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Colonization by Rhizobia of the seed and roots of legumes in relation to exudation of phenolics
COLONIZATION BY RHIZOBIA OF THE SEED AND ROOTS OF LEGUMES IN RELATION TO EXUDATION OF PH~NOLICS D.
KANDASAMY*
and N. N.
PRASAD
Department of Microbiology, Faculty of Agriculture. Annamalai C!niversity. An~amalaina~ar .- 608101, India
Summary-“-.~Astudy of the colonization pattern of R~~~~~~u~?i inoculated either in the soil or on to the seeds of green gram {~~~us~~~z~s uuwus Roxb.). black gram (P. mz~~z~o L.) and sun hemp (Crotafaria juncru L.) revealed a significant reduction of R~j~#~j~~~)~ in the spermosphere of sun hemp which was attributed to excretion of phenolic compounds by the seed. As the plants aged, the quantity of phenolics exuded bv the roots was reduced and there was a parallel increase in the Rhi~hium papulation in the rhi~~~pheres of the three plant species.
sphere soil samples were collected on days 7 and 9. ~~j~o~~~~z was estimated in the soil samples by a plant dilution technique (Clark, 1965). Seed diffusates were extracted (Kandasamy 01 al., 1974) and total phenol& in the seed diffusates were estimated (Bray and Thorpe. 1954). Phenolic compounds in the diff~lsat~s were separated by twodimensional paper chromatography (Block et al., 1958). Of the final fraction, 50 ~~!11 was spotted on Whatman No. 1 chromatographic paper (28 x 23 cm) and developed ascendingly in a solvent system CORsisting of n-butanol:aceti~ acid:water; 4: I:5 (vi@ for the first direction and 2% acetic acid for the second direction. To the air-dried chromatograms diazotized sulfanilic acid (DSA) at 0.5% in 20% Na&O, was used as a spray reagent to locate phenols. Phenols exuded by the roots of the three plant species were estimated using an exudation apparatus (Kandasamy rr al., 1973). Root exudates were collected on alternate days beginning on day 3 until day I1 from sowing and their total phenolic content was estimated. The influence of seed diffusates on the growth of rhizobia was tested (Yadav and Vyas, 1973).
INTRODUCTlON considerable attention has,been paid to the presence of anti-microbial substances in plants and active substances have now been found from at least 157 families of the plant kingdom. In some instances such substances have also been extracted from seeds (Bowen, 1961). Fottreil rr ul. (1964) observed that eertain leguminous seed diffusates were jnhibjtory to many Gram-positive and Gram-negative organisms. Extracts of certain weeds. and ieachates from soil of abandoned fields where those weeds occurred were found to inhibit half the ~~j~o~~~r~ strains tested (Rice. 1971). Dadarwal and Sen (1973) also observed variations in the inhibitory effect of seed diffusates of nine legume plant species tawards different strains of rhizobia, an ~~~tubuct~r and a ~u~illus, Little is known about the role of antimicrobial substances present in the seed in determining the survival and colonization of leguminous roots by Rhk&ium. We report the results of an investig~tjon on the establishment of rhizobia in the spermosph~res of green gram (~~~s~~~~.~UUI’YUS Roxb.), black gram (P. rnunyo L.) and sun hemp (Crotuluria ,juncm L.). The nature of the inbib~tory substances from the seed coats of the above three plant species. belonging to the same cross inoculation group was investigated.
RESULTS After I day the ~~~~~#~i~{~~~ inoc~llated in the soi had colonized the spermosphere of green gram more rapidly than that of biack gram. In the case of sun hemp, there were fewer rhizobia on the seed 24 h after sowing revealing a negative spermosphere effect. However, the cofonization pattern changed in the rhizospheres of the three plant species. The rhizosphere effect on the ~~i~o~j~~?zwas relatively similar in green gram and black gram while in sun hemp it was markedly low (Table 1). The Rhizohium inoculated on the seeds of green gram colonized the spermosphere and rhizosphere rapidly. A similar effect was observed in black gram, although there were fewer rhizobia. In the case of sun hemp. the initial colonization by rhizobia in the spermosphere was very poor, but it was found to proliferate rapidly later on in the rhizosphere (Table 2).
