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Effect of Phenolic Compounds on the Growth of Rhizobia
Zbl. Bakt. Abt.II., Bd. 128, 8.753-757 (1973)
[From Division of Microbiology, Indian Agricultural Research Institute, New Delhi-12, India]
Effect of Phenolic Compounds on the Growth of Rhizobia A. V. Rao and V.Iswaran
With 2 figures
Summary The effect of different phenolic compounds (catechol, cinnamic acid, vanillic acid, and p-coumaric acid) on the growth of Rhizobium sp., R. trifolii, R. legumirwsarum, and R. meliloti was examined by employing the paper disc method. All the phenolic compounds hav~. shown inhibitory action towards rhizobia, and the species differed in their sensitivity to the different phenolic compounds. The inhibitory action was not directly proportional to the concentration of the compound under test. It was observed in earlier studies (RAO 1972) that plants (Medicago sativa L. and Phaseolus mungo L.), inoculated with heterologous rhizobia, registered a high phenol content, compared to that present in plants inoculated with homologous rhizobia. SCHAAL and JOHNSON (1955), KOSUGE (1969), and RARE et a1. (1969) reported that various pathogenic fungi, bacteria, and other organisms were inhibited by different phenolic compounds. The present study was undertaken with a view to ascertain the effect of various phenolic compounds, such as cinnamic acid, vanillic acid, catechol, and p-coumaric acid on the growth of rhizobia.
Materials and Methods Filter paper discs of 8 mm. diameter were cut out from Whatman No.1 filter paper and sterilized at 70°C for 4 hours. With the help of a micro-pipette, each disc was enriched with a solution of the compound at the required concentration (0, 0.25, 0.5, 1.0, 2.0, a.nd 3.0 mg./disc). The bacteria under test were grown on yeast extract mannitol agar (FRED et al. 1932) slants for 4 days. Cell suspensions were prepared separately by suspending them in sterile 0.05 M potassium phosphate buffer at pH 7.0 (Loo et al. 1945). The YEMA medium was sterilized and cooled to 45-50°C. The cell suspension, prepared as above from two slants, was added to 150 ml. medium to achieve sufficient cell concentration in the medium. The seeded medium was poured in 20 ml. quantities into sterile Petri-dishes and allowed to solidify. Mter solidification, the filter paper discs, previously enriched with the phenolic compounds, were placed over the medium. The plates were incubated at 28 ± 2°C for 24 hours.
754
A. V. Rao and V. Iswaran
At the end of the incubation period, the inhibition zones were measured in millimeters and expressed in area by employing the formula of SMALE and KEIL (1966): A = n (R
+ r) (R -
r)
where A = Area of inhibition zone, R = Radius of the assay disc + radius of the inhibition zone, r = Radius of the assay disc.
Results and Discussion
The results are statistically significant at a 5% level. They are presented in Fig.1 and Table 1. The plates depicting the inhibition of rhizobia by phenolic compounds are shown in Figure 2. 50
VANILLIC
p-COUMARIC ACID
ACID
4·0
3·0
20
x-x
g d
'v"
o----a
RHIZOBIUM Sp R. TRIFOLlI
6 - 6 R. LEGUMINQSARUM
1·0
0 - 0 R MELiLarl
tj
~I
;;:;
0 5·0
CATECHOL
CINNAMIC ACID
t5
"a: "
UJ
4·0
3·0
2·0
1·0
0 MILLIGRAMM PER
DISC
Fig. I. Effect of phenolic compounds on the growth of Rhizobium spp.
On Rhizobium sp., p-coumaric acid produced maximum inhibition, followed by catechol, cinnamic acid, and vanillic acid. But taking the minimum concentration, the order was p-coumaric acid> cinnamic acid> catechol> vanillic acid. On R. trifolii, the effect at the maximum concentration was in the order p-coumaric acid > vanillic acid > catechol> cinnamic acid, while at the minimal level of 0.25 mg. the order was p-coumaric acid> cinnamic acid> vanillic acid> catechol.
*) Milligram per disc.
