Plant and Soil 46, 317-327 (1977) Ms.

2950

ROOT NODULES OF SOME TROPICAL

LEGUMES IN SINGAPORE
by G. LIM and H. L. NG
Botany Department, University of Singapore

SUMMARY

A survey of 35 legume species comprising 25 of Papilionoideae, 7 of Mimo-

soideae and 3 of Caesalpinioideae was made. Nodulation was found in all the
species except for 2 (Caesalpinnia pulcherrima and Cassia siamea) of Caesal-
pinioideae, both of which possessed dark coloured roots. Nodulation is re-
ported for the first time for A denanthera pavonina and Delonix regia. Nodule
shapes were described and classified into different types. The isolates of rhi-
zobia obtained belonged largely to the slow growing group (17 isolates)
isolated mainly from members of Papilionoideae; some belonged to the fast
growing group (14 isolates), and only 3 isolates belonged to the very slow
growing group. The slow growing group isolates were confirmed to be cow-
pea type rhizobia on the basis of positive nodulation with cowpea plants.

INTRODUCTION

Many workers have commented that our knowledge about the

nodulation of tropical leguminous species is limited 6 s 11 16 18 and
there is need for more fundamental data in this field. Members of
the Leguminosae form a predominant part of the flora in peninsular
Malaya and Singapore 15 ranging from herbaceous weeds, vegetable
crops and ornamentals, to large, woody trees. Some are indigenous,
others are introduced species. These leguminous plants have not so
far been seriously examined for their root nodules or nodulating
capacity. Two observations 14 17 on nodulation and its beneficial
effect had been reported in peninsular Malaya. This paper presents
data from a survey of leguminous species growing in Singapore.
318 G. LIM AND H. L. NG

MATERIALS AND METHODS

B o t h wild a n d c u l t i v a t e d legumes f r o m various areas in Singapore were

s u r v e y e d . A l t o g e t h e r 55 species were e x a m i n e d , t h e s e i n c l u d e d small weeds,
climbers, h e r b a c e o u s a n d w o o d y s h r u b s a n d trees.
T h e p r e s e n c e or a b s e n c e a n d degree of n o d u l a t i o n on t h e root s y s t e m s were
n o t e d . T h e m o r p h o l o g y of m a t u r e nodules was d e s c r i b e d a n d t h e i r sizes
( d i a m e t e r / l e n g t h / w i t h ) m e a s u r e d in m m .
R h i z o b i a isolates were o b t a i n e d in t h e usual m a n n e r 23 f r o m r o o t nodules,
m a i n t a i n e d on y e a s t m a n n i t o l agar (YMA) s l a n t s ( m a n n i t o l 10g, y e a s t ex-
t r a c t 0.5g, CaC12 0.01 g, FeCla 0.01 g, t a p w a t e r 1 litre, agar 15g), a n d t h e i r
g r o w t h p e r f o r m a n c e on Y M A observed. To c o n f i r m t h a t t h e isolates were
rhizobia, congo r e d m e t h o d is was used, in a d d i t i o n t o p l a n t i n o c u l a t i o n t e s t s .
The l a t t e r was t r i e d w h e r e seedlings of t e s t p l a n t s were available. Isolates
were also t e s t e d on seedlings of Vigna unguiculata (syn. sinensis) t o d e t e r m i n e
if t h e y b e l o n g t o t h e c o w p e a group.

OBSERVATIONS AND RESULTS

Among the leguminous plants growing in Singapore, members of

the sub-family Papilionoideae constitute the largest group. Conse-
TABLE 1

Nodule morphology and degree of uodulation on legume species

Legume species Nodule Mean Degree Remarks

morphology diameter size of
(of 10 nodules) nodulation

Subfamily Caesalpinioideae
Caesalpinia Nodules absent introduced
pulcherrima (Roots bright
(L.) Swartz. orange to
reddish brown
in eolour)
Cassia siamea Lain. Nodules absent native to
(Roots bright Peninsular
orange to Malaya
reddish brown
in colour)
Delonix regia Rafin. Globose, white, 2 mm + introduced
smooth surface

Subfamily Mimosoideae:
A casia auriculi- Globose to 2 mm X 12 mm q- + q- Jr introduced
]ormis (A. Cunn. elongate, lobed
ex. Benth. brown, smooth
surface
 ROOT N O D U L E S OF SOME TROPICAL L E G U M E S 319

