THE INFLUENCE OF THE LEGUME IN ROOT-NODULE

SYMBIOSIS
A COMPARATIVE STUDY OF HOST DETERMINANTS AND FUNCTIONS

B Y P . S. NUTMAN
Division of Plant Industry, C.S.I.R.O.,Canberra
(Received 27 3 n e 1955)

CONTENTS PAGE

I. Introduction . . . . . . . . . . . . 109
11. The physiology of infection . . . . . . . . . 1x0
( I ) Preconditions for infection . . . . . . . . I 10
(2) T h e route of infection .. . . . . . . . 1x2
(3) T h e mechanism of infection . . . . . . . . I 13
(4) T h e infection thread . . . . . . . . . I 15
111. The cross-inoculation groups . . . . . . . . . I 16
IV. Genetic resistance , . . . . . . . . . . 120
V. T h e determination of nodule sites . . . . . . . . IZO
( I ) T h e relation between nodule formation and rooting habit . . 120
(2) The association of infection with disomatic centres in the root . . 124
(3) Inhibitory effects of root secretions . . . . . . . 125
(4) Selective effect of host in rhizosphere . . . . . . 126
VI. The infected cell . . . . . . . . . . . 127
VII. Nodule form, growth and function . . . . . . . . 127
VIII. Nodule senescence . . . . . . . . . . . 128
IX. Genetic factors affecting intracellular host-bacterial compatibility . . 129
X. The influence of host species on compatibility . . . . . . 131
XI. The influence of the environment . . . . . . . . 134
(I) Nutritional factors . . . . . . . . . f I34
(2) Physical factors: light and temperature . . . . . * I37
XII. General conclusions . . . . . . . . . . . 138
XIII. Summary . . . . . . . . . . . . . 139
XIV. References . . . . . . . . . . . . 140
XV. Addendum . . . . . . . . . . . * I49

I. INTRODUCTION
T h e object of this article is to bring together information on the effect of host
variation in legume symbiosis, and to discuss the theories which have been advanced
to account for different aspects of the host's influence. A number of reviews have
appeared in recent years covering the whole field of the interrelation between the
leguminous plant and root nodule bacteria (Virtanen, 1947; Allen & Allen I950a;
Demolon, 1 9 5 1 ;Georlette, 1953; Allen & Allen, 1954; Allen & Baldwin, 1954;
Thornton, 1954; Vincent, 1 9 5 4 ~ ) .Each of these have included sections on
host susceptibility, cross-inoculation group specificity, host species and varietal
8 Biol. Rev. 31
II0 P. S. N U T M A N
differences affecting the efficiency of nitrogen fixation and environmental influences
acting through the physiology of the host. This list of topics serves also to define the
scope of this article, but it is proposed to consider them in the context of the life
history of the nodule.
For the purposes of this discussion the type of symbiosis exemplified by clover,
lucerne, or pea will be taken as typical and other types will be compared with it.
This approach has the advantage that at present most is known about symbiosis in
these genera; it is less objectionable than it might otherwise seem because there are
grounds, as will appear from this study, for regarding the clover and lucerne type of
symbiosis as the most specialized, and, as far as we know, the most highly developed.
It has, however, the disadvantage that it tends to conceal the very little at present
known about the great array of species outside the tribe Trifolieae. Except for
aspects of special interest the bibliography is selective.
Before going on to discuss host influences it may be useful to enumerate some
of the central problems in symbiosis which still await solution : ( I ) It is not known
why bacterial root nodule formation should be restricted to legumes or why some
members of the Leguminosae do not conform to this symbiotic habit. (2) T h e pro-
blem of the mechanism of entry of the bacteria into the host tissue by way of the root
hair or otherwise still remains unsolved. (3) The reasons are not known for the
varied degrees of specialization between host and bacteria displayed in the cross-
inoculation groups, and in the amount of nitrogen fixed by different associations of
plant and bacteria. (4)T h e biochemistry of the nitrogen fixation process remains
obscure. ( 5 ) The function of haemoglobin in the host cell is unknown.

11. T H E PHYSIOLOGY OF INFECTION

(I) Preconditions for infection
Prior to initial infection the host plant influences the processes taking place outside
the root in two ways: by the secretion of energy material and growth factors for
bacterial growth (Wilson, 1940), and by the secretion of some material essential for
root invasion by nodule bacteria. T h e growth-stimulating secretion is not restricted
to legumes, but these plants are more active in this respect than non-legumes
(Lochhead, 1952). The most active constituent of this secretion, so far as Rhizobium
is concerned, appears to be biotin, although other members of the B group of vita-
mins are also stimulatory for some strains (West, 1939). T h e second kind of secre-
tion was first demonstrated by Thornton (1929) for lucerne. This was confirmed
and extended to other species by Ludwig & Allison (1935) and by myself (1953),
and has been shown to be non-specific in its activity. Thornton deduced that an
effective accumulation of the exudate occurred by the time the seedling had
unfolded its first leaf, but experiments in which the first leaf (or plumule) was
removed showed that the substance concerned was not formed in the shoot. T h e very
rare formation of nodules on excised roots reported by Lewis & McCoy (1933) does
not exclude the possibility that nodule-promoting substances are produced in the
 Root-nodule symbiosis 111
top of the plant, since in their experiments the root systems examined were freshly
excised from 4-day-old seedlings.
Considerable variation occurs in the initial susceptibility of legumes, using this
term to denote simply the capacity of the host to form nodules. Some start to nodu-
late early in seedling growth, whereas in others nodule formation may be much
delayed. It has been shown(Nutman, 1953)that such differences in nodulating habit
in red clover are inherited and early-nodulating and late-nodulating lines have been
readily selected.
Using material of this kind, detailed studies of the effect of root exudates on
nodulation in red clover (Nutman, 1953 and in preparation) have shown that
the degree to which an individual plant can be induced to nodulate earlier by root
exudates depends wholly upon its normal nodulating habit in their absence. Those
plants which normally nodulate promptly are less affected by secretions than those
which habitually nodulate late in seedling growth; in the presence of secretions from
another plant early and late nodulating lines nodulate at the same time. Early and
late plants would therefore appear to differ only in their capacity to produce a sub-
stance essential for infection to take place. This is confirmed by selection experi-
ments which have shown not only that the early and late habits are inherited but
also that the early plants are more active in the production of root secretions.
The production of the stimulant in red clover, as in lucerne, takes place from the
outset of germination. This can be shown by preplanting agar cultures for varying
times with ‘donating’ seedlings, which can be removed and the test plant intro-
duced. A maximum stimulating effect is obtained with as little as 5 or 10 days’
preplanting. Comparison of inoculated and uninoculated preplantings show that
the bacteria are not involved in the production of the secretion. No information is
as yet available on the nature of the secretion, or whether the presence of the stimu-
latory secretion is a prerequisite for infection in all legumes. It would be of par-
ticular interest to examine this question with host plants in which a different mode
of infection occurs.
In red clover, however, even in the presence of an adequate amount of nodule-
promoting secretion, nodulation does not begin until about 12 days after germina-
tion. By delaying inoculation until the seedling is about 10 days old the interval of
time between inoculation and nodule appearance can be reduced to a minimum
of about 4 days. There is therefore about a week of obligatory host ‘resistance’,
and this corresponds in red clover to the period of root growth before branching
takes place, the root becoming susceptible to infection at about the same time that
laterals first arise.
Experiments carried out with lucerne have given similar results, even to defining
the same minimum time interval between inoculation and the visible results of
infection in nodule formation, viz. about 4 days compared with a s-day period
found by Thornton (197.9). Moreover, with large samples of plants it has been
shown that this minimum period is realized occasionally in the absence as well as in
the presence of secretions. This result shows that the secretion increases the
a-2
II2 P. S. N U T M A N
probability of infection taking place in the minimum time after adding the bacteria.
T h e significance of this relation will be discussed later in connexion with nodule
morphogenesis.
(2) The route of infection
I n the Trifolieae the bacteria enter the root by way of the root hair, as was first
demonstrated by Beijerinck in 1888. T h e infected root hair is usually, but not
invariably, deformed (Bieberdorf, 1938; Arora, 1954) owing to the secretion of
I.A.A. (indolyl acetic acid) by the bacteria (Kostermans, 1935; Thimann, 1939;
Georgi & Beguin, 1939). This reaction appears to be restricted to legumes; the root
hairs of other groups of plants are unaffected by concentrations of I.A.A. up to a
level sufficient to prevent growth (Meesters, 1936), although curling may be
induced by transferring roots from high to low osmotic solutions (Ekdahl,
'953)-
The action of this secretion is unspecific, all bacterial secretions acting on all hosts,
even those they cannot infect. Bacterial strains differ, however, in the amount of
I.A.A. they produce (Chen, 1938), but these differences do not seem to be related
to symbiotic behaviour (Georgi & Beguin, 1939). Curling may be prevented by the
addition of nitrate to the root solution, but occurs if sugar as well as nitrate is
present (Thornton, 1935).T h e former fact may be related to the well-known inhibit-
ing effect of nitrate on nodule formation.
Infection by way of curled root hairs occurs in the Trifolieae, the Vicieae and to a
lesser degree in other groups. Infection of the epidermal cell has been recorded in
an aquatic species, Neptunia oleracea, which does not form root hairs (Schaede,
1940), and through wounds and points of emergence of lateral roots in others
(McCoy, 1929; Arora, 1954). Infection in Arachis hypogea at points of lateral root
emergence (Allen & Allen, 1 9 4 0 ~results
) in the invariable location of nodules in
the mils of or subtending a lateral root. It is frequently observed that even in the
members of the Trifolieae nodules may subtend laterals, particularly in the young
seedlings, and it would be of interest to determine whether infection in these
instances also is through the ruptured epidermis.
Among those plants infected through the root hair the ratio of the number of root
hairs infected to the number curled or to the number of nodules formed varies
widely. Peirce (1902) showed that a great excess of infected root hairs over the
number of nodules formed occurred in lucerne growing on filter-paper. McCoy
(1932) found fewer infected hairs on this plant when grown in agar culture, but
these also much exceeded the number of nodules; only 5 % of the infected hairs
were associated with nodules. In Pisum also an excess of infected hairs are formed
(Wipf & Cooper, 1940; Bond, 1948a) of which a proportion continue their develop-
ment and form nodules. Sometimes a group of infection threads is associated in the
formation of a single nodule; these may arise in separate or in the same root hair
(Bieberdorf, 1938). Among species of Trifolium the number of root hairs infected
is very restricted. Purchase (1953) has shown that in red clover the number of
 Root-nodule symbiosis 1'3
infected root hairs closely corresponds to the number of nodules formed, a relation-
ship which was found to hold for selected genetic lines of red clover which differed
widely in the numbers of nodules developed upon them. T h e infected root hairs
show no other visibly distinguishable features from those which are not infected
and the nature of their physiological specificity remains to be determined.
These few examples show that the processes of initial infection are various, and
range from massive and apparently random infection in some species to the
specialized root-hair portal of entry general in the Trifolieae. Even within the last
group different degrees of restriction in the number of infected root hairs are found,
culminating in red clover and probably other species where there are little or no
superfluous infections.
(3) The mechanism of infection
It is not known whether entry into the host tissue (either of the root hair or other-
wise) takes place primarily by physical or chemical means. Tests for the bacterial
production of cellulase or pectinase (McCoy, 1932) have been negative, and the
closest examination at the point of entry has failed to reveal a break in the cell wall,
indeed, the wall may even appear somewhat swollen at this point, the hyaline or
amyloid spot of Viermann (1929). In the absence of a chemical explanation it is
generally supposed that entry is effected by mechanical means, as has been demon-
strated for many fungi (Gaumann, 1950).
Although the point of entry of the root hair is usually lateral in position, there
seems little doubt that penetration occurs at the site of active growth, the physio-
logical tip of the root hair which has become displaced because of distorted growth.
Moreover, in the event of entry taking place without curling, penetration can occur
at the tip (Viermann, 1929; McCoy, 1932). It is therefore probable that penetra-
tion takes place at a point, where the only barrier is the primary wall, which there-
fore assumes a special importance in this connexion. This was recognized by McCoy
(1932) who was, nevertheless, unable to obtain evidence from a thorough micro-
chemical examination of the host's cell walls, for either the theory of enzymatic
solution or physical penetration.
It is possible that neither of these proposed mechanisms of penetration is
employed, but that the bacteria are able to effect entry by taking part, in some way,
in the processes of growth at the root-hair tip. Recent chemical and electron micro-
scopic work on the composition and structure of the primary cell wall, and on the
physiology of extension growth, shows that such an alternative mode of infection is a
possibility worthy of examination.
The normal root hair grows at its tip where only the primary wall is present
(Lundegardh, I 946; Ekdahl, I 953). This consists of pectic substances-long-chain
polygalacturonides some of which are methylated (Kerr, 195I)-as well as arabans,
galactans and hemicelluloses, and waxes and polypeptides (Treiber, I 95 I ) . Chayen
( I 952) has also disclosed, following prolonged digestion with pectinases, a tenuous
protein membrane within the primary wall. These components form a continuous
17 4 P. S. N U T M A N
phase within which is deposited a discontinuous phase of cellulose fibrils (Kerr,
'951).
The cellulose layer of the primary plant wall differs markedly from that of the
secondary wall in that its fibrillae form a loose and open network, whereas those of
the secondary wall are closely woven and run in parallel series (Frey-Wyssling,
1948, 1952; Muhlethaler, 1949). I n the former the individual fibrillae are flat rib-
bons of mean diameter from IOO to 300A. (Frey-Wyssling, 1948; Preston, 1952)
and are thought to be separated by about 0-3p, the whole primary wall being
about o . 5 thick
~ (Frey-Wyssling, 1952).
Frey-Wyssling suggested that during the growth of the root hair the network of
cellulose becomes loosened and expanded so that new fibrils may be interwoven to
maintain the structure, a process first termed ' intussusception' by Nageli. Auxin
is thought to take part in this process by loosening the cellulosic cross-linkages
(Burstrom, 1951). Frey-Wyssling has also suggested that the loosening may be
aided by the increased production of intercalary material. As the primary wall
becomes older the cellulosic cross-linkages become fixed and the pectic substances
also become cross-linked to form calcium pectate (Cormack, 1954).
Quite minor changes in the external environment may lead to plasmoptysis at the
growing point (Frey-Wyssling, 1948; Ekdahl, 1953), and the problem of infection
may be not so much finding a mechanism for disrupting the structure of the
primary cell wall as finding a means for maintaining its structure and at the same
time allowing the bacteria to enter the root hair. Indeed, an overt rupture may lead
to the death of the root hair.
On the hypothesis that infection takes place by bacterial participation in root-
hair growth, it is possible to visualize the bacteria taking part in the processes of
intussusception and building themselves into and through the primary wall or
inducing a reorientated growth of the host cell wall to give an infection thread by
invagination. Both these possible paths of invasion may represent stages in the
process of infection, the bacteria first insinuating into the pectic layer, possibly by
the production of intercalary material, and then reorientating growth so that the
root hair becomes invaginated. In this way bacteria within the infection thread could
be considered to be still on the morphological outside of the root. It will be recalled
that no visible pore can be found at the point of supposed entry. This may arise by
the hardening of the pectic substances or by the reconstitution of the cellulose net
behind the bacteria.
The interesting fact that isolations from nodules are generally of single strains of
bacteria, even when an intimate mixture of strains is present on the outside of the
root, suggests that the initial penetration may be by one or a few bacteria from
the small colony near the point of entry (Dunham & Baldwin, 1931; Hughes &
Vincent, 1942; Vincent, 1954b). This would conform to the view that the initial
penetration is extremely localized in position and time.
This hypothesis of infection requires : ( I ) close chemical and physical affinity
between the bacterial surface and the primary wall and (2) a mechanism for
 Root-nodule symbiosis 115
perpetuating the redirected growth of the root hair as an infection thread. The com-
position of the bacterial slime varies largely from strain to strain, but all that have
been so far investigated contain polysaccharides which yield glucuronic acid on
hydrolysis (Hopkins, Petersen & Fred, 1931; Bray, Schluchterer & Stacey, 1944).
The peptic substances, on the other hand, mainly give galacturonic acids, but the
composition of both the bacterial surface and the primary cell wall of the plant is so
complex and incompletely known that there is room for as yet undetected affnities.
Demolon & Rozowska (195I) have drawn attention to the similarities between the
pectic substances of plant cell walls and the extracellular slime of nodule bacteria
as a medium for bacterial growth and suggest that an explanation of infection may
be found in this similarity. Similarities also occur in the physical structure, at a
molecular level, between the primary plant cell wall and the bacterial surface ; both
have a periodic structure of about the same dimensions (Labaw & Mosley, 1954).
With regard to the reorientated growth, this may be brought about by the I.A.A.
produced by the bacteria.
This general hypothesis that infection is a growth phenomenon offers an explana-
tion for a number of puzzling features associated with infection, and for the role of
I.A.A. It also suggests explanations, as will later appear, for other problems in the
symbiosis.