C.D. at 5% level
2.49 1.64 2.75 1.69 0.42
2.85 1.95 3.04 2.65
3.54 2.24 3.74 3.02
3.91 2.49 4.19 3.66
0.54 0.63 0.87 0.69
1.27 1.43 1.73 1.19 0.38
1.77 2.44 2.65 1.86
2.59 2.97 3.39 3.07
3.36 3.36 4.21 3.72
0.39 0.23 0.14 0.18
0.69 0.63 0.42 0.57
0.49
1.86 0.98 1.35 1.44
2.82 1.82 2.46 2.05
4.22 3.09 3.78 3.31
1.56 1.43 1.51 1.35
2.45 2.49 2.44 2.66
0.43
2.73 3.08 2.93 3.60
4.09 4.41 3.54 4.54
4.61 4.81 4.29 5.09
1.43 0.82 1.95 1.51
Rhizobium sp. R. trifolii R. legumino8arum R. meliloti
-~---
Area of Inhibition in cm Cinnamic acid Vanillic acid Catechol P·Coumaric acid 0.25 0.05 1.00 2.00 3.00 0.25 0.50 1.00 2.00 3.00 0.25 0.50 1.00 2.00 3.00 0.25 0.50 1.00 2.00 3.00 2•
Rhizobium spp.
Effect of phenolic compounds* on the growth of Rhiwbium spp.
'I'able 1
~
~
Ol Ol
-J
~.
~
~
a,
i
~
S·
t
~
(=j'
[
a,
g.
756
A. V. Rao and V. Iswaran
Fig.2. Effect of cinnamic acid (mg. ldis<,) on the growth of R. meliloti 0) 0.00 mg .. 1) 0.25 mg., 2) 0.50 mg., 3) 1.00 mg., 4) 2.00 mg., 5} 3.00 mg. In the case of R. leguminosarum, maximum inhibition at the lowest and highest levels came from cinnamic acid, followed by p-coumaric acid, vanillic acid, and lastly catechol. On R. meliloti, cinnamic acid was most inhibitory at the lowest level, followed in turn by p-coumaric acid, vanillic acid, and catechol. At the maximal range, however, p-coumaric acid exerted the maximum effect, followed by vanillic acid, cinnamic acid, and catechol. From the results it is evident that all the phenolic compounds tested are inhibitory towards rhizobia. SCHAAL and JOHNSON (1955) reported the inhibition of Streptomyces scabies, a potato scab organism, by the different phenolic compounds, and further observed that the inhibitory effect varied with the pH of the medium and the strain of the organism. This is in conformity with the present studies, where the inhibitory effect of the phenolic compounds vary with the species. A certain increase in the inhibitory effect was observed with increase in the concentration of the phenolic compounds, but it was not directly proportional to the concentration. Acknowledgement The authors wish to express thanks to Dr. N. S. SUBBA RAO, Head of the Division of Microbiology, Indian Agricultural Research Institute, for his interest and valuable suggestions in this work. ZusammenfassUDg Es wurde der Einflull einiger phenolischer Verbindungen (Katechol, Zimtsaure, Vanillinsaure, p-Kumarinsaure) auf das Wachstum von Rhizobium sp., Rh. trifolii, Rh. leguminosarum und Rh. meliloti mit Hilfe der Papierscheibenmethode untersucht. AIle gepriiften Verbindungen iibten cinen bemerkenswerten Effekt auf das Mikroorganismenwachstum aus. Dieser Effekt war bei einzelnen Mikroben unterschiedlich und der Konzentration nicht ganz proportional.
Effect of Phenolic Compounds on the Growth of Rhizobia
757
Literature FRED, E.B., BALDWIN, l.L., and McCoy, E.: Root nodule bacteria and leguminous plants. University of Wisconsin Press, Madison, Wisc., 343 (1932). - KOSUGE, T.: The role of phenolics in host response to infection. Ann. Rev. phytopathol. 7 (1969), 195-222. - Loo, Y.S., SKELL, P.S., THORNBERRY, H.H., EHRLIK, J., MCGe-IRE, J.M., SAVACE, G.M., and SYLVESTER, J.C.: Assay of streptomycin by the paper disc method. J. Bacter. 50 (1945), 701-709. - RAHE, J.E., Kuc, J., CHIE;';, M.C., and WILLIAMS, E.B.: Correlation of phenolic metabolism with histological changes in Phaseolus vulgaris, inoculated with fungi. Neth. J. PI. Path. 75 (1969), 58-71. RAO, A. V.: Physiology of leguminous roots in relation to inoculation with rhizobia. Ph. D. Thesis, P. G.School, LA. R. I., New Delhi 1972. - SCHAAL, L.A., and JOHXSON, G.: The inhibitory effect of phenolic compounds on the growth of Streptomyces scabies, as related to the mechanism of scab resistance. Phytopathology 45 (1955), 626-628. - SMALE, B.C., and KEIL, H.L.: A biochemical study of the intervarietal resistance of Pyrus communis fire blight. Phytochemistry 5 (1966), 1113 ~ ll20. Authors' address: Dr. A.V.Rao and Dr. V.Iswaran, Division of Microbiology, Indian Agricultural Research Institute, ~ew Delhi·12 (India)
[From Division of Microbiology, Indian Agricultural Research Institute, New Delhi-12, India]
Effect of Phenolic Compounds on the Growth of Rhizobia A. V. Rao and V.Iswaran
With 2 figures
Summary The effect of different phenolic compounds (catechol, cinnamic acid, vanillic acid, and p-coumaric acid) on the growth of Rhizobium sp., R. trifolii, R. legumirwsarum, and R. meliloti was examined by employing the paper disc method. All the phenolic compounds hav~. shown inhibitory action towards rhizobia, and the species differed in their sensitivity to the different phenolic compounds. The inhibitory action was not directly proportional to the concentration of the compound under test. It was observed in earlier studies (RAO 1972) that plants (Medicago sativa L. and Phaseolus mungo L.), inoculated with heterologous rhizobia, registered a high phenol content, compared to that present in plants inoculated with homologous rhizobia. SCHAAL and JOHNSON (1955), KOSUGE (1969), and RARE et a1. (1969) reported that various pathogenic fungi, bacteria, and other organisms were inhibited by different phenolic compounds. The present study was undertaken with a view to ascertain the effect of various phenolic compounds, such as cinnamic acid, vanillic acid, catechol, and p-coumaric acid on the growth of rhizobia.