Legume species Nodule Mean Degree Remarks

morphology diameter size of
(of 10 nodules) nodulation

A denanthera Globose, brown, 3 mm + native

pavonina L. smooth surface
A lbizzia ]alcataria Semi-globose, 3 mm + introduced
(L.) Fosb. single or in
cluster of 2
or 3, brown,
smooth surface
Calliandra Coralloid, 10 m m × 5 m m + + introduced
surinamensis brownish-pink,
Benth. smooth surface
Mimosa invisa } Elongate, single 2 m m × 7 111111 + + + introduced
Mart. ex. Colla or multi-lobed,
M. pudica L. brown, + + + introduced
smooth surface
Neptunia natans Elongated, 1 m m X 6 mm + + native
(L.f.) Druce bilobed,
brown, smooth
surface

Subfamily Papilionoideae
Andira inermis Globose to semi- nllTi q- introduced
H.B.K. globose,
brown~
smooth surface
Arachis hypogaea Semi-globose 3 mm + + introduced
L. reddish brown,
slightly rough
surface
Cajanus cajan Semi-globose, 4 mm + + introduced
(L.) Huth. brown, with
warty surface
Calopogonium Globose, brown 4 mm + + introduced
mucunoides Desv. with white
streaks on
surface
Canavalia gladiata Globose and 6 mm + + + native
DC irregular,
light brown,
with rough
surface
Centrosema Globose, light 3 mm + + introduced
plumieri Benth. brown, with
ribbed surface
Clitoria ternatea L. ] Globose and 3 mm + + introduced
C. lauri]olia Poir l some slightly 10mm × 5ram + introduced
bifurcate,
light brown,
smooth surface (to be continued)
320 G. LIM AND H. L. NG

(Table 1 continued)

Legume species Nodule Mean Degree Remarks

morphology diameter size of
(of 10 nodules) noduIation

Crotalaria Fan-shaped, 10 m m × 5 m m + + introduced

mucronata Desv. pinkish brown,
smooth surface
C. retusa L. Fan-shaped, base 10 mm × 5 m m + + native
of nodule pink,
smooth surface
C. usaramoensis Fan-shaped, 10 mm x 5 m m + + introduced
Baker pinkish brown,
smooth surface
Desmodium ] Globose, single 2 mm + + + + native
heterophyllum DC [ or in clusters,
D. ovali]olium Wall. !f brown, smooth + + native
D. triflorum DC J surface + + native
Dolichos lablab L. Globose, brown, 3 mm + introduced
rough surface
Glycine so]a (L.) Semi-globose, 4 mm + + introduced
Sieb. & Zucc. brown, smooth
(black and yellow surface
seeded varieties)
Phaseolus angularis ] Semi-globose, 2 mm + + introduced
F. W. Wright ] pale brown,
P. aureus Roxb. smooth surface + + introduced
P. lathyroides L. + + introduced
Psophocarpus Semi-globose, 6 mm + + introduced
tetragonolobus DC oblate, pale
brown, smooth
surface
Pueraria phaseo- Globose, brown, 4 mm + + + native to
loides (Roxb.) smooth to peninsular
Benth. warty surface Malaya
Stylosanthes Semi-globose, I mm + q- -t- introduced
gracilis H.B.K. light brown,
smooth surface
Tephrosia candida Young nodules 3 m m × 2 mm + + introduced
DC globose.
T. vogelii Mature ones + + introduced
Hook f. bifurcate,
brown, smooth
surface
Vigna unguiculata Globose, pale 4 mm + + introduced
(L.) Walp. brown, rough
surface

-- indicates absence of nodules. 2_ + + indicates heavy nodulation.

+ indicates sparse nodulation. + + + + indicates very heavy nodulation.
+ indicates moderate nodulation.
 ROOT N O D U L E S OF SOME TROPICAL LEGUMES 321

