather than Nif, to avoid confusion with bacterial genetic nomenclature.

PAGE 13

-5This review first examines the rhizobia and their contribution to the infection of legumes. The taxonomy of the Rhizobiaceae is discussed in light of its important role in defining research directions and its implications for better understanding of genetic data. Experimental genetic manipulation of the rhizobia is included to provide a background for discussion of the research presented in subsequent chapters. The literature which describes the phenotype of interaction of the bacterium and plant at the cellular level is reviewed. Plant genotypes known to have qualitative effects on the early stages of nodulation also are described. "Cross-inoculation Groups" and Rhizobium Taxonomy The taxonomy of Rhizobium is based on the range of host plants nodulated. Strains with similar host-range have been given species status. This idea was formalized in the landmark monograph of Fred, Baldwin, and McCoy in 1932. The authors of this monograph expounded the concept of "the crossinoculation group." This amounted to an exhaustive list of the legumes, species by species, which could be nodulated by rhizobia that were isolated from other species of legumes in that list but not by rhizobia from other groups of legumes. Although there were clearly some ambiguities, the concept of mutually exclusive internodulation within inoculation groups became an accepted paradigm, and thus the basis of Rhizobium taxonomy. As a result, a group of strains shared by an inoculation group

PAGE 14

-6was given species status. The species recognized by this criterion were I. Alfalfa group, R^_ me 1 i l oti ; II. Clover group, R^ t r if o 1 i i ; III. Pea group, R^_ le gumi nosarum ; IV. Bean group, R^_ phaseoli ; V. Lupine group, P~_ lupini ; and VI. Soybean group, R^_ japonicum . A seventh group, the cowpea group (or cowpea miscellany) was not given species status since the legumes that could be crossinocu lated were numerous and taxonomica 1 ly diverse. There also were apparent subgroups within the cowpea group which did crossinoculate some subgroups but not others. Fred et al. (1932) considered the cowpea group to be intermediate between the soybean group and the lupine group, because many of the rhizobia from the legume hosts in those groups formed nodules with many of the hosts for the cowpea group. RMzobium strains fall naturally into two main divisions on the basis of their physiology and morphology, those which produce rapid growth on rich media and have peritrichous flagella, and those which grow slowly on rich media and have a polar or subpolar flagellum (Jordan and Allen 1974). The fast-growing strains are generally those included in ^ leguminosarum , R. phaseol i, R. trifolii , and r. mel i l oti . The slow-growing strains are usually those included in R^ iu£^n_i, R^ ia£°ni_cum, and the cowpea miscellany (Jordan and Allen 1974). Several strains from the People's Republic of China nodulate soybean but are similar in growth response and physiology to the fastgrowing species of Rhizobium (Keyser et al. 1982). These

PAGE 15

-7strains were described taxonomica 1 ly as R^ japonicum and noted parenthetically to be fast-growing strains (Keyser et al. 1982, Jansen van Rensburg et al. 1983, Heron and Pueppke 1984). This taxonomic problem serves to underscore the overall deficiencies of a taxonomy based on host range. Recently, the taxon R^_ fredii was proposed for the fastgrowing rhizobia that infect soybean (Scholia and Elkan 1984) . In 1964, Graham (1964) revised the taxonomy of the Rhizobiaceae. He recognized previous criticism of the cross-inoculation group concept (Wilson 1944), and based his revision on a numerical taxonomy that compared 100 physiological characteristics. He proposed that R. phaseol i , R. trifol ii and R^_ leguminosarum be consolidated into a single species R^ l eguminosarum . Agr obacter ium tumef aciens and A. radiobacter were to be included in the genus Rhizobium as R^_ radiobacter . The fast-growing species r. mel i l oti was retained. Graham also proposed that the slow-growing strains be contained in a newly proposed genus Phy tomyxa . The taxonomic revision proposed by Graham was not widely accepted, but some of the general features of his system are included in recently proposed changes. The International Subcommittee on Agrobacter ium and Rhizobium recently proposed that the slow-growing strains of Rhizobium be transferred to a new genus Br adyrhi zobium gen. nov. (Jordan 1982). This taxon emphasizes the basic physiological difference between the fast-growing and slowgrowing strains. Jordan (1984) included most of Graham's

PAGE 16

proposed revisions in his recent description of the family Rhizobiaceae. The proposed species are R^_ mel i loti , R. l eguminosarum (with the biovars: tr if o li i , phaseo l i , and v iceae ) and R^_ l oti , which includes strains that nodulate Lotus spp. and related plants. The genus Br adyrhi zobium essentially represents the strains for which Graham proposed the name Phytomyxa (Graham 1964). The genus Aqrobacter ium was retained. This revision addresses many of the deficiencies in the former taxonomic treatments of the Rhizobiaceae. The new taxonomy should greatly facilitate discussions of the comparative genetics of strains of rhizobia, including the genetic basis of symbiotic interaction. However, for this literature review I will continue to use the nomenclature of Jordan and Allen (1974), because all of the literature to be examined follows that system. Nevertheless, the reader should consider the data on the genetics of symbiosis and host range and the comparisons in interaction phenotype in the framework of the biological relationships suggested by the taxonomy described by Jordan (1984). Genetics of Rhizobium Infection and Nodulation Much of what is known about the genetics of nodulation has been developed with the four classical species of fastgrowing Rh_i^ob_ium. The three allied species R. l eguminosarum , R. tr if ol i i , and R_;_ phaseo l i will be discussed together because similar procedures and genetic

PAGE 17

-9probes have been used to study them. Both Ljunggren (1961) and Beringer (1980) cite Krasilnikov (1941) as providing the first evidence for transfer of aodulating ability from strain to strain when he reported the transformation of nonnodulating strains with culture filtrates from nodulating strains. In 1961, Ljunggren reported the transformation of the nonnodulating R_^ tr i fol i i strain Bart A. The transformed strain, Bart A*2, nodulated clover, had a smooth colony morphology and produced a serological reaction unrelated to that of Bart A and only partially related to the transforming strain. However, one cannot rule out the selection of a contaminant capable of nodulation. The association of some of the symbiotic functions with plasmids and the development of procedures for genetic manipulation, including recombinant DNA techniques, have greatly increased experimentation on the genetics of nodulation. Plasmids were detected in various strains of several of the Rhizobium spp. (Tshitenge et al. 1975, Nuti et al. 1977). Higashi (1967) reported that R^ phaseo li acquired the ablility to nodulate clover with the transfer of an episomal factor from R_^ t r i f o 1 i i. Zurkowski et al. (1973) used chemical agents to cure strains of R. tr if o l ii of plasmids and reported concomitant loss of ability to nodulate clover. The introduction of the kanamycinresistance marker of the transposon Tn5_ into R . leguminosarum provided a means to both mutate and mark the location of the mutation. This enabled genetic linkage analysis and selection by DNA-DNA homology (Beringer et al.

PAGE 18

-101978). The presence of the Tn5^ marker in the conjugative plasmid, pRLlJI, enabled selection of transconjugants at a high frequency (Johnston et al. 1978). The ability to nodulate peas was restored to p lasmid-cured strains of R. leguminosarum by acquisition of pRLlJI. Ability to nodulate peas was transferred with pRLlJI to strains of R^_ trifol ii , a strain of R^ phaseo l i , and a slow-growing strain of Rhizobium from Cicer (chickpea). These strains retained their ability to nodulate their normal hosts, but the number of nodules per plant was somewhat reduced. In other strains, nodulation ability cotransferred at greater than 95% with bacteriocin production, a natural marker for pRLlJI (Brewin et al. 1980). R. l eguminosarum plasmids with genes for nodulation exist in several incompatibility groups (Brewin et al. 1982). When conjugated into the same cell, the plasmids either formed cointegrates or one of the plasmids was lost. Three sizes of hybrid plasmids were formed after conjugation of pJB5JI, which codes for pea nodulation and nitrogen fixation genes, into strain T37 of R^_ trif olii . The size of the cointegrate corresponded to a specific Nod and Fix phenotype on pea or clover (Christensen and Schubert 1983). Host-range specifying genes of R_^ l£2.umino sarum were localized in a 10 kb fragment of pRLlJI by using sequences adjacent to Tn5^ i nser ti oni nduced nodulation mutants to select cosmid clones with homology (Downie et al. 1983). The insertion of the 10 kb DNA clone enabled a plasmid-cured

PAGE 19

R. phaseo li strain to nodulate pea but not Phaseo lus vulgaris . The nodules produced on pea were normal appearing and contained typical bacteroids. At least some of the genes encoding enzymes for nitrogen fixatation are on plasmids in fast-growing species. Nuti et al. (1979) showed that cloned nitrogen fixation ( ni f ) genes from Kl ebsie l la pneumoniae , when used as DNA probes to Southern transfer blots of Eco RI -digested plasmid DNA, hybridized to 1 or 2 unique restriction fragments from several EU 1 e guminosarum strains. Hooykaas et al. (1981) reported that one particular plasmid in R^_ trifol ii encoded both nodulation functions and n j^ f genes. This was demonstrated by conjugating the plasmid, designated "Sym," into Nod" Fix" R^_ leguminosarum , which consequently became Nod + Fix + . The "Sym"-plasmid conjugated into a cured strain of A^ tumef acien s enabled it to form nodules on clover but nitrogen fixation did not occur. Similar experiments with "Sym" plasmids from other strains of R^ tr i f o l i i and from strains of R^ l eguminosarum yielded very similar results (Hombrecher et al. 1981, Prakash et al. 1981, Hooykaas et al. 1982). The restriction map of "Sym" plasmids from R. leguminosarum , and DNA-DNA homology studies with the "Sym" plasmid from R^_ trj^o^j^ and the Ti plasmid of A. tumef ac iens , show considerable conservation of sequences (Prakash et al. 1982b). Some of these conserved regions correspond to areas that are transcribed in bacteroids isolated from nodule tissue, and to regions with homology to nif probes (Prakash et al. 1982a). The use of the term

PAGE 20

-12"Sym" for plasmids which code for some of the functions necessary for symbiosis may serve to confuse the issue of the role of chromosomal genes in symbiosis. The plasmid genes have proven to be the most tractable, so have received the greatest attention. Evidence for the necessity of an appropriate chromosomal background for expression of the plasmid is clear (Beringer 1982), and some genetic evidence for the existence of specific chromosomal genes active in symbiosis is developing (Noel et al. 1984). The actual number of genes which are involved in coding for nodulation specific functions is unknown (Long 1984). Most of the products of the "symbiotic" genes are unknown. Zurkowski (1980) correlated the presence of the R. trifo l i i plasmid pWZ2 with the ability of strains to specifically adsorb to clover roots. Cured strains did not bind to the roots but a transcon j ugant did (Zurkowski and Lorkiewicz 1979). When strains with the plasmid were assayed for binding in the presence of 30 mM 2-deoxyg lucose (a hapten of the clover lectin) a reduction of adsorption was observed. Dazzo and Hubbell (1975) described antigenic differences between nodulating and nonnodulating strains of R. tr if ol ii; unfortunately, it is not clear that true sibling strains were used. The possibility of multiple gene differences make it difficult to evaluate the biological significance of the correlation of antigenicity to nodulating ability. Russa et al. (1982) correlated the occurrence of plasmid pUCS202 with differences in

PAGE 21

-13lipopolysaccharides of R^_ trifolii strains, an observation corroborated by Raleigh and Signer (1982), who selected nodulation-def icient R_;_ phaseo l i strains by enrichment of populations for altered surfaces. This was done by screening survivors refractory to infection by phage Fl. These data point to a problem not often addressed in other studies. Raleigh and Signer (1982) found considerable genetic changes in strain physiology in addition to those that had been selected, and it was difficult to identify which of the changes led to the inability to nodulate. Many of the studies reported here are based on the assumption that the only genetic effect of plasmid loss is the alteration of the component under study (.i.e., 1 ipopolysaccharide) , and the component is correlated only to the effect under study (i.e., nodulation), without careful consideration of what other genetic systems may be disrupted by loss of the plasmid. The plasmid may represent a substantial amount of the potential genetic information of the cell and many of the functions are, as yet, cryptic. Simple correlations with assumptions as to cause and effect in such complex systems with considerable potential for pleiotropic effects are injudicious until specific genes and gene products can be manipulated to test cause and effect unequivocally. Although much of the discussion has suggested the hostspecific nature of the genes encoded on the plasmids of strains of these three species of Rhizobium , some genes are clearly common to all of the strains. Djordjevic et al.

PAGE 22

-14(1983) tested the effect of two se 1 f transmi ss ible "Sym" plasmids, one (pJB5JI) from R^_ 1 eguminosar um , the other (pBRlAN) from R^_ tr if o l i i , on the phenotype of various strains of Rhizobium . When conjugated into a cured strain of either R^_ 1 eguminosarum or R^ tr i f o l i i , the pi asm id conferred the expected Nod + Fix + phenotype, j^.e. nodules formed, on the host of the strain from which the plasmid was derived. But either plasmid could restore the nodulating phenotype on clover to two R_;_ tr if o l li strains with Tn5induced "hair-curling" mutations. Neither plasmid restored the wild type to nonmucoid mutant strains of R. tri f o l i i , regardless of whether the mutant was spontaneous or Tn5^induced. The study of the genetics of R^ mel i l ot i has been somewhat slow to develop, but recently it has received more study than the genetics of the other rhizobia. Bechet and Guillaume (1978) provided the first evidence of very high molecular weight plasmids in R^ meliloti . These very large plasmids ( > 300 megadaltons) were visualized and characterized from several strains (Rosenberg et al. 1981). Rosenberg et al.(1982) extended this observation by demonstrating the presence of the very large plasmid in all of the 27 strains that were examined from diverse origins. Heat treatmentinduced deletion mutants that were Nod or Fix" or both and lacked all or part of the very large plasmid (Banfalvi et al. 1981). The nj^f genes of R. meliloti were cloned and selected by homology to n if

PAGE 23

-15sequences from K^ pneumoniae . These then were used to demonstrate by hybridization analysis that most of the Nod mutants lacked at least 24 kb of DNA. The fragments all included several of the nif structural genes as well as some functions essential for nodulation (Banfalvi et al. 1981, Ditta et al. 1980, Corbin et al. 1982). Transfer of sequences from the very large plasmid of R^_ me li loti coding for at least some of the nif genes and some nodulation genes into A_;_ tumef^acj^en^ or E^ coj.^. enabled the recipient bacteria to form nodules or nodule-like structures on alfalfa plants but not on clover (Hirsch et al. 1984, Truchet et al. 1984). Even quite small ( < 8.7 kb ) fragments were active (Hirsh et al. 1985). When the "Sym" plasmid from R^ 1 e guminosarum , which carries genes for uptake hydrogenase activity, was transferred to R^ me li loti , the ability of the recipient to form nodules on alfalfa was not impaired, but the uptake hydrogenase activity was expressed very little, if at all (Bedmar et al. 1984). Transposon mutagenesis has been used to study the genetics of nodulation and nitrogen fixation in R^_ mel iloti . Mead et al. (1982) examined 6000 strains with presumptive Tn5_ insertions for auxotrophy and screened the prototrophs on alfalfa plants. They detected 4 Nod" (0.07%) mutants, 46 Fix" (0.8%) mutants, and 20 (0.3%) auxotrophs, which suggests that there are either very few genes coding essential functions for nodulation or that Tn5_ insertion is not random. Long et al. (1982) described a system for cloning nodulation genes by direct complementation of Nod"

PAGE 24

-16mutants using members of an overlapping cosmid clone bank to map the mutations. Using this system they found that genes essential for nodulation in R^_ meli loti are located on the very large plasmid within 30 kb of nif K (Long et al. 1982, Zimmerman et al. 1983). The transfer of RP4 and R68.45 factors into R^_ meli loti enables the mobilization of the chromosome by conjugation (Kowalczuk and Lorkiewicz 1979). This procedure has allowed the development of linkage maps for chromosomal genes (Kiss et al. 1980, Forrai et al. 1983), which are essentially co linear with those of R^_ 1 eguminosarum and R^_ tr ifo l ii (Kondorosi and Johnston 1981, Beringer 1980). Of 13 Fixmutations mapped, 5 were localized to the chromosome and 8 were extrachr omosoma 1. The chr omosoma 1 ly located Fixmutations were not clustered. None of the Nod" mutants mapped to the chromosome (Forrai et al. 1983). There are several reports of generalized transduction in R^ mel iloti (Kowalski 1967, Sik et al. 1980, Finan et al. 1984, Martin and Long 1984), but transduction has not been used extensively in genetic studies of this bacterial species, perhaps because of the limitation in size of DNA segments which can be transferred (Beringer 1980). Very few of the Nod" mutants have been characterized. Hirsch et al. (1982) reported the infection phenotype of 4 mutants which had previously been derived by Tn5 mutagenesis (Meade et al. 1982). Two of the Nod" mutants did not induce root hair curling or penetrate host cells. The other two

PAGE 25

-17induced root hair curling, and entered root epidermal cells by some means other than infection thread formation. The mode of entry of the bacteria into epidermal cells was not determined, but it was clear that the process did not maintain cell membrane integrity, because bacteria were observed in cell cytoplasm. Understanding of the genetics of the slow-growing strains of R^_ japonicum has lagged behind that for the fastgrowing rhizobia, partly because of the truculence of the slow-growing rhizobia to most of the techniques developed for study of the fast-growing rhizobia. Maier and Brill (1976) treated strain 61A76 with n i tr osoguanidine and screened 2500 colonies on Corsoy soybean. Five mutants were selected on the basis of lack of nodule development or altered nodule appearance (including reduced leghemoglobin) . Two of the five were reported not to form nodules but were competent to fix nitrogen _rn v itro . Three of the five were Fix". It has since been reported that the two strains reported to be Nod are actually Nod + , but nodules are slow to appear (Stacey et al. 1982). Maier and Brill (1978) reported that two strains showed earlier nodulation greater nitrogen fixation, and one of the strains produced more nodules than the parent strain. It is not clear whether these selected strains demonstrated an increase in symbiotic efficiency through simple mutation or whether the changes were partly due to selection for tolerance to the specific growth conditions of the laboratory. Skogen-Hagenson and Atherly (1983) used elevated temperature and either SDS or

PAGE 26

-18ethidium bromide amendment of culture medium, procedures designed to cure plasmids, to produce mutants in strains USDA 74 and 61A76. They reported that none of the strains showed altered plasmid content, but that more than 50 of 133 isolates were symbiotical ly altered. Of those tested none was reported to be auxotrophic. Forty-four were Nod" and ninewere Fix". The exceptionally high ratios of symbiotic mutants to auxotrophs and of Nod" to Fix" make these results unique. If reproducible, the procedure should be very useful for study of Pw_ japonicum genetics. The role of plasmids in R^ japonicu m is not well understood. Gross et al. (1979) developed plasmid profiles for a group of "extras low-growing" strains indigenous to alkaline soils. Each strain had two to four plasmids, with sizes ranging from 48 to 130 megadaltons. Plasmids of 91 and 118 megadaltons were common to all of the extra-slowgrowing strains, but no phenotype was correlated with a particular plasmid. Plasmid content of several slow-growing strains was examined (Haugland and Verma 1981, Masterson et al. 1982). Strains were characterized as usually having one plasmid, but some strains had two or none. When cloned K. pneumoniae nif stuctural genes were used as a probe against r. japonicum plasmid DNA, no hybridization was detected. The nif genes with homology to cloned K^_ pneumoniae genes, nif KDH, have been localized in R^_ j aponicum strain USDA 110 (Hennecke 1981, Fuhrmann and Hennecke 1982, Kaluza et al. 1983, Fuhrmann and Hennecke 1983). Strain USDA 110

PAGE 27

-19is a plasmid-less strain. In contrast to the fast-growing strains of Rhizobium , where the nitrogenase structural genes are clustered in one operon ( nif KDH) , in this strain nif DK represents one operon and nif H is in another operon at least 12 kb away (Kaluza et al. 1983). Hadley et al. (1983) reported that the nif KDH region of K^ pneumoniae , when used as a probe on blots of restricted DNA from 17 slow-growing E.2li^°kiiil!! strains, hybridized to at least two EcoRI fragments from each strain, suggesting that the separation between the operons for nif structural genes is common among a number of strains and not unique to USDA 110. Hahn and Hennecke (1984) used a site-directed mutagenesis technique in which Tn5_ mutagenesis of the cloned R. japonicum nifDK operon was carried out in E^ co.l_i. The operon was transferred by conjugation to R^_ japonicu m by suicide vectors, and stable exconjugants were selected. Mutations within nif D or nif K caused a Nod + Fix" phenotype, whereas Tn5 insertions in the immediate area to either side of nif DK were Nod + Fix + . This is evidence that, unlike R^_ meli loti , the location of nodulation genes may not be closely linked to nif structural genes in R^ j aponicum . Horn et al. (1984) reported general mutagenesis of R^_ japonicum strain USDA 110 with Tn5_ carried on a suicide plasmid. Of ten thousand kanamycin resistant mutants, auxotrophs were detected at a frequency of 0.5%. Two hundred mutants were screened on plants and six Fix" mutants, but no Nod mutants, were detected.

