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Negative allelopathic effects of rutin and quercetin on fourteen soil and enteric microbes
BiochemicalSystematics and Ecology, 1978,Vol.6, pp. 1 to 3. PergamonPress.Printedin England.
Negative Allelopathic Effects of Rutin and Quercetin on Fourteen Soil and Enteric Microbes JOHN E. AVERETT, WILLIAM A. BANKS and DANIEL E. BOEHME Department of Biology, University of Missouri, Saint Louis, MO 63121, USA Abstract--The flavonoids rutin and quercetin were assayed separately and in combination for antimicrobial
activity against 14 soil and enteric microbes. Microbial inhibition was observed when rutin and quercetin (pH 5.7 and 5.8 respectively) solutions were not adjusted to pH 7.0. At neutral pH no antimicrobial activity was observed. The addition of flavonoids at neutral pH did not effect the bacterial growth curve of Agrobacterium tumefaciens or the exponential growth of Escherichia coil Both bacteria grew to turbidity from a Ioop-inoculum in medium containing rutin or quercetin as the sole carbon source. Furthermore,medium containing rutin and/or quercetin did not decrease either the number or relative frequency of several microbes isolated from open field and forest floor soil samples. It is concluded that microbial inhibition has not played a major evolutionary role in the production of flavonoids in plants. Introduction
Flavonoids have been implicated in a wide variety of physiological activities in plants (for reviews see [1, 2]). Few conclusions can be made regarding their function, but antibiotic activities are among the several roles suggested for flavonoids. Rice and Pancholy [3] found that condensed tannins at levels of only a few parts per million in soil suspension cultures of Nitrosomonas and Nitrobacter sp. completely inhibited nitrification. Several flavonoids, which included quercetin and quercetin glycosides, were found to have similar properties [4]. The potential significance of alielopathy to ecological succession is well known [ 5 - 7 ] and the work of Rice and Pancholy [3, 4] would suggest antibacterial properties of flavonoids might account, at least partially, for the widespread occurrence of these compounds in plants. To examine the question further, the flavonoids rutin and quercetin were assayed separately and in combination for antimicrobial activity against 14 soil and enteric microbes. Rutin, the 3-O-rutinoside of quercetin, is a widespread and frequently occurring flavonol in plants. Quercetin, and other flavonoid aglycones, occur rarely [8], but have been isolated from soil [4]. Because of this, and their suspected antimicrobial properties, these compounds were utilized in this study. Results
Zones of inhibition of microbial growth were observed with flavonoid-soaked discs. How-
ever, when the flavonoid solutions were adjusted to pH 7.0, no microbial inhibition was observed. Discs soaked with distilled water adjusted to pH 5.7-5.8 with 1N HCI produced zones of inhibition similar to the flavonoid solutions which were not adjusted to neutral pH. In addition, the number or relative distribution of microbes isolated from soil did not differ when results from NA or NA plus rutin, quercetin, or rutin and quercetin plates were compared. Furthermore, the growth curve of A. tumefaciens was not inhibited by the flavonoids (Fig. 1 ). A. tumefaciens grew in the vicinity of the flavonoid-soaked discs (pH 7.0) on MS agar plates. Bacterial growth was moderate and did not differ in the vicinities of discs soaked in either of the two or a mixture of the two flavonoids. These discs provided the sole source of carbon for these plates. When cultured in liquid MS medium with rutin as the only carbon source, A. tumefaciens Bom exhibited a normal growth pattern (Fig. 1 ). MS medium containing 100 I~g/ml rutin and MS medium containing 0.1% glucose (without flavonoids) supported growth of A. tumefaciens equally well under the conditions assayed. Much lower growth occurred with rutin at 10 IJg/ml. Thus, bacterial growth appeared dependent upon the concentration of rutin when the flavonoid provided the sole carbon source. E. coil E-26 grew in MS medium containing rutin or quercetin as the sole carbon source.
(Received 20 November 1976; received for publication 18 July 1977)
2
JOHN E. AVERETT, WILLIAM A, BANKS AND DANIEL E, BOEHME
2.0 /
1.0
08
//
.5 0.6
~o4 m.
I
0 2 ~'~AmAmAmA~A~AmA j
OI
t 5
I Time,
h
FIG. 1. BACTERIAL GROWTH CURVES OF A. TUMEFACIENS STRAIN B6m IN LIQUID MS MEDIA CONTAINING 0,1% D-GLUCOSE (w/v) A - - A AND 100 i~g/ml RUTIN WITHOUT GLUCOSE i - - i . All media were adjusted to pH 7.0. Absorbance was monitored with a Klett Summerson photoelectric colorimeter (green filter No. 54).
