Title: Interaction of Rhizobium japonicum with soybean isolines carrying unique genes which affect nodulation at the Rj1 locus
Full Citation
Permanent Link: http://ufdc.ufl.edu/UF00099339/00001
 Material Information
Title: Interaction of Rhizobium japonicum with soybean isolines carrying unique genes which affect nodulation at the Rj1 locus
Physical Description: viii, 151 leaves : ill. ; 28 cm.
Language: English
Creator: Payne, John Howard, 1949-
Copyright Date: 1985
Subject: Rhizobium japonicum   ( lcsh )
Soybean -- Roots   ( lcsh )
Plant Pathology thesis Ph. D
Dissertations, Academic -- Plant Pathology -- UF
Genre: bibliography   ( marcgt )
non-fiction   ( marcgt )
Statement of Responsibility: by John Howard Payne.
Thesis: Thesis (Ph. D.)--University of Florida, 1985.
Bibliography: Includes bibliographical references (leaves 137-150).
General Note: Typescript.
General Note: Vita.
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Bibliographic ID: UF00099339
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: alephbibnum - 000506652
oclc - 22864195
notis - ACS7002


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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


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


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




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

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

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


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


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

LOCUS............................ ................ .. 57

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



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

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

LOCUS .................................. ............. 120

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



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


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.



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.


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




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


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


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


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-


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


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-


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


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


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



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).


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


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


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


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


and soybeans when reciprocal tests of their rhizobia were


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


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


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


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


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


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


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.


The challenge remains to find the point at which the

ril-plant blocks infection and to elucidate the pathway of


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.



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


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


Breithaupt 1980b) of the effect of temperature on nodulation

of Clark and Clark-rji isolines to additional overcoming


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


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.


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.






15- 0


13 -

12 -

'11 -

-10- a

S 8 --

a- a,




22 27 32

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


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
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.


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


(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.


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.


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


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.

22 C 27 C 32 C

22 C 27 C 32 C












22 C 27 C 32 C




22 C 27 C 32 C

-= ONE

Figure 3.2 Continued


22 C 27 C 32 C











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.


3 I 3







-I o

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

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


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


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.


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


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


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


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




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



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


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.


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


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.


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.


0 30 60 90




" 50


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





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.


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

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


8.3 5.7a

0.1 0.4


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

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


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


(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




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


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


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


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.


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


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


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.


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.


I )I' i
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


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


V l(


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


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.


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