(4) The infection thread

In most legumes the continued infection of the developing cells of the nodule
takes place from ramifications of the original infection thread of the root hair. The
walls of infection threads so far studied are clearly of plant origin (McCoy, 1932;
Schaede, 1940,1941),consisting of cellulose deposited around the bacteria, con-
fining them within a narrow tube. Within this thread the bacteria occur sparsely,
sometimes as a single line of rods embedded in bacterial slime. The cells of the cor-
tex traversed by the infection thread have more densely granular contents. The
infection thread within these cells tends to grow towards the nucleus which shows
degenerative changes.
The growth of the infection thread is very rapid at first, as is also the early
development of the nodule. Chen & Thornton (1940)observed that the thread
passes down the root hair and induces proliferation in the cortex of clover within
24 hr. Its further development is less rapid and consists of irregular branching and
penetration into the newly formed cells of the central tissue of the nodule. The
cytochemical investigations of McCoy (1932)and others have shown that the
sheath of the infection thread is not continuous from cell to cell but is interrupted
at the middle lamella. This was elegantly demonstrated by macerating nodule tissue;
the individual cells separated, each with its own section of intact infection thread.
Thornton (personal communication) has also observed that the infection thread
appears to have no sheath when it first enters a new host cell.
McCoy suggested that bacterial zoogleae cross the cell wall by way of pits and are
later covered by the sheath of cellulose. This suggestion accords well with the
I 16 P. S. N U T M A N
mechanism of invasion outlined above. It is now known that the secondary cellu-
lose plant cell wall is interrupted at the pits which in the young cell consist only of
the primary wall and middle lamellar material. The growth of the thread from cell
to cell may therefore take place in an analogous manner to the initial penetration, a
fresh section of infection thread starting its development at each wall pit encoun-
tered. The dimensions of wall pits are such as to allow the passage of bacteria
provided that some mechanism was available to loosen the tenuous net of primary
wall micro fibrils (Muhlethaler, 1950).
I n some host plants progressive infection of the nodule is not from infection
threads, but from intercellular zoogleae or by the division of a few originally
infected cells (Milovidov, 1928) as in Lupinus (Schaede, 1932) and Phaseolus
(McCoy, 1929 and others). These less specialized modes of cell to cell transference
are in some instances associated with less specialized modes of original infection as
in Aeschynomene indica (Arora, 1954), in which infection threads are entirely absent,
the bacteria gaining access through ruptures in the root epidermis. In others, e.g.
Neptunia oleracea (Schaede, 1940), infection threads are present in the nodule
although root hairs are absent, or in Lupinus (Milovidov, 1928) they are present in
the root hair and absent in the nodule. In others again, as in serradella (Milovidov,
1928), nodule infection is both from threads and from intercellular bacteria.
The early workers, for example, Schneider (1892) regarded the infection thread
as a defence mechanism, but it would seem that much more needs to be known to
assign it its full significance. It appears that the partial or complete absence of
infection threads is more frequent in the cross-inoculation groups containing
larger numbers of species in which the route of original infection is more diverse.
In the more specialized groups, such as the clover group, infection threads appear
to be universal.
The release of the bacteria from the thread into the host cytoplasm generally
takes place from a special vesicle, which tends to develop near the host-cell nucleus
(Thornton, 193ob). Schaede (1941) considers, however, that in Vicia and Pisum
the deformation of the thread follows and is not antecedent to the release of the
bacteria. In soya bean and in cowpea the bacteria are released by the break up of the
threads (Bieberdorf, 1938). Threads may also pass through a cell without freeing
any bacteria (Schaede, 1940; Bergersen & Nutman, in preparation).
The vesicles on the thread are not enclosed in an envelope of secondarily deposited
cellulose (Bond, 1948a), and their formation may be a result of stimulated develop-
ment of the layer of primary cell-wall material which may occur on the inside of the
thread. In this way the final entry of the bacteria into the host cytoplasm can be
visualized as the culmination of a process begun at the surface of the root hair.