quently, of the 35 species of legumes examined, the majority, num-

bering 25 were from this sub-family, and only 7 from the sub-
family Mimosoideae and 3 from the sub-family Caesalpinioideae.
Observations on nodulation are presented in Table 1, which also
lists whether the plants are native or introduced species. Nodulation
was observed on all the species examined in the sub-families Mimo-
soideae and Papilionoideae. The majority of the species were moder-
ately to heavily nodulated, a few very heavily nodulated, in particu-
lar Acacia auriculi/ormis and Desmodium heterophyllum. Species that
were sparsely nodulated were Adenanthera pavonina and Albizzia
/alcataria of the sub-family Mimosoideae, and Andira inermis, Cli-
toria lauri/olia and Dolichos lablab of the sub-family Papilionoideae.
Of the 3 species of Caesalpinioideae examined, only Delonix regia
had a few nodules and these were found on young seedlings and never
encountered on mature trees; no nodules were found on Caesalpin-
nia pulcherrima and Cassia siamea although seedlings and mature
trees from different localities in Singapore were repeatedly examined.
Their roots however were a deep brown to orange eolour.
Generally, young nodules were round, white and smooth-surfaced,
whereas older ones assumed various shapes, were brown and rough-
surfaced. Nodule shapes were classified into different types as shown
in Fig. 1, ranging from globose (Fig. l a) to semiglobose (Fig. l b) to
elongate (Fig. 1c and d) to fan-shaped (Fig. 1e) to coralloid (Fig. 1f).
In Caesalpinioideae, nodules as represented by those on D. regia
were globose, white and smooth-surfaced. In Mimosoideae, members
had globose to elongate nodules. Coralloid types were only found in
Calliandra surinamensis (Fig. l f). The nodules found in A. auriculi-
/ormis and A. pavonina were globose to lobed, those on A./alcataria
were semi-globose, and those on Mimosa invisa, M. pudica and
Neptunia natans were elongate and lobed. In Papilionoideae, most
members had brown coloured, globose to semi-globose nodules of
various sizes and surface texture. Nodules of Calopogonium mucunoi-
des appeared different, in that they showed white, fluffy streaks on
the surface (Fig. 1g). Microscopic examination of these streaks show-
ed them to consistof a mass of elongate hyaline cells which may be
special aeration tissues. The 3 Crotalaria species bore fan-shaped
nodules (Fig. l e), and the 2 Tephrosia species had bifurcate nodules
(Fig. ld).
322 G. LIM AND H. L. NG

.g~e~: " " ~:...:':~

... • ..-,.,.-.-~,
• ,. ~.,_.~..~

.... " '~.%..~/ "~'~k,~':" ~': :.'..

cm

Fig. 1. S h a p e s of n o d u l e s , a. g l o b o s e ; b. s e m i - g l o b o s e ; c. e l o n g a t e a n d l o b e d ;
d. b i f u r c a t e ; e. f a n - s h a p e d ; f. c o r a l l o i d ; g. g l o b o s e a n d s t r e a k e d .
 ROOT NODULES OF SOME TROPICAL LEGUMES 323

Growth per/ormance o/Rhizobium isolates ore Y M A

The 34 isolates of rhizobia maintained on YMA slants showed some
differences. A broad classification into groups was attempted, based
on their growth performance, whether slow or fast growing. Three
groups were distinguished as follows: - group 1, fast growing, taking
less than 5 days to achieve abundant growth; group 2, slow growing,
taking 7-10 days to achieve similar abundant growth; and group 3,
very slow growing, taking more than 12 days to achieve similar
growth (Table 2).

TABLE 2

Classification of Rhizobium isolates into groups according to rate of growth (29°C)

Fast growing (< S days) Slow growing (7-10 days) Very slow growing
(> 12 days)

A denanthera pavonina Acacia auriculi]ormis Glycine soja (black variety)

Albizzia/alcataria A rachis hypogaea G. soja (yellow variety)
A ndira inermis Cajanus cajan
Calliandra surinamensis Calopogon~um mucunoides Phasedus lathyroides
Centrosema plumieri Canavalia gladiata
Desmodium heterophyllum Clitoria ternatea
D. ovali/olium C. lauri/olia
D. tri]lorum Crotalaria mucronata
Mimosa invisa C. retusa
M. pudica C. usaramoensis
Neptuni~ natans Delonix regia
Phaseolus angularis Dolichos lablab
P. aure~s Phosphocarpus tetragonolobus
Pueraria phaseoloides Stylosanthes gracilis
Tephrosia candida
T. vogelii
Vigna unguiculata
Total isolates: 14 Total isolates: I7 Total isolates: 3

Plant inoculation tests

All the isolates obtained were confirmed to be Rhizobium. Some
of the isolates were tested against various species of seedlings to
determine their nodulating ability. The results are shown in Table 3.
A few isolates appeared able to nodulate other legume species, while
others could not. All the isolates tested on C. pulcherrima and C.
siarnea did not produce nodules on these 2 species.
Inoculation tests carried out on seedlings of V. unguicuIata (cow-
324 G. LIM A N D H. L. NG

TABLE 3

Inoculation tests with Rhizobium isolates

Host isolations Test pl a nt s Nodulation

A denanthera pavonina Caesalpinia pulcherrima --

Calliandra s u r i n a m e n s i s C. mucunoides +
Calopogonium mucunoides D e s m o d i u m ovali]olium --
C. mucunoides V i g n a unguiculata +
Clotoria ternatea V. unguiculata +
Crotalaria retusa Crotalaria usaramoensis +
Delonix regia C. pulcherrima --
D. regia Cassia siamea --
Glycine so]a C. pulcherrima --
G. so]a C. mucunoides +
G. so]a C. siamea --
M i m o s a pudica C. pulcherrima --
M . pudica C. siamea --
N e p t u n i a natans C. mucunoides +
N . natans M i m o s a pudica --
V i g n a unguiculata C. mucunoides +
+ indicates effective nodules formed.
- - indicates no nodules formed.

pea) showed that of the 34 isolates tested, only those belonging to

the slow growing group gave positive results of nodulation.
The inoculation experiments also revealed that irrespective of
whichever isolates were used as inocula, the nodules formed on each
respective host species were characteristic for that species.