MATERiALS AND METHODS Healthy seed was surface sterilized with O.lTi HgC12 and washed in several changes of sterile distilled water. The seed was sown in garden soil contained in 15 cm pots already inoculated with a cell suspension (25 mljpot) of I-week old culture of Rhtohim sp. (I 0’ ml- ‘) or inoculated on the surface such that each seed carried 6 x I Oh.ml - r. Spermosphere soils in triplicate, were sampled to count Rhi~ohium at the end of 24 and 72 h after sowing and the rhizo--~ * Present address: Associate Professor of Microbiology. Tarnil Nadu Agricultural University. Coimbdtore-641M)3. India. 73
74
D. Table
I. Establishment
KANDASAMY
and N. N.
PRASAD
of soil-inoculated Rhizohium in the spermosphere of green gram, black gram and sun hemp
and
rhizosphere .-
Sampling time after sowing (days)
Soil alone
Black gram Green gram (Rhizobium x 10 ‘g-’ oven-dry soil)
Sun hemp
Spermosphere
I
18.4
3
9.5
7
42.4 (2.3)* 21.2 (2.2)
23.2 (1.3) 12.9 (1.4)
3.4 (0.2) 11.4 (1.2)
2.2
16.3
2.2
(7.6) 9.4
10.8 (5.0) 9.7 (4.4)
6.3 (2.9) 4.9 (2.2)
Rhizosphere
9
(4.3) * Figures 2.0 Table
in parentheses
2. Establishment
denote
the spermosphere
in the spermosphere the seed of green gram,
Sampling time after sowing
Soil alone
or rhizosphere
effect S.E. = 0.7; CD.
and rhizosphere by Rhizobium inocuiated black gram and sun hemp
Green
gram
Black gram
(Rhizobium x 10m6gg’ oven-dry
.(days)
=
on
Sun hemp soil)
Spermosphere 1
0.7
3
0.9
7
0.8
13.3 (18.4)* 11.9 (2 1.O)
12.8 (17.8) 15.4 (18.1)
18.2 (22.5) 14.4 (18.2)
(1:::) 7.5 (9.5)
2.3 (3.1) 6.7 (7.8)
Rhizosphere
9
0.8
* Figures in parentheses denote the snermosphere 0.8.
Studies carried out to determine the presence of inhibitory substances in the seed coat and root exudates revealed that although there was not much varic-----c c&en gram C---O Block gicen
‘60
--y--n
140
i
Sunhemp
%
Fig. 1. Total phenolics in the root exudates black gram and sun hemp.
of green gram,
5.4 (:,a’ (6:4)
or rhizosphere effect S.E. = 0.3; C.D. =
ation in the quantity of total phenol& diffused from the seeds (green gram, 6.4, black gram 7.5 and sun hemp 6.6 mg. 1OOg’ of dry seed material) DSA reagents revealed the presence of two positive spots in green gram, three in black gram and six in sun hemp. Similarly the total quantity of phenolics exuded through the roots varied with plant species. The quantity of phenolics exuded decreased as the plants aged. Marked differences in the phenolic content in the exudates of the three plant species were observed only on day 3 (Fig. 1). The sun hemp seed diffusates caused a marked reduction in the growth in liquid culture of the Rhizohium isolated from sun hemp as well as green gram and black gram. The seed diffusates of green gram and black gram did not however, show much inhibitory effect on the three rhizobial cultures (Fig. 2).