111. T H E CROSS-INOCULATION GROUPS

A solution of the problem of the mechanism of entry of the bacteria would throw
light upon the differentiation of the cross-inoculation groups. A cross-inoculation
group comprises a number of host species any one of which will form nodules when
 Root-nodule symbiosis "7
inoculated with bacteria isolated from nodules occurring on any other members of
the group. Between such groups cross-inoculation is absent or a rare event. Since
this concept was first introduced it has been much modified in the light of increas-
ing knowledge largely provided by American work. Fred, Baldwin & McCoy (1932)
proposed about twenty cross-inoculation groups and designated as bacterial species
the strains which were thought to be restricted to each group, e.g. Rhizobium
trzjolii, R. legurninosarum, R. meliloti. Many exceptions were later found to this
grouping which has now been reduced to some half a dozen, within the larger of
which subdivisions have been proposed depending upon host susceptibility and
response within the group (Allen & Baldwin, 1954).
The Trifolium cross-inoculation group is the most restricted of those which have
been studied and consists of species of Trifolium only. There are no fully authenti-
cated records of failure of cross-inoculation, although a recent report of Baird
(1955)to the contrary requires examination. The possibility has not been excluded
that her results may be explained in terms of irregular development of bacterial
avirulence. T. ambiguum occupies a very special position in rarely forming nodules
with most strains of Rhizobium trifolii (Parker, Allen & Ahlgren, 1949;Parker &
Allen, 1952). Only with strains obtained from the region of this species' natural
distribution in the Caucasus is nodulation regularly found (Erdman, private com-
munication). Recent work (Hely & Brock, unpublished data) indicates that this
species consists of a number of different races varying in their chromosomal comple-
ment. Further investigation on cytological lines may give an explanation of this
species' sporadic resistance. Hely, Bonnier & Manil (1953)have shown that the
resistance of Trifolium ambiguum may be broken by grafting T. repens scions on to
T. ambiguum stock.
The Pisum, Vicia and Lathyrus cross-inoculation group is also coherent and few
irregularities have been recorded within this group. T h e experiments of Hauke-
Pacewiczowa (1952)suggest the inclusion of Galega oficinalis in this group as it
will nodulate with strains from Viciafaba and V . sativa, although no symbiosis
occurred with strains from Pisum sativum. A case of cross-inoculation between
a pea strain and a clover host confirmed by plant passage has been recorded
(Kleczkowska, Nutman & Bond, 1944). A relation between the pea and clover
group is also indicated by common antigens in their respective bacteria, although in
the example of cross-invasion noted above no serological relationship was found
(Kleczkowski & Thornton, 1944), and by similar patterns of phage susceptibility
(Kleczkowska, 1945).
The Medicago, Melilotus and Trigonella group is also clearly differentiated with
only two species, Medicago laciniata and M . hispida, occupying an exceptional
position. Each of these species is resistant to strains isolated from the other and
also to some strains from other species of Medicago (Purchase, Vincent & Ward,
1951;Vincent, 1954~).
Because of their economic importance the soya-bean and cowpea groups have been
extensively studied. These two plants at one time formed the type species of two
118 P. S. N U T M A N
cross-inoculation groups (Fred et al. 1932) which are now generally considered
together. Isolates from soya-bean cultures usually nodulate both these species,
whereas most cowpea bacteria do not nodulate soya bean (Leonard, 1923; Sears &
Carroll, 1927; and others). In general, however, strains isolated from soya bean
nodulated fewer species than those isolated from cowpea, lima bean or Lespedeza.
Included in this group of plants is a great variety of subtropical and tropical
legumes (Allen & Allen, 1936a, 1939; Burton, 1952; and others). Many cases of
non-reciprocal inoculation have been described, thus : all strains from Sesbania
have been shown to nodulate cowpea and soya bean but none from cowpea and
soya bean nodulate Sesbania (Johnson & Allen, 1952); similarly strains from Les-
pedeza fail to nodulate Phaseolus lunatus, whereas strains isolated from P. lunatus
nodulate Lespedeza. Phaseolus lunatus appears to occupy in the cowpea group a
position analogous to Medicago laciniata in being nodulated by very few strains
isolated from other members of the group. Bushnell & Sarles (1937) and Reid (1936)
have demonstrated some slight degree of cross-inoculation between the cowpea,
soya-bean and lupin groups, the degree of cross-inoculation depending upon the
time of year the test was made. Richmond (1926) and Hofmann (1927) in experi-
ments, in which reciprocal grafts were made between Phaseolus vulgaris and
P. lunatus, claim to have broken the resistance of the P . lunatus to bacteria isolated
from the P. vulgaris, and also that this effect of grafting was transmitted to seeds
formed on the scion. These claims were not substantiated by Hansen & Tanner
(1931) working on the same species and also with Dalea and Phaseolus. Bonnier,
Hely & Manil (1952) by grafting have induced nodule formation on soya bean with
some strains from other cross-inoculation groups.
Not much is known of the cross-inoculation relationships within the Phaseolus
group (which consists of some species of Phaseolus but not P . lunatus). A case of
non-reciprocal cross-inoculation has been demonstrated between Dalea alopecu-
roides and Phaseolus vulgaris (Sears & Clark, 1929).
Insufficient work has as yet been carried out on the Lupinus-Ornithopus group
and the other small groups proposed by Fred et al. (1932) for definite conclusions to
be drawn. As noted above the soya-bean and lupin groups appear to be related.
Much remains to be done before the cross-inoculation interrelationships have
been fully explored. Of some twelve thousand species of legumes distributed in
about 450 genera only 487 species in fifteen genera had been employed in cross-
inoculation tests up to 1947 (Allen & Allen, 1947). However, the main pattern of
variation in host susceptibility is clear. Most legume species so far examined belong
to the cowpea group within which a subsidiary separation may be made of species
into groups and individual plants depending upon their susceptibilities to strains
within that group, and it is probable that the complexity of this subsidiary pattern
will increase as more bacterial strains are examined, but there is no reason to doubt
the broad identity of the cowpea group. Other groups show less and less diversity
both with respect to their botanical composition and the host-bacterial interrelation-
ships found within them. Some of these groups are connected by rare but un-
 Root-nodule symbiosis 119
doubted cases of cross-inoculation, but the extensive claims of promiscuous cross-
inoculation between the majority of legumes (Wilson, 1931 to 1946) have not been
confirmed with the exception of the work of Aughtry (1948) and Gregory & Allen
(1953). Aughtry was able to obtain nodules on selected lines of Medicago falcata
and M . sativa with isolates from species of Astragalus, and claims to have been able
to select for susceptibility. The experiments were carried out with cuttings and are
not wholly conclusive because of possible contamination. The claims of promis-
cuous cross-inoculation by Gregory & Allen concern Caragana arborescens,
isolates from which were found to nodulate species belonging to the cowpea, bean,
lotus and clover groups. This result again runs counter to general experience and
awaits confirmation.
The nature of cross-inoculation group specificity has not yet been determined,
presumably because of the difficulty in analysing the metabolic or chemical affinities
between bacteria and host. Serological investigations have been carried out on the
bacteria (see review by Allen & Allen, 1 9 5 0 ~and
) on the seed proteins of a number
of legumes (Baldwin, Fred & Hastings, 1927). Both on the side of bacteria and
host a broad correlation was found between antigenicity and cross-inoculation
grouping. Many exceptions, however, were encountered, so that it could not be
said that specificity was causally determined by an antigen or a group of antigens.
No experiments appear to have been made to examine relations between host plant
and bacterial antigens.
The hypothesis suggested above that the initial infection of the root depends
upon some chemical correspondence between primary cell wall and bacterial
surface opens fresh fields for the investigation of this problem. On this view the
absence of infection of the host plant of one cross-inoculation group when inoculated
by bacteria from another would be due to considerable dissimilarity between
primary cell-wall substance and the exterior of the bacteria. The known chemical
complexity of the primary cell wall on one hand and the bacterial envelope on the
other provides ample scope for the development of intricate patterns of host sus-
ceptibility and strain specificity. Reports that nodule bacteria are subject to trans-
formation of the kind first established by Avery, McLeod & McCarty (1944)for the
capsular polysaccharides of Pneumococcus are of special interest in this connexion.
Israil’skii (1954)was unable, using simple sterile filtrates as the transforming agent,
to confirm Krasilnikov’s (1941) original claim for transformation in nodule bacteria.
Recent work by Balassa (1954),using heat killed suspensions, extracts and treatment
with ultrasonics, describes transformation in a variety of characteristics of the
bacteria including virulence and host specificity.
This concept may also eventually provide an explanation of the restriction of root-
nodule bacterial symbioses to the Leguminosae with a few doubtful exceptions
(Sabet, 1946; Mostafa & Mahmoud, 1 9 5 1 ;Allen & Allen, 194ob, rosob), and also
for the absence of symbiosis in a minority of genera.
I20 P. S. N U T M A N
IV. GENETIC RESISTANCE
Completely non-nodulating legumes with three recorded exceptions are confined
to the subfamilies Caesalpinioideae and Mimosoideae (McDougall, 1921; Allen &
Allen, 19363; Allen & Baldwin, 1954; BanZdos & Fernandez, 1954). These species
belong to a primitive group which may not have completely acquired the nodulating
habit. Elsewhere, viz. in certain strains of Trifolium ambiguum, T . pratense
(Nutman, 1 9 4 9 ~and) Soya max (Lynch & Sears, 1951; Williams & Lynch, 1954),
resistance represents a change in some property of the host. T h e case of Trifolium
ambzguum has been discussed above; its resistance is not complete, since it can be
broken by grafting and does not occur in all strains of the host (Hely et al. 1953),or
with all strains of the bacteria. T h e resistant line of red clover differs from T. am-
bzguum in that its resistance is complete and unaffected by grafting with a suscep-
tible scion. The inheritance of resistance in this instance was found to depend upon
a simple recessive factor associated with a cytoplasmic component. T h e responsible
gene was shown to be widely distributed as the heterozygote but very rare as the
homozygote. This was because the homozygote was lethal in the absence of the
cytoplasmic factor. Genetic evidence suggested that the production of the cyto-
plasmic factor was itself controlled by the resistance gene, a residual amount of the
factor being necessarily present in the embryo sac for the gene to mediate its
increase. On the basis of these relations the speculation was advanced (Nutman,
1949 a) that the nodulating habit in legumes has a cytoplasmic basis and may have
arisen and spread through the family like a virus. T h e recently recorded case of
resistance in soya bean has also been shown to depend upon a simple recessive.

V. THE DETERMINATION OF NODULE SITES

Whatever the portal of entry, there appears to be a limitation to the number of
successful infections, i.e. those that give rise to nodules. This limitation, whether it
takes place at the root hair or subsequently, seems to be determined by the host.
The facts on which this conclusion is based derive from work in the clover group,
summarized by Nutman (1952a),but as they are cogent to a number of other aspects
of symbiosis to be discussed it is not inappropriate to recall them.