PAGE 28

-20These data suggest that the slow-growing strains have a much different genome arrangement than the fast-growing rhizobia. The strains included in R^ japonicum have not been shown to have the class of plasmids greater than 300 megadaltons which is nearly ubiquitous in the fast-growing rhizobia. In fact, several strains of R^ japonicum seem to have no plasmids at all, and in at least one strain, the genes which are commonly plasmid borne in the fast-growing rhizobia have been mapped to the chromosome. The results of the genetic studies of the fast-growing rhizobia, although providing insight and guidance in the development of suitable experimental approaches for study of R^_ japonicum genetics, should not be indiscr iminant ly generalized to the slow-growing strains without critical evaluation of their broader applicability. Infection and Nodulation of Legumes The colonization of roots by rhizobia (Roc) is generally believed to be a nonspecific phenomenon. At one time it was assumed that a legume specifically enhanced the growth of nodulating rhizobia, and that such enhancement was one of the underlying causes of the observed specificity of nodulation. The results of experiments measuring root colonization have not. yielded clear-cut evidence for such a specific plant effects on colonization. These studies have been reviewed extensively by Fahraeus and Ljunggren (1968) and Vest et al. (1973).

PAGE 29

-21Debate continues as to the role of adsorption of bacteria to roots (Roa) in the specific selection of strains by the plant. The hypothesis that the control of bacterial host range is mediated by the ability of the bacteria to bind to plant surfaces is mostly supported by indirect evidence. Support for this theory is principally in the form of the correlation between the ability of a plant component to bind _in vi tro to strains of rhizobia and the ability of those strains to nodulate the plant. Such experiments have given rise to the "lectin hypothesis" (reviewed by Dazzo and Hubbell 1982). The hypothesis suggests that plant lectins act as highly selective molecules on or near the surface of roots, where'they "recognize" potential microsymbionts by attaching them to reactive sites on the root surface, initiating infection. The ability of the lectin to discriminate between even very closely related rhizobia is thus believed to be the basis of the observed host range of the rhizobia. Correlations of ability of the microsymbiont to bind the host lectin are far from perfect. Even correlations of 5 strains which bind lectin out of 7 strains which nodulate have been cited as validating the lectin hypothesis (Law and Strijdom 1984)! Rarely is an explanation provided for the selective host range of those strains which do not bind lectin in a hostspec i fie manner or those which bind the lectin in a hapten-reversible manner yet do not infect the plant that produces the lectin. Data are less conclusive in relating bacterial host range to ability to adsorb to roots in experiments where

PAGE 30

-22actual numbers of bound bacteria are determined. Experiments designed to determine the amount of bacterial binding are of three general types: i_. measurement of radioactivity of roots to which radiolabled bacteria have been bound, j^i. microscopic estimation of the number of bacteria attached to roots, and ii i . determination of the number of bound bacteria by grinding root segments, plating the grindate, and extrapolating the number of resulting colonies to the number of bacteria bound to the root segments. The relative advantages and drawbacks of these experimental approaches were reviewed by Pueppke (1984a) . A comparison is made in the introduction to Chapter Four of this dissertation of several of those studies as they relate to bacterial host range determination. The "Hac" phenotype refers to curling of root hairs by infective strains of rhizobia. The curled root hairs are often the ones that contain infection threads in soybean (Ranga Rao and Keister 1978, Turgeon and Bauer 1982, Pueppke 1983). Callaham and Torrey (1981) demonstrated in white clover that most infections arise at the inner curve of strongly curled root hairs, but others occur in nearly straight root hairs. Some have postulated the existence of separate "curling factors," although no such factor is well characterized, and experiments using crude preparations have been ambiguous (Ervin and Hubbell 1985). Hubbell (1981) has proposed a model of root hair curling that explains the curling in terms of asymetric disruption of the elongating

PAGE 31

-23root hair cell wall by bacterial enzymatic degradation, suggesting that curling is an immediate consequence of the incipient infection. This is corroborated in studies of infection of alfalfa by A^_ tumef aciens containing cloned genes from a region of the R^ mel i l ot i "Sym" plasmid shown to have functions essential for nodulation (Hirsch et al. 1984). The strains which had a root hair curling phenotype like the wild-type were those which formed infection threads. The strains which produced various deformations of root hairs, readily discriminated from the wild-type phenotype, did not form infection threads. Sutton et al. (1984) cloned genes from R^ japonicum which cause distortion of root hairs of G l ycine soja. From the data they present it is impossible to evaluate whether the root hair "curling" is similar to that seen in root hairs with infections or is some other type of distortion, although their photomicrographs suggest the later. The process of infection thread formation (Inf) was examined in some detail in the small-seeded legumes using techniques of light microscopy which allow direct examination of living plants under nodulating conditions (Fahraeus and Ljunggren 1968, Ljunggren 1969, Li and Hubbell 1969, Callaham and Torrey 1981). In those plants infection thread formation is highly specific; strains which are capable of nodulating a plant are found to form infection threads; conversely, those strains which can form infection threads, with rare exceptions, produce nodules.

PAGE 32

-24The formation of infection threads in soybean was first described by Bieberdorf in 1938. He noted that infection leading to nodulation generally was by means of infection threads in root hairs, though he stated that infection directly through root epidermis occurred on occasion. Direct penetration of soybean root epidermis has not been corroborated, though infection by direct penetration or through natural wounds leads to nodulation of some tropical legumes (Allen and Allen 1940, Ranga Rao 1977, Chandler 1978, Chandler et al. 1982). Infection threads have been described in soybean by Ranga Rao and Keister (1978), Newcomb et al. (1979), Turgeon and Bauer (1982), Pueppke (1983), and Heron and Pueppke (1984). Turgeon and Bauer (In press) described root hair infection of soybean at the ultrastructural level. Pueppke (1983) demonstrated that when eight lines of soybean, four lines of wild soybean, and one cowpea cultivar were inoculated separately with 18 Rhizobium strains, infection threads were formed in all combinations which also formed nodules but were not formed in any nonnodu 1 ati ng combination. The infection threads almost exclusively were formed in root hairs which were distal to the region with mature root hairs at the time of infection . The formation of nodules, the Nod phenotype, though not an early infection event, is often used as a quick and easily observed assay for mutations affecting infection. Although this is understandable, particularly in large screening experiments, too often the presence of nodules is

PAGE 33

-25assumed, prim a faci e, to be evidence that all of a set of specific infection events ha ve occurred. Hirsch et al. (1982, 1984) have demonstrated that nodules or "pseudonodules" are produced on alfalfa by mutant strains of r. meli l oti which have altered infection phenotypes, including Hac" and Inf. In many studies the reason for the relatively small number of Nod" mutants compared to Fix" mutants may be that some mutants had altered infection phenotypes but still produced nodules. Most or all of the steps described above, including additional phenotypical ly defined steps such as release of the bacteria from the infection thread, are essential in legume-microsymbiont combinations with only the root hair infection mechanism. Symbiotica 1 ly deficient nodules that are superficially normal may form even though all of the steps do not occur. It is not yet clear which of the infection steps are necessary for the production of a macroscopic nodule. An additional problem with using nodulation as a screen for infection events is the assumption that infection proceeds only by means of infection threads in root hairs. For several tropical legumes nodulated by slow-growing rhizobia this is not the case (Allen and Allen 1940, Ranga Rao 1977, Chandler 1978, Chandler et al. 1982). In an uncorroborated report Bieberdorf (1938) suggested that soybean could likewise be infected by direct penetration of the root epidermis in addition to infection threads in root hairs. If two pathways were to exist for infection and they

PAGE 34

-26depended on separate sets of genes in the bacterium, screening for mutation in infection by nodule production would cause many of the mutations to be missed due to nodulation by the remaining pathway even when one pathway was blocked. Phenotypic Nodulation Response of Soybean Variation in nodulation phenotypes of soybean cultivars was noted early in the century by Vorhees (1915), who reported that in plots which had been inoculated with either of two commercial soybean inocula, the soybean variety Haberlandt had no nodules while five other varieties were well nodulated. Haberlandt had no nodules, even in plots where it was interplanted with a cultivar which was well nodulated. Vorhees concluded that different varieties of soybean carried different levels of resistance to association with symbiotic bacteria. In an addendum to Vorhees' report, Morse (1915) notes that in subsequent years he observed efficient nodulation of the variety Mammoth in plots that did not support nodulation of the varieties Acme and Tokio. Morse commented that in tests other than that reported by Vorhees, Haberlandt nodulated as well as other varieties, suggesting that Vorhees 1 data were indeed best explained on the basis of strain-specific resistance rather than basic incompatibility of the cultivar to nodulation. In addition, Briscoe and Andrews (1938) observed that differences between the nodulation responses of varieties of soybeans were as great as the differences between cowpeas

PAGE 35

-27and soybeans when reciprocal tests of their rhizobia were made . In a study covering three consecutive years (Caldwell and Vest 1968, Vest et al. 1973), bacteria from nodules on 17 soybean genotypes were serotyped. The cultivar Lee was used as a check variety. The population of strains nodulating the cultivar Pickett was not significantly different from the backcross parent Lee, but many of the soybean lines less closely related were significantly different when the serogroups of strains which formed nodules were compared. This was true even for varieties grown side-by-side. The cultivar Peking was included in this study. Even though it was planted in a field known to have a natural population of mixed strains of R. j aponicum of which strain 110 was a major component, fewer than 1% of the nodules on Peking contained strain 110. And yet, when Peking was inoculated with strain 110 in pots of sterile soil, the plants were nodulated very efficiently. Of course, competition between the rhizobia may account for some of these differences. Nodulation Restrictive Soybean Genotypes In 1954, Williams and Lynch reported the inheritance of a nonnodulating character in soybean. The trait was found in 1947 among breeding selections from a cross between Lincoln and a line selected from a Lincoln X Richland cross. Resistance to nodulation segregated as a single recessive character, with the homozygous recessive plant expressing

PAGE 36

-23the resistance to nodulation. Selfs of both parents and test crosses between the parents all gave the normal nodulating phenotype, indicating that the characteristic apparently had arisen as a mutant. Williams and Lynch (1954) called the gene no and its dominant allele No . Caldwell (1966) renamed these genes r j i and Rjj to conform with general soybean genetic nomenclature. Genotypes conditioning resistance to Rhj.zobj.um infection have been characterized in several legumes, other than soybean. The best characterized phenotypic expression of such a genotype is in red clover (Nutman 1949). Resistance is expressed as lack of any infection threads, although some root hair deformation occurs upon inoculation with R. trif olii. The resistant genotype is described as homozygous recessive, r_r_. A cytoplasmic factor, £, interacts with rr and is inherited in a complex manner. A genotype resulting in a Fix" phenotype is designated ii. The gene _i segregates independently of r (Nutman 1949). Two independently segregating genes affect symbiosis in field peas. The genotype sym ? sym ? conditions resistance to nodulation, and s_y_m3s_y_m3 prevents nitrogen fixation (Holl 1975). A Fix" phenotype is produced in crimson clover ( T. incarnatum ) , irrespective of strain, by the single recessive gene pair rtj^rt^, with possible modifiers (Smith and Knight 1983). The genetic constitution of phenotypica 1 ly Nod" peanut (Arachj.s h yj) P. g_ a_ e_a_ ) requires the independently segregating double recessive gene complement, Hin.in2H2

PAGE 37

-29(Nigam et al. 1980). None of these nodulation resistant genotypes has been characterized at the cellular or biochemical level. It is interesting to note that even peanut, which is infected through natural wounds (Allen and Allen 1940, Chandler 1978), often considered a passive mode of infection, expresses resistance in the recessive genotype. Several soybean genes condition strain-specific Fix" phenotypes. In each case the Fix" phenotype is conditioned by the dominant allele. The R_2 2 allele conditions against strains of the 122 and cl serogroups (Caldwell 1966), the Rj 3 allele conditions against strain USDA 33 (Vest 1970), and the Rj 4 allele conditions against strain USDA 61 (Ham et al. 1971). One or both of the alleles Rj 2 or JL24 are present in 30% of the plant introduction lines, but have generally been selected against in breeding programs and are present in only a few named cultivars (Devine and Breithaupt 1980c, 1981, Devine 1984a). When plants carrying the dominant allele are inoculated with the restricted strains, they produce small nodules or nodule-like proliferations on the roots. Pueppke (1983) showed that the cultivar Hardee, which carries the genotype RJ.2H23 • developed infection threads which appeared to be normal with strain USDA 138 (cl serogroup) , so the block in nodule function appears to occur late in development. Although Rj.2' *Ll3 ' and HJ.4 are often described with rj -i as a group of "nodulation restrictive genes," clear differences are apparent. Whereas the r j-| r j 1 genotype conditions against most strains of R^ japonicum

PAGE 38

-30with no nodules or nodule-like structures formed by restricted strains; the other "nodulation restrictive genes" are restrictive only to a few strains or specific serotypes, and nodules or nodule-like proliferations are formed. Rj.2' Rj 3 , and R J 4 restrict symbiotic effectiveness not nodulation so the term "nodulation restrictive genes" is a misnomer for them. The £2 i^Ji" soybean was originally described as nonnodulating (Williams and Lynch 1954). Clark (1957) found that a few nodules were formed by a few strains when plants were grown hydroponical ly using sand as the support medium, but the plants were incapable of being nodulated in soil. The typical nodulation response in sand culture was about one nodule per plant. Nodulation response was reduced for plants grown in sand amended with soil, and no nodules were formed when the sand was amended with 10% bentonite clay. Isolines of soybean differing at the Rj]^ locus were found in one study of root colonization to harbor approximately equal numbers of rhizobia (Clark 1957). Elkan (1962) later reported larger numbers of rhizobia in rhizospheres of rj^rj^soybean than in its isoline for approximately the first 40 d of plant growth in the field. Clark (1957) reported no differences in the kinds or amounts of amino acids in the two isolines, but Hubbell and Elkan (1967b) noted that roots of uninoculated Rj^-plants contained larger amounts of protein and reducing sugars and smaller amounts of free amino acids than did uninoculated

PAGE 39

-31rj-Lrj^-plants. The biological significance of this latter finding is not apparent in light of Elkan's (1962) earlier data on root colonization, which certainly suggests no basic growth inhibition of rhizobia. Elkan (1961) suggested that the 2L2i _r.il -soybean produced a nodulation-inhibiting excretion capable of a highly significant reduction of nodulation on the Rj-^-genotype. The amount of nitrate added to culture medium for container-grown plants, however, was sufficient to have a potential effect on nodule number. Eskew and Schrader (1977) reexamined the putative nodulation-inhibiting excretion from r_i -^soybean using modifications of Elkan's (1961) experimental design. They found no statistically significant reduction in nodule number due to co-cultivation of nodulating plants with r j-jisolines, but a strong inhibitory effect of nitrate was noted . Hubbell and Elkan (1967a) compared thephys io logica 1 characteristics of strains of R^_ japonicum with differential abilities to nodulate isogenic lines of soybean differing at the Rji locus. High measurable indoleacetic acid formation, low indoleacetic acid destruction, formation of large amounts of capsular material, failure to metabolize nitrate, and failure to reduce triphenyl tetrazolium chloride were properties associated with ability to nodulate both normal and mutant soybean. Stains with the opposite properties generally were able to nodulate only the normal soybean. A mode of infection could not be educed from correlations

PAGE 40

-32between physiological characteristics and nodulation phenotype. Devine and Weber (1977) observed that many R^ japonicum strains capable of overcoming rj 1 -condi tioned resistance to nodulation produced a previously reported soybean foliar chlorosis (Erdman et al. 1956, Johnson and Means 1960). The chlorosis symptoms were ascribed to the formation of rhizobitoxine, 2-amino-3-hydroxypropoxy v i ny lg lyci ne by the bacteria (Owens and Wright 1965, Owens 1969, Giovanelli et al. 1971, Owens et al. 1972). Devine and Weber (1977) suggested that the production of rhizobitoxine might enable the infection of rjiijl" soybean by overcoming strains. This was examined indirectly by Devine and Breithaupt (1980b), who tested the effect of three temperature regimes on nodulation and chlorosis. The effects were opposite, in that the chlorosis symptoms were greatest at the highest temperature (32 C) , but the most nodules were formed at the low and intermediate temperatures (21 C and 27 C). No evidence was found for a diffusible compound capable of endowing the rj^incompatible strains with ability to nodulate rj^rj^-soybean (Devine et al. 1981). The ethoxy analog of rhizobitoxine, when added to bacteria and used to inoculate soybeans in Leonard jars, did not modify the nodulating ability of strains of R^ japonicum on rji £2 \ ~ soybean (Devine and Breithaupt 1980a). Devine (1984a) concludes from these data that rhizobitoxine probably has no enabling role in infection. Devine suggests that the rhizobitoxine is only correlated with the r j L r j 1 -overcoming

PAGE 41

-33strains due to fixation of the separate genetic factors in the same population by "random drift." Devine et al. (1980) determined that the L2iLll~ resistance was not a basic i ncompa t i b 1 i ty with the nonnodu 1 at ing stains. When the strains capable of nodulation and those which were not were mixed and used as inoculum, 32% of the resulting nodules on EJ.iLll~ soybean contained both strains, 36% contained only the usually nonnodulating strain, and 32% contained only the usually nodulating stain. The mode of infection of rj_i.Lii" so y bean has not been determined. Nutman (1981) notes that the rr phenotype of red clover conditions inability of the plant to form infection threads; and, since the soybean resistance to nodulation is likewise a recessive trait, it seems likely to condition a similar block early in infection. Devine (1984a) notes that no nodules or nodule-like proliferations are formed on roots inoculated with incompatible strains, and likewise suggests that the block is early in infection. Tanner and Anderson (1963) examined soybean roots for infection in root hairs but unfortunately were unable to find infection threads in either the rj^rj^-soybean or the normally nodulating line. Perspective The challenge remains to find the point at which the rjj-plant blocks infection and to elucidate the pathway of

PAGE 42

-34infection for those strains which can overcome the r j^r j-] resistance to nodulation. These problems are the focus for the studies reported in this dissertation. The function of temperature on nodulation number and pattern was studied to find the conditions under which infections were most likely to be observed in the rj^rj^-soybean. Tne hypothesis that bacterial adsorption has a role in determining differential nodulation ablilty of strains between normally nodulating and restrictive lines of soybean was tested. The plasmid content of strains was determined and attempts were made to alter the genetic complement of strains, in search of clues to the genetic basis for infection. Finally, roots were examined using light and scanning electron microscopy to determine the phenotype of infection at the cellular level.