DISCUSSION

In this survey, all the members of Mimosoideae (7 species) and

Papilionoideae (25 species) examined possessed root nodules. In
Caesalpinioideae, of the 3 species examined, 2 were non-nodulating
and only D. regia possessed root nodules, but only in the seedling
stage. A l l e n and A l i e n 2 working on legumes of Hawaii reported
however that D. regia lacked nodules. This paper therefore gives the
first report of occurrence of nodules in this species.
Observations as recorded in Table 1, indicate that there was no
correlation between nodulating ability and whether a species was
native or introduced. This confirms M a s efi e 1d's is explanation and
also bears out the observations of other workers 7 8 17. In this study,
absence of nodules in the natural habitat is not regarded as conclusive
 ROOT NODULES OF SOME TROPICAL LEGUMES 325

evidence of lack of nodulating ability, though the inoculation tests

carried out (Table 3) tended to give supporting evidence on the in-
ability of C. pulcherrima and C. siamea to develop root nodules with
the isolates so far tested. These two legume species possessed colour-
ed roots both in their natural habitats and under laboratory condi-
tions. All the other nodulating species did not have similarly colour-
ed roots. These observations agree with reports 10 21 on the relation-
ship between root colour and nodulation.
C o r b y 10 also recorded that among the sub-families and tribes of
Leguminosae, a large proportion of Caesalpinioideae were non-nod-
ulating. S u b b a R a o 22 also commented that 65% of Caesalpinioi-
deae in India do not possess nodules. More members of this sub-
family here need to be examined for further data and analysis. In
their survey and appraisal on the scope of nodulation in the Legu-
minosae, A l l e n and A l l e n 1 5 mentioned that nodules were not
found in A. pavonina despite growing hundreds of these plants over
a period of 15 years and using diverse soil types and treatments with
rhizobia inocula. But ill this current survey, nodules were found on
this species, though the degree of nodulation was sparse (Table 1).
This therefore is the first report as far as we know that records the
presence of nodules in A. pavonina.
Nodule colours generally varied from pale brown to brown.
Reports 9 12 on the occurrence of black nodules in D. lablab due to
certain strains of Rhizobia and in Centrosema pubescens (plurnieri)
were noted. In this survey, black nodules were not encountered in
these species or in other legumes.
Nodule shapes were found to be characteristic of legume species
and independent of Rhizobimn strains. Many workers recognise this
among whom Corby's work 10 is noted, and his description of nodule
shapes was used as a guide for the observations made in this paper.
Among the 3 groups of rhizobia isolates differentiated on their
growth characteristics on YMA, the fast growing group comprised of
isolates from all the members of Mimosoideae examined except A.
auriculi/orrnis, and from some of Papilionoideae (Tables 1 and 2).
The latter sub-family yielded isolates which were mainly slow grow-
ing, and a few very slow growing. The sole isolate from Mimosoideae
belonged to the slow growing group. The results further showed that
isolates from different species of the same genus did not necessarily
belong to the same group, as for example among Phaseolus species,
326 G. LIM A N D H. L. NG

isolates from P. angularis and P. aureus were fast growing, while

isolates from P. lathyroides were very slow growing.
Literature reports that the cowpea type rhizobia is culturally
slow growing and symbiotically promiscuous, and according to
N o r r i s 19 represents the ancestral type of the organism, and com-
mon to all tropical legume species 20. On the basis of reciprocal
cross inoculation with V. unguiculata (cowpea), A l l e n and A l i e n 3 4
gave a list of host isolations belonging to the cowpea group. On a
similar basis, results from the inoculation tests indicated that only
those isolates listed as slow growing (Table 2) belonged to the cow-
pea group, and these results confirm in some cases but not complete-
ly A l l e n and A l l e n ' s reports 8 4. More legume genera and species
need to be examined and more isolations made and tested before
tropical legumes and their nodulating capacity can be understood
completely.

ACKNOWLEDGEMENTS

W e w o u l d like t o t h a n k A s s o c i a t e P r o f e s s o r H s u a n K e n g for h i s k i n d a s s i s -
t a n c e w i t h t h e b o t a n i c a l n a m e s of l e g u m e s p e c i e s .

Received 29 August 1975

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