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S H O R T C O M M U N I C A T I O N

Mixture of endophytic Agrobacterium and Sinorhizobium melilotistrains could induce nonspecific nodulation on some woody

legumes

Jie Liu • En Tao Wang • Da Wei Ren •

Wen Xin Chen

Received: 20 March 2009/ Revised: 1 October 2009/ Accepted: 4 January 2010 / Published online: 23 January 2010

Ó Springer-Verlag 2010

Abstract Agrobacterium sp. II CCBAU 21244 isolated

from root nodules of Wisteria sinensis was verified as anendophytic bacterium by inoculation and reisolation tests.

However, inoculation with a mixture of this strain and a

Sinorhizobium meliloti strain could induce root nodules on

W. sinensis and two other woody legumes, which do not

form a symbiosis with S. meliloti alone. Rod-shaped and

irregular nodules were found on the inoculated plants, in

which the S. meliloti strain was detected in all of the

nodules; while the Agrobacterium strain was inside of the

rod-shaped nodules, or occupied only the nodule surface

of the irregular globe-shaped nodules. These findings

revealed novel interactions among the symbiotic bacteria,

endophytic bacteria and the legume plants, although the

mechanisms are still unknown.

Keywords Nodulation Á Agrobacterium Á Sinorhizobium Á

Endophyte Á Woody legume Á Nodule morphology

Introduction

Recently, many Agrobacterium strains have been isolated

from root nodules (Gao et al. 2001; Han et al. 2005;

Mhamdi et al. 2005; Sohail et al. 2004). Most of the strains

were non-symbiotic bacteria belonging to Agrobacterium

tumefaciens (de Lajudie et al. 1999; Wang et al. 2006), and

some of them may represent novel biovars (Tiwary et al.

2007). Further studies revealed that the Agrobacterium

strains could coexist with the rhizobial strains in the nod-

ules (Mhamdi et al. 2005; Wang et al. 2006) and they were

also endophytic bacteria in roots and stems (Wang et al.

2006). These nodule endophytic strains may have no

effects on nodulation and growth of host plants (Wang

et al. 2006), or could specifically inhibit the nodulation of

Rhizobium gallicum on common bean (Mrabet et al. 2006).

However, the impact of nodule endophytic Agrobacterium

strains is unclear in most cases.

In our previous study, ten bacterial strains isolated from

root nodules of Wisteria sinensis were identified as Agro-

bacterium sp. II by amplified 16S and 23S rDNA restric-

tion analysis (Liu et al. 2005). The nodulation capacity or

nodule occupancy and the existence of symbiotic genes in

these Agrobacterium strains were not investigated. In the

present study, further investigation was performed in order

to verify the questions mentioned above, and to estimate

the impact of the novel endophytic Agrobacterium strains

on nodulation of the host legumes.

Communicated by Ursula Priefer.

Electronic supplementary material The online version of thisarticle (doi:10.1007/s00203-010-0543-2 ) contains supplementarymaterial, which is available to authorized users.

J. Liu (&)

Department of Bioengineering and Biotechnology,

Qingdao University of Science and Technology,

266042 Qingdao, Chinae-mail: [email protected]

E. T. Wang

Departamento de Microbiologıa, Escuela Nacional de Ciencias

Biologicas, Instituto Politecnico Nacional,

11340 Mexico D.F., Mexico

e-mail: [email protected]

J. Liu Á D. W. Ren Á W. X. Chen

State Key Laboratories for Agro-biotechnology,

College of Biological Sciences, China Agricultural University,

100193 Beijing, China

123

Arch Microbiol (2010) 192:229–234

DOI 10.1007/s00203-010-0543-2

http://dx.doi.org/10.1007/s00203-010-0543-2

http://dx.doi.org/10.1007/s00203-010-0543-2





confocal microscope. The inoculants of (1) GFP-labeled

Agrobacterium strain alone; (2) the reisolate S. meliloti

30N1 alone and (3) a mixture of the GFP-labeled Agro-

bacterium strain and 30N1 were, respectively, inoculated

to W. sinensis, C. racemosa and A. fruticosa as mentioned

above. In each treatment, 16 seedlings were inoculated and

8 seedlings without inoculation were included as blank

control. The obtained nodules were sectioned and observedunder a confocal microscope and nitrogenase activity was

determined as mentioned above.

Results and discussion

In the nodulation tests, regular and reddish nodules (Sup-

plementary Fig. A) were formed on the W. sinensis seed-

lings inoculated with Rhizobium sp. I strain CCBAU21218

(positive control) 12 weeks after the inoculation. The

control seedlings and the herbaceous legumes inoculated

with Agrobacterium sp. II CCBAU 21244 did not formnodules. In 10 of the 32 seedlings of W. sinensis and in

about one-third of the C. racemosa and A. fruticosa plants

inoculated with Agrobacterium sp. II CCBAU 21244, 1–5

small (1 mm in diameter), white, irregular and highly lig-

nified nodules (Supplementary Fig. A) per plant were

obtained after 3 months of inoculation. Low activity of

nitrogenase (20.7 nmol C2H4 g-1 h-1) was detected from

these nodules and a small red region in the center of each

nodule was observed (Supplementary Fig. A), indicating

that the nodules were partially effective.