Our experiments revealed that the germinating seeds of green gram, black gram and sun hemp exuded inhibitory compounds which were phenolic in nature. Such an exudation of phenohc compounds from seeds has been reported by Knapp (1954), Borner (1958) and Kandasamy et al. (1974). Although there was not much difference between
Phenolic
compounds
in seed
Fig. 2. Inhibitory activltk of the seed diffusates towards rhizobia: Tube I. Broth (unmoculated); 2. Green gram seed diffusatc + Rhirohium (CG6i: 3. Green gram seed diffusate + Xhizohiurn (CB2); 4. Green gram seed diffusate + Rhkohiuul (S9): 5. Rh;:ohiuru (CC@ isolate alone; 6. Black gram seed diffusate + CG6 isolate; 7. Black grant seed difusate + CB2 isolate: 8. Black gram seed diffusate + S9 isolate: 9. Rhi~~~bi~~~?? (CB2) alone; 10. Sun hemp seed diffusate + CG6 isolate; Il. Sun hemp seed diGsate t CB2 isolate: 12. Sttn hemp seed diffusate + S9 isolate; 13. R/~;~~~~l~~~~ (S9 alone).
the total phenolic contents of the seed diffusates of green gram, black gram and sun hemp seeds. their anti-rhizobial properties varied. The growth of Rhkobium isolates from the nodules of green gram, black gram and sun hemp was inhibited more by sun hemp seed diffusates than by green gram and black gram seed diffusates. This might, perhaps, be due to the qualitative differences in the phenolics exuded by the seeds of these three plant species. The selective nature of seed exudates towards rhizobia was reported by Thompson (1960) and Bowen (1961) who showed that seeds of both ~r~~oii~~?zs~bi~rran~~il~ and ~entro~~~rn~ puhescens exuded water-soluble and thermostable substances active against several bacterial species including rhizobia, while exudates from lucerne seeds had no antimicrobial activity. The prevalence, in general. of an enhanced spermosphere effect in the second sampling of spermosphere soil and rhizosphere effect in the rhizosphere soils may, perhaps, be due to a decrease in the quantity of phenolics present in the root exudates of the plants as they aged. We have evidence to corroborate the inhibition of rhizobia by a variety of phenolic compounds, Conceivably, the antimicrobial phenolicdiffusates from the seeds might have played a role in the early colonization of R~i~o~~~~7 in the spermosphere. REFERENCES BLCKK R. J., DURRUM E. L. and ZWEIG G. (1958) A Manual UJ Paper Chromatography and Paper Electrophorrsis. Academic Press. New York.
BOHN~R H. (1958) Nachweis phenohschen Vrrbindungen in Leinsamen und i bre Abgabe wahrend der Quellung. Floru 143. 479-496. BOWEIU’G. D. (1961) The toxicity of legume seed dill’usates toward rhizobia and other bacteria. P/. Soi/ 15, 155-I 65. BRAY 8. G. and THORPE W. V. (1954) Plnalysis of phenolic compounds of interest in metabolism. Mettz. Biochern. Anal,~. I. 27-m52. CLARK F. E. (1965) Rhizobia: In Merhods of Soil Anal~~sis. Part II (C. A. Black. D. D. Evans. J. L. White. L. E. Ensminger and F. E. Clark, Eds). pp. 1487-1492. American Society of Agronomy. Madison, Wisconsin. DADARWAL K. R. and SEN A. N. (1973) Inhibitory effect of seed diffusates of some legumes on rhizobia and other bacteria. Indian J. agric. Sci. 43, 82-87. FUTTRELL P. F.. O’CONNOR S. and MASTIXSON C. L. (1964) Identification of the flavonol mpricetin in legume seed and its toxicity to nodule bacteria. Irish J. Agric. Rex 3. 246249. KANDASAIMYD., V~~PHHAJVV. and PRASAU N. N. (1973) Apparatus for aseptic culturing of plants. fttdictn J. e.up. Bioi. 11, 256-257. KANDASAMY D., KESAVAI*‘R., RAMASAMYK. and PRASAD N. N. (1974) Occurrence of microbial inhibitors in the exudates of certain leguminous seeds. Indian 1. Microbiol, 14, 25-30. KNAPP R. (1954) E.xperrimenteile Soziologie der Hoheren P&mxn. Verlag, Stutrgart. RICX E. L. (1971) Inhibition of nodulation of inoculated legumes by Ieaf leachates from pioneer plant species from abandoned fields. Am. J. Bat. 58, 368-371. THOMPSON J. A. (1960) Inhibition of nodule bacteria by an antibiotic from legume seed coats. Nature. 187, 619. YADAV N. K. and VYAS S. R. (1971) Effect of salts on the growth of Rhcohium straits. Indian J. Microhioi. II, 95-99.