A loop-sized inoculum from an overnight MS plus 0.1% glucose culture produced turbid cultures within 24 h in glucose-free media containing rutin or quercetin. Bacterial growth was moderate and was apparently dependent upon the flavonoid concentration when these compounds provided the sole carbon/energy source. The addition of rutin or quercetin (pH 7.0) to exponentially growing E. coil E-26 (bacterial generation time 38.3 4- 0.8; avg. 4- s.e., N=12) produced only an expected dilution effect. Exponential growth was not significantly altered (bacterial generation times: 100 I~g/ml rutin 38.5 4- 0.9, N=6 and 100 pg/ml quercetin 38.1 4- 1.5, N=6, avg. 4- s.e.). Discussion Growth in minimal salts medium necessitates the de novo synthesis of all organic molecules required for cellular growth from exogenous supplements. Presumably, cells cultured in such medium would have a large number of biosynthetic processes available for inhibition by rutin or quercetin. However, it is conceivable that rutin or quercetin may have inhibited a variety of nonessential metabolic processes. In any case, the bacteria were not pre-adapted to flavonoid containing media nor were flavonoid-resistant colonies isolated prior to these studies. These data indicate that the flavonoids
rutin and quercetin have no apparent antimicrobial activity at neutral pH. In fact, the only antimicrobial feature of these compounds was their acidic properties. The ability of A. tumefaciens and E. coil to grow in minimal salts medium where rutin and quercetin provided the only source of carbon indicates bacteria can even metabolize flavonoids if no alternative carbon source is available. However, flavonoids may have a role as contributors to soil acidity, along with a variety of other plant constituents. Crude leaf extracts (acidic pH) are known to be bacteriocidal and it would appear that plants contain several compounds including flavoholds, which display synergistic antimicrobial acidosis. Hence, flavonoids may have an allelopathic effect as suggested by Rice and Pancholy [4] and others; but the effect is from the acidic properties of the compounds. The allelopathic role of flavonoids is probably not major, especially compared to such compounds as tannins. Flavonoids are widespread in plants and their occurrence does not appear to be correlated with successional stages. If this is true, their impact on inhibition of nitrification would be as great in pioneer as in climax communities. This study did not examine the process of nitrification or the possible involvement of flavonoids in this process. Likewise, a physiological influence of flavonoids on nitrifying bacteria was not examined. Rather, a general antimicrobial effect of rutin or quercetin on soil microflora was analyzed. While the microflora from open field and forest floor samples differed, the addition of rutin or quercetin or both flavonoids to the culture medium did not alter these microbial populations. Caution is required when interpreting inhibition of soil microbes by plant and soil constituents. These microbial populations may have evolved a variety of resistance mechanisms to protect against any adverse effects of flavonoids, The absence of flavonoid-induced growth inhibition did not warrant identification of these microbes. However, the ability of rutin and quercetin to support the growth of A. tumefaciens and E. coil suggests that the ability to utilize these compounds could confer a selective advantage for soil microbes by providing an additional nutrient source and it is conceivable that some flavonoids under certain cultural conditions could exert an adverse effect on microbial growth or survival. However, we do not believe that selection stemming from such
NEGATIVEALLELOPATHICEFFECTSOF RUTINAND QUERCETINON FOURTEENSOILAND ENTERICMICROBES a c t i v i t y has played a major e v o l u t i o n a r y role in the p r o d u c t i o n of f l a v o n o i d s in plants.