Materials and Methods Filter paper discs of 8 mm. diameter were cut out from Whatman No.1 filter paper and sterilized at 70°C for 4 hours. With the help of a micro-pipette, each disc was enriched with a solution of the compound at the required concentration (0, 0.25, 0.5, 1.0, 2.0, a.nd 3.0 mg./disc). The bacteria under test were grown on yeast extract mannitol agar (FRED et al. 1932) slants for 4 days. Cell suspensions were prepared separately by suspending them in sterile 0.05 M potassium phosphate buffer at pH 7.0 (Loo et al. 1945). The YEMA medium was sterilized and cooled to 45-50°C. The cell suspension, prepared as above from two slants, was added to 150 ml. medium to achieve sufficient cell concentration in the medium. The seeded medium was poured in 20 ml. quantities into sterile Petri-dishes and allowed to solidify. Mter solidification, the filter paper discs, previously enriched with the phenolic compounds, were placed over the medium. The plates were incubated at 28 ± 2°C for 24 hours.
754
A. V. Rao and V. Iswaran
At the end of the incubation period, the inhibition zones were measured in millimeters and expressed in area by employing the formula of SMALE and KEIL (1966): A = n (R
+ r) (R -
r)
where A = Area of inhibition zone, R = Radius of the assay disc + radius of the inhibition zone, r = Radius of the assay disc.
Results and Discussion
The results are statistically significant at a 5% level. They are presented in Fig.1 and Table 1. The plates depicting the inhibition of rhizobia by phenolic compounds are shown in Figure 2. 50
VANILLIC
p-COUMARIC ACID
ACID
4·0
3·0
20
x-x
g d
'v"
o----a
RHIZOBIUM Sp R. TRIFOLlI
6 - 6 R. LEGUMINQSARUM
1·0
0 - 0 R MELiLarl
tj
~I
;;:;
0 5·0
CATECHOL
CINNAMIC ACID
t5
"a: "
UJ
4·0
3·0
2·0
1·0
0 MILLIGRAMM PER
DISC
Fig. I. Effect of phenolic compounds on the growth of Rhizobium spp.
On Rhizobium sp., p-coumaric acid produced maximum inhibition, followed by catechol, cinnamic acid, and vanillic acid. But taking the minimum concentration, the order was p-coumaric acid> cinnamic acid> catechol> vanillic acid. On R. trifolii, the effect at the maximum concentration was in the order p-coumaric acid > vanillic acid > catechol> cinnamic acid, while at the minimal level of 0.25 mg. the order was p-coumaric acid> cinnamic acid> vanillic acid> catechol.
*) Milligram per disc.
C.D. at 5% level
2.49 1.64 2.75 1.69 0.42
2.85 1.95 3.04 2.65
3.54 2.24 3.74 3.02
3.91 2.49 4.19 3.66
0.54 0.63 0.87 0.69
1.27 1.43 1.73 1.19 0.38
1.77 2.44 2.65 1.86
2.59 2.97 3.39 3.07
3.36 3.36 4.21 3.72
0.39 0.23 0.14 0.18
0.69 0.63 0.42 0.57
0.49
1.86 0.98 1.35 1.44
2.82 1.82 2.46 2.05
4.22 3.09 3.78 3.31
1.56 1.43 1.51 1.35
2.45 2.49 2.44 2.66
0.43
2.73 3.08 2.93 3.60
4.09 4.41 3.54 4.54
4.61 4.81 4.29 5.09
1.43 0.82 1.95 1.51
Rhizobium sp. R. trifolii R. legumino8arum R. meliloti
-~---
Area of Inhibition in cm Cinnamic acid Vanillic acid Catechol P·Coumaric acid 0.25 0.05 1.00 2.00 3.00 0.25 0.50 1.00 2.00 3.00 0.25 0.50 1.00 2.00 3.00 0.25 0.50 1.00 2.00 3.00 2•
Rhizobium spp.
