Biochem. Physiol. Pflanzen 187, 331-336 (1991) Gustav Fischer Verlag Jena

Influence of Rhizosphere Application of Syringaldehyde on Rhizobia - Mungbean (Vigna radiata) Symbiosis

VEENA JAIN, NISHA GARG and HIMMAT S. NAINAWATEE

Department of Chemistry and Biochemistry Haryana Agricultural University, Hisar, India

Key Term Index: Mungbean, naringenin, symbiosis, syringaldehyde; Rhizobium sp. (Vigna), Vigna radiata.

Summary

The efficiency of mungbean (Vigna radiata) - rhizobia symbiosis was influenced by the rhizosphere application of syringaldehyde and naringenin. Syringaldehyde caused increase in nodule number (6-26%), nodule weight (9-36%) and nitrogenase activity (16-38%). Naringenin had an inhibitory effect on various parameters of symbiosis. There was increase in the plant biomass production by syringaldehyde treatment. These results open up a possibility of enhancement of biological nitrogen fixation by rhizosphere application of nod gene regulators.

Introduction

Root-nodule formation in pea, clover and alfalfa has been demonstrated to be linked with the expression of rhizobial nod genes, which are induced by some components present in the root exudates (DOWNIE et al. 1985; INNEset al. 1985; MULLIGAN and LONG 1985). Both

inducers and antagonists of nod genes of fast growing species such as R. meliloti, R. trifolii, R. leguminosarum have been identified in the root exudate of host plants (FIRMIN et al. 1986;

PETERS et al. 1986; REDMOND et al. 1986). Exogenous application of nod regulators has been reported to influence the early event of symbiosis (ROLFE and GRESSHOFF 1988; ZAAT et al. 1987 b). However, not much information is available on the induction of nod genes of slow growing species of rhizobia. Results reported here concern the effect of application of nod regulators on the mungbean (Vigna radiata) - rhizobia symbiosis.

Materials and Methods

Seeds of mungbean (Vigna radiata) cultivar K 851 were obtained from the Department of Plant Breeding of this University. Seeds were surface sterilized and treated with the inoculum of Rhizobium sp. Vigna S 24 prepared in yeast extract mannitol medium (FRED et al. 1932). The seeds were sown in modified Leonards jars (DAHlYA and KHURANA 1981) and the crop was raised under nitrogen dependent conditions using N-free nutrient solution (SLOGER 1969). The experiment was conducted in a green house following complete randomized design for layout. In each Leonard jar, two plants were allowed to grow and for each observation ten plants were sampled. Different concentrations of syringaldehyde and naringenin were applied to the mungbean rhizosphere at the time of sowing or at early vegetative (10 DAS: days after sowing) and pre-flowering (30 DAS) stages of plant growth. Sampling for various estimations was done at 30 DAS and 45 DAS stages which respectively corresponded to the preflowering and post-flowering stages. The nitrogenase activitiy of intact root nodule system was assayed by incubation in 10% acetylene atmosphere for 2h at 28°C. Ethylene

BPP 187 (1991) 4 331

produced was analysed on Shimadzu mini-2 gas chromatograph using Porapak-N column. Plant biomass was determined after drying the whole plant to a constant weight at 80°C. Total nitrogen analysis was done using micro Kjeldahl method (ANONYMOUS 1970). Analysis of variance, least significant difference (LSD) and Tukey's test have been used for the statistical analysis of data.

Results

Application of syringaldeyde and naringenin to the rhizsophere of mungbean crop affected various parameters of symbiosis. Naringenin in general had'negative effect, while the syringaldehyde had a positive influence on symbiosis . Of the various concentrations, 10 IJ.M naringenin and 700 fA.M syringaldehyde concentration had the maximum effect. Treatments given at the pre-flowering stage had no significant effect on symbiosis (results not shown). Results presented in Table 1 and 2 show that syringaldehyde application significantly enhanced the nodule number per plant. The maximum increase in nodule number (38.6%) was observed when the application was done at the time of sowing and sampling was done at the post-flowering stage (Table 2). Even application at the early vegetative stage resulted in about 25 % enhancement in nodule number. Application of naringenin caused a reduction in nodule number. The number of nodules per plant at pre­flowering stage was 28 in 10 IJ.M naringenin treated plants as against 47 in control. Application of syringaldehyde also significantly increased the weight of nodules (Table 1-2). The increase in nodule weight was 36% at post-flowering stage in sowing time syringaldehyde treatment. At the post-flowering stage , the nodule weight was 191 mg/plant in the absence of any treatment while it was 261 mg/plant in response to sowing time treatment of700 flM syringaldehyde. Naringenin treatments caused significant reduction in the weight of nodules of plants particularly at the pre-flowering stage (Table 1). At post flowering stage, nodule weight per plant was unaffected in the naringenin treatment given at early vegetative stage. Application of naringenin did not significantly affect acetylene reduction activity (ARA) of nodules while syringaldehyde resulted in the enhancement of ARA. In sowing time syringaldehyde (700 IJ.M) treatment, nodules at post-flowering stage had 38% higher ARA as compared to the nodules of untreated plants. The effect of 10 flM syringaldehyde on ARA was negligible. Naringenin treatment caused reduction in plant weight, the maximum reduction being 38% observed at the pre-flowering stage in 10 flM naringenin treatment given at the time of sowing (Table 1). Syringaldehyde treatment caused significant increase in the plant dry weight. The maximum increase (13 %) in weight was in the plants sampled at post-flowering stage which received 700 IJ.M syringaldehyde treatment at sowing time (Table 2). The total nitrogen content of plants was also affected by the naringenin and syringaldehyde treatments. Application of syringaldehyde (700 flM) at sowing time resulted into 20% and 15% increase in the nitrogen content per plant at pre­flowering and post-flowering stages, respectively. Naringenin (10 IJ.M) application at sowing time resulted in 36 % and 20 % reduction in N-content of plants respectively atthe pre­flowering and post-flowering stages of growth.