The formation of nodules on plants inoculated with

Agrobacterium sp. II CCBAU 21244 was interesting

because all the previously reported Agrobacterium strains,

such as those studied by de Lajudie et al. ( 1999), could not

induce nodules on their host of origin. To confirm if the

nodules were induced by the inoculated strain, a total of 40

nodules were randomly selected from the seedlings of

W. sinensis and inoculated with Agrobacterium strain were

used in bacteria reisolation. The results showed that about

50% of the nodules contained only one bacterium and the

colony morphology was similar for all the isolates but was

different from that of CCBAU 21244. A colony representing

these isolates was named as 30N1. The other 50% of nodules

contained two bacteria: one had colonies the same as 30N1

and a representative colony was named as 30N2; the other

showed colonies the same as for CCBAU21244 and a rep-

resentative colony was named as 30D. The reisolation

results were similar for the nodules in the seedlings of

A. fruticosa and C. racemosa (data not shown).

In BOX-PCR and ERIC-PCR (Fig. 1), the reisolated

strain 30D had fragments identical to CCBAU 21244, and

both were very similar to Agrobacterium sp. II strains

CCBAU 31079 and CCBAU 31169. They were different

from the reference strains of A. tumefaciens IAM13129T,

A. rhizogenes IAM13570T, A. vitis IAM14140T and A. rubi

IAM13569T. These results confirmed the previous obser-

vations based upon the 16S and 23S rRNA gene RFLP

patterns (Liu et al. 2005), therefore they may represent

novel species or biovars as suggested in another case

(Tiwary et al. 2007), but more data are needed to define

their exact taxonomic position.In addition, 30D showed the 16S rRNA gene sequence

(GenBank accession number GQ337862) to be identical

with that of CCBAU 21244 (AY555768), confirming the

existence of the inoculated Agrobacterium strain in 50% of

the nodules.

The isolates 30N1 and 30N2 were different from the

inoculated Agrobacterium strain and from Rhizobium sp.

(Wisteria) CCBAU21218 (positive control). Both isolates

had identical or very similar patterns in BOX- and ERIC-

PCR analyses, while their 16S rRNA gene sequences

(accession numbers GQ337863 for 30N1 and GQ337864

for 30N2) were identical to that of S. meliloti USDA 1002T

(X67222), indicating that all the isolated nodules contained

a S. meliloti strain.

The reisolation and identification results made the no-

dulating ability of Agrobacterium sp. II CCBAU 21244

uncertain, the nodulation might be caused by the S. meliloti

contaminant in the watering procedure, since nodulation

tests of S. meliloti on alfalfa were realized in the same

period and the same place. However, the normal micros-

ymbionts of the three woody legumes used in this study

were species of Rhizobium, Mesorhizobium and Brady-

rhizobium and no S. meliloti was isolated from these plants

grown in fields (Liu et al. 2005). In this case, detection of

symbiotic genes and nodule occupation by the Agrobac-

terium stain CCBAU 21244 were performed to verify if

this bacterium participates in the nodulation procedure.

Using the primers specific to nifH and nodA genes, some

bands corresponding to the size of target fragments were

amplified from the three Agrobacterium sp. II strains by PCR

(Supplementary Fig. B). When the re-annealing temperature

was increased from 55 to 58°C, these bands disappeared

while the positive control (S. meliloti USDA1002T) main-

tained the products, indicating that the amplified fragments

from Agrobacterium sp. II strains were not highly specific to

the primers. By purifying these bands from gels and

sequencing, we found that the amplified fragments from the

Agrobacterium strains were similar to some function-

unknown genes reported in other Agrobacterium strains

(results not shown). These results did not demonstrate the

existence of symbiotic genes in the nodule-originated

Agrobacterium strains. In this case, the Agrobacterium sp. II

strains were different from those of A. rhizogenes, from

which both the symbiotic and pathogenic genes were found

(Velazquez et al. 2005).

Arch Microbiol (2010) 192:229–234 231

123



About ten nodules were obtained from each of the

seedlings inoculated with the mixture of S. meliloti 30N1

and GFP-labeled CCBAU21244 after 3 months of growth

under aseptic conditions. There were no nodules obtained

from the blank control plants or the plants inoculated

separately with each of these strains. These results dem-

onstrated that Agrobacterium sp. II CCBAU 21244 or

S. meliloti 30N1 alone could not induce the nodulation of

W. sinensis, but their mixture could, implying the existence

of cooperation between these two bacteria. This coopera-

tion was further indicated by the observation of GFP-

labeled CCBAU 21244 in nodules.