PAGE 43

CHAPTER THREE EFFECT OF TEMPERATURE ON NODULATION OF SOYBEAN ISOLINES DIFFERING AT THE Rj^ LOCUS Introduction The nodulation restrictive genotype of soybean, r j]_rj^, identified as a spontaneous mutant in a soybean breeding, program, was reported by Williams and Lynch in 1954. Initial work determined that one genetic locus is involved in conditioning the restrictive phenotype and that the homozygous recessive genotype is required for expression of the trait. The alleles originally were named no and NO for nonnodulating, but since have been redesignated rjj_ and Rj_]_ to conform to currently accepted terminology for soybean genetics (Caldwell 1966). It once was believed that the nodulation restrictive plants are unable to be nodulated (Williams and Lynch 1954, Caldwell 1966). Now it is clear that although most strains of Rhizobium japonicum are unable to nodulate these plants, several strains produce a small number of nodules on plants grown in hydroponic culture (Clark 1957). These strains are called the "overcoming" strains because they overcome the plant resistance. Devine and Breithaupt (1980b) reported a temperature effect on nodulation of the soybean cultivar Clark and its nodulation-restr icti ve isoline Clark-r ji by -35-

PAGE 44

-36two overcoming strains. The trends in nodulation response of both isolines to temperature are similar. The two bacterial strains have different temperature optima for nodulation with the greatest number of nodules per plant formed at 27 C and 21 C, respectively, for the two strains. Fewer nodules were formed at 32 C for both strains (Devine and Breithaupt 1980b). Bhuvaneswari and colleagues (Bhu v aneswar i 1981, Bhuvaneswari et al. 1980, 1981) developed a model for nodulation of soybean. The model predicts that most nodules will be clustered near the point that represents the position of the root tip at the time of inoculation. The model is supported by a correlation between nodule distribution and the position of areas that had immature root hairs or had not yet developed root hairs at the time of inoculation. This developmental model of nodulation is extended by the observations of Pueppke (1983) and Calvert et al. (1984), who demonstrated that the formation of infection threads is the developmental ly restricted event in soybean and two other legumes. In accordance with this model, no nodules are expected to form above the zone of developing root hairs on the primary root (Bhuvaneswari et al. 1980). The objectives of my study were to _i. find the temperature optima for overcoming strains, _H. test the appropriateness to J£J.iL2l~ soybean of the Bhuvaneswari model of transient susceptibility of root cells to infection leading to nodulation, and iii . extend the study (Devine and

PAGE 45

-37Breithaupt 1980b) of the effect of temperature on nodulation of Clark and Clark-rj^ isolines to additional overcoming strains . Materials and Methods The bacteria all were obtained from the U. S. Department of Agriculture, Nitrogen Fixation and Soybean Genetics Laboratory, Beltsville, MD , courtesy of H. H. Keyser, D. F. Weber, and R. Griffin. All bacteria are USDA strains of R. japonicum . The overcoming strains used were 61, 84, 94, and 119. The nono vercoming strain 110 was used as a control in all experiments. The bacteria were maintained at 4 C on yeast extract-manni to 1 agar slants (Vincent 1970). Seeds of Gly cine max (L.) Merr. cultivar Clark-Ll (Rji Rji ) and the nodulation restrictive isoline of Clark-Ll, L63-1889 (r Jirji ) , were obtained from R. L. Bernard, USDA Regional Soybean Laboratory, University of Illinois, Urbana, and D. A. Phillips, Agronomy and Range Science Department, University of California, Davis. The isolines are designated Clark and Clark-rj^ according to the nomenclature of Devine and Breithaupt (1980b). Seeds were surface disinfested by soaking in 50% ethanol for 2 min with agitation, rinsing in deionized water, and then shaking in 0.5% aqueous sodium hypochlorite for 2 min. Seeds were washed for 20 min in running deionized water and were germinated in the dark on water agar plates for 4 to 5 d at

PAGE 46

-3822 C, 27 C, or 32 C, depending on the temperature to be used for nodulation experiments. Inoculum was produced from 50 ml log-phase cultures grown in liquid gluconate-mannitol medium (Bhu vaneswari et al. 1977) at 28 C with rotory shaking at 120 rpm. Bacterial suspensions were centrifuged at 7500 x £ for 10 min and the bacteria were resuspended in sterile nitrogen-free Jensen's plant-growth solution (Vincent 1970). Cell concentration was adjusted turb id ime t r i ca 1 1 y to 5 x 10 8 cells/ml. Seedlings with roots approximately 4 cm long were inoculated by dipping the roots for 10 min in the bacterial suspension. Inoculated seedlings were placed, 2 per pouch, in autbclaved plastic growth pouches (Northrup King Seed Co, Minneapolis, MN) containing 15 ml of Jensen's solution. The surface of the growth pouch was marked at the location of the primary root tip of each plant (Bhu vaneswar i et al. 1980b). This mark was designated the root tip mark (RTM). Plants were grown for 30 d at continuous temperatures of 22, 27, or 32 C with a 12 hr light/dark cycle in a Conviron E-15 growth chamber with 900 uE/m 2 /sec (400-700 nm) irradiance at canopy height. Each temperature experiment was repeated 3 times with 6 plants of Clark and 10 plants of Clark-rj^ tested for each R^ japonicum strain in each experiment. Appropriate control plants sham-inoculated with sterile Jensen's solution were included in each experiment. Plants were watered as needed with deionized water. The distance (to the nearest mm) from the RTM to the root crown and from the RTM to each nodule on the primary

PAGE 47

-39root was measured on each plant at harvest. The number of nodules on secondary roots was counted. Data were analyzed by analysis of variance using the general linear models (GLM) procedure of the Statistical Analysis System (SAS Institute Inc., Cary, NC). Computing was done utilizing the facilities of the Northeast Regional Data Center of the State University System of Florida. Results The mean number of nodules per plant on Clark ranged from 2 to 8 at 32 C, up to 15 to 19 at 22 C (Figure 3.1, Table 3.1). On Clark-rj^ inoculated with overcoming strains, the mean number of nodules ranged from to about 0.2 nodules per plant at 32 C, to from 0.7 to 2.2 nodules per plant at 22 C (Table 3.2, Figure 3.1). Nodules were not formed on Clark-r j 1 inoculated with the nonovercoming strain 110 or on plants sham-inoculated with plant-growth medium. Analysis of variance was conducted using the number of nodules per plant as the dependent variable. In a preliminary test, the responses of Clark and Clark-r jj_ were demonstrated to be significantly different. All subsequent analysis thus was conducted separately for the two genotypes to avoid heterogeneity of variance. Temperature significantly (p < 0.01) influenced nodule number for each plant genotype. F-values for strain, replication, and the interactions of each of the independent variables were

PAGE 48

Figure 3.1. The mean number of nodules formed per plant at three temperatures with five strains of Rhizobium japonicum . Plants were dip inoculated in suspensions (5 x 10 8 cells/ml) of one of the strains indicated, placed in plastic growth pouches, and grown at constant temperature for 30 d. For each strain at each temperature the experiment was replicated three times with six plants per treatment for Clark and ten plants per treatment for Clark-rjy Treatments with Clark are indicated by the solidline, Clark-rj 1 with the broken line. D= strain 61, = strain 84, • = strain 94, 0= strain 110, and A= strain 119.

PAGE 49

-4120-i 19181716151413z 12 —•11 BU --10oo hi 9. _j a 8 65^4321 0o22 TEMPERATURE (C 32

PAGE 50

-42Table 3.1. The effect of temperature on the nodulation of Clark soybean by Rhizobium japonicum Strain Temperature 22 C 27 C 32 C 61 15 ± 6 a 12 i 4 8 ± 3 84 16 = 7 10 ± 3 7 ± 3 94 18 i 5 15 6 2 ± 2 110 16-9 12 i 5 7 ± 3 119 19 ± 7 15 i 6 6 ± 3 a Mean number of nodules per plant for 3 replications with 6 plants per replication ± standard deviation.

PAGE 51

-43Table 3.2. Effect of temperature on the nodulation of Clarkr j ^ Rhizobium japonicum Temperature Strain 3 22 C 27 C 32 C 61 2.2 2.0 b 1.0 ± 1.2 0.1 ± 0.3 84 0.8 ± 1.4 0.9 ± 1.7 0.2 ± 0.7 94 0.9 ± 1.4 0.1 ± 0.4 110 119 0.7 ± 1.1 a Strain 110 is a nonovercoming control. All others are overcoming strains. b Mean number per plant for 3 replications with 10 plants per replication standard deviation.

PAGE 52

-44not significant. A dramatic indication of the effect of temperature on nodulation of Clark-rJ! is provided when the data are expressed as the percentage of plants developing at least one nodule per plant after inoculation with an overcoming strain (Table 3.3). At each temperature a total of 120 plants was inoculated with one of the four overcoming strains; of those, 4% of the plants were nodulated at 32 C, 23% at 27 C and 44% at 22 C. The ratio of primary to secondary nodules ranged from 1:0.6 to 1:0.9 on Clark. On Clark-rjj the ratios were 1:9 at both 32 C and 27 C, and 1:4 at 22 C. Histograms were developed for each interaction of bacterial strain x isoline for each temperature as described for various other legume x microsymbi ont combinations (Bhu vaneswar i 1981, Bhuvaneswari et al. 1980, 1981, Halverson and Stacey 1984, Heron and Pueppke 1984). These are shown in Figures 3.2 and 3.3. In most interactions with overcoming strains, nodules formed well above what has been considered the zone of inf ectibi 1 ity, defined as that area which has only emerging root hairs or no root hairs at the time of inoculation. This type of anomolous nodulation is seen at 22 C and 27 C for combinations of Clark with the strains 61, 84, and 94, each an overcoming strain. A pattern of nodulation similar to those described as fitting the nodulation model of Bhuvaneswari (1981) is seen in the combination of Clark with strain 110, a nono vercoming strain, at 22 C and 27 C. From the nodule profiles

PAGE 53

-45(Figure 3.2), it can be seen that the pattern of nodulation at 32 C of Clark by all of the tested bacterial strains produced flattened peak and a population of nodules displaced downward with respect to the profiles observed at 22 C and 27 C. This downward displacement is clearly evident when the data are expressed as the mean distance of all primary root nodules from the RTM (Table 3.4). The nodule profiles of the overcoming strains on Clark-r j^ showed sparse nodulation down the length of the root from just above the RTM. Discussion The soybean cultivar Clark and its isoline, Clark-r jt , were used by Devine and Breithaupt (1980b) to study the effect of temperature on nodulation. They tested the two overcoming strains USDA 61 (used in this study) and 76. Strain 76 produced the most nodules on Clark-rji at 27 C with few nodules formed at 21 C or 32 C. The combination of Clark-r Jt with strain 61 developed the most nodules at 21 C (7.5), with 6.4 and 3.1 nodules at 27 C and 32 C, respectively. In my study the slope of the regression of a plot — number of nodules versus temperature — was similar to that observed by Devine and Breithaupt (1980b), but the absolute numbers of nodules per plant were lower (Figure 3.1). All of the overcoming strains that I tested responded similarly to strain 61 in the previous study, except strain 84 which had slightly fewer nodules at 22 C than at 27 C.

PAGE 54

-46Table 3.3. The percentage of Clark-rj^ soybean plants nodulated by overcoming strains of Rhizobium japonicum at three temperatures Temperature 22 C 27 C 32 C Strain pri sec any a pri sec any pri sec any 61 23 73 77 10 50 53 3 3 7 84 3 30 30 7 30 30 10 10 94 13 27 40 10 10 119 3 27 30 000 000 Total b 11 39 44 4 23 23 13 a Percentage of 30 plants nodulated (3 replications x 10 plants) at the locations indicated. Pri = percentage of plants with at least one nodule on the primary root. Sec = percentage of plants with at least one nodule on the secondary roots. Any = percentage of plants with at least one nodule. b Percentage of all plants in each temperature experiment with at least one nodule (120 plants per temperature).

PAGE 55

X. U JC 4J U CD u o to 4-> (0 o r-4 «-l W U o 4J ro O O W M O >i » M r(0 CM e W CN CUCM Q! £ 4-> (0 CD O.S H ^J e o c o «J I0 1 •n| en C CD o JZ u o o O JZ ^ jQ 4J K •H 3 WO 1 '-' 4-> >-i O >i X c « CD CD d CD CD W . 0> — a. I! i t u 3 CD U O 4J C (t! CD "3 e .h «8 o o w en en TD (J C *^4J W CD

PAGE 56

-48" III 1H1I lillll'NHIIIIIIIII llillil in II in ill nun ni i ii i l I I— '""I IIMIlMilinilllm ii i urn n e\4 CO i i n i ml hi n l iiltnlil Inn 1 1 I ill L I I Hlll lllllllllllMlllllllllll'hllllll II Mill mi III I ' i ' '"" ii umiinwi nit i i ii ii ii li mi i In ! ""' 'I mil nil in I" i in — i — l— "llllliillllll III llll I I I III iH HIHIIIIIIiIiiIiiiiiiii iliiliih ii i ill CM *

PAGE 57

-49evi as e\i «* ' 3 '"""" ' ' i i i liln i in li I n In liil in i ill in 1 1 l_l_ esi I 'I Hliill iiilliliiililil li i in i i ii es4 CM I llll ll ll lllllll III! 1 1 II I I I i i i ll 1 1 i 1 1 1 il i i ill I I I ill — I I II IN LL evj llililli » 'I I II I II" 'Hill lllllll III UNI llll H I

PAGE 58

U-l

PAGE 59

-51i i i i i i i i i i i CM CO CM CM CM I ii "I I I L

PAGE 60

-52Table 3.4. Mean distance of primary root nodules on Clark soybean from the root tip mark at time of inoculation Temperature Strain 22 C 27 C 32 C 61 16 ± 2 a 13 i 2 61 i 5 84 16 ± 2 16 ± 2 56 ± 4 94 11 ± 2 7 ± 2 51 b 110 16 i 1 14 ± 1 53 ± 7 119 9 ± 1 9 i 1 43 ± 5 a Mean nodule distance in millimeters for 18 plants (3 replications x 6 plants) ± standard error of the mean. Measurements for nodules above the RTM were given a negative value. b Only one nodule produced on the primary root of a plant in 3 replications of this treatment.

PAGE 61

-53At 22 C only the combination of the nonovercoming strain 110 and Clark-r ji failed to produce nodules on at least some of the plants. At 27 C, Clark-r j l failed to form nodules with strain 119 as well as with strain 110, and at 32 C no nodules were formed by strains 94 and 119, as well as 110. The effect of temperature on nodulation of both Clark and Clark-r j L had the same trend (Figure 3.1), although the number of nodules per plant was much different for the two plant types. Nodulation of nonovercoming strain 110 on Clark was affected by temperature in a manner similar to the overcoming strains, but strain 110 did not nodulate the Clark-r j-) isoline at any temperature. Reports have been made on the effect of temperature on numbers of soybean nodules per plant both in the greenhouse and field, as well as under controlled conditions (Devine and Breithaupt 1980b, Munevar and Wollum 1982, Weber and Miller 1972), but this study is the first to examine the effect of temperature on the pattern of nodulation. The pattern of nodulation of strain 110 on Clark soybean at 27 C and 22 C was similar to the pattern reported by others for compatible interactions and follows that predicted by the model of transient susceptibl i ty to nodulation (Bhuvaneswar i 1981, Bhuvaneswari et al. 1980, 1981). The general shape of the profiles for the overcoming strains on Clark is similar to strain 110 on Clark, suggesting that at least most of the nodules that arise are the result of infections constrained by developmental processes to areas of susceptibility, as

PAGE 62

-54defined by the region of the root on which the root hairs are immature or are not yet formed at the time of inoculation. Overcoming strains 61, 84, and 94 produced some nodules well above that region known to be infectible by the model of infection. The presence of these nodules can be explained in two ways. First, immature root hairs may exist in this region and remain infectible after the surrounding root hairs have matured. Alternatively, these overcoming strains may have an additional infection sequence that is not restricted to areas with developing root hairs. If the first is true, one would expect that strain 110 and other nonovercoming strains also would produce nodules in this area, at least occasionally. Since nodules are produced in this area only by overcoming strains, an alternative infection mechanism is suggested. The pattern of nodulation at 32 C of Clark soybean with the five strains of R^ japonicum gave a more flattened curve which was displaced downward relative to those at the lower temperatures. Nodulation generally was lower on the primary root, and some nodules were very far below the RTM. The pattern of nodulation obtained at this temperature looks much like that reported (Halverson and Stacey 1984, Heron and Pueppke 1984) for interactions with fewer nodules relative to other inter act i-ons tested in those studies. Heron and Pueppke (1984) reported a similar pattern on the soybean cultivar Vicoja inoculated with the fast growing R^_ japonicum strain 191. Halverson and Stacey (1984) reported that the delayed nodulating mutant strain HS111 produced a

PAGE 63

-55similar scattered and downwardly displaced pattern on Essex soybean as compared to the pattern obtained with strain 110. An explanation for the striking similarity in the nodulation pattern for all of these interactions is that the they are merely diagnostic for any interaction in which the initial number of successful infections is reduced and plant regulation of additional nodulation is not triggered (Pierce and Bauer 1983). That is, the similarities of the patterns may be coincidental. But the surprising similarity of those nodulation patterns to the ones in this study resulting from restrictive temperature, brings up the question as to what effect temperature might have on those interactions. The soybean cultivar Vicoja was developed at the Universidade Federal de Vicosa, Vicosa, Minas Gerais, Brazil, specifically for local Brazilian conditions, including high temperature. Thus the observed inefficiency of nodulation is perhaps due to assay temperatures below those to which Vicoja is adapted. Similarly, the possiblity that HSlll is a temperature sensitive mutant that has been tested only at restrictive temperatures cannot be ruled out. The nodulation of Clark-r_j 1 is so sparse that interpretation of the nodule profile is difficult. There is certainly no clear peak in the histogram near the point corresponding to the RTM. Whether such a peak would become evident if much greater numbers of plants were examined is uncertain, but seems unlikely; the pattern appears to be scattered and random. Although these data are insufficient

PAGE 64

-56to either validate or invalidate the application of the model of Bhuvaneswari (1981) to Clark-rj^, these data show only limited correspondence with the expected pattern of nodulation predicted by the Bhuvaneswari model. The scattered and sparse nodulation and the reduced correlation of nodulation with the region near the RTM are likely to make the elucidation of early infection events in the nodulation restrictive rJLlJLil" soybean that much more challenging.