332 BPP 187 (1991) 4

Tab

le 1

. E

ffec

t of r

hizo

sphe

re a

pplic

atio

n o

f nar

inge

nin

and

syri

ngal

dehy

de o

n R

hizo

bia-

mun

gbea

n (V

igna

rad

iata

) sy

mbi

osis

at p

re-f

low

erin

g st

age

(30

DA

S).

Dif

fere

nt c

once

ntra

tion

s of

nar

inge

nin

and

syri

ngal

dehy

de w

ere

appl

ied

at t

he t

ime

of s

owin

g (S

) or

at t

he e

arly

veg

etat

ive

stag

e (V

) w

hich

cor

resp

onde

d to

10

DA

S (d

ays

afte

r so

win

g) s

tage

of

plan

t gr

owth

.

Stag

e T

reat

men

t

Nil

N

arin

geni

n

10

0n

M

Nod

ule

num

ber/

plan

t

S 47

.01

± 2.

12

40.4

2 ±

2.30

* V

47

.01

± 2.

12

44.4

3 ±

2.61

L

SD a

t 5

% 1

.81,

HSD

at

5%

2.7

2

Nod

ule

fres

h w

eigh

t (m

g/pl

ant)

S 17

1.2

± 15

.6

V

171.

2 ±

15.6

13

9.4

± 8.

7*

149.

0 ±

11.1

* L

SD a

t 5

% 6

.69,

HSD

at

5%

10.

09

Nit

roge

nase

act

ivity

(n

mol

C2H

dmin

lmg/

fres

h w

t.)

S 35

.04

± 2.

83

34.1

4 ±

1.98

V

35

.04

± 2.

83

LSD

at

5%

1.9

1, H

SD a

t 5

% 2

.87

Bio

mas

s (g

dry

wei

ght/p

lant

)

S 0.

70 ±

0.0

5 V

0.

70 ±

0.0

5

34.6

7 ±

4.21

0.67

± 0

.05

0.69

± 0

.06

1 f.t

M

32.0

3 ±

1.00

* 37

.09

± 1.

41*

116.

0 ±

8.8*

13

7.0

± 9.

0*

34.7

4 ±

2.19

34

.98

± 3.

14

0.56

± 0

.05*

0.

58 ±

0.0

5*

10 f

.tM

28.2

1 ±

0.84

* 33

.63

± 2.

07*

104.

2 ±

6.9*

13

4.4

± 9.

9*

34.3

6 ±

1.80

34

.61

± 1.

50

0.43

± 0

.04*

0.

49 ±

0.0

4*

Syri

ngal

dehy

de

lOf.

tM

50.2

3 ±

0.84

* 49

.48

± 1.

67*

173.

8 ±

12.6

17

1.2

± 12

.1

35.5

3 ±

2.41

36

.31

± 2.

41

0.70

± 0

.06

0.69

± 0

.06

lOO

f.tM

51.4

2 ±

2.01

*

53.4

6 ±

1.69

*

186.

8 ±

12.4

* 17

7.6

± 12

.8

40.5

±

3.08

* 39

.44

± 2.

53*

0.69

± 0

.06

0.72

± 0

.D7

700

f.tM

56.8

4 ±

1.78

* 55

.83

± 3.

27*

224.

8 ±

19

.1*

20

2.4

± 14

.7*

40.6

3 ±

2.56

* 39

.80

± 3.

12*

0.72

± 0

.06

0.72

± 0

.06

1:0 ::g

LSD

at

5%

0.0

46,

HSD

at

5%

0.0

69

... ~

~

...... ~

...... +- w

w

w

Tot

al n

itro

gen

(mg/

plan

t)

S 11

.71

± 1.