(I) The relation between nodule formation and rooting habit

The classical view of infection is that the number of nodules formed on the root
is primarily a function of the virulence of the bacterial strain, that is, the capacity of
the bacteria to enter the root and induce nodule formation thereon. This view
gained much support from the great variations recorded in the apparent virulence of
strains and from the fact that the alternative thesis that nodules were metamor-
phosed roots was open to serious anatomical objections (see Allen & Allen, 1 9 5 0 ~ ) .
The alternative view has, however, been upheld by a number of workers since the
earliest days of investigation of nodule origin, and received experimental support
from Thimann’s (1936) experiments which indicated that the initiation of the
 Root-nodule symbiosis I21
lateral primordium giving rise to the nodule was induced from outside the root by
the secretion of I.A.A. by the nodule bacteria.
Support for this hypothesis was obtained in experiments on injection of growth
substances into roots. T h e growth of the lateral root primordium was restricted
and came to assume a superficially nodule-like appearance. Also growth-factor
assays of nodules showed that auxin was formed in a concentration sufficient to
explain the different growth patterns of nodules and roots, though not their
structures (Thimann, 1939; Link & Eggers, 1940; Link, Eggers & Moulton, 1941).
Bond (19483) and Allen, Allen & Newman (1953) have examined the outgrowths
produced on roots by the external application of a variety of growth substances.
These were unlike nodules in arising from the pericycle. Thimann’s view of nodule
initiation fails to account for some of the salient facts concerning infection and for
the apparent differences in strain invasiveness or virulence.
A different approach has been made to this problem by following the develop-
mental physiology of roots and nodules in selected lines of red clover which were
very different in their nodulating habits (Nutman, 1948). These studies revealed a
very close relation between lateral root production and infection in spite of the
different apparent sites of origin of laterals and nodules. Plants selected and bred
for abundant nodule formation always gave rise to larger numbers of lateral roots
than plants selected for sparse nodulation, even when uninoculated with bacteria.
It was also found that in the early stages of seedling development the number of
lateral roots was larger on uninoculated than on inoculated plants, so that as a
result of inoculation a replacement of laterals by nodules occurred. This result
strongly suggested that infection occurred at sites or foci which ordinarily give rise
to lateral roots.
In support of this hypothesis it was also established (Nutman, 19493) that in the
very young seedlings differences in bacterial strain virulence were not at first evident
but developed later when morphological differences between the nodules arose,
strains giving larger nodules appearing less virulent. This dependence of virulence
on nodule size suggested that the larger nodules of the less virulent strains may be
inhibitory for further infection, and the hypothesis was advanced that the develop-
ment of the infectable foci in the root, i.e. lateral primordia, was itself determined
by the activity of the primordia already present. If inhibition of this kind occurs
in the root, the nodule as well as the lateral root will influence the development of
further infectable foci in the root.
This view provides an explanation for the apparent differences in strain virulence,
since it is clear that a small and degenerate nodule produced by a virulent strain
will have a transient inhibitory effect compared with that of a large nodule formed
by a less virulent strain. This explanation accounts for the general relationship
that large nodules tend to be sparse and small nodules abundant.
Many instances of the association of abundant nodulation with ineffectiveness
have been described (Dkherein & Demoussy, 1900; Ruf & Sarles, 1937; and others),
but this correlation is by no means without exception (McKnight, 1949; Burton,
 I22 P. S. N U T M A N
1952;and others). Studies on a wide range of subterranean clover varieties and
Rhixobium strains (unpublished data) are of particular interest in this connexion.
In this species neither nodule size nor abundance are related to effectiveness; some
ineffective strains give rise to few large nodules and others to many small nodules,
and even between effective strains wide variations occur in the number and size
of nodules found. A closer examination of the relation between nodule size and
number disclosed the following relations: (I)for each host variety the product of
average nodule length and number per plant tends to a constant value (i.e. these
determinations are the parameters of an hyperbola); (2)the clearly defined hyper-
bolic curve of this relationship is characteristic for a host variety and differs between
varieties. Thus, points on a curve for a given host variety given by a freely nodulat-
ing bacterial strain are confined to that end of the curve corresponding to small
average nodule size, and those given by a sparsely nodulating strain to the opposite
end of the curve. Moreover, with some strains of bacteria the points on the curve
may be restricted to a small segment, whereas with others they may extend over its
whole length. These different dispositions of points for bacterial strains do not
correspond in the hyperbolae of different varieties of the host. Thus with the
variety Bacchus Marsh a particular bacterial isolate gives a restricted group of
points, whereas with the variety Dwalganup the same isolate gives points on all
parts of the curve.
I n this species therefore infection and nodule growth are closely interrelated and
are determined by a distinctive interaction between plant and bacteria. This
allows, on the one hand, great variation in nodule density and nodule size, and
on the other it assures that the total mass of nodular tissue (here proportional to
number x length) remains the same.
Red clover in contrast shows a much less well-defined relation connecting size
and number (Nutman, 1949b) which may reflect the heterozygosity of this species
compared to subterranean clover. Indeed, if the individual curves corresponding
to each variety of subterranean clover are plotted together a similar indefinite
relationship appears.
The nodulating habit of a species is broadly related to its rooting habit; those
with strongly developed tap roots, such as, for example, species of Medica,no,
nodulate less abundantly than the fibrous rooting species such as the clovers
(Jensen, 1948;Nutman, 1953). Aberrant nodulation in the field may also be
explained in terms of this relation. For example, it has been frequently observed
in clover sown in areas unsuitable for the spread of the bacteria inoculated on to the
seed that nodulation is restricted. Only one or two nodules may be produced in the
immediate vicinity of the seed coat; such nodules may attain grotesque proportions.
Experimental confirmation of the hypothesis of the limitation of infection by the
activity of meristematic foci already present was obtained in experiments on
excision of nodules and lateral root tips (Nutman, 1952b). Removal of nodules or
root tips or nodule tips each led to a stimulation of infection during the period
immediately following the operation. This would suggest that the inhibitory effect
 Root-nodule symbiosis '23
came from the growing point of the root or nodule rather than from tissues con-
taining bacteria. It was also found that the amount of stimulation obtained per
nodule excised was greater for families of plants producing few rapidly growing
nodules than for families with many small nodules.
This hypothesis may also explain the differences found in the rate of infection
following delayed inoculation (Nutman, 1949b). If initial infection is delayed
for a short period by withholding the inoculum, nodules arise subsequently at a
much increased rate. This may be because the lateral roots which would have been
infected give rise instead to more roots which themselves originate further pri-
mordia, thus raising the potential number of infectable foci. With a moderate delay
this stimulating effect on nodulation is permanent, but if inoculation is delayed until
severe nitrogen starvation has set in no stimulation appears. Instead, only a few
nodules are initially formed, after which infection ceases altogether for a period of
some weeks until the fixation of nitrogen allows a resumption of root growth.
With delayed inoculation by ineffective strains no stimulation occurs in red clover,
presumably because the ineffective nodule is small, has no active growing point
and is not inhibitory; even on plants inoculated at sowing such nodules do not
influence the number of foci appearing on the root.
It will be recalled that the interval between inoculation and nodule formation in
the young seedling of red clover can be reduced by delayed inoculation and has a
minimum value of about 4 days, and also that the presence of additional stimulatory
root secretions does not shorten this minimum interval but increases the probability
of its being found for a particular infection. This result is also in conformity with
the view that infection is limited to definite foci on the root and is an event which
may or may not take place at a given time; the presence of stimulatory secretions
merely increases its probability.
The virulence theory of infection raises the question as to why the whole root is
not completely covered with nodules, whereas the view that infection occurs at
meristematic foci on the root raises the question as to why all lateral primordia are
not infected. The intervention of the mechanism of root-hair infection may provide
for concurrent development of both roots and nodules, since infection would be
restricted to foci arising within the narrow zone of active root-hair growth. It is
well known that nodules do not arise on the mature parts of the root even when root
hairs are present.
As remarked above, in some species the number of root hairs infected much
exceeds the number of the nodules formed, whereas in others the numbers of
infected root hairs and nodules are about the same. In the latter the foci of infection
must exert an influence on the susceptibility of neighbouring root hairs. This may
reflect a corresponding restriction of the secretion of substance necessary for infec-
tion to take place to a few points on the root.
This morphogenetic view of infection is rejected by Allen & Allen ( ~ g s o a )who
,
consider that the close relationship between nodulation and root formation is
incidental.
 P. S. N U T M A N

( 2 ) Association of infection with disomatic centres in the root

A discussion of factors determining the siting of nodules is incomplete without
considering the origin of nodule disomaty. The abnormal cytological situation in
nodules was first noticed by Fred in 1908 and was commented upon by Dangeard
and others during the following two decades, and more recently the studies of Wipf
(1939)and Wipf & Cooper (1938, 1940) and Fujita & Mitsuishi (1953)have added
much to our knowledge. They found for a wide range of legumes inoculated with
effective or ineffective strains of bacteria, that the host cells in the central tissue of the
nodule contained double the number of chromosomes present in the rest of the root.
In the nodule itself the infected central region is disomatic and the uninfected
cortex derived from the original root cortex monosomatic, the vascular traces are of
mixed diploid and tetraploid cells; occasional triploid cells have been found in the
meristem (Bond, 1 9 4 8 ~ )These
. relationships were found for polyploid as well as
for diploid hosts, so that with, for example, a tetraploid host the infected nodule cells
are octaploid.
The disomatic cells of a nodule arise from single disomatic cells already present
in the root cortex before infection and indeed also in the uninoculated root. These
and neighbouring cells appear to be stimulated into division by the near approach
of the infection thread, the diploid cells showing, however, pathological symptoms
in the immediate vicinity of the thread. Wipf & Cooper (1940) observed in the pea,
where infection of the root hair is fairly frequent, that those threads penetrating the
cortex away from the disomatic cells cause no stimulation and do not give rise to
nodules. They also observed that the disomatic cells are commonest in the root-
hair zone and are often associated with lateral roots. Bonnier (1954) has further
investigated the connexion between infection and polyploidy in experiments with
clover and lucerne grown in colchicine solution. Infection was found to be both
more frequent and occurred earlier in presence of high dilutions of the mitotic
poison.
Comparison of a number of diploid and tetraploid races of red clover (Nilsson &
Rydin, 1954) and of subterranean clover (Nutman, unpublished data) has shown
that nodulation is invariably sparser in the latter. This suggests that the disomatic
meristem is more apically dominant in the physiological sense than the diploid.
A similar conclusion was also suggested by experiments on the effect of nodule and
lateral root-tip excision which showed that the nodule meristem, consisting largely
of disomatic cells, was always slightly but appreciably more apically dominant than
the lateral root tip. Nilsson, Bengsten & Mellander (1954)have analysed chromato-
graphically the amino-acid content of z n and 4 n lines of red clover in an endeavour
to discover the cause of differences between them in performance and nodulation.
With the exception of arginine, which was only present in the 4 n plants, the same
amino-acids occurred in all plant parts of both lines.
The disomatic cells seem to be the actual predetermined sites of infection, but the
reason for restriction of intracellular infection to cells containing twice as many
 Root-nodule symbiosis 125
chromosomes as their neighbours remains quite obscure. T h e relation between these
centres and associated lateral primordia is worthy of closer study, in particular as to
whether this association is obligatory. T h e original disomatic cells may legitimately
be described as meristematic initials, and the developing nodule functions in many
respects as a lateral root.