PAGE 65

CHAPTER FOUR ADSORPTION OF STRAINS OF RHIZOBIUM JAPONI_CUM WITH DIFFERENTIAL NODULATING ABILITY TO ROOTS OF SOYBEAN ISOLINES THAT DIFFER AT THE Rj_ 1 LOCUS Introduction The ability of rhizobia to bind to toots of their legume hosts has long been assumed to have a principal role in the specificity of the infection process and is believed to be a major determinant of host range (Bhuvaneswar i 1981, Dazzo 1981). These assumptions often are based on the correlation between binding of some component of a plant to a microbe _in v itro , and the ability of that microbe to infect the plant (Dazzo and Hubbell 1975, Robertson et al. 1981). These and other indirect tests have been interpreted as evidence for a direct role of binding in determining host range of microsymbionts. The tests have included studies of cross-reacting antigens on plant and bacterial surfaces (Bishop et al. 1977, Dazzo and Hubbell 1975), the determination of number of nodules formed after various substances were added to plant roots with inoculum (Halverson and Stacey 1984), and tests for lectin binding to bacteria and the search for those lectins on or in plant roots (Bohlool and Schmidt 1974, Bhuvaneswari et al. 1977, Dazzo et al. 1978, Stacey et al. 1980, Gade et al. 1981, Law et al. 1982, Law and Strijdom 1984). Although these studies -57-

PAGE 66

-58have not always addressed the subject of bacterial binding to plant roots, they have been interpreted in terms of the role of adsorption. The underlying assumptions are U that the examined process is a necessary antecedent to adsorption, or ji^. that a tested factor has a direct intermediary role in adsortion. Unfortunately, these assuptions have not been tested. Measurement of bacterial binding per se has been reported with mixed results in terms of its perceived role in host-range determination. Broughton et al. (1980) tested binding of 3 5 S r ad i o i so tope1 abe 1 1 ed strains of R. leguminosarum to roots of Pi sum sativum . Their results are difficult to evaluate due to the very low specific activity of labelling attained and the high variability between experiments. They concluded that ability to adsorb to plant roots was not a determining factor in the differential ability of the bacteria tested to nodulate cultivars of pea. This conclusion was corroborated by microscopic examination of inoculated pea roots (Broughton et al. 1982). Chen and Phillips (1976) used 32 P-radioisotope-label 1 ing of several rhizobia to test adsorption to severed root segments in vitro . Considerable radioactivity was taken up by the root segment tissues during incubation with the radioi sotopelabelled bacteria. This fact and the artificiality of their assay conditions confound the conclusions. Their data tend to discredit the role of adsorption in host range

PAGE 67

-59determination, because binding between their bacterial strains and plant roots was rather nonspecific. Light microscopic examination of binding, either with transmitted light or fluorescence labelling techniques, has been used as an assay for adherence. From such studies qualitative rather than quantitative data are generally reported. Dazzo and Brill (1979) reported specific adherence of R^ trifo lii strain 0403 to root hairs of Trifolium repens . A strain of Azotobacter vinelandii that had been transformed with R. tri fol ii DNA and selected for antigenic cross-reactivity with Tj_ repens also bound. They reported that an A^ vinela ndii revertant "did not adhere." Chen and Phillips (1976) reported that no differences were apparent between the binding of f luoresescentlabe 1 led R^ leguminosarum to roots of pea, which it normally nodulates, and the roots of several legumes which it does not nodulate. Conversely, a direct role in adsorption has been inferred by Stacey et al. (1980) from their light microscopic and scanning electron microscopic examination of the binding of rhizobia to soybean (Gly cine max ) and wild soybean (Gj_ soja ). Unfortunately, they tested various haptens of lectins for their effect on binding but not on nodulation. The assays involved adsorption of rhizobia to the elongated root hairs, which are not believed to be normally infected in soybean (Bhuvaneswar i 1981, Pueppke 1983, 1984a). Using similar binding assays in another study, Stacey et al. (1982) reported that, of a number of

PAGE 68

mutant R^_ japonicum strains with genetic lesions affecting nodulation, only two failed to bind to soybean root hairs. Two studies have measured bacterial binding to roots of soybean directly (Law et al. 1982, Pueppke 1984b). Law et al. (1982) determined the number of cells of mutant isolates which bound to 1 cm segments of excised soybean root. The root pieces were incubated for 1 hr in a dilute bacterial suspension, gently washed, ground, diluted, and plated. Between 1000 and 2300 bacteria bound per root segment. Unfortunately, nodulation cannot be compared to binding using inoculation conditions similar to those used in this assay since the binding assay uses severed roots. The loss of plant sap from the cut ends may also affect the number of bacteria bound. These problems were answered in the procedure devised by Pueppke (1984b), in which intact seedlings were suspended with their roots dangling in bacterial suspensions. After timed incubation the plants were removed and rinsed vigorously. A root segment was removed, ground, and plated for determination of colony forming units. Adsorption to seedling roots of soybean and cowpea by one fast-growing and four slow-growing rhizobia was independent of plant species and of the ability of the strains to nodulate these hosts. This procedure (Pueppke 1984b), although more cumbersome than the previous assay (Law et al. 1982), allows examination of root adsorption in a system similar to a commonly used inoculation protocol. The effect of the binding and rinsing conditions on

PAGE 69

nodulation can be tested by creating seedlings and then transferring them to plastic growth pouches. The objective of ray research was to compare the binding of rhizobia to soybean roots, with the known abilities of the Rhizobium strains to nodulate specific genotypes of soybean (Devine 1984a). The study used the procedures developed Pueppke (1984b) to enable direct assay of bacterial binding to living plants. The assay conditions were similar to the customary inoculation procedure used for nodulation studies. Nearisogenic lines of soybean that have differential ability to form nodules with the R^_ japonicum strains were utilized. Two temperatures known to affect the number of nodules formed on these isolines (Devine and Breithaupt 1980b, Chapter Three) were tested. Materials and Methods The USDA strains 94 and 110 of Rhizobium japonicum were obtained from the USDA Nitrogen Fixation and Soybean Genetics Laboratory, Beltsville, MD , courtesy of H. H. Keyser, D. F. Weber, and R. Griffin. They were maintained on yeast extract mannitol (YEM) agar (Vincent 1970) slants at 4 C. Cultures for adsorption studies were grown at 28 C with shaking in liquid defined g luconate-mann i to 1 medium (Bhuvaneswari et al. 1977). Bacterial concentration was estimated turbid imetr ica 1 ly. Bacteria were pelleted by centrifugation at 7500 x g_, washed once in sterile filtered nitrogen-free Jensen's plant growth solution (Vincent 1970), repelleted, and resuspended in Jensen's solution at

PAGE 70

-62approximating 1 x 10 4 bacteria/ml. Aliquots were plated on YEM agar for determination of colony forming units to quantify viable bacteria in the inoculum. Seeds of Gl ycin e max (L.) Merr. cultivar Clark-Ll (RJlRJj) and its isoline, L63-1889 (rj 1 rj 1 ) y were obtained from R. L. Bernard, USDA Regional Soybean Laboratory, University of Illinois, Urbana, and D. A. Phillips, Department of Agronomy and Range Science, University of California, Davis. In this study the terminology of Devine and Breithaupt (1980b) is followed; the term Clark is used for the parent cultivar Clark-Ll, and Clark-r j^ designates the isoline L63-1889 carrying the nodu 1 at ion-restr icti ve genes. The nodu 1 at ion-restr ict i ve genotype conditions resistance to nodulation by most strains of R. japonicum , including strain 110. Strain 94 is one of the "overcoming" strains which overcome rj^-res istance and form a few nodules. Clark is nodulated abundantly by both strains. Seeds were surface disinfested by soaking them in 50 percent ethanol for 2 min, rinsing, shaking in 0.5% aqueous sodium hypochlorite for 2 min, and rinsing in running deionized water for 20 min. Seeds were germinated on water agar for 4 d at 28 C in the dark. Seedlings lacking bacterial or fungal growth were placed, three per pouch, in autoclaved plastic growth pouches (Northrup King Seed Co, Minneapolis, MN) containing 15 ml of Jensen's solution. Pouches were covered with plastic sleeves to maintain sterility. Seedlings were placed under fluorescent lights

PAGE 71

-63with a 12 hr light/dark cycle at 450 uE/m~2/sec irradiance for 1 d to allow root elongation. The adsorption assays ^ere completed as previously reported (Pueppke 1984b). All assays were done in a laminar flow hood under aseptic conditions. Bacterial inoculum (25 ml) was placed in each of sixteen 100 x 25 mm sterile test tubes. Two bent paper clips were hooked on each test tube rim, each to support a seedling. Seedlings were suspended with the roots immersed in the bacterial suspension. After 30, 60, 90, and 120 min, four sets of two seedlings were harvested. The roots of each seedling were washed vigorously with 25 ml of sterile filtered Jensen's solution delivered from a Brinkman Dispensette. The solution was delivered with the maximum possible stream that would still run down around the root when the seedling was held intersecting the stream. Each root was severed 2 cm from the root tip. The distal segments of the two roots from each tube were ground together in a ground-glass tissue grinder in 1 ml of sterile Jensen's solution. The resulting pulp was appropriately diluted and five 0.1 ml aliquots were plated on YEM agar plates. The plates were incubated in the dark at 28 C, and the colonies were counted after 7 to 10 d. In each experiment, for each plant type at each time, there were four replications represented by four tubes. Each experiment was repeated at least five times for each strain x plant combination. The assays described above were completed at an ambient air temperature of approximately 27 C. Additional assays

PAGE 72

-64were completed for the strain 94 x Clark-r j-^ combination at 22 C. All procedures were completed as described, except that the test tubes with inoculum were placed in a water bath and equilibrated to 22 C for 30 rain before seedlings were added. Temperature was monitored carefully during the assay to maintain 22 0.5 C. Data were collected as the mean number of colonies formed on five replicate plates spread with suspensions resulting from grinding each set of root segments. The data were normalized to correct for experiment-to-experiment variation in actual inoculum density. This was accomplished by dividing treatment means by a ratio representing the turbidimetr ical ly estimated inoculum (1 x 10 4 ) divided by the actual colony forming units in the inoculum. Normalized data are expressed as the number of bacteria adsorbed per plant. Two kinds of control experiments were conducted. Known amounts of bacteria were ground with root tissue to determine the effect of grinding and of plant tissue constituents on numbers of bacteria producing colonies on YEM. The other control tested the effect of adsorption assay conditions on nodulation. Seedlings that had been incubated with bacteria were washed as described above and then directly placed into prepared growth pouches and maintained in a Conviron E-15 growth chamber at 22 C for 20 d with 900 uE/m 2 /sec irradiance (400-700 nm) with a cycle 12 hr light and 12 hr dark.

PAGE 73

-65Results In the adsorption assay, both bacterial strains bound to Clark soybean roots in roughly similar numbers with a near linear increase with time. Approximately 100 bacteria were bound per plant at 2 hr (Figure 4.1). The capacity of Clark-r j-. to adsorb overcoming strain 94 was very similar to that of Clark. Somewhat surprising was the binding of greater numbers of bacteria in the nonnodulating combination of Clark-rj-, x strain 110. In this combination, the mean number of bacteria bound per plant was nearly 100 at 30 min, about twice the number bound in the Clark x 110 combination. A reduction in assay temperature from 27 to 22 C markedly decreased the binding of strain 94 to the isoline Clark-rj^ (Figure 4.2). After 1 hr the number bound at 27 C was 59 ± 4 (i SE), but at 22 C was 15 1, about a 75% reduction. After 2 hr, the number bound at 27 C was 105 8 but for 22 C was 21 2, an 80% reduction. The effect of these temperatures on adsorption was opposite to their effect on nodulation. At 22 C this plant-strain combination had a greater number of nodules, a greater percentage of the plants with nodules, and a greater number of nodules on the primary root than at 27 C (Tables 4.1 and 4.2 [data from Chapter Three] ) . Controls in which inoculum with known numbers of bacteria was ground with either type of root tissue produced colony counts which were not significantly different from

PAGE 74

-66each other or from those plated directly from the inoculum. Controls to test the suitability of the conditions of the adsorption assay for nodulation were examined 20 d after placing seedlings in growth pouches. The seedling had been incubated in inoculum for 120 min and washed in parallel treatments to seedlings in adsorption assays. The mean number of nodules per plant for each combination is presented in Table 4.3. Only the combination of strain 110 x Clark-rj-L and the seedlings incubated in sterile Jensen's solution without bacteria failed to form nodules. The following number of plants were nodulated in the other combinations: Clark-rj 1 x strain 94, 1 of 8; Clark x strain 110, 8 of 8; and Clark x strain 94, 8 of 8. Thus, it is clear that adsorption was assayed under conditions which were conducive to infection leading to nodulation. Discussion One objective of this study was to determine whether the nodulation restrictive genotype, Clark-JQi/ reduces the ability of the plants to adsorb nono vercomi ng strains of Rhizobium , thus influencing bacterial host range. This is not the case, because after 2 hr Clark-rj^ adsorbed more of either R. japonicum strain tested that did Clark. In fact, the ranking of plant type x strain combinations based on numbers of nodules is precisely opposite to their ranking by numbers of bacteria adsorbed at 2 hr. Although no biological significance is apparent, this inverse

PAGE 75

Figure 4.1. Adsorption of cells of Rhizobium japonicum to soybean roots. The experiments were completed at 27 C. Each point represents the mean from five experiments with four pairs of plants tested at each time for each plant-strain combination at each replication of the experiment. The roots of soybean seedlings were incubated in bacterial suspensions (1 x 10^ cells/ml) for the times indicated and rinsed vigorously. The terminal 2 cm of the primary roots were excised, ground and plated for determination of colony forming units. = Clark x strain 110, = Clark x strain 94, O = Clark-rJ^ x strain 110, and • = Clark-r j^ x strain 94. Bars represent the standard error of the mean.

PAGE 76

-68150 30 60 90 120 MINUTES

PAGE 77

Figure 4.2. Adsorption of cells of Rhizobium japonicum strain 94 to roots of Clark-rj^ soybean at two temperatures. Each point represents the mean of four pairs of plants tested at each time for each temperature from each of five replications of the experiment. The roots of seedlings were incubated in bacterial suspensions (1 x 10 4 cells/ml) equilibrated at 22 C or 27 C for the times indicated and rinsed vigorously. The terminal 2 cm of the primary roots were excised, ground and plated for determination of colony forming units. V = 22 C, and • = 27 C (The 27 C curve is duplicated from Figure 3.2.). Bars represent the standard error of the mean.

PAGE 78

150 100 30 60 MINUTES

PAGE 79

-71Table 4.1. Percentage of plants nodulated at 27 C and 22 C Combination Primary Secondary Any Root 27 C 22 C 27 C 22 C 27 C 22 C Clark x 94 19 a 100 89 100 100 100 Clark x 110 89 100 50 89 100 100 Clark-r j x x 94 Clark-rj-L x 110 13 10 27 10 40 a Data in table from Chapter Three. Percentage of plants nodulated at each indicated location. Data are for all plants from 3 replications at each temperature with 6 to 10 plants per replication.

PAGE 80

-72Table 4.2. Mean number of nodules per plant at 27 C and 22 C Primary Secondary Any Root Combination 27 C 22 C 27 C 22 C 27 C 22 C Clark x 94 |3 7.9 5.8 10.2 14.6 18.1 Clark x 110 8.3 8.9 3.6 6.8 11.9 15.7 Clark-r j l x 94 Clark-rj-L x 110 0.3 0.1 0.5 0.1 0.9 Data in table from Chapter Three. Mean number of nodules per plant on primary roots, secondary roots, or on any root of the plant as indicated. Each value represents the average for 3 replications at each temperature with 6 to 10 plants per replication.

PAGE 81

-73Table 4.3. Nodulation of plants inoculated with Rhizobium japonicum under the conditions of the adsorption assay Bacterial strain Soybean isoline 94 110 Clark 8.3 ± 5.7 a 6.5 ± 2.1 Clark-rji 0.1 ± 0.4 Seedlings were inoculated by dipping the roots in a bacterial suspension containing approximately 10 cells/ml for 2 hr. The roots were rinsed vigorously. Plants were grown for 20 d in plastic growth pouches (see Materials and Methods for detailed description of procedures) .

PAGE 82

-74relationship underscores the rejection of the hypothesis that host range is primarily dependent on ability of rhizobia to adsorb to host roots, at least for the differential nodulation of Clark and its isoline, Clark-r j-^. Clark (1957) reported that similar numbers of rhizobia were recovered from the roots of plants carrying the r j ^ r j [ genotype and plants that carried a nonrestr icti ve genotype whether they were grown in a greenhouse or in the field. Elkan (1962) demonstrated that Clark-r j-^ actually maintained substantially higher populations of rhizobia in rhizosphere soil in the field than did Clark for 45 out of the first 60 d of plant growth. These observations, in conjunction with the results of the present study, suggest that the limiting step in nodulation of these plants occurs post-adsorption. Pueppke (1984b) showed that adsorption of R^_ japonicum strain 138 to roots of the soybean cultivar Hardee is temperature sensitive; when that combination was subjected to assay temperatures of 4 C, 27 C, and 37 C, the optimum binding temperature was 27 C. The number of bacteria bound per plant after coi ncuba t i on for 1 hr was reduced approximately 90% at 4 C and approximately 65% at 37 C, compared to the number bound at 27 C. In the present study, the binding of strain 94 to roots of Clark-r j-^ similarly was temperature-sensitive. The reduction in the number of bacteria bound per plant in 1 hr with a drop in assay temperature from 27 C to 22 C was about 75%. The large reduction in adsorption with only a 5 degree temperature

PAGE 83

-75difference suggests that this combination is either more highly temperature sensitive than the combination used in the previous study (1984b), or that perhaps the curve of temperature sensitivity for both combinations is very steep on either side of an optimum temperature. The shape of the response curve has not yet been determined, and would require testing adsorption at considerably more temperatures than have been used as treatments in either report. For the combination of Clark-rj^ with strain 94, the response of adsorption to temperature is opposite to that of nodulation to temperature for the same (Tables 4.1 and 4.2 [data from Chapter Three], Devine and Breithaupt 1980b). Although 40% of the plants were nodulated at 22 C, only 10% were nodulated at 27 C. The number of bacteria bound per plant after 2 hr at 22 C was 21 2. At 27 C 105 8 bacteria were bound, a five-fold increase. Clearly the temperature effect on nodulation of these plants is independent of its effect on binding. The following conclusions are drawn from the data presented in this report: ^. Compared to 27 C, 22 C favors nodulation in the Clark-rjj^ x strain 94 combination, whereas, the effect of temperature on adsorption is precisely the opposite. _i_i. There is not a qualitative difference between the adsorption of strains 94 and 110 to Clark and Clark-rj^ soybean roots. _ii__i. The rates of adsorption in these combinations are similar to the rates reported for other strain x soybean cultivar combinations

PAGE 84

-76(Pueppke 1984b). _iv. Under these experimental conditions, there is no correlation between the number of rhizobia bound to roots and the extent of nodulation of Clark or Clark-r j-^ soybean .