KANDASAMY*
and N. N.
PRASAD
Department of Microbiology, Faculty of Agriculture. Annamalai C!niversity. An~amalaina~ar .- 608101, India
Summary-“-.~Astudy of the colonization pattern of R~~~~~~u~?i inoculated either in the soil or on to the seeds of green gram {~~~us~~~z~s uuwus Roxb.). black gram (P. mz~~z~o L.) and sun hemp (Crotafaria juncru L.) revealed a significant reduction of R~j~#~j~~~)~ in the spermosphere of sun hemp which was attributed to excretion of phenolic compounds by the seed. As the plants aged, the quantity of phenolics exuded bv the roots was reduced and there was a parallel increase in the Rhi~hium papulation in the rhi~~~pheres of the three plant species.
sphere soil samples were collected on days 7 and 9. ~~j~o~~~~z was estimated in the soil samples by a plant dilution technique (Clark, 1965). Seed diffusates were extracted (Kandasamy 01 al., 1974) and total phenol& in the seed diffusates were estimated (Bray and Thorpe. 1954). Phenolic compounds in the diff~lsat~s were separated by twodimensional paper chromatography (Block et al., 1958). Of the final fraction, 50 ~~!11 was spotted on Whatman No. 1 chromatographic paper (28 x 23 cm) and developed ascendingly in a solvent system CORsisting of n-butanol:aceti~ acid:water; 4: I:5 (vi@ for the first direction and 2% acetic acid for the second direction. To the air-dried chromatograms diazotized sulfanilic acid (DSA) at 0.5% in 20% Na&O, was used as a spray reagent to locate phenols. Phenols exuded by the roots of the three plant species were estimated using an exudation apparatus (Kandasamy rr al., 1973). Root exudates were collected on alternate days beginning on day 3 until day I1 from sowing and their total phenolic content was estimated. The influence of seed diffusates on the growth of rhizobia was tested (Yadav and Vyas, 1973).
INTRODUCTlON considerable attention has,been paid to the presence of anti-microbial substances in plants and active substances have now been found from at least 157 families of the plant kingdom. In some instances such substances have also been extracted from seeds (Bowen, 1961). Fottreil rr ul. (1964) observed that eertain leguminous seed diffusates were jnhibjtory to many Gram-positive and Gram-negative organisms. Extracts of certain weeds. and ieachates from soil of abandoned fields where those weeds occurred were found to inhibit half the ~~j~o~~~r~ strains tested (Rice. 1971). Dadarwal and Sen (1973) also observed variations in the inhibitory effect of seed diffusates of nine legume plant species tawards different strains of rhizobia, an ~~~tubuct~r and a ~u~illus, Little is known about the role of antimicrobial substances present in the seed in determining the survival and colonization of leguminous roots by Rhk&ium. We report the results of an investig~tjon on the establishment of rhizobia in the spermosph~res of green gram (~~~s~~~~.~UUI’YUS Roxb.), black gram (P. rnunyo L.) and sun hemp (Crotuluria ,juncm L.). The nature of the inbib~tory substances from the seed coats of the above three plant species. belonging to the same cross inoculation group was investigated.
RESULTS After I day the ~~~~~#~i~{~~~ inoc~llated in the soi had colonized the spermosphere of green gram more rapidly than that of biack gram. In the case of sun hemp, there were fewer rhizobia on the seed 24 h after sowing revealing a negative spermosphere effect. However, the cofonization pattern changed in the rhizospheres of the three plant species. The rhizosphere effect on the ~~i~o~j~~?zwas relatively similar in green gram and black gram while in sun hemp it was markedly low (Table 1). The Rhizohium inoculated on the seeds of green gram colonized the spermosphere and rhizosphere rapidly. A similar effect was observed in black gram, although there were fewer rhizobia. In the case of sun hemp. the initial colonization by rhizobia in the spermosphere was very poor, but it was found to proliferate rapidly later on in the rhizosphere (Table 2).