(3) Inhibitory ejfects of root secretions

Although root secretions are at first stimulatory, this effect is transient, and at a
later stage their action is to inhibit infection, as may be demonstrated by growing
more than one plant together in the same container. Under certain conditions the
number of nodules produced in a culture of red clover is independent of the number
of plants present, so that, for example, each plant of a pair growing together will
produce half the number of nodules as plants grown singly in the same volume of
medium. If the volume of the medium is altered the number of nodules also alters
proportionately (Nutman, 1945).
Inhibitory effects can also be demonstrated by preplanting experiments similar
to those described above. By this means it has been shown that the inhibitor is
produced continuously by the seedling, different species and even strains of host
differing in the rate at which the inhibitor is produced. Within a host species the
most striking differences are between lines which nodulate early or late. T h e early
lines are more inhibitory than the late ones, a result which suggests that the
root secretion stimulatory at the initiation of infection becomes inhibitory as its
concentration increases. Relationships of this kind are common in physiology; it is
only necessary to cite plant growth-promoting substances in this connexion.
Individual hosts vary in their sensitivity to inhibition as to stimulation. For
example, nodulation on lucerne is as much inhibited by a preplanting period of
10or 20 days as by one of 40 or even 80 days, whereas clover nodulation is increas-
ingly inhibited by preplanting periods of up to about 60 days; a straight-line rela-
tionship of inhibition against preplanting time suggests a constant rate of secretion.
Some species appear to be relatively insensitive to inhibition, for example, Vicia
hirsuta is stimulated in associated growth with another legume to produce nodules
earlier than it otherwise would, but is not afterwards inhibited (Nutman, 1953).
Very little information is to hand on the chemical nature of the stimulatory or of
the inhibitory exudate. Substances responsible for these effects can be transferred
from one culture system to another (Nutman, 1952a), and in so far as inhibition is
concerned, activity is proportionately reduced by dilution. All attempts at chemical
concentration have failed and efforts are being directed to adsorption of the active
material on to a variety of substances. Of these, activated charcoal, fuller's earth
and bentonite were most potent in stimulating nodulation in older plants, pre-
sumably because of the adsorption of the inhibitor. This effect was more notable in
a medium of p H above 7, suggesting that the active material may be acidic. Efforts
at the recovery of the inhibitor from the adsorbents have so far been inconclusive
(Nutman, 1 9 5 2 a ; Turner, 1955), indicating either that it is very strongly adsorbed
9 Biol. Rev. 31
126 P. S. N U T M A N
or readily inactivated by even mild chemical treatment. Turner’s (1955) observa-
tion that earlier nodulation is promoted in the presence of adsorbent is of particular
interest in view of the observation, noted above, that stimulation produced by added
root secretions appears to be independent of concentration.
The effect of adding chemical adsorbents to culture media has also been examined
by Vantsis & Bond (1950) on peas and by Gukova & Butkevich (1941)with soya
beans. The latter authors found an increased number of nodules in presence of
charcoal, but Vantsis & Bond obtained fewer nodules with charcoal although nitro-
gen fixation and growth were much stimulated.
It may be mentioned that in the course of these experiments it was observed that
roots developing in bentonite frequently became outlined with a zone of coloured
clay, the colour produced depending upon the species, blue-green for clover,
orange for vetch, and pink for Melilotus. Whether this interesting phenomenon has
any relevance to the problem of infection remains to be discovered, but it would
seem to be of some significance that legumes appear to be more regular and active
producers of colour than non-legumes (Nutman, 195 I ) .
(4) Selective effect of host in rhizosphere
With a single species of host grown in aseptic culture no selective effect of the
host has been observed in determining infection from mixtures of bacterial strains
present in the immediate surroundings of the root (the rhizosphere) ; strains isolated
from the nodule occurring in the same proportions in which they are found in the
rhizosphere (Nicol & Thornton, 1942;Kalnin’sh, 1951; Harris, 1954). Under less-
defined conditions this proportionality does not hold (Virtanen & Linkola, 1944).
Nodule formation may also be affected by other organisms present in the rhizo-
sphere (Krasilnikov & Koreniako, 1949;Harris, 1953). In mixed host and bacterial
cultures nodulation appears to be influenced by a specific host effect (Vincent &
Waters, 1953),so that a strain, not dominant in the rhizosphere, will form a higher
proportion of the nodules on the roots of one host species than on another. Selec-
tive host influences of this kind may serve to explain the conflicting evidence on the
effect of plant passage (Allen & Baldwin, 1931;Nutman, 1946b;Virtanen & Linkola,
1944; Fedorov & Svitych, 1954). It would only be necessary to assume that where
such effects are found the bacterial strains are intrinsically unstable for the charac-
teristic, usually virulence or effectiveness, thought to be changed by plant passage.
Explanations of the selective influences of the host in the rhizosphere must await
further investigation. They may be due to localized differential stimulation or
suppression of the bacteria, to interference with the synthetic processes which may
take place at the point of infection or to an indirect effect on the determination of the
nodule sites.
It appears, however, from the experiments of Purchase (1953), using heavy
inocula of mixtures of normal clover nodule bacteria and its avirulent variant (in
which the proportions of the two types remained constant), that a very heavy
dilution of the virulent by the avirulent is required to have any effect on nodulation.
 Root-nodule symbiosis 127
From her results Purchase concluded that a population of a few thousand
virulent nodule bacteria in the rhizosphere of a clover plant in agar culture is
sufficient to give maximum infection. For Phaseoh rudiutus Bhaduri (195I ) found
that infection is related to size of inoculum over a much wider range.

VI. T H E INFECTED CELL

After their release from the infection thread or otherwise into the host cytoplasm of
the large tetraploid parenchymatous cells of the centre of the nodule, the bacteria
rapidly increase in number and size. T h e detailed changes which take place in
bacterial form and staining reactions as the short rods give rise to the ‘ bacteroids’
have been described in a number of species (Fred et al. 1932). These are still con-
sidered by some authors to represent stages in a life cycle (Bissett & Hale, 1951;
Bissett, 1952) rather than the product of special conditions in the host cytoplasm.
Recent work by Bergersen (1955) on clover bacteroids has shown that with effective
strains the increase in size of the bacterial cell is accompanied by a division of the
nuclear material and by the gradual development of perinuclear areas. As the nuc-
lear centres become smaller and break up, the perinuclear area enlarges to occupy
most of the bacteroid. Surrounding the perinuclear area are mitochondria1 granules
which are the centres of oxidative activity. These granules are more prominent
in ineffective bacteroids and with some strains are associated with the accumulation
of glycogen.
Certain investigators have attributed bacteroid formation to alkaloids, glucosides,
etc., present in the nodule sap (Sembrat, 1934; Itano & Matsuura, 1936, 1938).
and claims have been made for bacteroid formation in artificial media supplemented
in various ways (Nilsson, Bjalfve & Burstrom, 1939; Norndorf & Nilsson, 1943;
Heumann, 1952). These should be re-examined cytologically, since change in form
may be not of primary significance.
Bergersen’s observation that the nuclear apparatus of the bacteroid is degenerate
may account for the interesting suggestion, first made by Beijerinck and later con-
firmed by the single cell studies of Almon (1933), that bacteroids isolated from the
host cell are no longer able to multiply on artificial media, although they react to
vital stains (Spicher, 1954). It is also of interest to note that the host cell nucleus
is also degenerate at this stage and no Ionger capable of division (Schaede, 1940). I

T h e significance of these remarkable sequences of changes in the host and bacterial

cell as the symbiosis is consummated is quite obscure. T h e cytology and division of
nuclear material in cells grown on artificial media are normal (Voets, 1949).

VII. NODULE FORM, GROWTH AND FUNCTION

T h e detailed morphology and anatomy of the nodule may show interesting diver-
gencies from the well-known Trifolium type. Its exterior may be smooth as in the
soya bean, scurfy as in lucerne or sculptured and ridged as in Lotus. It may have
the colour of the neighbouring root or may be brightly coloured with green and
9-2
I 28 P. S. N U T M A N
brown pigments, anthocyanin or in some cases chlorophyll so as to mask the
haemoglobin colour of the interior.
In its relation to infection the factors determining the size of the nodule have
already been discussed, and are related to the inherent meristematic potential of the
root. Nodule form depends upon the shape and size of the meristem and its ten-
dency towards bifurcation. Among the Trifoliae the nodule is usually cylindrical
and unbranched, though in exceptional circumstances branching may be very
extensive, as in subterranean clover growing under conditions restricting the
number of infections which take place.
In structure all nodules consist of a central bacterial zone and an outer cortex
within which develops a vascular system which is very complex in some species
(Lechtova-Trnka, 193 I ; Gregory, 1949). In many though not in all host plants, for
example peanut (Allen & Allen, 194oa), the central bacterial tissue as well as the
vascular system is enclosed in an endodermis, the function of which is unknown
(Fraser, 1942).T h e cortex may also contain pigment cells, tannin cells and sclerides
and may also develop a periderm (Allen, Gregory & Allen, 1955). The exact dis-
position of the vascular traces varies and has been used as basis for the classification
of nodules. Spratt (1919)recognized a number of main and subsidiary types; the
Trifolium type with a basal strand which remains undivided for some time is among
the simpler.
T h e nodule is an organ of relatively short life, usually renewed season by season.
In some woody species it persists and is perennial, developing annual rings of
growth (Jimbo, 1927). In Sesbania (Harris, Allen & Allen, 1949), Caragana (Allen
et al. 1955) and on rare occasions in red clover (author’s observation), renewed
growth takes the form of the development of rootlets from the periphery of the old
nodule meristem.
This versatility in morphology and structure, which may fit the nodule to function
under a great variety of conditions, would repay systematic study, but it should be
stressed that underlying this diversity there is a remarkable regularity in basic
structure. This uniformity applies in particular to the tissue containing the bacteria
which in all species studied consists of large cells, the cytoplasm of which is densely
packed with the bacteria.

VIII. NODULE SENESCENCE

T h e infected tissue of the nodule ordinarily shows no change for some weeks or
months before it eventually breaks down and disappears. In the species most
studied in this connexion nodule degeneration begins by the multiplication of the
bacteria which have remained in the infection thread and their spread into the inter-
lammellar and intercellular spaces (Thornton, 1 9 3 0 ~ The
) . contents of the host cells,
including the bacteroids, become lysed in the process. Lysis of the bacteroids is
very rapid, no stages in the process being discernible. Tissue breakdown starts at the
base of the nodule and progresses apically until the whole of the nodule is destroyed.
Gaumann (1946) considers that this digestion and elimination of the intracellular
 Root-nodule symbiosis 129
bacteria culminates the anti-infectional plasmatic reactions of the host. This inter-
pretation is not in accord with the facts, since it is the host plant cell as well as its
contents which are lysed. At one time it was thought that the host received benefit
from the release of nitrogen chiefly or entirely at lysis, but studies of the nitrogen
content and transfer of nitrogen from the nodule at different stages of its growth
have shown this view to be erroneous (Bond, 1936;Jensen, 1948;Tsujimura, 1951).
The nodule may enter the degenerative phase at an early stage if the conditions of
growth of the host are unsuitable, or if the bacteria and the host are inherently
incompatible. I n many cases this incompatibility can be ascribed in respect of a
particular host to a change in the bacterial strain (Stevens, 1925; and others), such
ineffective strains usually giving rise to numerous small nodules. I n red clover
Chen & Thornton (1940)have shown that the ineffectiveness can be wholly ascribed
to the early degeneration of the bacteroid tissue of the nodule and the involvement of
the nodular meristem, which in turn may be due to the induced production by the
host plant of a substance harmful to the bacteria (Thornton, 1939). Breakdown of
the nodular meristem is not invariably the cause of ineffectiveness, as, for example,
with some soya-bean strains (Chen & Thornton, 1940), where the degeneration of
the dispersed infected tissue takes place without the collapse of the nodule. Some
strains ineffective on subterranean clover are known in which ineffective nodular
tissue is persistent, in others no organized infected tissue is formed (Bergersen,
unpublished observations).