PAGE 85

CHAPTER FIVE INFECTION OF SOYBEAN ISOLINES DIFFERING AT THE Rjj LOCUS BY RHIZOBIUM JAPONICUM STRAINS WITH DIFFERENTIAL NODULATING ABILITY Introduction The nodulation-restricti ve genotype of soybean, r j -^ r j p originated as a field mutant in a soybean breeding program (Williams and Lynch 1954). The phenotype was first characterized as nonnodu 1 a t i ng with Rhi zobium j aponicum (Williams and Lynch 1954), but Clark (1957) reported that a few strains, called overcoming strains, form a few nodules on plants growing in sand or vermiculite but not soil. Several investigators have sought to identify the factor that differentiates rj^rjj-plants from t hose that nodulate normally. The amino acid content of both plant types has been compared and found to be similar (Clark 1957, Hubbell and Elkan 1967b). The restrictive step is not the inability of the r j-| r ji -plants to support rhizobia in the rhizosphere; such plants support populations equal to (Clark 1957) or greater than (Elkan 1962) those supported by a nearisogenic but normally nodulating soybean line. Devine and Weber (1977) determined that successfully nodulated xJllAl P lants were capable of fixing nitrogen. From these studies, Devine (1984a) inferred that the incompatibility conditioned by the -77-

PAGE 86

-78JLi]_r_j_]_ genotype is not a general antagonism to Rhi zobium metabolism and function, but that the answer to incompatibility is likely to be found in early stages of the infection process. A number of investigators have attempted to elucidate the bacterial property that enables a few strains to overcome the plant's resistance and produce some nodules. Hub bell and Elkan (1967a) reported that several physiological characteristics of R^ japonicum strains were correlated with the ability to nodulate r j^r j-| -soybean, but none of the factors was implicated in infection. Devine and Weber (1977) noted that the ability of bacteria to nodulate the £J_ -|_ r_ j_ ]_ genotype was highly correlated with the production of a bacterial metabolite that induces chlorosis in several soybean lines. The bacterial product was latter shown to be rhizobitoxine (2-amino-3-hydroxypropoxyv iny lg lyci ne) (Owens et al. 1972). They postulated that this chlorosis-causing agent had an enabling role in infection of r^r^soybean. Devine and Breithaupt (1980b) showed that processes leading to nodulation and to the expression of chlorosis had different temperature optima. When added to a Rhizobiura inoculum, an analog of rhizobitoxine did not enhance nodulation of the restrictive soybean (Devine and Breithaupt 1980a). No diffusible compound was detected in tests for a factor to endow r j incompatible strains with the ability to nodulate the rj^rj^-soybean (Devine et al. 1981). Devine concluded that the correlation of rhi zobitox ineinduced chlorosis and

PAGE 87

-79ability to form nodules on the restrictive soybean is likely incidental and "not the result of an intrinsic physiological relationship" (Devine 1984a [p 150]). Infection of the small-seeded temperate legumes has been studied in the most detail due to the development of the Fahraeus slide technique enabling study of the root surface of living plants with the light microscope (Nutman 1981). These plants, which are are nodulated by fastgrowing rhizobia, are believed to be infected exclusively through infection threads in root hairs (Fahraeus 1957, Nutman 1959, Ljunggren 1969, Callaham and Torrey 1981). Some tropical legumes, such as peanut ( Arachis hypogaea L.) and several species of Sty lo santhe s , are infected through natural wounds caused by emergence of lateral roots, with no infection thread formation in root hairs (Allen and Allen 1940, Ranga Rao 1977, Chandler 1978, Chandler et al. 1982). In the tropical genus Lotus , one species (L. cornic ul atus L.) was reported to be infected only by means of infection threads in root hairs, but in another (L^ hi spidus Desf.) most nodules originated by bacterial penetration directly through the epidermis, and infected root hairs were rare (Ranga Rao 1977) . In 1938, Bieberdorf described the infection process in soybean in considerable detail. He was the first to note that infections usually progress via infection threads in soybean, but he states that rhizobia also infect directly through root epidermal cells. Apparently, corroboration of

PAGE 88

-80the initial report of root hair infection of soybean was not made until fifty years later (Ranga Rao and Keister 1978) and direct infection processes have not been corroborated. Several studies of nodule structure have included some details of early infection (Cabezas de Herrera and Fernandez 1982, Goodchild and Bergersen 1966, Newcomb et al. 1979). Pueppke (1983) recently examined soybean in nodulating and nonnodulating combinations with eighteen strains of rhizobia for signs of early infection. He showed that infection threads were formed exclusively in nodulating combinations and that infection threads were restricted to locations distal to the region of the root on which root hairs were fully elongated at the time of inoculation. A thorough description of root hair infection at the u 1 t r as tr uc tura 1 level was recently reported by Turgeon and Bauer (Turgeon and Bauer 1982, In press). Neari sogen i c lines of soybean were examined in this study to determine whether the difference between the phenotypes of normally nodulating soybean and nodulationrestrictive (r j-i r ji ) soybean were expressed at the level of infection. Both Nutman (1981) and Devine (1984a) have suggested that the r_ jirj_i soybean might be infected exclusively by means other than infection threads in root hairs. The mode of infection of rj.li.2l soybean has not previously been reported.

PAGE 89

Materials and Methods The bacteria utilized were R^_ japonicum strains 94 and 110 obtained from the U. S. Department of Agriculture Nitrogen Fixation and Soybean Genetics Laboratory, Beltsville, MD, courtesy of H. H. Keyser, D. F. Weber, and R. Griffin. Strain 94 is an overcoming strain that forms a few nodules on the nodulation-restr icti ve isoline, but forms abundant nodules on Clark (Chapter Three). Strain 110 nodulates Clark abundantly but does not nodulate the restrictive isoline (Chapter Three). Rhizobia were maintained on yeast ex tr act-mann i to 1 (YEM) agar (Vincent 1970) slants at 4 C, and stock cultures were transferred about every three months. Bacteria for inoculation were grown to mid-log phase at 28 C with shaking in 50 ml liquid defined g luconate-manni to 1 medium (Bhu vaneswar i et al. 1977). Cell number was estimated tur b i d ime t r i ca 1 1 y . Cultures then were centrifuged at 7500 x £ for 10 min and bacteria were resuspended at a concentration of approximately 5 x 10 8 cells/ml (except where noted) in sterile Jensen's solution (Vincent 1970). The plants were two neari sogenic lines of soybean Glycine max (L.) Merr., the cultivar 'Clark-Ll' (R^R^) and its isoline L63-1889, which carries the nodulationrestriction genotype, rj^ r j] . The isolines will be referred to as Clark and Clark-rj^, respectively, following the terminology of Devine and Breithaupt (1980b). Seeds were obtained from R. L. Bernard, USDA Regional Soybean Laboratory, University of Illinois, Urbana, and D. A.

PAGE 90

-82Phillips, Department of Agronomy and Range Science, University of California, Davis. Seeds were soaked in 50% aqueous ethanol for 2 min with agitation, rinsed with water, soaked in 0.5% aqueous sodium hypochlorite for 2 min with agitation, and rinsed for 20 min under running deionized water. Surface disinfested seeds were germinated at 22 C in the dark for 5 d on water agar plates. Inoculation and seedling transfer were completed in a laminar flow hood using procedures designed to maintain sterility. Germinated seedlings were carefully selected for lack of any sign of contaminating microorganisms and for uniform size (ca. 4 cm, excluding cotyledons). Seedlings were inoculated by immersing the roots in the bacterial suspension for 10 min. Seedlings were placed, 2 per pouch, in plastic growth pouches (Northrup King Seed, Co., Minneapolis, MN). Growth pouches had been prepared by adding 15 ml nitrogen-free Jensen's plant growth solution to each and autoclaving. Control plants were sham-inoculated with sterile Jensen's solution only. A mark, called the root tip mark (RTM), was made on the growth-pouch surface at the position of the primary root tip. Except where noted, the tops of growth pouches were covered with plastic sleeves for several days to prevent contamination. Plants were grown at 22 C in a Conviron E-15 growth chamber at an irradiance of 900 uE/m 2 /sec (400-700 nm) with 12 hr light, 12 hr dark.

PAGE 91

-83Root surfaces were examined by light microscopy for curled root hairs of the type referred to previously as "question mark shaped" (Pueppke 1983), and for the presence of infection threads. Plants used in light microscopy experiments were grown essentially as stated above, except that seeds occasionally were germinated and plants grown at other temperatures and light intensities. Growth pouches were not covered during plant growth. Plants were sampled for light microscopy as previously described (Pueppke 1983) by stripping away very thin strips of tissue from root surfaces for several centimeters on either side of a point corresponding to the RTM. Two to four strips were made for each plant. These collectively included almost all of the root surface in the region of the RTM. Root surfaces of 18 Clark plants inoculated with strains 94 or 110, and 27 plants of Clark-r ji inoculated with strain 94 were examined. These strips were mounted in phosphate-buffered saline (Bhuvaneswar i et al. 1977) with or without prior staining with toluidine blue and were examined using bright-field or interference-contrast optics (Hoffman modulation). The entire strips were scanned for short, curled root hairs and infection threads. Light microscopy also was used to examine the surface of nodules for residual curled root hairs and infection threads. Plants were examined for very young developing nodules. The nodules were removed from the plants and a thin layer of the nodule surface distal to the plant was sliced away with a sharp scalpel. These thin

PAGE 92

-84disks of tissue were mounted and examined as described for root surface strips. Samples for scanning electron microscopy (SEM) were obtained from 10 day-old plants by severing the primary root with a scalpel at the RTM and at 10 mm above the RTM. Secondary roots attached to these sections were severed at a line parallel to the primary root, at 5 mm perpendicular from the primary root. The root sections immediately were placed into a fixing solution consisting of 4% g lutaraldehyde (Eastman Kodak Co.) in 50 mM sodium cacodylate buffer (pH 7.4). After fixation overnight at 4 C, samples were rinsed with distilled water and post-fixed overnight at 4 C in 2% aqueous osmium tetroxide. Sections were washed in 5 changes of distilled water and dehydrated progressively in a graded water-ethano 1 series with never more than a 10% increase in ethanol concentration per step or less than 15 min per step. A faster dehydration or steeper series caused distortion. The samples were transferred in anhydrous ethanol to a critical point dryer (Balzers Model H) and dried from liquid carbon dioxide. Dried samples were attached to Cambridge mounts (Ernest F. Fullam, Inc.) with double-sided tape, sputter-coated with ionized gold, and examined with a Hitachi S-450 scanning electron microscope at 20 kV. Two or more groups of 5 plants were sampled and fixed separately for each combination of Clark x strain 110, Clark x strain 94, and Clark-rj^ x strain 94. Additional plants were examined for some combinations. Two uninoculated Clark

PAGE 93

-85control plants and two of the Clark-r^ x strain 94 combination that had been inoculated at 1 x 10 10 cells/ml were examined. Root sections were examined using SEM at low magnification (50x) and photomicrographs were made of the entire root segment to enable the mapping of higher magnification photomicrographs for interpretation in context. Nodule surfaces and root surfaces were examined. All samples were scanned at 150x and 250x for curled root hairs of the type previously identified by light microscopy to harbor infection threads. Results Clark soybean inoculated with either of the fully compatible strains 110 or 94 at 5 x 10 8 cells/ml had short, tightly curled root hairs (Figure 5.1), many containing infection threads. The tightly curled root hairs with infection threads sometimes occurred in clusters. A number of root surface strips were examined from 27 plants for the combination Clark-rj^ x strain 94, which was previously demonstrated to produce a few nodules (Chapter Three). No short, tightly curled root hairs similar to those seen in the fully compatible combinations were found and no infection threads were ever observed. Light microscopy was used to examine nodule surfaces for residual root hairs with infection threads. Thirteen of twenty sections of nodule surface tissue examined for the

PAGE 94

Figure 5.1. Curled root hair on Clark soybean 10 days after inoculation with Rhizobium japonicum strain 110. Phase contrast optics.

PAGE 95

-87-

PAGE 96

Clark x strain 94 combination had curled root hairs, and in 9 the infection thread(s) was still visible. None of twelve nodules examined similarly from the Clark-rj-^ x 9 4 combination had curled root hairs or visible infection threads. Primary root segments extending from the RTM to 10 mm above the RTM were examined using SEM. Uninoculated plants developed long root hairs that were free of detectable bacteria (Figure 5.2). No short, curled root hairs were apparent except where it was obvious that the root had come in contact with a solid surface such as the plastic of the growth pouch. In these areas the individual deformed root hairs were easily discriminated from root hairs characteristic of inoculated roots of compatible combinations. Root segments from Clark soybean inoculated with 5 x 10 8 cells/ml of fully compatible strains of R. japonicum were examined using SEM. The root segments from Clark soybean inoculated with strain 110 had clusters of short curled root hairs similar in appearence to the root hairs shown by light microscopy to contain infection threads (Figure 5.3). Clark inoculated with strain 94 produced short curled root hairs very similar to those seen in the the combination Clark x strain 110 (Figures 5.4 and 5.5). There were differences in the general appearence of root segments from these two combinations. The root surfaces of Clark were smooth (almost waxy appearing in SEM micrographs)

PAGE 97

Figure 5.2. Clark soybean root hairs on a control plant sampled and fixed 10 days after sham inoculation with plant growth solution. The area sampled was the 10 cm of the primary root immediately above the point representing the root tip at the initiation of the experiment.

PAGE 98

-90m JF>>;?i^»i#]0

PAGE 99

Figure 5.3. Clark soybean root hairs on plants sampled 10 days after inoculation with Rhizobium j aponicum strain 110. Clusters of tightly curled root hairs characteristic of this plant-strain combination are marked by arrows.

PAGE 100

-92-

PAGE 101

Figure 5.4. Curled root hair on Clark soybean sampled 10 days after inoculation with Rhizobium j aponicum strain 110. Note the bacterial cells adsorbed to the root hair and epidermal surface.

PAGE 102

-94-

PAGE 103

Figure 5.5. Curled root hair on Clark soybean sampled 10 days after inoculation with Rhizobium japonicum strain 94.

PAGE 104

-96-

PAGE 105

-97in the combination with strain 110, whereas when inoculated with strain 94, Clark roots often had a roughened appearance in localized patches that frequently were associated with presence of bacteria (Figure 5.6). Remnants of tightly curled root hairs were seen with SEM on surfaces of nodules produced from both interactions (Figure 5.7). Clark-rjj roots which had been inoculated with strain 94 (5 x 10 8 cells/ml or 10 10 cells/ml) were examined using SEM. No short, tightly curled root hairs of the type observed in the fully compatible interactions of Clark with strains 94 and 110 were seen for either of these treatments. Roughened areas were observed on root surfaces inoculated with the lower cell concentration similar to those noted for the combination Clark x strain 94. For Clark-rj^ soybean which had been inoculated with strain 94 at 10 10 cells/ml, there was evidence of degradation of a surface component, presumably mucigel (Foster et al. 1983), associated with the presence of bacteria on the root surface (Figure 5.8). The cell walls of some epidermal cells appeared to be perforated (Figures 5.9, 5.10, and 5.11). These perforations usually were heavily populated with bacteria. The perforations were most often seen at the base of short, uncurled root hairs; yet some epidermal cells which had no evidence of root hair development were apparently perforated and colonized by bacteria. The breached epidermal cells were associated with those areas where roughening of the mucigel-like material was most evident.

PAGE 107

-99-

PAGE 108

Figure 5.7. Residual root hairs on the Clark soybean nodule surface 10 days after inoculation. One of the root hairs is tightly curled (arrow).

PAGE 109

-101-

PAGE 110

Figure 5.8. Note the extensive colonization of the root surface by bacteria and the degradation of a surface component, presumably mucigel (arrows). ClarkLll soybean root surface sampled 10 days afte^ inoculation with Rhizobium j aponicum strain 94 (10 cells/ml) .

PAGE 111

-103-

PAGE 112

Figure 5.9. Extensive colonization of the root surface of Clark-r j^ by bacteria 10 days after inoculation with Bllil^^AiilB i a .E°I!A.£y.IH strain 94 (10 1 cells /ml). Apparent degradation and penetration of the epidermal cells is apparent (arrows). The boxed areas are shown at higher magnification in the following two figures.

PAGE 113

-105-

PAGE 114

Figure 5.10. Clark-rj^ soybean root surface sampled 10 days after inoculation with Rhizobium j aponicum strain 94 (l(r-° cells/ml). Arrows indicate locations of apparent penetration of the epidermal cells. This view is a higher magnification of an area shown in Figure 5.9.

PAGE 115

-107-

PAGE 116

Figure 5.11. Clark-r j-^ soybean root surface sampled 10 days af_ter inoculation with Rhizobium japonicum strain 94 (lCD 1 " cells/ml). Arrows indicate locations of apparent penetration of the epidermal cells. This view is a higher magnification of an area shown in Figure 5.9.

PAGE 117

-109-

PAGE 118

-110Discussion When Clark soybean was inoculated with either of the fully compatible strains 110 or 94 (5 x 10 8 cells/ml), clusters of tightly curled, short root hairs were formed by the tenth day of growth. These curled root hairs were detected both by light microscopy of thin strips of root surface tissues and by SEM of root segments. They were similar to those described by Pueppke (1983) as resembling "question marks." As reported in the previous study (Pueppke 1983), many of the question mark-shaped root hairs contained infection threads readily resolved at a magnification of 400x with either bright field or interference contrast optics (Hoffman modulation). Scanning electron micrographs were produced of the tightly curled root hairs for both of these compatible combinations. The curled root hairs appeared similar to those represented by the scanning electron micrographs of the compatible strain 110 x Williams combination (Turgeon and Bauer In press). In my study, the tightly curled hairs often were free-standing (Figures 5.4 and 5.5), although curled over almost to the epidermal surface or even touching it. Root hairs that were tightly appressed to the epidermal cells (Turgeon and Bauer In press) were less common. Ranga Rao and Keister (1978) published light micrographs of infected root hairs of the soybean cultivar Beeson infected by several Rhizobium strains. Some of their micrographs showed the tightly curled infected root hairs of

PAGE 119

-111the type exclusively seen in this study and in other reports (Pueppke 1983, Turgeon and Bauer In Press), but most showed elongated root hairs with curling only at the ends with the infection threads clearly visible. Turgeon and Bauer (1982) saw only the short, tightly curled hairs when they examined that same cultivar with a different strain. Therefore, it seems more likely that the growth conditions rather than specific cultivar x strain interactions induce the different response. Ranga Rao and Keister (1978) used a hydroponic culture, as have the other studies (Turgeon and Bauer 1982, In Press, Pueppke 1983), but they used sand as a support medium rather than the paper support used in the growth pouch system. Bieberdorf (1938) also reported that infections occurred in elongated root hairs. He claimed, additionally, that those elongated root hairs with infection threads were uncurved. But, if the illustration he cites as showing such an infection (Bieberdorf 1938 [Figure l.a]) is compared with his other illustrations of infection, it can be conjectured that the root hair he describes as "without curvature," is actually curved out of the focal plane (Compare also Pueppke 1983 [Figure 5]). Although he includes photomicrographs in his report, illustrations showing infection all were produced by means of a camera lucida. Perhaps the lack of sufficient depth-of-f ield and resolution of his microscope, and the extra simplification created by the camera lucida, caused him to overlook curling which was actually present. No other report has claimed infection of soybean in fully elongated root hairs without

PAGE 120

-112curvature. The other illustrations of infection of soybean that Beiberdorf provides are in general agreement with this study. No tightly curled root hairs of the type shown to have infection threads in nodulating combinations were observed on strips of root epidermis from Clark-r j^ plants inoculated with strain 94. The procedures in which root surfaces were examined for infection structures might not be adequate to detect a rare event. For instance, since the average is less than one nodule formed per Clark-r j 1 plant, if Clarkrj, were infected through root hairs by infection threads but only one infection occurred per nodule, that infected root hair would be unlikely to be observed. Since investigators of early infection processes in soybean have noted curled root hairs on surfaces of expanding nodules (Bieberdorf 1938, Ranga Rao and Keister 1978), and since nodules are the only marker for a successful infection, it is logical to use them as a means to localize the search for root hair infection. When free-hand sections of nodules formed by strain 94 were examined microscopically, curled root hairs with infection threads were found on nodules from Clark, but curled root hairs were not observed on nodules from Clark-r j^. I provisionally conclude that Clark-r j^ is not infected through root hairs as has been reported for other soybean genotypes. It appears that rj^^r j i -resistance to infection by most strains of rhizobia is expressed as resistance to infection via root hairs.