Effect of phenolic compounds* on the growth of Rhiwbium spp.
'I'able 1
~
~
Ol Ol
-J
~.
~
~
a,
i
~
S·
t
~
(=j'
[
a,
g.
756
A. V. Rao and V. Iswaran
Fig.2. Effect of cinnamic acid (mg. ldis<,) on the growth of R. meliloti 0) 0.00 mg .. 1) 0.25 mg., 2) 0.50 mg., 3) 1.00 mg., 4) 2.00 mg., 5} 3.00 mg. In the case of R. leguminosarum, maximum inhibition at the lowest and highest levels came from cinnamic acid, followed by p-coumaric acid, vanillic acid, and lastly catechol. On R. meliloti, cinnamic acid was most inhibitory at the lowest level, followed in turn by p-coumaric acid, vanillic acid, and catechol. At the maximal range, however, p-coumaric acid exerted the maximum effect, followed by vanillic acid, cinnamic acid, and catechol. From the results it is evident that all the phenolic compounds tested are inhibitory towards rhizobia. SCHAAL and JOHNSON (1955) reported the inhibition of Streptomyces scabies, a potato scab organism, by the different phenolic compounds, and further observed that the inhibitory effect varied with the pH of the medium and the strain of the organism. This is in conformity with the present studies, where the inhibitory effect of the phenolic compounds vary with the species. A certain increase in the inhibitory effect was observed with increase in the concentration of the phenolic compounds, but it was not directly proportional to the concentration. Acknowledgement The authors wish to express thanks to Dr. N. S. SUBBA RAO, Head of the Division of Microbiology, Indian Agricultural Research Institute, for his interest and valuable suggestions in this work. ZusammenfassUDg Es wurde der Einflull einiger phenolischer Verbindungen (Katechol, Zimtsaure, Vanillinsaure, p-Kumarinsaure) auf das Wachstum von Rhizobium sp., Rh. trifolii, Rh. leguminosarum und Rh. meliloti mit Hilfe der Papierscheibenmethode untersucht. AIle gepriiften Verbindungen iibten cinen bemerkenswerten Effekt auf das Mikroorganismenwachstum aus. Dieser Effekt war bei einzelnen Mikroben unterschiedlich und der Konzentration nicht ganz proportional.
Effect of Phenolic Compounds on the Growth of Rhizobia
757
Literature FRED, E.B., BALDWIN, l.L., and McCoy, E.: Root nodule bacteria and leguminous plants. University of Wisconsin Press, Madison, Wisc., 343 (1932). - KOSUGE, T.: The role of phenolics in host response to infection. Ann. Rev. phytopathol. 7 (1969), 195-222. - Loo, Y.S., SKELL, P.S., THORNBERRY, H.H., EHRLIK, J., MCGe-IRE, J.M., SAVACE, G.M., and SYLVESTER, J.C.: Assay of streptomycin by the paper disc method. J. Bacter. 50 (1945), 701-709. - RAHE, J.E., Kuc, J., CHIE;';, M.C., and WILLIAMS, E.B.: Correlation of phenolic metabolism with histological changes in Phaseolus vulgaris, inoculated with fungi. Neth. J. PI. Path. 75 (1969), 58-71. RAO, A. V.: Physiology of leguminous roots in relation to inoculation with rhizobia. Ph. D. Thesis, P. G.School, LA. R. I., New Delhi 1972. - SCHAAL, L.A., and JOHXSON, G.: The inhibitory effect of phenolic compounds on the growth of Streptomyces scabies, as related to the mechanism of scab resistance. Phytopathology 45 (1955), 626-628. - SMALE, B.C., and KEIL, H.L.: A biochemical study of the intervarietal resistance of Pyrus communis fire blight. Phytochemistry 5 (1966), 1113 ~ ll20. Authors' address: Dr. A.V.Rao and Dr. V.Iswaran, Division of Microbiology, Indian Agricultural Research Institute, ~ew Delhi·12 (India)