IX. GENETIC FACTORS AFFECTING HOST-BACTERIAL

COMPATIBILITY
Examples of ineffectiveness have also been described which are more appropriately
accounted for in terms of a defect in the host. I n some of these the factors in the
host giving rise to ineffectiveness have been analysed genetically. T h e results
obtained in these studies will be discussed in some detail as they fundamentally
concern the influence of the host plant in the symbiosis.
As yet this analysis has been undertaken only for red clover which is an obligatory
cross-pollinated species and rich in symbiotic variability. In the variety Late-
flowering Montgomeryshire Red, about 2 yo of plants respond ineffectively with the
standard effective strain (Rothamsted Strain A) used in these studies (Nutman,
19464 1954a, b), and of these a genetic basis could be demonstrated in most
instances.
Two recessive genes are now known (Nutman, I 954b, and in preparation) conferring
ineffectiveness on the host. One of these (called il) gives rise to typical ineffective-
ness, the homozygotes bearing nodules which are small, numerous and ephemeral.
This incompatibility, however, is specific to the particular combination of gene i,
and strain A, other effective strains of bacteria giving rise to normal effective sym-
bioses with this host genotype. Moreover, the incompatibility between A and i,
can be overcome by the action either of suppressor genes in the host or by mutative
change in strain A. The suppressor gene (m)is also recessive and segregates
130 P. S. N U T M A N
independently of i,, so that ineffective symbioses can be represented by the following
genetic constitutions: A (ilil,M m )or A (ilil,M M ) ; and the effective by: A (ilil,mm)
or A’ (ilil,Mm),etc., where A’ is a variant of A or an unrelated strain.
The second gene ie segregates independently of i, (Nutman, in preparation). It
seems to be associated with a similar system of suppressors but its effect on symbiosis
is different. Its specificity with respect to strain A is not marked. With a number of
strains of average effectiveness this genotype is ineffective, as with strain A, but with
two strains of more than average effectiveness on unselected host material it confers
a normal degree of effectiveness on some individual plants but not on others. I n
such cases the proportion of effectives to ineffectives often correspond to simple
genetic segregating ratios, suggesting that here also the symbiotic phenotype depends
upon associated pairs of host factors interacting with specific factors in the bacterial
strain which are also hereditable.
The primary gene ie also differs from i, in giving rise to relatively few nodules of
average to large size. Anatomical studies (Bergersen & Nutman, in preparation)
show that ineffectiveness in ie nodules can be attributed to the failure to develop
bacteria-containing tissue. Early degeneration of the central tissue of the nodule
does not take place as with typical ineffective strains of bacteria or with the gene i,.
These relations therefore conform to the hypothesis of infection outlined above that
the nodule meristem has an inhibitory influence on further infection, As far as the
physiology of the host is concerned it is immaterial whether the degeneration of the
nodule is caused by changes in the bacterial strain or host.
The primary modes of action of these host and strain factors is quite unknown.
They do not appear to affect the growth of the host in any other way, and they only
reveal themselves in the final stages of the symbiosis. This suggests that they
ordinarily play some essential part in normal metabolic processes which may be
similar in bacteria and plant. In the very intimate association in the bacteroid tissue
such processes may interfere one with the other, and thus be the cause of the simul-
taneous degeneration of the host cell and bacteroid. With this in mind a model
scheme for the interactions leading to ineffectiveness has been proposed (Nutman,
1949c, 1 9 5 2 ~in
) which two related reaction chains are involved in host-bacterial
interactions leading to nitrogen fixation instead of the single-chain series generally
favoured.
A discussion of the mechanism of nitrogen fixation and excretion is outside the
scope of. this article, but it might be pointed out that the failure after many
attempts to isolate a system from the nodule (or from Azotobacter or Clostridium)
which is able to fix nitrogen in vitro (Wilson & Burris, 1953; Wilson & Roberts,
1954) is the biochemical expression of the complexity of the reactions involved.
Fedorov (1946) considers from studies on the inhibiting effect of narcotics on
iixation that the surface upon which the reactions proceed is used only once and
has constantly to be renewed; the surface does not have the true catalytic behaviour
of an enzyme. From this he concludes that fixation can only take place in the living
cell.
 Root-nodule symbiosis 131

X. T H E INFLUENCE OF HOST SPECIES ON COMPATIBILITY

In view of the complex pattern that appears in the symbiosis of a single species it
is not surprising to find very considerable variation in host-strain interrelations
between species.
Helz, Baldwin & Fred (1927) were the first to show that host species within a
cross-inoculation group varied in their symbiotic responses. They examined patterns
of effectiveness in Viciafaba, V . villosa, Lathyrus odoratus and Lens esculenta with
a number of strains of bacteria and found that whereas one strain would be effective
on one species and ineffective on another, a second strain would give rise to con-
trary responses in the same pair of hosts. In general, strains were effective on the
host species from which they were isolated and less effective on others, effectiveness
being associated in these species with the production of large nodules grouped
around the tap root and ineffectiveness with numerous well distributed nodules.
Later work has confirmed this kind of variation in the pea group (Bjalfve, 1933,
1935; Razumovskaia, 1937; Erdman & Burton, 1938; Strong, 1940) and demon-
strated it in others. In Pisum, Bjalfve (1935) showed also that differences occurred
in the response of pea varieties. Using other material Virtanen & Hausen (1932)
were unable to detect varietal differences in peas.
In the Trifolium cross-inoculation group the pattern is complex and illustrates the
difficulty in attempting to make any rigid classification of host species on the basis
of their responses with different strains of bacteria, the number of classes often
depending upon the number and source of the strains of bacteria used in making the
tests. Erdman (1946), using four host species and fifteen strains of bacteria, was
unable to detect a pattern or relation depending upon the host species from which
the bacteria were isolated, Burton & Briggeman (1948), using seven species and ten
isolates, were also unable to find any correlation, although three of the species
tended to give similar reactions. Earlier, however, Strong (1937) with the three
species-T. subterraneum, T . repens and T . pratense, subsequently included in
Erdman, Burton and Briggeman’s experiments-and isolates from these hosts, was
able to demonstrate a pattern. Four out of five isolates from subterraneanclover
responded effectively on this host and ineffectively on the others, whereas all red
and white clover isolates were effective on their parent hosts and less effective or
ineffective on subterranean clover. It is of interest that the exceptional isolate from
subterranean clover was wholly effective on white and partially effective on both
subterranean and red clovers. This suggested that strains of bacteria become adapted
to host, the validity of a host grouping depending as much on the source of the
bacteria as on taxonomic affinities between the hosts. Vincent (1945), in tests of
isolates from T. repens, T. glomeratum, T. subterraneum, T . frageriferum and
T . pratense, detected seven patterns of effectiveness on three of these species;
furthermore, with other material he was able (Vincent, 1 9 5 4 ~to ) confirm Strong’s
separation of subterranean from red and white clovers. Additional work has been
carried out on species of Trifolium by Purchase & Vincent (r949), Parker et al.
132 P. S. N U T M A N
(1949), and Bond & McGonagle (1951). From this Vincent ( 1 9 5 4 ~ )concluded
that the species tested fall into three main groups showing broadly similar re-
sponses: ( I ) T. repens, T. pratense, T. hybridum, T. procumbens and T.jragerijerum ;
(2) T. subterraneum, T. incarnatum, T . glomeratum and T . alexandrinum; and
( 3 ) T. ambiguum, which until recently had not been known to nodulate effectively
with any strain. My unpublished observations of T. ornithopodoides suggest the
inclusion of this species in the second group.
It may be significant that the species within the above group tend to be more
related ecologically than taxonomically. T h e association of botanically diverse
species in the same habitat would allow cross-inoculation to occur regularly and
may have led to their parallel adaptation. A degree of adaptation between local
races of red clover and local strains of nodule bacteria has been demonstrated in
Sweden, where the close association of host and bacteria has been fostered over long
periods (Nutman & Read, 1952). Here differences in effectiveness tend to be small
but nevertheless significant. However, there remain examples of similarities
between species which cannot be explained in these terms.
T h e Medicago, Melilotus, Trigonella group provides further examples of host
specificity in symbiotic effectiveness as between: ( I ) genera (Burton & Erdman,
1940; Strong, 1940; Purchase et al. 1951); ( 2 ) species of Medicago (Bond, Wilson &
Wagner, 1936; Burton & Wilson, 1939; Purchase et al. 1951); (3) species of
Melilotus (Wilson, Burton & Bond, 1937) and (4) varieties of lucerne (Jordan &
Garrard, 1951; Erdman & Means, 1953). As in the Trifolium group a great variety
of patterns of effectiveness have been found, and it is not possible from the available
data to point to any significant relationships. Medicago laciniata seems to differ
from other species of this genus in failing to give a completely effective symbiosis
with any strain of bacteria (Purchase et al. 1951)~and in this respect resembles
Trifolium ambiguum. Recent work by Hely (unpublished data) has shown that
effective strains with Medicago laciniata are found in certain areas in its Australian
range which resembles the normal habitat of this species.
In the Lotus group Erdman & Means (1949) have shown that L. uliginoszis and
L. corniculatus vary in effectiveness and in the number of nodules produced. Some
bacterial strains are effective with one species only, others with both. These authors
were also able to demonstrate host specificity in the L. corniculatus varieties arvensis
and tenuifolius.
Of the other recognized cross-inoculation groups only the cowpea-soya bean
group has been much studied in this regard, This group includes a great variety of
genera from each of the three subfamilies of the Leguminosae.
T h e first comprehensive examinations of host specificity in this group was made
by Carrol(1934a, b) and Allen & Allen (1936a), who showed that with a few excep-
tions bacterial strains were more effective on the hosts from which they were
isolated than on others. T h e most notable example of differences in reaction was
that between Samanea saman and Vigna sinensis; all cultures from both hosts were
effective on Samanea, but all isolates from Samanea were ineffective on cowpea.
 Root-nodule symbiosis ‘33
In a later paper, Allen & Allen (1939) examined a large number of isolates with
about thirty host species and found a great variety of reactions. At one extreme
Vigna sinensis was benefited by all strains, Clitoria ternatea by about half and
Phaseolus lunatus responded ineffectively with all, including those isolated from
P . lunatus itself. A further interesting observation was also made with P . aconiti-
folius, Samanea saman and Mucuna utilis (Stizolobium utile), on which some strains
of bacteria gave at first a wholly ineffective reaction which later changed to a
normally effective one. This ‘latent or delayed state of effectiveness’ was not due
to contamination and may have reflected a critical change in the environment affect-
ing symbiosis in these particular combinations of host and bacteria. Alternatively,
adaptation between the symbionts may have occurred in the course of the experiment.
Reisolations were not made to test this point. This finding is similar to that already
described for red clover in a previous section.
Burton ( I 952) carried out similar and extensive host-specificity tests using
fifteen host species and fifteen strains of bacteria previously shown to be effective
on their parent hosts. Although a complex pattern of responses was found, two
clear relationships appeared: ( I ) all strains were more or less effective on Vignu
sinensis and V . sesquipedalis, (2) Canaaalia ensiformis and Phaseolus lunatus were
sharply distinguished from the remaining species in responding effectively only
with isolates from either of these species which were however ineffective on all
other hosts. This relationship recalls the separation of Trifolium subterraneum,
T . glomeratum, etc., from other species of Trifolium.
The experiments of Johnson & Allen (1952) showed that Sesbania also occupies
an exceptional place in the group in that all the strains (thirty-nine tested) isolated
from and effective with this genus were uniformly ineffective on both soya bean and
cowpea.
Other studies on host specificity have been carried out with Australasian members
of this group and their bacteria by Steindl (1941)and McKnight (1949), the former
author showing that locally developed races of cowpea bacteria were more effective
on cowpea than introduced ones, though whether this is an expression of a high
degree of adaptation as has been shown for other groups is a matter for speculation.
Vorhees (1915),Erdman & Wilkins (1928)and Shulyndin (1953) have also described
varietal differences in nodulation in soya bean.
Mutual adaptation between host and bacteria leading to efficient fixation of
nitrogen may involve host factors common to a group of genera or confined to a
genus, or which may be specific, varietal or even concern single genetic loci having
no other expression than in the symbiosis. In view of the fact that phenotypic
effects are so complex it is surprising that there should be any discernible patterns
to invite speculation.
One may distinguish the following components in this pattern: ( I ) isolations of
bacteria from a particular species tend to be more effective on this species than on
others; ( 2 ) groups of host species showing similar patterns of response occur in each
cross-inoculation group ; these may be taxonomically diverse: (3) the response
'34 P. S. N U T M A N
patterns in such groups may be either ill defined or exclusive; (4) the number of
species comprising a well-defined group tends to be small.
The first of these relations may reflect the normal processes of adaptation within
a group, the remainder are less readily explained. They may represent stages in
increasing specialization during which the host acquires characteristics of special
significance with reference to a particular bacterial genotype which tend towards
incompatibility with others.
This summary of work on host specificity shows how small a part of a large field
of comparative study in the Leguminosae has as yet been explored. Nevertheless,
the same kinds of results have recurred in each cross-inoculation group; the
principal outlines may already be clear and further work will only provide more
examples. Insufficient data is as yet available to suggest the origin of the symbiotic
habit or to discuss phylogenetic relationships.