PAGE 121

-113La Favre and Eaglesham (1981) suggested in a preliminary report that a very high inoculum concentration could increase the nodule number on sand-grown rjj r Ji ~ soybean. The effect in this study of increasing the concentration of strain 94 to approximately 1 x 10 cells/ml for inoculation of Clark-rj^ was observed using SEM. The extensive roughening of the root surface and the apparent perforation of epidermal cells in Clark-rjj plants suggests enzymatic degradation of plant surface and cell wall components resulting from inoculation. It appears, therefore, that the ability of overcoming strains to infect r j^rj^soybean could be due to their ability to penetrate epidermal cells directly. Such an infection mechanism has been described for several other legumes (Chandler et al. 1982, Ranga Rao 1977). Several rhizobia are known to produce the potential wall degrading enzymes cellulase and pectolytic enzymes (Hubbell et al. 1978, Morales et al. 1984). The earliest desciption of soybean infection included direct infection through epidermal cells as one of the alternative infection mechanisms, but that report (Bieberdorf 1938) has not been corroborated and often has been discounted. An alternative to root hair infection, such as direct penetration, would potentially explain the anomalous nodulation of Clark soybean in the zone of mature root hairs by the overcoming strains of R^_ ia^or^icum (Chapter Three), assuming that the alternative pathway of direct infection is occurring at a low frequency and without the developmental constraints observed for root hair

PAGE 122

-114infection. Such anomalous nodulation was not observed for strain 110, which correlates with its inability to overcome the r j-irj^resistance and presumed inability to infect by direct penetration. I should quickly add the caveat that these observations, though they suggest direct penetration as an infection mechanism for the overcoming strains, are merely correlative and not direct evidence for such a mechanism. Compelling evidence for such a mechanism would be provided by genetic alteration of the bacterium affecting a single gene which caused either a gain or loss of both the ability to infect zjl rj^-soybean and the ability to penetrate soybean root surfaces directly. Persuasive evidence for direct penetration leading to infection could also be obtained though transmission electron microscopy, as it was for infection mechanisms of peanut and Stylosanthes (Chandler 1978, Chandler et al. 1982). No simple procedure has yet been devised to localize infections leading to nodules in Clark-rj^ before the actual appearance of those nodules. That difficulty is underscored by the amount of effort that has been invested with incipient infections being located (Tanner and Anderson 1963). The suggested additional infection pathway for overcoming strains — through direct penetration — is one more distinguishing characteristic, in addition to those noted by Devine (1984a), between these rhizobia and the other strains that infect soybean. He suggests, based on several lines of

PAGE 123

-115evidence, that the overcoming strains constitute a separate genetic population that should be separated phylogenetical ly from the other strains now included in R_^ japonicum . My conclusion that the rj^ r j^-geno type of soybean is characterized as lacking infection through root hairs but is nodulated, albeit sparsely, by certain strains of R^_ j aponicum , leads to a unique description of the symbiotic phenotype. Following the terminology of Vincent (1980), the interaction of Clark-rJ! with the overcoming strain 94 would be characterized as Inf" (no infection threads formed) Nod (nodules formed). All other soybean x Rhizobium combinations that have been characterized have been either Inf" Nod" or Inf + Nod + . This is the first report of a combination characterized as Inf" Nod .

PAGE 124

CHAPTER SIX SUMMARY The interaction phenotype was examined for the soybean isolines, Clark and Clark-rj^, with strains of Rhizobium japonicum possesing differential ability to nodulate. Various factors were examined for their role in the infection and nodulation process. The effect of temperature on nodulation was tested to find optimum conditions for study of infection processes. Temperature had statistically significant effects on nodulation of both plant types irrespective of bacterial strain. The largest number of nodules per plant and the highest percentage of nodulated plants was at 22 C. When inoculated Clark plants were grown at 32 C, nodulation on the primary root was displaced downward and was more random. The nodule profile lacked a definite peak representing nodule production near the location of the root tip of the primary root at inoculation. This peak was present for plants grown at 27 C and 22 C. The role of adsorption of rhizobia to roots of the soybean isolines was tested for strains with and without the ability to overcome the Clark-r j-^ resistance to nodulation. When isoline x strain combinations were ranked by the number of bacteria bound to roots, the order was opposite to their rank by nodules formed per plant. Thus the hypothesis that -116-

PAGE 125

-117the differential ability of the two strains to nodulate Clark-rj^ soybean was mediated by selective adsorption to plant roots was rejected. Reduction of temperature from 27 C to 22 C reduced the number of bacteria bound to roots of Clark-rj^ by 20%. This is precisely opposite the effect of those temperatures on nodule number. Clearly, the temperature effect on nodulation also occurs at some step subsequent to adsorption of bacteria to roots. Roots of the soybean isolines were examined microscopically 10 days after inoculation with either overcoming strain 94 or nonovercoming strain 110. Although curled root hairs, some with infection threads, were observed by light microscopy on Clark soybean inoculated with either strain 110 or 94, no infection threads and no curled root hairs of the type associated with infection were ever found on Clark-rj^ root surfaces. Curled root hairs were seen on surfaces of a majority of the nodules from Clark but not on nodule surfaces from Clark-rj^. Curled root hairs were observed by scanning electron microscopy on Clark roots that resembled those with infection threads visualized by light microscopy. Similar curled root hairs were never seen on uninoculated plants or on inoculated Clark-r ji . Several lines of evidence for a possible alternative infection process for the overcoming strains of R^_ japonicum are suggested. When very high inoculum concentrations of an overcoming strain were used with Clark-rj^, apparent

PAGE 126

-118epidermal cell perforation was observed. This implies that the bacteria had the potential zz> degrade the plant surface directly, and it seems that this might provide an infection pathway. Other evidence of the potential for the overcoming strain's ability to degrade plant surfaces was provided by the roughening of Clark roots by the overcoming strain which was not noted when Clark was inoculated with the nonovercomi ng strain. Overcoming strains, unlike nonovercoming strains, produced a few nodules in the region of the root which had well developed root hairs at the time of inoculation, suggesting an alternative pathway for infection that is not developmental ly limited. Vincent (1970) described initiation of 1 egumeRhi zob ium symbioses in terms of discrete interaction phenotypes. The interaction of Clark with both overcoming and nonovercoming strains would be described as Roc + , Roa + , Hac + , Inf , Nod , and Fix + . That is, roots are colonized, root adsorption of rhizobia occurs, root hair curling results, infection threads are formed, leading to nodulation, and nodules are capable of nitrogen fixation. The Clark-r j L is Roc , Roa , Hac", Inf", Nod", Fix"; demonstrating the expected pattern for an interaction with a block at one of the steps. The interaction is progressive so it is generally assumed that if one is blocked the subsequent phenotypes will be null. The interaction phenotype of Clark-rJ! with overcoming strains, by this reckoning, is an anomaly; Roc , Roa , Hac", Inf", Nod + , Fix + . This is the first report of such an interaction phenotype for soybean. The hypothesis that an

PAGE 127

-119additional infection mechanism is present in these combinations is consistent with this reaction type.

PAGE 128

APPENDIX A PLASMIDS OF RHIZOBI UM J APONICUM STRAINS THAT NODULATE SOYBEAN ISOLINES DIFFERING AT THE Rj_ ] _ LOCUS Plasmids of fast-growing strains of Rhizobium carry genes essential for nodulation, including genes affecting host range, and genes that encode enzymes for nitrogen fixation (Sadowsky and Bohlool 1983, Long 1984). These symbiotic functions are generally carried on the very large plasmid usually found in these strains (Casse et al. 1979, Rosenberg et al. 1982). Less is known about the plasmids and their functions in the slow-growing strains of R. japonicum . The studies in which techniques were used that produce point mutation do not lend themselves to analysis of plasmid involvement, because adequate genetic markers to which linkage can be measured have not been described for R. japonicum plasmids (Maier and Brill 1976, Stacey et al. 1982). The plasmid profiles of a few slow-growing strains of R. japonicum have been reported, and some strain-tostrain similarities were noted (Masterson et al. 1982). The results from most studies of R^_ jap onicum plasmid content have not shown the similarities reported in that study (Haugland and Verma 1981, Cantrell et al. 1982). Neither are the similarities seen between slow growing strains that are apparent between plasmid profiles for strains of some species of the fast-growing rhizobia -120-

PAGE 129

-121(Broughton et al. 1984), nor indeed, even the similarities seen between strains of the fast growing rhizobia from soybean (Sadowsky and Bohlool 1983, Broughton et al. 1984, Heron and Pueppke 1984). Certain strains of R^_ japonicum , referred to as the "overcoming strains," have the ability to form a few nodules on lines of soybean that carry the genotype r j L r j lf which is resistant to nodulation by other strains of rhizobia. The objectives of this study were to examine some overcoming strains of R^ japonicum for plasmid content, including determination of whether they harbor very large plasmids (> 300 megadaltons, the so-called "megaplasmids") , and whether there is a correlation between the ability of a strain to nodulate r ji r j^-soybean and that strain's plasmid profile. The overcoming strains of R^ japonicum used were USDA 61, 84, 85, 94, 117, and 119; USDA 110 was used as a nonovercoming control. The bacterial strains used for plasmid molecular weight standards were those reported by Heron and Pueppke (1984) with the additional strain R. japonicum 14C carrying plasmids of 49, 74, 91, and 118 megadaltons (Gross et al. 1979) obtained from A. K. Vidaver, University of Nebraska, Lincoln. Plasmids were visualized by a modification, similar to that reported by Heron and Pueppke (1984), of the in-gel lysis procedure described by Eckhardt (1978) for electrophoretic detection of bacterial plasmids. The bacteria were cultured to midlog phase at 27 C with shaking

PAGE 130

-122in liquid defined gluconate-mannitol medium (Bhu vaneswar i et al. 1977). For each strain a volume of culture equivalent to 1 x 10 9 cells (determined turbidimetr ical ly) was placed in a Corex centrifuge tube. The subsequent steps were carried out at 4 C. All centr ifugations were for 10 min at 7500 x g_. R^ japonicum strains were washed in 5-10 ml of TE (50 mM Tris, 20 mM EDTA; pH 8) containing 3.0% (w/v) NaCl and 1.0% (w/v) Sarkosyl, followed by centr if ugation. Cells were washed in 5-10 ml of TE containing 1.0% Sarkosyl and centrifuged. The pelleted cells were carefully suspended in 2 ml of TE by shaking the tubes gently on a Vortex shaker and then sedimented by centr if ugation. Bacteria other than r. japonicum were treated similarly except that the first wash was deleted and the subsequent wash contained 0.1% Sarkosyl. Immediately after the centr i fugati on following the last wash, the liquid was decanted from the tubes, excess liquid was removed with an adsorbant paper swab, and the tubes were placed on ice. Cells were resuspended in 250 ul of lysis mixture (Heron and Pueppke 1984) by slow shaking on a Vortex shaker. A 25 ul aliquot was immediately transferred to a 10-mm well formed in a 0.7% agarose gel (DNA grade agarose, Sigma Chemical Co., St. Louis, MO; gel apparatus, 160 x 140 x 3 mm, BioRad, Richmond, CA) in TBE (89 mM Tris, 2.5 mM EDTA, 8.9 mM borate; pH 8.2). After 10 min, the cell lysis mixture was overlaid with 25 ul of TBE containing 10% Ficoll 400 (Pharmacia, Uppsala, Sweden) and sodium dodecyl sulfate (SDS) and then with 100 ul of TBE

PAGE 131

-123containing 2.5% Ficoll and SDS. The SDS concentration in the overlay solutions for R_;_ japonicum strains was 1.0%, and 0.1% for all other strains. The wells were sealed with molten 0.7% agarose in TBE. Electrophoresis was carried out in TBE buffer at a constant current of 8 mA for 30 min and then 15 mA until the bromphenol blue marker dye reached the bottom edge of the gel (8-12 hr). Gels were stained in aqueous ethidium bromide (5 ug/1) and were visualized and photographed with transmitted UV light (Maniatis et al. 1982). A photograph of a representative gel is provided in Figure A.l. The logarithms of the reported molecular weights of plasmids from strains used as standards were plotted against the migration distance of those plasmids on the gel. This plot was used to estimate molecular weights of unknown plasmids by calculating the log molecular weight for their migration distance. Estimated molecular weights are expressed as the mean value from 4 to 9 independent observations per strain. The sizes estimated for plasmids are given in Table A.l. Two strains described here, USDA 110 (= 3Ilbll0) and USDA 94 ( = 3llb94), also were examined by Masterson et al. (1982). They reported two plasmids of 58 and 118 megadaltons in strain USDA 94. The 118 megadalton plasmid was obtained consistently in this study, but the 58 megadalton plasmid was never observed. Masterson et al. (1982) also detected a 184 megadalton plasmid in strain USDA 110. In this study no plasmid was visualized for strain USDA 110, even though that strain was included on more than

PAGE 132

-12415 gels on which plasmids were clearly evident for other strains. Strain USDA 110 is reported not to have plasmids by Haugland and Verma (1981), and by Cantrell et al. (1982). Both Gross et al. (1979) and Cantrell et al. (1982) reported a plasmid in USDA 117 but none was observed in this study. No correlation was observed between plasmid content and the ability of these strains to nodulate Clark-rjj soybean. The overcoming strains USDA 61, 85, and 94, each had one plasmid and USDA 84 had two. The overcoming strains USDA 117 and 119, like the nono vercoming strain USDA 110, had none. Although the lack of plasmids has been correlated with the presence of a functional hydrogen uptake system (Cantrell et al. 1982), the plasmids of R^_ japonicum remain cryptic with no fuctions yet associated with their presence.

PAGE 133

Figure A.l. Agarose gel electrophoresis of plasmid DNA from strains of Rhizobium japonicum . Lanes A through I are, respectively, R^ japonicum strain 14C and strains USDA 61, 84, 85, 94, 84, 110, 117, and 119. Agrobacter ium tumef aciens strain C58 is in lane J. Numbers in the margin represent the size of the reference plasmids in megadaltons.

PAGE 134

-126-

PAGE 135

-127Table A.l. Plasmids from strains of Rhizobium japonicum USDA 84 Strain Molecular mass 3 (megadaltons) USDA 61 61-4 97 ± 9 153 ± 23 USDA 85 HO ± I 7 USDA 94 118 ± 10 USDA 110 N pb USDA 117 NP USDA 119 NP a Molecular mass is expressed as the mean value SD for 4 to 9 independent observations per plasmid. b For strains denoted "NP," no bands character is tic of plasmids were present on at least 15 gels on which plasmids were observed in other strains.

PAGE 136

APPENDIX B EVALUATION OF PROCEDURES REPORTED TO INDUCE HIGH-FREQUENCY MUTATION IN STRAINS OF RHIZOBIUM JAPONICUM The results of preliminary research designed to evaluate a report (Skogen-Hagenson and Atherly 1983) of techniques for high-frequency mutation in strains of Rhizobium japonicum are presented here. When this study was undertaken, the literature outlining the role of plasmids in the fast-growing Rhizobium species was developing rapidly, but little was known of the role of plasmids in symbiotic functions of R^ japonicum . The report of a method for inducing high frequency mutations in nodulation and nitrogen fixation genes (Skogen-Hagenson and Atherly 1983) led to evaluation of similar methods for induction of nodulation mutants in the overcoming strains of R^_ japonicum . The overcoming strains produce a few nodules on plants of an isoline of the soybean cultivar Clark that has the nodulation restrictive genotype, EJ-lLll (Chapter Three, Devine and Breithaupt 1980). This isoline will be referred to as Clark-r j l . The objectives of this study were _i. to determine whether procedures reported to induce plasmid loss or deletion mutations also produce nodulation mutants in the overcoming strains of R^_ japonicum , and jj^. to test the usefulness of prescreening mutagenized rhizobia for lack of •128-

PAGE 137

-123homology to nif sequences as a means of enriching populations for nodulation mutants. Modifications of the procedures of Skogen-Hagenson and Atherly (1983) were tested. Bacteria were cultured at elevated temperature with either SDS or ethidium bromide added to the bacterial growth medium. In the first procedure 10 6 cells of R. japonicum strain 94 were added per ml of liquid gluconate-manni tol (G-M) medium (Bhuvaneswar i et al. 1977) containing various concentrations of SDS (Table B.l). The cultures were incubated at 37 C in the dark without shaking for 32 d and then were diluted and plated on yeast extract-manni to 1 agar (YEM) (Vincent 1970). One hundred colonies were selected at random, plated in duplicate, and each was inoculated onto seedlings in a plastic growth pouch with 2 seedlings in each of either Clark or Clark-rj^. The seedlings had been grown for one day in plastic growth pouches from seed disinfested and germinated as described in Chapter Three. Each colony was scraped from YEM with the broad end of a flat, sterile toothpick and suspended in 1 ml of sterile water. Bacteria from each of the duplicate plates were inoculated onto plants of one of the isolines. The bacterial suspension was dripped over the roots of the plants in a pouch with a sterile pipette. Inoculated plants were grown at an ambient temperature of about 27 C under fluorescent lights with a 12 hr on-off cycle at an irradiance of 450 uE/m 2 /sec (400-700 nm). Plants were screened for nodulation at approximately

PAGE 138

-130Table B.l. Survival of Rhizobium japonicum strain USDA 94 after 32 days at 37 C in gluconate-mannitol medium amended with sodium dodecyl sulfate (SDS). Sds (%) Colony forming units / ml 0.00 7.7 x 10 3 a 0.01 3.1 x 10 4 0.10 1.2 x 10' a The initial bacterial concentration was 10 cells/ml. Cultures were kept in the dark without shaking.

PAGE 139

-1312 wk after inoculation and subsequently at weekly intervals up to 30 d. Strains inoculated on Clark were scored as nonnodulating (0 nodules per pouch after 30 d) or nodulating (1 or more nodules formed within 30 d). Strains inoculated on Clark-rji were rated for enhanced nodulating ability (arbitrarily designated 2 or more nodules per plant) at 30 d. Any strains rated nonnodulating on Clark or rated for enhanced nodulating ability on Clark-r_j^ were retested by inoculating 5 growth pouches, each with 10 plants of the appropriate host. In addition to the strains rated directly on plants for nodulation, 75 colonies were selected at random for colonyhybridization preselection. Colonies were plated in a 5 x 5 array on YEM agar in standard 100 mm dia plates. Colony hybridization was carried out using 95 mm dia circles of 0.45 urn nitrocellulose filter (Schleicher & Schuell, Keen, NH) following the procedures outlined by Maniatis et al. (1982 [p. 326]) for binding DNA from bacterial colonies to nitrocellulose. The nitrocellulose filters were probed with nif HDK sequences cloned from K lebsie lla pneumoniae (Ruvkin and Ausubel 1980). Escherichia col i strain HB101 containing the cloned nif genes in plasmid pACYC184 as recombinant plasmid pSA30, was obtained courtesy of K. T. Shanmugam, Department of Microbiology and Cell Science, University of Florida, Gainesville. The nif sequence was purified by standard procedures (Maniatis et al. 1982) for plasmid purification, restriction, gel electrophoresis, and recovery on activated DEAE-cel lulose. The probe was labelled with

PAGE 140

-132cytidine 5 '[ 32 P ] tr iphospha te (Amersham Corp. Arlington Heights, IL) using nick translation (Maniatis et al. 1982 [p. 109]). Hybridization of the probe to the nitrocellulose filters bearing the colony replicas as bound DNA was accomplished by the procedure described in Maniatis et al. (1982 [p. 326]) using a hybridization temperature of 68 C for 24 hr. Colonies identified by reduced binding of the nif HDK probe in colony hybridization were cultured individually in 50 ml of liquid G-M medium, harvested and inoculated on seedlings grown in pouches as described in Chapter Three. A total of 10 plants in 5 pouches were rated for each putative mutant screened. Plants were rated for ability of the bacteria to nodulate and for evidence of nitrogen fixation. Nitrogen fixation was assumed to be efficient if the plant was a healthy green color and nodules were large and had leghemoglobin (on the basis of internal red color on visual examination) . The second experiment for high-frequency induction of mutation was as described by Skogen-Hagenson and Atherly (1983). The media used were YEM and the Skogen-Hagenson and Atherly medium (SHAM), which is a yeast-extract medium containing high iron. R. j aponicum strains USDA 74 and 76 were cultured in the dark at 36 C with shaking in each of the media amended with ethidium bromide at 50 ug/ml. Control flasks included the 2 strains in each of the unamended media grown at the treatment temperature, and the 2 strains in amended media grown at 28 C. Every 4 d an aliquot was

PAGE 141

-133removed from each flask, diluted, and plated for determination of colony forming units. Two-hundred-fifty colonies were tested for ability to nodulate Clark; 100 of those colonies also were tested for enhanced ability to nodulate Clark-rj^. The tested bacteria were from colonies of strain USDA 76 selected from dilution plates of cells removed from the amended SHAM treatment on day 10. As seen in Table B.l the survival rate of R^ japonicum strain 94 is high after 32 d in SDS-amended G-M medium incubated at 37 C. The 100 colonies selected from the SDS treatment nodulated Clark soybean and none produced two or more nodules per plant on Clark-rj^. Of the 75 colonies hybridized to the nif probe, 7 were selected on the basis of greatly reduced hybridization and were screened on plants. All nodulated both Clark and Clark-rjx soybean with no obvious alteration in nodulation pattern. Five of those strains appeared to have normal nitrogen-fixation phenotype, but two produced smaller nodules which had white-green centers and the plants showed symptoms consistent with nitrogen difficiency. Figure B.l represents the survival curves for the strains USDA 74 and 76 in both YEM and SHAM, each amended with ethidium bromide at 50 ug/ml. The rates of bacterial death are approximately the same as reported by SkogenHagenson and Atherly (1983) for strains USDA 74 and 61A76 in similarly amended SHAM. No changes in nodulation phenotype were detected in the 250 selections of treated USDA 76 on Clark or the 100 of those tested on Clark-r ji .