Two nodule types were found in the plants inoculated

with mixture: 70% were rod shaped and 30% were irreg-

ular small spherical (2 mm in diameter) (Fig. 2). Both

types showed low nitrogenase activity as mentioned above.

Five representative nodules for each type were assayed

under the confocal microscope. GFP-labeled Agrobacte-

rium cells were observed inside the rod-shaped nodules,

mainly in the meristem zone surrounding the nodule and a

little in the nodule center (Fig. 3). The scattered distribu-

tion of gfp-labeled CCBAU 21244 inside the nodules was

clearly different from the symbiotic rhizobia reported

previously (Gage et al. 1996) that almost filled the whole

nodule. The small spherical nodules only showed green

fluorescence on the surface, but not inside the nodules. The

distribution of GFP inside or on the surface of nodules

could explain why the strain CCBAU 21244 was reisolated

only from 50% of the nodules, since the bacteria adhered

on the surface of nodules could be killed by the surface

sterilization in the isolation procedure. These results also

implied that the Agrobacterium strain has been involved in

all the nodules, either inside or on the surface of the

nodules.

1 0 0

9 5

9 0

8 5

8 0

7 5

7 0

6 5

6 0

5 5

A. tumefaciens IAM13129

A. rubi IAM13569

30N1

A. rhizogenes IAM13570

CCBAU21218

A. vitis IAM14140

CCBAU2123930N2

30D

CCBAU21244

CCBAU31079CCBAU31169

1 0 0

9 8

9 6

9 4

9 2

9 0

8 8

8 6

8 4

CCBAU21244

30N2

CCBAU31169

30D

CCBAU31079

CCBAU21218CCBAU21239

A. tumefaciens IAM13129

A. vitis IAM14140

A. rubi IAM13569

30N1

A. rhizogenes IAM13570

BOX-PCR

ERIC-PCR

Rhizobium sp.

Agrobacterium sp. II

Agrobacterium sp. II

Rhizobium sp.

S. meliloti

S. meliloti

Similarity (%)

Similarity (%)

Fig. 1 UPGMA dendrogram

showing the relationships

among the bacteria reisolated

from root nodules, the

inoculated Rhizobium and

Agrobacterium strains, and the

Agrobacterium reference strains

Fig. 2 Two types of nodules formed on the roots of W. sinensis

seedlings inoculated with Agrobacterium sp. II CCBAU21244 and

S. meliloti strain 30N1. The rod-shaped nodules contained the

GFP-labeling Agrobacterium sp. II strain inside the nodules, while the

small spherical nodules were occupied by Agrobacterium on surface

only

232 Arch Microbiol (2010) 192:229–234

123



In addition, the green fluorescence also appeared in root

tissues (Fig. 3) and the existence of the Agrobacterium

strains in plants did not affect the growth of the hosts (data

not shown), as reported previously (Wang et al. 2006).

The nodulation tests with the GFP-labeled CCBAU

21244 confirmed that the Agrobacterium strains could be

endophytic bacteria in the nodules (Mhamdi et al. 2005;

Wang et al. 2006). However, our results were different

from the previous reports on two points:

1. the coexistence of Agrobacterium and S. meliloti

strains was essential for the nonspecific nodulation

on the involved woody legumes;

2. the Agrobacterium sp. II strains seemed to participate

in the nodule formation with some unknown mecha-

nism, because the seedlings inoculated with the

mixture formed two types of nodules corresponding

to the Agrobacterium occupation on the nodule surface

or inside the nodules.

We conclude that the nodulation of W. sinensis and the

other two woody legumes by the mixture of Agrobacterium

and S. meliloti strains was nonspecific and was caused by

cooperation between them because neither of them alone

could induce nodulation on these plants. Although we do

not understand the mechanism yet, this finding was

significant because it could be the reason why some sym-

biotic gene containing rhizobia isolated from nodules of

various legumes failed to induce nodules on their original

hosts (Gu et al. 2007; Lin et al. 2007; Tian et al. 2007).In conclusion, the strains of Agrobacterium sp. II

isolated from root nodules could occupy the nodules and

roots, and could induce the nonspecific nodulation of

coexisting S. meliloti strain and affect the nodule

morphology.

Acknowledgments This work was supported by the Foundation of

National Basic Research Program of China (2006CB100206) and

Specialized Research Fund for the Doctoral Program of Higher

Fig. 3 Colonization of GFP-labeled Agrobacterium sp. II

CCBAU21244 in nodules of W. sinensis. a Horizontal section of a

rod-shaped nodule. b Green fluorescence observed in the nodule

section in a indicating that the Agrobacterium strain mainly

distributed in the epidemic zone inside the nodules. c Vertical section

of nodules showing indicating the existence of Agrobacterium strain

(green fluorescence) mainly in the meristem zone surrounding the

nodule and in root tissues (color figure online)

Arch Microbiol (2010) 192:229–234 233

123



Education (20050019017). E.T.W. was financially supported by the

grants of SIP 20080322 and SIP 20090179 authorized by IPN,

Mexico.

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