XI. T H E INFLUENCE O F T H E ENVIRONMENT

We have been concerned so far with host variability and functions without reference
to the environment. This can have a decisive influence on the progress of sym-
biosis, acting primarily through the host's physiology. The environment can be
considered broadly in its chemical (i.e. nutritional) and physical aspects, both of
which have been covered in fairly recent reviews (Anderson, 1949; Wilson &
Burris, 1953),and I do not propose to do more than to summarize the main findings
and their interpretations and to relate them to the views above expressed on nodule
morphogenesis.
(I) Nutritional factors
The nitrogen nutrition of the nodulated legume occupies a special place because
of the marked depressing effect of combined nitrogen on infection, nodule size and
function, the reduction in nodule number and size and activity being generally
proportional to concentration (Wilson, 1917;Hopkins, Wilson & Peterson, 1932;
and others) and varying greatly with species of host (Nowotny-Mieczyfiska &
Ruszkowska, 1952). At low levels of added nitrogen Fred & Wilson (1934) and
Orcutt & Wilson (1935) found that some stimulation of infection and nitrogen
fixation occurred which was thought to be due to a stimulatory effect on photosyn-
thesis. The influence of added nitrogen was not confined to nitrate, other inorganic
and organic forms of nitrogen having similar if less'marked effects (Wilson, 1917;
Hopkins & Fred, 1933) and could be largely overcome by encouraging increased
photosynthesis with high light and supplementary carbon dioxide (Fred & Wilson,
1934;Weber, 1930; Georgi, 1935; and others) or providing the plant with added
carbohydrate in the form of sucrose, dextrose or levulose (Wilson, 193I a ; Allison &
Ludwig, 1934; Nowotny-Mieczyliska & Ruszkowska, I ~ S Z ) ,or mannitol (Hopkins
& Fred, 1933), although the sparse nodulation of etiolated peas in the dark was
found to be inhibited rather than stimulated bv added sucrose (McGonagle,
'949).
 Root-nodule symbiosis ‘35
The interpretation of the effect of combined nitrogen has been various. Among
the early workers Hiltner considered that the nitrogen reduced the virulence of the
bacteria outside the plant, whereas Suchting thought nitrogen favoured the host
plant in its ‘struggle’ with the bacteria. Thornton & Rudorf (1936) showed that the
nodules of plants provided with nitrogen were abnormal in showing thickening of
the cell walls of the meristematic areas and endodermis, absence of starch and
reduced cell contents and abnormal bacterial form, but were unable to offer an
explanation. In so far as infection is concerned it has been shown that the time of
the initial infection of the young seedling is unaffected by added nitrogen (Weber,
1930). Also if nitrate is supplied slowly to the plant at a rate equivalent to fixation,
nodule number is stimulated in some species (Fedorov & Kozlov, 19543) and
unaffected in others. In the latter the nodules produced are smaller and fix nitro-
gen at a decreased rate (Jensen, 1948). Reduction in nodulation has been found to
depend to some extent upon seasonal and climatic conditions which may be related
to nutrition (Lyons & Early, 1952;Masefield, 1952). Virtanen (1953) has suggested
that nitrate inhibits nodulation through the formation of nitrite from a nitrite-
leghaemoglobin complex. Thornton & Nicol(1936) have shown that the inhibiting
effect of nitrate is more noticeable on nodule size than on nodule number and have
suggested that preliminary curling reaction of the root hair may be affected.
Thornton (1935) showed that nitrate prevented the normal curling of root hairs in
the presence of bacterial secretions, but that sucrose in the medium restored the
activity of the secretions; he considered that the supply of carbohydrate to the
piliferous layer was an important factor. Whatever the mechanism, a local reaction
at the root surface is involved, since it has been shown that nitrate supplied to one
part of a divided root system has no influence on infection elsewhere (Gaumann,
Jaag & Roth, 1945; Fedorov & Kozlov, 19543).
The very general correlation between nodulation and nitrogen fixation and the
carbohydrate status of the host plant has led to the formulation of an hypothesis
relating the carbohydrate-nitrogen ratio to various aspects of the symbiosis (Wilson,
1935; Wilson & Wagner, 1935; Allison, 1935). This relation, however, only sum-
marizes experience and does not provide an explanation. Moreover, since its
formulation many exceptions have been found-e.g. both low or very high levels of
carbohydrate inhibit fixation (Fred & Wilson, 1934). Too little is as yet known of
the details of the interrelations of carbohydrate and nitrogen metabolism and auxin
activity to formulate an alternative hypothesis in terms of growth. It may be more
profitable to seek an explanation in changes in the growth-regulating mechanisms
in the host, in particular, in those factors affecting root morphogenesis and cell
physiology.
For normal functioning of the symbiosis all the elements ordinarily necessary for
plant growth, other than nitrogen, are required. With the exception of molybdenum
(Anderson & Thomas, 1946; Anderson & Spencer, 195oa) and possibly sulphur
(Anderson & Spencer, 1949, 1950a, b), calcium and magnesium (Albrecht, 1937;
Wilson, 1917),all these function at the normal levels required for plant growth.
136 P. S. N U T M A N
Anderson & Spencer drew an interesting distinction between the effects of molyb-
denum and sulphur, deficiency in molybdenum leading to increased demands by
the plant for nitrogen which cannot be met by further nodulation, whereas shortage
of sulphur leads to smaller host requirements due to impairment of protein synthesis.
For this reason plants supplied with sulphur have both more nodules and a higher
percentage of nitrogen in their tissues than those without.
Deficiencies in mineral nutrients affect nodule development and function in
various ways, although it is not often possible to distinguish specific from general
effects on plant growth. Phosphorus is essential for normal nodule growth (Ander-
son & Thomas, 1946) and increases the rate of infection, and may increase nodule
density (Poschenrieder, Sammet & Fischer, 1940;Wilson, 1917)and nodule growth
(Stalder, I 952; Diener, 1950). Under conditions of adequate phosphorus nutrition,
potassium stimulates infection and nitrogen fixation (Lynch & Sears, I ~ S I ) but
, is
inhibitory if phosphorus is low. T h e results obtained by Wilson and Diener were
found with plants with divided root systems, and with the exception of potassium
no transfer of the influence of the nutrient was found. It was also observed that at
low levels of phosphate, infection of the root took place but that nodules were not
formed, possibly because of the absence of meristematic foci. Wilson (1917) found
chlorides stimulating and sulphates depressing for nodulation in water culture.
Lynch & Sears (1951) claim that the response of Lotus to nutrients depends upon
the strain of nodule bacteria.
Legumes have high requirement for zinc and copper, but these elements do not
appear to have specific effects on nodulation (Mulder, 1948b). Deficiency in boron,
on the other hand, affects nodule development by preventing infection in its
absence (Mulder, 1948b) and normal development of the vascular tissue when
deficient (Brenchley & Thornton, 1925). With inadequate boron the nodule remains
small with incomplete development of the bacteroid tissue, but nitrogen is fixed
and the plant is green although dwarf.
Molybdenum occupies a special position in that this element is required by the
nodulated plants at a much higher level than by a plant supplied with combined
nitrogen and has been shown to be concerned in the fixation process (Anderson &
Thomas, 1946;Jensen, 1948; Anderson & Spencer, 195ob; Mulder, 1948a, 1950).
Nodulation, however, is not superficially influenced. The molybdenum require-
ment for optimum nitrogen-fixation differs among legumes (Andrew & Milligan,
1954). The recent observation that nodule bacteria are a rich source of vitamin B,,
and that this vitamin occurs in the nodule (Bertrand & Wolf, 1954;Levin, Funk &
Tendler, 1954) may foreshadow a cobalt requirement for symbiosis. This is also
suggested by the studies of Nowotny-Mieczyliska (1952) on the stimulating effect
of added cobalt chloride and ascorbic acid upon fixation and haemoglobin produc-
tion. An explanation of the mechanism of the action of these and other elements
must await a fuller understanding of their normal function in the plant.
 Root-nodule symbiosis '37
(2) Physical factors: light and temperature
The effect of light intensity, day length and temperature have been examined by
a number of workers, and have been shown to influence nodulation. I n the dark,
nodules form on some species and not on others. On lucerne and on vetch seedlings
only a very few nodules are formed in the complete absence of light, and if nodulat-
ing plants are placed in the dark nodule formation ceases and those already formed
degenerate (Wilson, 193I a ; Thornton, 1 9 3 0 ~ )Leucaenaglauca,
. red clover and soya
bean are quite resistant to infection in the dark unless provided with sugar
(Schweizer, 1932), whereas the number of nodules formed on peas in the dark is
reduced by sugar although their size is increased (McGonagle, 1949). On the other
hand, Diener (1950) reported that nodule formation on the pea grown in light ceases
altogether even if the light is reduced to a low level, but that moderate reduction (to
50 yo normal light intensity) reduced nodule size without affecting number. Fred &
Wilson (1934), Orcutt & Fred (1935), Fred, Wilson & Wyss (1938) and Raju
(1936, 1939) found an optimum light intensity for maximum nodulation and
nitrogen fixation. At very high light intensity fixation was completely inhibited in
the young seedling which remained in the seedling nitrogen hunger phase. This
depressing influence of high light intensity could be overcome either by a short
period of shading or by adding nitrate. Unpublished observations on subterranean
clover have confirmed this relationship, and it has also been observed that the few
nodules formed at high light intensities combined with high temperature are large
but ineffective.
With the exception of an incidental comparison of long and short days on soya
bean by Eaton (1931),Orcutt & Wilson (1935) and Orcutt & Fred (1935), in which
no effect was noted on yield or nitrogen content, and the work of Moshkov (1939)
and Borodulina (1950) (quoted by Rudakov, 1951),there appear to be no records
of the influence of day length on nodulation. This latter author, working with
Phaseolus, found that long- and short-day varieties responded more effectively
when grown in their appropriate length of day. In view of the photoperiodic
control of morphogenesis in many plants a close study of the effect of length of day
is overdue, in particular with regard to nodule number and size.
Information on the effect of temperature on nodulation is similarly sparse and
little has been done since the early researches of Jones & Tisdale (1921). These
authors applied a range of temperatures to the roots of alfalfa, red clover, peas and
soya beans grown in soil, maintaining the air temperature fairly uniform at between
14 and 20' C. For all species an optimum temperature of 24' C. was found for
nodule size and number. Plant growth was adversely affected at the higher tempera-
tures, presumably through the effect of symbiosis ; this occurred at lower tempera-
tures for peas and red clover than for lucerne and soya bean. Stalder (1952) has
shown that the temperature optimum for growth of Pisum sativum and for its
nodule bacteria are the same, and that over a wide range the infection of the root is
unaffected by temperature (between 6.5 and 27' C.). With red clover, on the other
138 P. S. NUTMAN
hand, it has been found that nodules form less readily during the winter, and this
has been largely attributed to lower temperatures (Nutman, 1949b).
Other factors in the environment which have received recent attention are p H
(Jensen, 1948; Lynch & Sears, 1951), aeration (Bond, 1951; Egle & Munding,
1954; Ferguson & Bond, 1954) and the presence of surface-active substances in the
medium surrounding the root (Vantsis & Bond, 1950; Nutman, r g 5 2 a ; Turner,
1955). The first two factors generally show optimum relationships, depending
upon the host examined, whereas the effect of adsorbents are generally stimulatory,
either on the rate of nitrogen fixation or on infection. T h e stimulatory influence
noted by Bond (1951)of the insoluble constituents of the culture solution may be
explained similarly.
The mechanism of the action of adsorbents in removing inhibitory root exudate is
discussed above and has been related to a general hypothesis of infection. An
explanation of other environmental effects may be sought similarly in terms of
their influence on the physiology of the host rather than on the bacteria outside the
nodule.
XII. GENERAL CONCLUSIONS
The formation of nodules on leguminous plants takes place in most species in a
series of well-defined stages: (I) stimulation of rhizosphere growth, ( 2 ) root-hair
curling, (3) host-controlled penetration at defined points on the root or root hair,
(4) development of the infection thread, ( 5 ) organization of the nodule’s growth,
(6) invasion of the disomatic tissue, and finally (7) the release of the bacteria into the
host cytoplasm. The first stages require no direct contact between plants and
bacteria; they are unspecific and tentative in nature. T h e remainder involve an
increasing degree of interaction between the symbionts, culminating in the intra-
cellular phase. It may be argued that until this last stage the nodule bacteria, like
ectotropic mycorrhizal fungi, are still outside the host cell. Certain aspects of the
host’s physiology-response to growth factors, root morphogenesis, mechanism of
cell-wall formation, root secretion-are implicated in many if not all of the pre-
liminary phases, and on fuller examination may provide explanations for them. I n
particular, the importance of the plant meristem may be emphasized as the focus of
infection (in its disomatic form), as the organ by which nodule growth is promoted
and as a morphogenetic centre determining the general pattern of growth. Much
variation occurs in the expression of these preliminary phases, even to their
omission in some groups; this also would repay physiological investigation on
comparative lines.
In contrast much less morphological variation is apparent in the intracellular phase
of the symbiosis, the physiology of which is obscure, in spite of the accumulation of
much cytological data and information on the metabolism of the whole nodule.
Very little is known about the essential processes taking place which lead to actual
nitrogen fixation. Genetic investigations of the host show that a complex system of
inherent factors is concerned in the interrelationships within the infected cell, no
single element of which as yet appears to be acting independently but is always
 Root-nodule symbiosis '39
directly associated with a specific factor or group of factors present in the bacteria,
which factors are in their turn also inherently determined.
As none of these genetically determined host or bacterial factors have any effect
outside the symbiosis (and in this they are in marked contrast to those determining
the preliminary stages) nothing is as yet known of their physiological or biochemical
modes of action. That they may be concerned in some way with cell multiplication
and nuclear activity is suggested by the fact that incompatibility in the examples
quoted leads to cell and nuclear degeneration. It is in the closer study of the intra-
cellular phase of symbiosis that our understanding is most likely to be advanced.