PAGE 142

-134D A Y S Figure B.l. Survival curves for Rhizobium japonicum strains in media amended with ethidium bromide at 50 ug/ml . • -strain 76 in Skogen-Hagenson and Atherly (1983) medium (SHAM) , strain 76 in yeast extract-mannitol (YEM) medium (Vincent 1977), O -strain 74 in SHAM, Q-strain 74 in YEM.

PAGE 143

-135The number of putative mutants screened for lack of binding to K^ pneumoniae ni £ sequences and tested for nodulation are inadequate to demonstrate that nif and nodulation genes are not linked in these strains, although the limited data are consistent with the apparent lack of linkage in R_j_ ia.£on.icum (Hennecke 1981, Fuhrmann and Hennecke 1983, 1984). Given these results and the growing evidence for lack of linkage of these functions this method for enrichment of Nod" mutants of slow-growing R^ japonicum seems not to have the utility that it has for the fastgrowing rhizobia (Hirsch et al. 1982, 1984). The use of heat and curing agents did not induce nodulation mutants in high frequency; none of 350 isolates selected from treatments reported to induce high-frequency mutation in symbiotic functions showed an obvious change in nodulation phenotype. This contrasts sharply with the previous report that 44 of 133 isolates (33%) were Nod (Skogen-Hagenson and Atherly 1983). Classical mutagenesis with chemical agents yielded 2 nodulation mutants in R^ japonicum out of 2500 isolates tested (Maier and Brill 1976). Horn et al. (1984) reported no Nod" mutants out of 200 isolates carrying Tn5. Although these procedures require extensive screening of isolates on plants and produce mutants at low frequencies, the apparent randomness of mutations produced, rather than tending to be specific for plasmid encoded genes, make them more suitable for screening for production of nodulation mutants in R. japonicum . The use of transposoninduced mutation in

PAGE 144

-136particular holds promise because of the ability to use positive selection for introduced markers _in vivo and for homology to introduced DNA sequences _in vitro .

PAGE 145

LITERATURE CITED Allen, 0. N., and E. K. Allen. 1940. Response of the peanut plant to inoculation with rhizobia, with special reference to morphological development of the nodules. Bot. Gaz. 102:121-142. Banfalvi, Z., V. Sakanyan, C. Koncz, A. Kiss, I. Dusha, and A. Kondorosi. 1981. Location of nodulation and nitrogen fixation genes on a high molecular weight plasmid of Rhizobium meliloti . Mol. Gen. Genet. 184:318-325. Bechet, M., and J. B. Guillaume. 1978. Mise en evidence d'ADN extrachromosomique chez Rhizobium mel i l oti . Can. J. Microbiol. 24:960-966. Bedmar, E. J., N. J. Brewin, and D. A. Phillips. 1984. Effect of plasmid pIJ1008 from Rhizobium 1 eguminosar urn on symbiotic function of Rhizobium meli l oti . Appl. Envir. Microbiol. 47:876-878. Beringer, J. E. 1980. The development of Rhizobium genetics. J. Gen. Microbiol. 116:1-7. Beringer, J. E. 1982. The genetic determination of host range in the Rhizobiaceae. Israel J. Bot. 31:89-93. Beringer, J. E., J. L. Beynon, A. V. Buchanan-Wo 1 laston , and A. W. B. Johnston. 1978. Transfer of the drug-resistance transposon Tn5_ to Rhizobium . Nature 276:633-634. Bhuvaneswar i , T. V. 1981. Recognition mechanisms and infection process in legumes. Econ. Bot. 35:204-223. Bhuvaneswari, T. V., A. A. Bhagwat, and W. D. Bauer. 1981. Transient susceptibility of root cells in four common legumes to nodulation by rhizobia. Plant Physiol. 68:11441149. Bhuvaneswari, T. V., S. G. Pueppke, and W. D. Bauer. 1977. Role of lectins in p lant-microoganism interactions. I. Binding of soybean lectin to rhizobia. Plant Physiol. 60:486-491. -137-

PAGE 146

-138Bhuvaneswari, T. V., B. G. Turgeon, and W. D. Bauer. 1980. Early events in the infection of soybean (G l ycine max (L.) Merr.) by Rhizobium japonicum . I. Localization of infectible root cells. Plant Physiol. 66:1027-1031. Bieberdorf, F. W. 1938. The cytology and histology of the root nodules of some Leguminosae. J. Amer. Soc. Agron. 30:375-389. Bishop, P. E., F. B. Dazzo, E. R. Appelbaum, R. J. Maier, W. J. Brill. 1977. Intergeneric transfer of genes involved in the Rhizobium legume symbiosis. Science 198:938-940. Bohlool, B. B., and E. L. Schmidt. 1974. Lectins: a possible basis for specificity in the Rhizobium legume root nodule symbiosis. Science 185:269-271. Brewin, N. J., J. E. Beringer, A. V. Buchanan-Wo 1 laston , A. W. B. Johnston, and P. R. Hirsch. 1980. Transfer of symbiotic genes with bacter iocinogenic plasmids in Rhizobium leguminosarum . J. Gen. Microbiol. 116:261-270. Brewin, N. J., E. A. Wood, A. W. B. Johnston, N. J. Dibb, and G. Hombrecher. 1982. Recombinant nodulation plasmids in Rhizobium leguminosarum . J. Gen. Microbiol. 128:1817-1827. Briscoe, C. F., and W. B. Andrews. 1938. Effect of strains of nodule bacteria and lime on the response of soybeans to artificial inoculation. J. Amer. Soc. Agron. 30:711-719. Broughton, W. J., N. Heycke, H. Meyer Z. A., and C. E. Pankhurst. 1984. P lasmid1 i nked nif and " nod " genes in fast-growing rhizobia that nodulate GJ.^cj.ne maj£, Ps ophocarpus tetragonolobus , and Vigna unguiculata . Proc. Natl. Acad. Sci. U.S.A. 81:3093-3097. Broughton, W. J., U. Samrey, and B. B. Bohlool. 1982. Competition for nodulation of Pisum sativum cv. Afghanistan requires live rhizobia and a plant component. Can. J. Microbiol. 28:162-168. Broughton, W. J., A. W. S. M. van Egeraat, and T. A. Lie. 1980. Dynamics of Rhizobium competition for nodulation of Pisum sati vum cv. Afghanistan. Can. J. Microbiol. 26:562565. Cabezas de Herrera, E., and M. Fernandez P. 1982. Ultrastructural changes produced during the development of soybean root nodules. Phytopath. Z. 103:173-185. Caldwell, B. E. 1966. Inheritance of a strain-specific ineffective nodulation in soybeans. Crop Sci. 6:427-428.

PAGE 147

-139Caldwell, B. E., and G. Vest. 1968. Nodulation-interactions between soybean genotypes and serogroups of Rhizobium japonicum . Crop Sci. 8:680-682. Callaham, D. A., and J. G. Torrey. 1981. The structural basis for infection of root hairs of Trifo l ium repens by Rhizobium . Can. J. Bot. 59:1647-1664. Calvert, H. E., M. K. Pence, M. Pierce, N. S. A. Malik, and W. D. Bauer. 1984. Anatomical analysis of the development and distribution of Rhizobium infections in soybean roots. Can. J. Bot. 62:2375-2384. Cantrell, M. A., R. E. Hickok, and H. J. Evans. 1982. Identification and characterization of plasmids in hydrogen uptake positive and hydrogen uptake negative strains of Rhizobium japonicum . Arch. Microbiol. 131:102-106. Casse, F., C. Boucher, J. S. Julliot, M. Michel, and J. Denarie. 1979. Identification and characterization of large plasmids in Rhizobium meli,loti using agarose gel electrophoresis. J. Gen. Microbiol. 113:229-242. Chandler, M. R. 1978. Some observations on infection of Arachis hypogaea L. by Rhizobium . J. Exper. Bot. 29:749-755. Chandler, M. R., R. A. Date, and R. J. Roughley. 1982. Infection and root-nodule development in Styl osanthes species by Rhizobium . J. Exper. Bot. 33:47-57. Chen, A. -P. T., and D. A. Phillips. 1976. Attachment of R llil.°^iHI!l to le g ume roots as the basis for specific Interactions. Physiol. Plant. 38:83-88. Christensen, A. H., and K. R. Schubert. 1983. Identification of a Rhizobium trifolii plasmid coding for nitrogen fixation and nodulation genes and its interaction with pJB5JI, a Rhizobium leguminosarum plasmid. J. Bacteriol. 156:592-599. Clark, F. E. 1957. Nodulation responses of two near isogenic lines of the soybean. Can. J. Microbiol. 3:113-123. Corbin, D., G. Ditta, and D. R. Helinski. 1982. Clustering of nitrogen fixation ( nif ) genes in Rhizobium me 1 il oti . J. Bacteriol. 149:221-228. Dazzo, F. B. 1981. Bacterial attachment as related to cellular recognition in the Rhizobium legume symbiosis. J. Supramol. Struct. Cell. Biochem. 16:29-41. Dazzo, F. B., and W. J. Brill. 1979. Bacterial polysaccharide which binds Rhizobium trifolii to clover root hairs. J. Bacteriol. 137:1362-1373.

PAGE 148

-140Dazzo, F. B., and D. H. Hubbell. 1975. Antigenic differences between infective and noninfective strains of Rhizobium trifolii . Appl. Microbiol. 30:172-177. Dazzo, F. B., and D. H. Hubbell. 1982. Control of root hair infection. I_n: Ecology of Nitrogen Fixation, Vol 2, Rhizobium , W. Broughton, ed., Oxford University Press, New York, NY. Dazzo, F. B., W. E. Yanke, and W. J. Brill. 1978. Trifoliin: a Rhizobium recognition protein from white clover. Biochim. Biophys. Acta 539:276-286. Devine, T. E. 1984a. Genetics and breeding of nitrogen fixation. I_n: Biological Nitrogen Fixation, M. Alexander, ed., Plenum Publishing Corp., New York, NY. Devine, T. E. 1984b. Inheritance of soybean nodulation response with a fast-growing strain of Rhizobium . J. Hered. 75:359-361. Devine, T. E., and B. H. Breithaupt. 1980a. Effect of the ethoxy analog of rh izobi tox ine on nodulation of soybeans. Crop Sci. 20:819-821. Devine, T. E., and B. H. Breithaupt. 1980b. Phenotypic thermal stability of rhizobitoxine-induced chlorosis and the nodulation controlling gene, r j-^ Crop Sci. 20:394-396. Devine, T. E., and B. H. Breithaupt. 1980c. S igni f icance_ of incompatibility reactions of Rhizobium j aponicum strains with soybean host genotypes. Crop Sci. 20:269-271. Devine, T. E., and B. H. Breithaupt. 1981. Frequencies of nodulation response alleles, Rl 2 and PJ4, in soybean plant introduction and breeding lines. U. S. Dept. Agric, Tech. Bull. 1628. Devine, T. E., B. H. Breithaupt, and L. D. Kuykendall. 1981. Tests for a diffusable compound endowing r j -j -incompatible strains of Rhizobium japonicum with the ability to nodulate the r ji r j-| soybean genotype. Crop Sci. 21:696-699. Devine, T. E., L. D. Kuykendall, and B. H. Breithaupt. 1980. Nodulation of soybeans carrying the nodulation-restr ictive gene, r j i, by an incompatible Rhizobium japonicum strain upon mixed inoculation with a compatible strain. Can. J. Microbiol. 26:179-182. Devine, T. E., and D. F. Weber. 1977. Genetic specificity of nodulation. Euphytica 26:527-535.

PAGE 149

-141Ditta, G. f S. Stanfield, D. Corbin, and D. R. Helinski. 1980. Broad host range DNA cloning system for gram-negative bacteria: Construction of a gene bank of Rhizobium me 1 i 1 o t i . Proc. Natl. Acad. Sci. U. S. A. 77:7347-7351. Djordjevic, M. A., W. Zurkowski, J. Shine, and B. G. Rolfe. 1983. Sym plasmid transfer to various sybiotic mutants of Rhizobium t rifo l ii, FL_ 1 e guminosarum , and R_^ melj^oti. J. Bacterid. 156:1035-1045. Downie, J. A., G. Hombrecher, Q.-S. Ma, C. D. Knight, and B. Wells. 1983. Cloned nodulation genes of Rhizobium l eguminosarum determine host-range specificity. Mol. Gen. Genet. 190:359-365. Eckhardt, T. 1978. A rapid method for the identification of plasmid deoxyribonucleic acid in bacteria. Plasmid 1:584588. Elkan, G. H. 1961. A nodu 1 at ioninhibi t ing root excretion from a non-nodu lat i ng soybean strain. Can. J. Microbiol. 7:851-856. Elkan, G. H. 1962. Comparison of rhizosphere microorganisms of genetically related nodulating and non-nodulating soybean lines. Can. J. Microbiol. 8:79-87. Erdman, L. W. , H. W. Johnson, and F. Clark. 1956. A bacterial-induced chlorosis in the Lee soybean. Plant Dis. Rep. 40:646. Ervin, S. E., and D. H. Hubbell. 1985. Root hair deformations associated with fractionated extracts from Rhizobium trifolii . Appl. Env. Microbiol. 49:61-68. Eskew, D. L., and L. E. Schrader. 1977. Effect of rj_ 1 rj_ 1 (non-nodulating) soybeans on nodulation of near isogenic Rj-Rj, plants in nutrient culture. Can. J. Microbiol. 23:"588-993. Fahraeus, G. 1957. The infection of clover root hairs by nodule bacteria studied by a simple glass slide technique. J. Gen. Microbiol. 16:374-381. Fahraeus, G., and H. Ljunggren. 1968. Preinfection phases of the legume symbiosis. I_n: The Ecology of Soil Bacteria, An International Symposium, T. R. G. Gray and D. Parkinson, eds., University of Toronto Press, Toronto, ON. Finan, T. M., E. Hartwieg, K. LeMieux, K. Bergman, G. C. Walker, and E. R. Signer. 1984. General transduction in Rhizobium meliloti. J. Bacteriol. 159:120-124.

PAGE 150

-142Forrai, T., E. Vincze, Z. Banfalvi, G. B. Kiss, G. S. Randhawa, and A. Kondorosi. 1983. Localization of symbiotic mutations in Rhizobium meliloti . J. Bacterid. 153:635-643. Foster, R. C., A. D. Rovira, and T. W. Cock. 1983. Ultrastructure of the Root-Soil Interface, The American Phytopathological Society, St. Paul, MN. Fred, E., I. Baldwin, and E. McCoy. 1932. Root Nodule Bacteria, University of Wisconsin Press, Madison. Fuhrmann, M., and H. Hennecke. 1982. Coding properties of cloned nitrogenase structural genes from Rhizobium japonicum . Mol. Gen. Genet. 187:419-425. Fuhrmann, M., and H. Hennecke. 1983. Rhizobium jap onicum nitrogenase Fe protein gene ( nifH ). J. Bacterid. 158:10051011. Gade, W., M. A. Jack, J. B. Dahl, E. L. Schmidt, and F. Wold. 1981. The isolation and characterization of a root lectin from soybean (Gl ycine max (L.) , cultivar Chippewa). J. Biol. Chem. 256:12905-12910. Giovanelli, J., L. D. Owens, and H. Mudd. 1971. Mechanism of inhibition of spinach beta-cystathi onase by rhi zobi toxine. Biochim. Biophys. Acta 227:671-684. Goodchild, D. J., and F. J. Bergersen. 1966. Electron microscopy of the infection and subsequent development of soybean nodule cells. J. Bacteriol. 92:204-213. Graham, P. H. 1964. The application of computer techniques to the taxonomy of the root-nodule bacteria of legumes. J. Gen. Microbiol. 35:511-517. Gross, D. C, A. K. Vidaver, and R. V. Klucas. 1979. Plasmids, biological properties and efficacy of nitrogen fixation in Rhizobium japonicum strains indigenous to alkaline soils. J. Gen. Microbiol. 114:257-266. Hadley, R. G., A. R. J. Eaglesham, and A. A. Szalay. 1983. Conservation of DNA regions adjacent to nifKDH homologous sequences in diverse slow-growing Rhizobium strains. J. Mol. Appl. Genet. 2:225-236. Hahn, M., and H. Hennecke. 1984. Localized mutagenesis in Rhizobium japonicum . Mol. Gen. Genet. 193:46-52. Halverson, L. J., and G. Stacey. 1984. Host recognition in the Rhizobium soybean symbiosis. Detection of a protein factor in soybean root exudate which is involved in the nodulation process. Plant Physiol. 74:84-89.

PAGE 151

-143Ham, G. E., L. R. Frederick, and I. C. Anderson. 1971. Serogroups of Rhizobium japonicum in soybean nodules sampled in Iowa. Agron. J. 63:69-72. Hanson, A. A. 1981. Ways that we'll continue to be well fed. rn: Will There Be Enough Food? U. S. Dept. Agric. Beltsville, MD. Haugland, R., and D. P. S. Verma. 1981. Interspecific plasmid and genomic DNA sequence homologies and localization of nif genes in effective and ineffective strains of Rhizobium japonicum . J. Mol. Appl. Genet. 1:205-217. Hennecke, H. 1981. Recombinant plasmids carrying nitrogen fixation genes from Rhizobium japonicum . Nature 291:354-355. Heron, D. S., and S. G. Pueppke. 1984. Mode of infection, nodulation specificity, and indigenous plasmids of 11 fastgrowing Rhizobium japonicum strains. J. Bacterid. 160:10611066. Higashi, S. 1967. Transfer of clover infectivity of Rhizobium trifol ii to Rhizobium phaseoli as mediated by an episomic factor. J. Gen. Appl. Microbiol. 13:391-403. Hirsch, A. M., D. Drake, T. W. Jacobs, and S. R. Long. 1985. Nodules are induced on alfalfa roots by Agrobacter ium tumefaciens and Rhizobium trifol ii containing small segments of the Rhizobium meli loti nodulation region. J. Bacteriol. 161:223-230. Hirsch, A. M., S. R. Long, M. Bang, N. Haskins, and F. M. Ausubel. 1982. Structural studies of alfalfa roots infected with nodulation mutants of Rhizobium meli loti . J. Bacteriol. 151:411-419. Hirsch, A. M., K. J. Wilson, J. D. G. Jones, M. Bang, V. V. Walker, and F. M. Ausubel. 1984. Rhi^ob^um me.l.i_lotj L nodulation genes allow Ag_r obact er__ium tumefaciens and Esche richia coH to form pseudonodu les on alfalfa. J. Bacteriol. 158:1133-1143. Holl, F. B. 1975. Host plant control of the inheritance of dinitrogen fixation in the Pisuni-Rhj.zobiurn symbiosis. Euphytica 24:767-770. Horn, S. S. M., S. L. Uratsu, and F. Hoang. 1984. Transposon Tn5_-induced mutagenesis of Rhizobium japonicum yielding a wide variety of mutants. J. Bacteriol. 159:335-340. Hombrecher, G., N. J. Brewin, and A. W. B. Johnston. 1981. Linkage of genes for nitrogenase and nodulation ability on plasmids in Rhizobium 1 eguminosarum and R^ phaseo l i. Mol. Gen. Genet. 182:133-136.