MATERiALS AND METHODS Healthy seed was surface sterilized with O.lTi HgC12 and washed in several changes of sterile distilled water. The seed was sown in garden soil contained in 15 cm pots already inoculated with a cell suspension (25 mljpot) of I-week old culture of Rhtohim sp. (I 0’ ml- ‘) or inoculated on the surface such that each seed carried 6 x I Oh.ml - r. Spermosphere soils in triplicate, were sampled to count Rhi~ohium at the end of 24 and 72 h after sowing and the rhizo--~ * Present address: Associate Professor of Microbiology. Tarnil Nadu Agricultural University. Coimbdtore-641M)3. India. 73
74
D. Table
I. Establishment
KANDASAMY
and N. N.
PRASAD
of soil-inoculated Rhizohium in the spermosphere of green gram, black gram and sun hemp
and
rhizosphere .-
Sampling time after sowing (days)
Soil alone
Black gram Green gram (Rhizobium x 10 ‘g-’ oven-dry soil)
Sun hemp
Spermosphere
I
18.4
3
9.5
7
42.4 (2.3)* 21.2 (2.2)
23.2 (1.3) 12.9 (1.4)
3.4 (0.2) 11.4 (1.2)
2.2
16.3
2.2
(7.6) 9.4
10.8 (5.0) 9.7 (4.4)
6.3 (2.9) 4.9 (2.2)
Rhizosphere
9
(4.3) * Figures 2.0 Table
in parentheses
2. Establishment
denote
the spermosphere
in the spermosphere the seed of green gram,
Sampling time after sowing
Soil alone
or rhizosphere
effect S.E. = 0.7; CD.
and rhizosphere by Rhizobium inocuiated black gram and sun hemp
Green
gram
Black gram
(Rhizobium x 10m6gg’ oven-dry
.(days)
=
on
Sun hemp soil)
Spermosphere 1
0.7
3
0.9
7
0.8
13.3 (18.4)* 11.9 (2 1.O)
12.8 (17.8) 15.4 (18.1)
18.2 (22.5) 14.4 (18.2)
(1:::) 7.5 (9.5)
2.3 (3.1) 6.7 (7.8)
Rhizosphere
9
0.8
* Figures in parentheses denote the snermosphere 0.8.
Studies carried out to determine the presence of inhibitory substances in the seed coat and root exudates revealed that although there was not much varic-----c c&en gram C---O Block gicen
‘60
--y--n
140
i
Sunhemp
%
Fig. 1. Total phenolics in the root exudates black gram and sun hemp.
of green gram,
5.4 (:,a’ (6:4)
or rhizosphere effect S.E. = 0.3; C.D. =
ation in the quantity of total phenol& diffused from the seeds (green gram, 6.4, black gram 7.5 and sun hemp 6.6 mg. 1OOg’ of dry seed material) DSA reagents revealed the presence of two positive spots in green gram, three in black gram and six in sun hemp. Similarly the total quantity of phenolics exuded through the roots varied with plant species. The quantity of phenolics exuded decreased as the plants aged. Marked differences in the phenolic content in the exudates of the three plant species were observed only on day 3 (Fig. 1). The sun hemp seed diffusates caused a marked reduction in the growth in liquid culture of the Rhizohium isolated from sun hemp as well as green gram and black gram. The seed diffusates of green gram and black gram did not however, show much inhibitory effect on the three rhizobial cultures (Fig. 2).
Our experiments revealed that the germinating seeds of green gram, black gram and sun hemp exuded inhibitory compounds which were phenolic in nature. Such an exudation of phenohc compounds from seeds has been reported by Knapp (1954), Borner (1958) and Kandasamy et al. (1974). Although there was not much difference between
Phenolic
compounds
in seed
Fig. 2. Inhibitory activltk of the seed diffusates towards rhizobia: Tube I. Broth (unmoculated); 2. Green gram seed diffusatc + Rhirohium (CG6i: 3. Green gram seed diffusate + Xhizohiurn (CB2); 4. Green gram seed diffusate + Rhkohiuul (S9): 5. Rh;:ohiuru (CC@ isolate alone; 6. Black gram seed diffusate + CG6 isolate; 7. Black grant seed difusate + CB2 isolate: 8. Black gram seed diffusate + S9 isolate: 9. Rhi~~~bi~~~?? (CB2) alone; 10. Sun hemp seed diffusate + CG6 isolate; Il. Sun hemp seed diGsate t CB2 isolate: 12. Sttn hemp seed diffusate + S9 isolate; 13. R/~;~~~~l~~~~ (S9 alone).