XIII. SUMMARY
I . Secretions produced by the legume root stimulate unspecifically the rapid
growth of strains of nodule bacteria in the rhizosphere. Secretions from non-
legume roots are less stimulatory.
2. Secretions from the root also affect nodule initiation. At low concentrations
they stimulate nodule formation and at higher concentrations are inhibitory.
3. The bacterial secretion of indolylacetic acid causes root-hair deformation
(curling) in legumes but not in other families of plants.
4. The usual mode of infection of the root is through the curled root hair.
Elsewhere entry is through epidermal cells, wounds or points of emergence of
lateral roots. The number of infected root hairs compared with the number of
nodules formed varies widely between species.
5 . The mechanism of entry into the root hair is not known. The hypothesis is
advanced that bacteria at the growing tip of the root hair induce reorientated growth
in the primary cell wall to give rise to an infection thread by invagination.
6. Complete failure in nodule formation with any bacterial strain is restricted
(with the exception of rare variants of normally susceptible hosts) to few genera and
species in the primitive tribes Mimosoideae and Caesalpinioideae.
7. Resistance in the host to infection by bacteria isolated from other species is
widespread. Those species which will nodulate with the same strains of bacteria
comprise a cross-inoculation group.
8. Six well-defined cross-inoculation groups are established, which include,
however, only a small proportion of leguminous species. The remainder are pro-
visionally included in a seventh group (cowpea group), within which complex
patterns of susceptibility are found, depending upon bacterial strain.
9. A few examples of resistance dependent upon bacterial strain are also known
in the established cross-inoculation groups.
10. Cell to cell infection in root cortex and nodule usually takes place by ramifi-
cation of the original infection thread within the tissues of the host. I n some species
it is by division of primarily infected cells or from intercellular zoogleae, or by a
combination of these modes of spread.
I I . Intracellular infection by the bacteria is confined to tetraploid cells origi-
nating from tetraploid initials already present in the uninfected root cortex.
 P. S. N U T M A N
12. Nodule density on the root is related to the number of preformed foci in the
root. The development of these foci is determined by factors affecting root morpho-
genesis, viz. ( I ) the inherent capacity of the root to produce lateral meristems,
(2) inhibition from meristems already present. Apparent differences in bacterial
strain virulence are explicable in these terms.
13. T h e cytology of the nodule cell containing effective bacteria appears to be
uniform throughout the Leguminosae. It is characterized by: (I) massive develop-
ment of swollen forms of the bacteria (bacteroids) in large isodiametric cells of host,
(2) hypertrophy and degeneration of bacteroid and host-cell nucleus, (3) formation
of haemoglobin.
14. The following forms of intracellular incompatibility (ineffectiveness) have
been described: (I) failure in proliferation of bacteria in host cytoplasm, (2)failure
in bacteroid formation, (3) early lysis of infected cells, (4)failure in nitrogen fixation
in nodules containing bacteroid tissue.
15. Symbiotic ineffectiveness in a single species of host may be due to primary
defects in bacteria or host. These defects, which are inherited, appear to affect only
the symbiosis and not the phenotype or metabolism of host or bacteria grown
separately.
16. Genetic factors for ineffectiveness in the host may be simple or polygenetic
and may interact specifically or unspecifically with bacterial strain factors. Ineffec-
tiveness involving primary host factors may be overcome by changes in secondary
plant genetic factors as well as by mutative change in the bacteria.
17. Within the cross-inoculation groups the symbiotic effectiveness of cross-
inoculated associations of host and bacteria varies widely. I n general: ( I ) strains of
bacteria tend to be more effective on the host species from which they were isolated
than on others; (2)groups of host species showing similar responses are taxonomi-
cally diverse; (3) well-defined groups contain few host species.
18. The rare symbiosis between a host of one cross-inoculation group and
bacteria of another is ineffective.
19. The external environment appears to affect symbiosis largely through the
host’s physiology and metabolism.
For helpful criticism of this manuscript I am much indebted to Dr H. G. Thornton, F.R.S.,
Prof. J. M. Vincent, Dr D. 0. Norris, Dr K. 0. Muller and Mr F. W. Hely and to other colleagues
both at Rothamsted Experimental Station, England, and the Division of Plant Industry, C.S.I.R.O.,
Canberra.
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XV. ADDENDUM
A number of recent papers deal with host differences in the nodulation of legumes
in the field. Mostert (1955) describes contrasted types of nodulation on a range of
species of the South African veld, some of which, belonging to the Papilionaceae
and Caesalpinioideae, are devoid of nodules. Masefield (1955) has shown that
nodulation is not affected by the nearby presence of a plant of the same or another
species; he also observed that the nodules of Dolichos lablab and Phaseolus trilobus
grown in Sudan were active until the peak of the flowering season. The nodulating
capacity of the ground-nut grown in Senegal has been shown by Jaubert (1955) to
be related to varietal habit, the erect varieties forming nodules less readily than the
recumbent. Varietal differences were also demonstrated in response to inoculation
with specific strains of bacteria. Fox & Lipps (1955) have traced the relation
of nodulation of lucerne and sweet clover to characteristics of the soil profile,
particularly cation composition, exchange capacity and phosphorus availability.
Abundant nodules and root formation were correlated and were affected more by
nutritional than by physical factors per se. The specific effect of fertilizers on
nodulation and nitrogen fixation is the subject of papers by Villax & Alves (1954)
and Allos & Bartholomew (1955).
Among studies on rhizosphere activities bearing on nodulation Peterson (1954)
has shown that some organisms are antagonistic to nodulation, some are synergistic
and others neutral. Peterson (1955) also claims that changes in host-plant specificity
follow the treatment of a strain of one cross-inoculation group with bacterial
filtrates from another. Further transformation experiments on Rhizobium are
reported by Balassa (1955). Filippov (1955) confirms the secretion of biotin into
the rhizosphere of leguminous as well as non-leguminous plants. Other root
secretions which give rise to colour complexes with bentonite clay have been shown
by Turner (1955) to vary considerably between species of the leguminous host
plant. His results indicate that clay-iron may be implicated in colour formation and
suggests that indole derivatives may be concerned.
I 50 P. S. N U T M A N
The physiological significance of the assimilating apparatus for nodule formation
has been investigated in papers employing varying degrees of illumination, different
photoperiods and grafting.
Virtanen, Moisio & Burris (1955) report that nodules excised from darkened pea
plants fix even less nitrogen than those from plants remaining in the light.
Fedorov & Uspenskaya (1955) have demonstrated that the reproductive type of
growth found in peas in long days and in soya beans in short days is associated with
the production of fewer nodules. The rate of nitrogen fixation per gram of such
nodules was higher than in nodules of plants in the vegetative phase. Bonnier &
Sironval(1956) have examined the nodulation of soya bean grown under controlled
conditions of temperature, light intensity and photoperiod. Large pink nodules
were readily formed in a 16 hr. day, but in an 8 hr. day the nodules were small,
infrequent or altogether absent. Kvasnikov & Dolgikh (1955) attribute a similar
effect of day length on early and late varieties of peas to its influence upon vegetative
growth and carbohydrate content of the plant, in particular that of the root mono-
saccharides (which also stimulates bacterial growth in the rhizosphere).
Rudin, Popapov & Germanova (1953) have endeavoured to determine the
physiological basis of the legumes’ susceptibility to infection by grafting experi-
ments. A number of grafts were successfully established between legumes and
non-legumes. Nodules were invariably formed on the leguminous stock, whatever
the scion, but in no instance was nodulation induced upon a non-legume root.
It was also observed that the number of nodules formed on the legume root was
influenced by the scion. Grafting experiments made by Kvasnikov & Dolgikh
(1955) were extended to include an examination of cross-inoculation group
specificity, and species and varietal differences in susceptibility. They used the
following kinds of grafts : (I) Glycine (cowpea cross-inoculation group) with
Phaseolus vulgaris (Phaseolusgroup), ( 2 ) P . acutifolius and P. aureus (cowpea group)
with P. vulgaris, and (3) abundantly and sparsely nodulating varieties of P . vulgaris.
With the exception of a single nodule produced by a Phaseolus strain of bacteria
upon a soya-bean root grafted with a P . vulgaris top (confirmed by isolation and
inoculation on to Phaseolus) the cross-inoculation group specificities were un-
affected by grafting, the root retaining its specific susceptibility. The intensity of
nodulation was, however, markedly influenced by the scion. In the intergeneric
and interspecific grafts more nodules were found on the roots of those combinations
giving the largest top growth. In the intervarietal grafts also, more nodules occurred
on the hybrid grafts than on the self grafts, which were less vigorous in their
growth.
The relation between inoculum size and nodule formation on clover has been
shown by Purchase & Nutman (in preparation) to conform to a form of Mitscherlich
equation, and therefore to support the hypothesis of infection at discrete foci on
the root.
 Root-nodule symbiosis '5'
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