01

V

11.7

1 ±

1.01

11

.37

± 0.

98*

11.6

1 ±

0.89

L

SD a

t 5

% 0

.876

, H

SD

at

5%

1.3

16

9.64

± 0

.95

10.3

4 ±

0.94

7.

41 ±

0.7

2*

8.27

± 0

.83*

11

.98

± 1.

19

11.9

0 ±

1.20

* V

alue

s ar

e si

gnif

ican

tly

diff

eren

t fr

om t

he c

ontr

ol a

t 5

% H

SD

lev

el o

f si

gnif

ican

ce (

Tuk

ey's

mul

tipl

e ra

nge

test

).

12.9

6 ±

1.09

* 13

.07

± 1.

05*

13.7

9 ±

1.40

* 13

.70

± 1.

25*

,~

\;l

.j:>.

to :g ... ~

~ ... ~ ~

.j:>.

Tab

le 2

. E

ffect

of

rhiz

osph

ere

appl

icat

ion

of

nari

ngen

in a

nd s

yrin

gald

ehyd

e on

Rhi

zobi

a-m

ungb

ean

(Vig

na

radi

ata)

sy

mbi

osis

at

post

-flo

wer

ing

stag

e (4

5 D

AS

).

Dif

fere

nt c

once

ntra

tion

s o

f na

ring

enin

and

syr

inga

ldeh

yde

wer

e ap

plie

d at

the

tim

e o

f so

win

g (S

) or

at

the

earl

y ve

geta

tive

sta

ge (

V)

whi

ch

corr

espo

nded

to

10 D

AS

(day

s af

ter

sow

ing)

sta

ge o

f pl

ant

grow

th.

Sta

ge

Tre

atm

ent

----

----

----

----

----

----

----

----

----

----

----

----

----

----

----

----

----

----

----

----

----

----

---

Nil

Nod

ule

num

ber/

plan

t

S 64

.01

± 3

.63

V

64.0

1 ±

3.6

3

Nar

inge

nin

l00

nM

5sm

± 4

.04*

60

.07

± 3

.07

LS

D a

t 5

% 2

.95,

HS

D a

t S

% 4

.42

Nod

ule

fres

h w

eigh

t (m

g/pl

ant)

S 19

1.2

± 1

3.2

V

191.

2 ±

13.

2 16

7.2

± 1

0.7*

18

6.0

± 1

2.7

LS

D a

t 5

% 9

.47,

HS

D a

t 5

% 1

4.21

Nit

roge

nase

act

ivit

y (n

mol

C2J

4!m

inlm

g fr

esh

wt.

)

S 14

.27

± 0

.87

13.4

9 ±

0.9

4 V

14

.27

± 0

.87

14.1

1 ±

0.3

0 L

SD

at

5%

0.9

45,

HS

D a

t 5

% 1

.419

Bio

mas

s (g

dry

wei

ght/

plan

t)

S 1.

26 ±

0.1

1 1.

24 ±

0.1

0 V

1.

26 ±

0.1

1 1.

24 ±

0.1

2 L

SD

at

5%

0.1

17,

HS

D a

t 5

% 0

.176

Tot

al n

itro

gen

(mg/

plan

t)

S 27

.23

± 2

.24

26.4

7 ±

2.5

0 V

27

.23

± 2

.24

27.5

1 ±

0.3

8 L

SD

at

5%

1.9

9, H

SD

at

5%

2.9

9

1 !-1

M

54.0

7 ±

2.74

* 54

.05

± 4

.64*

151.

6 ±

12.

7*

186.

0 ±

13.

4

13.5

8 ±

0.4

6

14.0

4 ±

0.3

7

1.20

± 0

.10

1.

19 ±

0.1

0

25.8

6 ±

1.9

6 25

.16

± 0

.46

10 !-

1M

44.0

8 ±

1.87

* 49

.69

± 3.

21 *

119.

6 ±

8.8

9*

189.

8 ±

12.

0

13.2

9 ±

0.9

5 13

.08

± 0

.93

1.02

± 0

.09*

1.

04 ±

0.1

0*

21.8

0 ±

1.7

0*

21.5

1 ±

1.9

3*

Syr

inga

ldeh

yde

lOf!

M

65.0

7 ±

2.5

9 72

.03

± 4

.39*

22S

.6

± 7

.7*

186.

0 ±

11.

9

13.8

5 ±

1.0

1 14

.49

± 1

.66

1.34

± 0

.12

1.26

± 0

.10

28.1

8 ±

2.4

S

27.1

6 ±

2.5

5

* V

alue

s ar

e si

gnif

ican

tly

diff

eren

t fr

om t

he c

ontr

ol a

t 5

% H

SD

lev

el o

f si

gnif

ican

ce (

Tuk

ey's

mul

tipl

e ra

nge

test

).