the total phenolic contents of the seed diffusates of green gram, black gram and sun hemp seeds. their anti-rhizobial properties varied. The growth of Rhkobium isolates from the nodules of green gram, black gram and sun hemp was inhibited more by sun hemp seed diffusates than by green gram and black gram seed diffusates. This might, perhaps, be due to the qualitative differences in the phenolics exuded by the seeds of these three plant species. The selective nature of seed exudates towards rhizobia was reported by Thompson (1960) and Bowen (1961) who showed that seeds of both ~r~~oii~~?zs~bi~rran~~il~ and ~entro~~~rn~ puhescens exuded water-soluble and thermostable substances active against several bacterial species including rhizobia, while exudates from lucerne seeds had no antimicrobial activity. The prevalence, in general. of an enhanced spermosphere effect in the second sampling of spermosphere soil and rhizosphere effect in the rhizosphere soils may, perhaps, be due to a decrease in the quantity of phenolics present in the root exudates of the plants as they aged. We have evidence to corroborate the inhibition of rhizobia by a variety of phenolic compounds, Conceivably, the antimicrobial phenolicdiffusates from the seeds might have played a role in the early colonization of R~i~o~~~~7 in the spermosphere. REFERENCES BLCKK R. J., DURRUM E. L. and ZWEIG G. (1958) A Manual UJ Paper Chromatography and Paper Electrophorrsis. Academic Press. New York.
BOHN~R H. (1958) Nachweis phenohschen Vrrbindungen in Leinsamen und i bre Abgabe wahrend der Quellung. Floru 143. 479-496. BOWEIU’G. D. (1961) The toxicity of legume seed dill’usates toward rhizobia and other bacteria. P/. Soi/ 15, 155-I 65. BRAY 8. G. and THORPE W. V. (1954) Plnalysis of phenolic compounds of interest in metabolism. Mettz. Biochern. Anal,~. I. 27-m52. CLARK F. E. (1965) Rhizobia: In Merhods of Soil Anal~~sis. Part II (C. A. Black. D. D. Evans. J. L. White. L. E. Ensminger and F. E. Clark, Eds). pp. 1487-1492. American Society of Agronomy. Madison, Wisconsin. DADARWAL K. R. and SEN A. N. (1973) Inhibitory effect of seed diffusates of some legumes on rhizobia and other bacteria. Indian J. agric. Sci. 43, 82-87. FUTTRELL P. F.. O’CONNOR S. and MASTIXSON C. L. (1964) Identification of the flavonol mpricetin in legume seed and its toxicity to nodule bacteria. Irish J. Agric. Rex 3. 246249. KANDASAIMYD., V~~PHHAJVV. and PRASAU N. N. (1973) Apparatus for aseptic culturing of plants. fttdictn J. e.up. Bioi. 11, 256-257. KANDASAMY D., KESAVAI*‘R., RAMASAMYK. and PRASAD N. N. (1974) Occurrence of microbial inhibitors in the exudates of certain leguminous seeds. Indian 1. Microbiol, 14, 25-30. KNAPP R. (1954) E.xperrimenteile Soziologie der Hoheren P&mxn. Verlag, Stutrgart. RICX E. L. (1971) Inhibition of nodulation of inoculated legumes by Ieaf leachates from pioneer plant species from abandoned fields. Am. J. Bat. 58, 368-371. THOMPSON J. A. (1960) Inhibition of nodule bacteria by an antibiotic from legume seed coats. Nature. 187, 619. YADAV N. K. and VYAS S. R. (1971) Effect of salts on the growth of Rhcohium straits. Indian J. Microhioi. II, 95-99.