PAGE 152

-144Hooykaas, P. J. J., F. G. M. Snijdewint, and R. A. Schi lperoort. 1982. Identification of the Sym plasmid of Rhizobium leguminosarum strain 1001 and its transfer to and expression in other rhizobia and Agrobacter ium tumef aciens . Plasmid 8:73-82. Hooykaas, P. J. J., A. A. N. van Brussel, H. den Dulk-Ras, G. M. S. van Slogteren, and R. A. Schi lperoort. 1981. Sym plasmid of Rhizobium trifo lii expressed in different rhizobial species and Agrobacter ium t umef aciens . Nature 291:351-353. Hubbell, D. H. 1981. Legume infection by Rhizobium : A conceptual approach. BioScience 31:832-837. Hubbell, D. H., and G. H. Elkan. 1967a. Correlation of physiological characteristics with nodulating ability in Rhizobium japonicum . Can. J. Microbiol. 13:235-241. Hubbell, D. H., and G. H. Elkan. 1967b. Host physiology as related to nodulation of soybean by rhizobia. Phytochemistry 6:321-328. Hubbell, D. H., V. M. Morales, and M. Uma 1 i-Garc ia. 1978. Pectolytic enzymes in Rhizobium . Appl. Envir. Microbiol. 35:210-213. Johnson, H. W. , and U. M. Means. 1960. Interactions between genotypes of soybeans and genotypes of nodulating bacteria. Agron. J. 52:651-654. Johnston, A. W. B., J. L. Beynon, A. V. Buchanan-Wo 1 laston, S. M. Setchell, P. R. Hirsch, and J. E. Beringer. 1978. High frequency transfer of nodulating ability between strains and species of Rhizobium . Nature 276:634-636. Jordan, D. C. 1982. Transfer of Rhizobium japonicum Buchanan 1980 to Bradyrhizobium gen. nov., a genus of slow-growing, root nodule bacteria from leguminous plants. Int. J. Syst. Bacteriol. 32:136-139. Jordan, D. C. 1984. Rhizobiaceae Conn 1938. In: Bergey's Manual of Systematic Bacteriology, Vol. 1, N. R. Krieg and J. G. Holt, eds., Williams & Wilkins, Baltimore, MD. Jordan, D. C, and 0. N. Allen. 1974. Rhizobiaceae Conn 1938. _I_n: Bergey's Manual of Determinative Bacteriology, 8th ed., R. E. Buchanan and N. E. Gibbons, eds., Williams & Wilkins, Baltimore, MD. Jansen van Rensburg, H., B. W. Strijdom, and C. J. Otto. 1983. Effective nodulation of soybeans by fast-growing strains of Rhizobiu m japonicum . S. Afric. J. Sci. 79:251252.

PAGE 153

-145Kaluza, K., M. Fuhrmann, M. Hahn, B. Regensburger , and H. Hennecke. 1983. In Rhizobium japonicum the nitrogenase genes nifH and nifDK are separated. J. Bacteriol. 155:915-918. Keyser, H. H., B. B. Bohlool, T. S. Hu, and D. F. Weber. 1982. Fast-growing rhizobia isolated from root nodules of soybean. Science 215:1631-1632. Kiss, G. B., K. Dobo, I. Dusha, A. Breznovits, L. Orosz, E. Vincze, and A . Kondorosi. 1980. Isolation and characterization of an R-prirae plasmid from Rhizobium meliloti . J. Bacteriol. 141:121-128. Kondorosi, A., and A. W. B. Johnston. 1981. The genetics of Rhizobium . In : Biology of the Rhi zobi acea e, K. L. Giles and A. G. Atherly, eds., Academic Press, New York, NY. Kowalski, M. 1967. Transduction in Rhizobium meliloti . Acta Microbiol. Polon. 16:7-12. Kowalczuk, E., and Z. Lorkiewicz. 1979. Transfer of RP4 and R68.45 factors to Rhizobium . Acta Microbiol. Polon. 28:221229. Krasilnikov, N. A. 1941. Variability of nodule bacteria. Doklady Akademii Nauk. S.S.S.R. 31:90-91. LaFavre, J. S., and A. R. J. Eaglesham. 1981. Chlorosisinducing toxins produced by Rhizobium and their relationship to nodulation o'f non-nodu lat ing soybean. I_n: Advances in Nitrogen Fixation Research, C. Veeger and W. E. Newton, eds., Ni jhof f/Junk, Pudoc, Wageningen. Law, I. J., and B. W. Strijdom. 1984. Role of lectins in the specific recognition of Rhizobium by Lotononi s b ainesi i . Plant Physiol. 74:779-785. Law, I. J., Y. Yamamoto, A. J. Mort, and W. D. Bauer. 1982. Nodulation of soybean by Rhizobium japonicum mutants with altered capsule synthesis. Planta 154:100-109. Li, D., and D. H. Hubbell. 1969. Infection thread formation as a basis of nodulation specificity in Rhi zobi um -strawberry clover associations. Can. J. Microbiol. 15:1133-1136. Ljunggren, H. 1961. Transfer of virulence in Rhizobium trifolii . Nature. 191:623 Ljunggren, H. 1969. Mechanism and pattern of Rhizobium invasion into leguminous root hairs. Physiol. Plant. Suppl. V. Long, S. R. 1984. Genetics of Rhizobium nodulation. I_n: Plant-Microbe Interactions, T. Kosuge and E. W. Nester, eds., Macmillan Publishing Company, New York, NY.

PAGE 154

-146Long, S. R., W. J. Buikema, and F. M. Ausubel. 1982. Cloning of E.lllzobj^um me_l_i_lot_i nodulation genes by direct complementation of Nod" mutants. Nature 298:485-483. Maier, R. J., and W. J. Brill. 1976. Ineffective and nonnodulating mutant strains of R h i z o b i um ja£onj L cum. J. Bacteriol. 127:763-769. Maier, R. J., and W. J. Brill. 1978. Mutant strains of Rhizobium japonicum with increased ability to fix nitrogen for soybean. Science 201:448-449. Maniatis, T., E. F. Frisch, and J. Sambrook. 1982. Molecular Cloning, Cold Spring Harbor Laboratory, Cold Spring, NY. Martin, M. 0., and S. R. Long. 1984. Generalized transduction in Rhizobium meliloti . J. Bacteriol. 159:125129. Masterson, R. V., P. R. Russell, and A. G. Atherly. 1982. Nitrogen fixation (n_if_) genes and large plasmids of Rhizobium japonicum . J. Bacteriol. 152:928-931. Mead, H. M., S. R. Long, G. B. Ruvkun, S. E. Brown, and F. M. Ausubel. 1982. Physical and genetic characterization of symbiotic and auxotrophic mutants of Rhizobium mel i l oti induced by transposon Tn5_ mutagenesis. J. Bacteriol. 149:114-122. Morales, V. M., E. Martinez-Molina, and D. H. Hubbell. 1984. Cellulase production by Rhizobium . Plant Soil 80:407-415. Morse, W. J. 1915. Note I_n: Variations in soy bean inoculation. J. Amer. Soc. Agron. 7:140. Munevar, F., and A. G. Wollum, II. 1982. Response of soybean plants to high root temperature as affected by plant cultivar and Rhizobium strain. Agron. J. 74:138-142. Newcomb, W., D. Sippell, and R. L. Peterson. 1979. The early morphogenesis of Glycine max and Pisum sativum root nodules. Can. J. Bot. 57:2603-2616. Nigam, S. N., V. Arunachalam, R. W. Gibbons, A. Bandyopadhyay, and P. T. C. Nambiar. 1980. Genetics of nonnodulation in groundnut ( Arachi s hypogaea L.). Oleagineux 35:453-455. Noel, K. D., A. Sanchez, L. Fernandez, J. Leemans, and M. A. Cevallos. 1984. Rhizobium phaseo l i symbiotic mutants with transposon Tn5_ insertions. J. Bacteriol. 158:148-155.

PAGE 155

-14' Nuti, M. P., A. M. Ledeboer, A. A. Lepidi, and R. A. Schi lperoort. 1977. Large plasmids in different Rhizobium species. J. Gen. Microbiol. 100:241-248. Nuti, M. P., A. A. Lepidi, R. K. Prakash, R. A. Schilperoort, and F. C. Cannon. 1979. Evidence for nitrogen fixation ( nif ) genes on indigenous Rhizobium plasmids. Nature 282:533-535. Nutman, P. S. 1949. Nuclear and cytoplasmic inheritance of resistance to infection by nodule bacteria in red clover. Heredity 3:263-294. Nutman, P. S. 1959. Some observations on root-hair infection by nodule bacteria. J. Exper. Bot. 10:250-263. Nutman, P. S. 1981. Hereditary host factors affecting nodulation and nitrogen fixation. In: Current Perspectives in Nitrogen Fixation, A. H. Gibson and W. E. Newton, eds., Australian Acad. Sci., Canberra. Owens, L. D. 1969. Toxins in plant disease: Structure and mode of action. Science 165:18-25. Owens, L. D., J. F. Thompson, R. G. Pitcher, and T. Williams. 1972. Structure of rhizobitoxine, an antimetabol ic enol-ether amino-acid from Rhizobium japonicum . J. C. S. Chem. Comm. p714. Owens, L. D., and D. A. Wright. 1965. Production of the soybean-chlorosis toxin by Rhizobiu m j aponicum in pure culture. Plant Physiol. 40:931-933. Pierce, M., and W. D. Bauer. 1983. A rapid regulatory response governing nodulation in soybean. Plant Physiol. 73: 286-290. Prakash, R. K., R. A. Schilperoort, and M. P. Nuti. 1981. Large plasmids of fast-growing rhizobia: Homology studies and location of structural nitrogen fixation ( nif ) genes. J. Bacteriol. 145:1129-1136. Prakash, R. K., A. A. N. van Brussel, A. Quint, A. M. Mennes, and R. A. Schilperoort. 1982a. The map position of Sym-plasmid regions expressed in the bacterial and endosymbiotic form of Rhizobium 1 eguminosar urn . Plasmid 7:281-286. Prakash, R. K., R. J. M. van Veen, and R. A. Schilperoort. 1982b. Restriction endonuclease mapping of a Rhizobium leguminosarum Sym plasmid. Plasmid 7:271-280.

PAGE 156

-148Pueppke, S. G. 1983. Rhizobium infection threads in root hairs of Glycine max (L.) Merr., Glycine soja Sieb. & Zucc, and V igna unguicu l ata (L.) Walp. Can. J. Microbiol. 29:6976. Pueppke, S. G. 1984a. Adsorption of bacteria to plant surfaces. I_n: Plant-Microbe Interactions, Molecular and Genetic Perspectives, V 1, T. Kosuge and E. W. Nester, eds., Macmillan Publishing Company, New York, NY. Pueppke, S. G. 1984b. Adsorption of slowand fast-growing rhizobia to soybean and cowpea roots. Plant Physiol. 75:924928. Raleigh, E. A., and E. R. Signer. 1982. Positive selection of nodulation-def icient Rhizobium phaseo l i. J. Bacteriol. 151:8 3-88. Ranga Rao, V. 1977. Effect of root temperature on the infection processes and nodulation in Lotus and Stylosanthes . J. Exper. Bot. 28:241-259. Ranga Rao, V., and D. L. Keister. 1978. Infection threads in the root hairs of soybean (G l ycine max ) plants inoculated with Rhizobium japonicum . Protoplasma 97:311-316. Robertson, J. G., P. Lyttleton, and C. E. Pankhurst. 1981. Preinfection and infection processes in the legumeRhizobium symbiosis. _In: Current Perspectives in Nitrogen Fixation, A. H. Gibson and W. E. Newton, eds., Australian Acad. Sci., Canberra . Rosenberg, C, P. Boistard, J. Oenarle, and F. CasseDelbart. 1981. Genes controlling early and late functions in symbiosis are located on a megaplasmid in Rh^zobj^um meliloti . Mol. Gen. Genet. 184:326-333. Rosenberg, C, F. Casse-De lbar t, E. Dusha, M. David, and C. Boucher. 1982. Megaplasmids in the plant-associated bacteria lill-i zoMum inel._il.otJ. and Pseudomonas so 1 anacea rum. J. Bacteriol. 150:402-406. Russa, R., T. Urbanik, E. Kowalczuk, and Z. Lorkiewicz. 1982. Correlation between the occurrence of plasmid pUCS202 and 1 ipopolysacchar ide alterations in Rhizobium . FEMS Microbiol. Lett. 13:161-165. Ruvkin, G. B., and F. M. Ausubel. 1980. Interspecies homology of nitrogenase genes. Proc. Natl. Acad. Sci. U.S.A. 77:191-195. Sadowsky, M. J., and B. B. Bohlool. 1983. Possible involvement of a megaplasmid in nodulation of soybeans by fast-growing rhizobia from China. Appl. Environ. Microbiol. 46:906-911.

PAGE 157

-149Scholla, M. H., and G. H. Elkan. 1984. Rhizobium fredii sp. nov., a fast-growing species that effectively nodulates soybeans. Int. J. Syst. Bacterid. 34:484-486. Sik, T., J. Horvath, and S. Chatterjee. 1980. Generalized transduction in Rbj^zob j^um me_l j^oJLL* Mol « Gen. Genet. 178:511-516. " "" " Skogen-Hagenson, M. J., and A. G. Atherly. 1983. Highfrequency induction of nodulation and nitrogen fixation mutants of Rhizobium japonicum . J. Bacteriol. 156:937-940. Smith, G. R., and W. E. Knight. 1983. Inheritance of ineffective nodulation in crimson clover. Crop Sci. 23:601605. Stacey, G., A. S. Paau, and W. J. Brill. 1980. Host recognition in the Rhizob i urn soybean symbiosis. Plant Physiol. 66:609-614. Stacey, G., A. S. Paau, K. D. Noel, R. J. Maier, L. E. Silver, and W. J. Brill. 1982. Mutants of Rhizobium japonicum defective in nodulation. Arch. Microbiol. 132:219224. Sundquist, W. B. 1981. Farming and U. S. well-being through the years. I_n: Will There Be Enough Food? U. S. Dept. Agric, Beltsville, MD. Sutton, B. C. S., J. Stanley, M. G. Zelechowska, and D. P. S. Verma. 1984. Isolation and expression of Rhizobium j aponicum cloned DNA encoding an early soybean nodulation function. J. Bacteriol. 158:920-927. Tanner, J. W. , and I. C. Anderson. 1963. Investigations on non-nodulating and nodulating soybean strains. Can. J. Plant Sci. 43:542-546. Truchet, G., C. Rosenberg, J. Vasse, J.-S. Julliot, S. Camut, and J. Denarie. 1984. Transfer of Rhizobium mel i loti pSym genes into Agrobacter ium tumef aciens : Host-specific nodulation by atypical infection. J. Bacteriol. 157:134-142. Tshitenge, G., N. Luyindula, P. F. Lurquin, and L. Ledoux. 1975. Plasmid deoxyribonucleic acid in Rhizobium v igna and Rhizobium trif olii . Biochim. Biophys. Acta 414:357-361. Turgeon, B. G., and W. D. Bauer. 1982. Early events in the infection of soybean by Rhizobium japonicum . Time course and cytology of the initial infection process. Can. J. Bot. 60:152-161.

PAGE 158

-150Turgeon, B. G., and W. D. Bauer. In press. U 1 1 r as tructure of infectionthread development during the infection of soybean by Rhizobium japonicum . Planta. Vest, G. 1970. RJ3--A ge ne conditioning ineffectiveness in soybean. Crop Sci. 10:34-35. Vest, G., D. F. Weber, and C. Sloger. 1973. Nodulation and nitrogen fixation. I_n: Soybeans: Improvement, Production, and Uses, B. E. Caldwell, ed., American Society of Agronomy, Madison, WI. Vincent, J. M. 1970. A Manual for the Practical Study of Root-nodule Bacteria, Blackwell Scientific Publications, Oxford. Vincent, J. M. 1980. Factors controlling the legumeRhizobium symbiosis. I_n: Nitrogen Fixation, Vol II, W. E. Newton and W. H. OrmeJohnson , eds., University Park Press, Baltimore, MD. Voorhees, J. H. 1915. Variations in soy bean inoculation. J. Amer. Soc. Agron. 7:139-140. Weber, D. F. 1981. The fix is in. I_n: Will There Be Enough Food? U. S. Dept. Agric, Beltsville, MD. Weber, D. F., and V. L. Miller. 1972. Effect of soil temperature on Rhizobium japonicum serogroup distribution in soybean nodules. Agron. J. 64:796-798. Williams, L. F., and D. L. Lynch. 1954. Inheritance of a non-nodulating character in the soybean. Agron. J. 46:28-29. Wilson, J. K. 1944. Over five hundred reasons for abandoning the crossinocu lation groups of the legumes. Soil Sci. 58:61-69. Zimmerman, J. L., W. W. Szeto, and F. W. Ausubel. 1983. Molecular characterization of Tn5_-induced symbiotic (Fix ) mutants of Rhizobium meliloti . J. Bacterid. 156:1025-1034. Zurkowski, W. 1980. Specific adsorption of bacteria to clover root hairs, related to the presence of the plasmid pWZ2 in cells of Rhizobium trifol ii . Microbios 27:27-32. Zurkowski, W., M. Hoffman, Z. Lorkiewicz. 1973. Effect of acriflavine and sodium dodecyl sulphate on inf ecti veness of R. trifolii . Acta Microbiol. Polon. 5:56-60. Zurkowski, W., and Z. Lorkiewicz. 1979. P lasmid-medi ated control of nodulation in Rhj^obj^um t r__i_fo 1 i^i. Arch. Microbiol. 123:195-201.

PAGE 159

BIOGRAPHICAL SKETCH John Howard Payne was born in Kansas City, Missouri, June 4, 1949. He attended schools in various communities in Kansas and Missouri, graduating from high school at Mt. Zion Bible School in Ava, Missouri. John attended Kansas City College and Bible School, and the University of Missouri — Kansas City. In 1970, he enlisted in the United States Navy and was on the crew that commissioned the USS Cook (FF 1083) in 1972. He served on board her until his honorable discharge in 1976. At the time of his discharge he held the rate, first class sonar technician, and served as watch conning officer of the ship. John attended Chapman College, Orange, California, and Northeast Missouri State University, Kirksville, each for a short time, before returning to the University of Missouri — Kansas City where he received a B.S. degree in botany and a B.A. degree in chemistry in 1978. He has pursued graduate training at the University of Florida, Gainesville, since fall 1979, and anticipates conferral of the Ph.D. in May 1985. John is a member of several professional and scientific organizations and of the agriculture honor fraternity, Alpha Zeta. -151-

PAGE 160

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. --JlUtMA Steven G. Ptfeppke, Chairman Associate Professor of Plant Pathology 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. Raghavan Charudattan Professor of Plant Pathology 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. J^z^^l--i'—7h Thomas E. Freeman Professor of Plant Pathology

PAGE 161

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. £ 1 t 7 c~r$ > , ' , y i ire ^_-J_ -CS2L.-JL. Seorge ETl Bowes Associate Professor of Botany 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. William B. Gurle] Assistant Professor of Microbiology and Cell Scienqe This dissertation was submitted to the Graduate Faculty of the College of Agriculture and to the Graduate School, and was accepted as partial fulfillment of the requirements for the degree of Doctor of Philosophy. #. Dean, College of Agriculture May 1985 Yi^~~~~ 77 lege of AqricMt Dean, Graduate School

PAGE 162

UNIVERSITY OF FLORIDA 3 1262 08285 176 6