100

!-1M

68.6

4 ±

6.8

2 71

.06

± 6

.55*

230

.8

± 1

6.0*

20

3.4

± 1

3.7

15.4

8 ±

1.1

8 14

.65

± 0.

74

1.41

± 0

.13

1.32

± 0

.12

30.2

8 ±

2.4

7 28

.78

± 2

.68

700

f!M

89.0

0 ±

6.4

2*

80.0

3 ±

7.1

8*

261.

2 ±

19.1

* 21

2.4

± 1

6.9*

19.7

5 ±

1.6

9*

17.2

8 ±

1.03

*

1.42

± 0

.13*

1.

26 ±

0.1

3

34.5

1 ±

3.0

3*

30.9

2 ±

2.8

1*

Discussion

Root exudates oflegume crops have been reported to contain inducers and anti-inducers of nod genes of rhizobia (FIRMIN et al. 1986; MULLIGAN and LONG 1985). The nod gene products are required forthe early event oflegume-rhizobia symbiosis (ROSSEN et al. 1987). It is not known , whether the process of nodulation is limited by the concentration of either nod inducers or the nod gene products. However, in the present investigation, rhizosphere application of syringaldehyde caused significant enhancement in nodule number, nodule weight, nitrogenase activity and biomass and nitrogen content of plants. Several plant flavones are known to induce nod-lac Z translational fusions in fast growing Rhizobium species (REDMOND et al. 1986). Flavone limitations to root nodulation and symbiotic nitrogen fixation in alfalfa have also beenreported. Addition of 10 !J.M luteolin (KAPULNIK et al. 1987) and naringenin (JAIN et al. 1990) to the rhizosphere of alfalfa seedlings increased nodulation, N2 fixation, total N and total dry weight. The nod genes of slow growing Bradyrhizobium species are reported to be induced by isoflavones, daidzein and genistein (KOSSLAK et al. 1987; GOTTFERT et al. 1988). In the present investigation, in mungbean rhizobia which is also a slow growing species, acetophenone analogue syringaldehyde increased the nodulation while naringenin which is known to be an inducer of nod genes of Rhizobium leguminosarum (ZAAT et al. 1987 a) decreased the nodulation. In nod-lac Z translational fusions of Bradyrhizobium japonicum also, naringenin was reported not to induce B-galactosidase activity (KOSSLAK et al. 1987). The acetophenones have been reported to be antagonist of nod genes inR. leguminosarum (FIRMIN et al. 1986). Therefore, it appears that the mechanism of regulation of nodulation in fast growing rhizobia is different from the slow growing rhizobia. It is quite likely that nod genes of mungbean rhizobia are induced by several compounds including syringaldehyde. The very fact that nodulation and nitrogen fixation has been enhanced by the application of syringladehyde opens up another technology for maximizing symbiotic nitrogen fixation. However, a detailed study on the relationship between Rhizobium and nod induction signals of host plant is required.

Acknowledgement

VJ (SRF) and NG (Pool-Officer) are thankful to the Council of Scientific and Industrial Research, New Delhi for financial support.

References

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BPP 187 (1991) 4 335

GOTTFERT, M., WEBER, J., and HENNECKE, H. : Induction of a nod A-lac Z fusion in Bradyrhizobium japonicum by an isoflavone. J. Plant. Physiol. 132,394-397 (1988).

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Received January 23, 1990; revised form accepted December 20, 1991

Authors' address: Prof. Dr. H . S. NAINAWATEE, Dr. (Mrs .) VEENAJAIN and Dr. (Mrs.) NISHA GARG. Department of Chemistry and Biochemistry, Haryana Agricultural University, Hisar 125004, India.

Biochemie und Physiologie der Pflanzen Verlag: Gustav Fischer Verlag Jena, Villengang 2, D-0-6900 lena, Telefon 27332; Geschaftsftihrer: Johanna Schluter und Bernd Rolle Verantwortlich ftir die Redaktion: Prof. Dr. Klaus Muntz, Redaktion BPP, Institut ftir Genetik und Kulturpflanzenforschung, CorrensstraBe 3, D-0-4325 Gatersleben Satz, Druck und Buchbinderei: Druckhaus Jena GmbH Anzeigenannahme: Gustav Fischer Verlag Jena, Anzeigenverwaltung, Villengang 2, D-0-6900 Jena; Telefon 27332, Telex 588676. Zur Zeit gilt Anzeigenpreisliste Nr. I vom 1. 1. 1990. Alle Rechte beim Verlag . N achdruck (auch auszugsweise) nur mit Genehmigung des Verlages und des Verfassers sowie mit Quellenangabe gestattet. Printed in Germany

336 BPP 187 (1991) 4