Experimental This study was conducted with strains of Agrobacter/um tumefaciens, Bacillus subti/is, Erwinia amylovora, Escherichia coil, Pseudomonas aeruginosa, Rhizobium leguminosarum, R. meliloti, R. trifoli, Salmonella typhimurium, Staphylococcus aureus, Saccharomyces cereviciae, Penicillium sp. and two unidentified fungi isolated from soil. The organisms were grown on nutrient agar and minimal salts media. Nutrient agar (NA) contained 23g Difco nutrient agar, 5g D-glucose, and l g Difco yeast extract per 1 H20, adjusted to pH 7.0. Minimal salts (MS) medium contained 5g NH4CI, l g NH4NO3, 2g Na2SO3 (anhydrous), 3g K2HPO4 (anhydrous), l g KH2PO4 and O.lg MgSO4 .7H20 per 1 H20, pH 7.0. Where indicated, MS medium was solidified with Difco purified agar, 1.5%. K phosphate buffer. 0.1 M pH 7.0 was used for all dilutions and cell suspensions. Distilled water suspensions (100 pg/ml) were pH 5.8 for quercetin and pH 5.7 for rutin. Where indicated, these solutions were adjusted to pH 7.0 with 1 N NaOH. Flavonoid solutions were filter sterilized before use (0.45 #m Millipore filter). Antimicrobial A cdvity Assay The sensitivity disc method as described in the "Manual of Microbiological Methods" (Society of American Bacteriologists, 1957 [9]) was utilized. Before use, discs were soaked in water saturated with rutin and/or quercetin (100 IJg/ml). A. tumefaciens B6m and E. coil E-26 growth curves were assayed. Overnight bacterial cultures in MS media plus l g / I glucose were inoculated into MS medium plus l g/I glucose and supplemented with 100 l~g/ml rutin and/or quercetin and compared to
3
growth curves obtained in MS medium plus l g / I glucose. Soil microflora was isolated from open field and forest floor soil samples, l g of soil was suspended in 10 ml potassium phosphate buffer. The suspension was centrifuged and the supernatant was collected and diluted to obtain between 100 and 300 colonies per plate. The soil isolates were plated on NA containing 100 t~g/ml rutin, quercetin or rutin and quercetin (100 I~g/ml each) and on NA without flavonoids. Flavonoid Metabolism Flavonoid-soaked discs were placed on MS agar and covered with a lawn of bacteria or fungi. Flavonoids provided the only source of carbon for growth. In addition, 10-100 t~g/ml rutin was added to MS cultures of A. tumefaciens B6m and 100 pg/ml quercetin or rutin to E. coil E-26 as the only carbon source.
References 1. Levin, D. A. (1971) Am. Natur. 105, 157. 2. McClure, J. W. (1976) Physiology and function of flavonoids, in The Flavonoids (Harborneo J. B., Mabry, T. J. and Mabry, H., eds.), Chapman ~ Hall, London. 3. Rice, E. L. and Pancholy, S. K. (1973) Am. J. Botany 60, 691. 4. Rice, E. L. and Pancholy, S. K. (1974) Am. J. Botany 61, 1095. 5. Muller, C. H. (1966) Bull. Torrey Dot. Club 95, 432. 6. Rice, E. L. (1967) Bios 38, 67. 7. Lodhi, M. A. K. and Rice, E. L. (1967) Bull. Torrey Dot. Club 98, 83. 8. Harborne, J. B. (1967) Comparative Biochemistry of the Flavonoids, Academic Press, London. 9. Society of American Bacteriologists, (1957) Manual of Microbiological Methods, McGraw-Hill, New York.
Negative Allelopathic Effects of Rutin and Quercetin on Fourteen Soil and Enteric Microbes JOHN E. AVERETT, WILLIAM A. BANKS and DANIEL E. BOEHME Department of Biology, University of Missouri, Saint Louis, MO 63121, USA Abstract--The flavonoids rutin and quercetin were assayed separately and in combination for antimicrobial
activity against 14 soil and enteric microbes. Microbial inhibition was observed when rutin and quercetin (pH 5.7 and 5.8 respectively) solutions were not adjusted to pH 7.0. At neutral pH no antimicrobial activity was observed. The addition of flavonoids at neutral pH did not effect the bacterial growth curve of Agrobacterium tumefaciens or the exponential growth of Escherichia coil Both bacteria grew to turbidity from a Ioop-inoculum in medium containing rutin or quercetin as the sole carbon source. Furthermore,medium containing rutin and/or quercetin did not decrease either the number or relative frequency of several microbes isolated from open field and forest floor soil samples. It is concluded that microbial inhibition has not played a major evolutionary role in the production of flavonoids in plants. Introduction
Flavonoids have been implicated in a wide variety of physiological activities in plants (for reviews see [1, 2]). Few conclusions can be made regarding their function, but antibiotic activities are among the several roles suggested for flavonoids. Rice and Pancholy [3] found that condensed tannins at levels of only a few parts per million in soil suspension cultures of Nitrosomonas and Nitrobacter sp. completely inhibited nitrification. Several flavonoids, which included quercetin and quercetin glycosides, were found to have similar properties [4]. The potential significance of alielopathy to ecological succession is well known [ 5 - 7 ] and the work of Rice and Pancholy [3, 4] would suggest antibacterial properties of flavonoids might account, at least partially, for the widespread occurrence of these compounds in plants. To examine the question further, the flavonoids rutin and quercetin were assayed separately and in combination for antimicrobial activity against 14 soil and enteric microbes. Rutin, the 3-O-rutinoside of quercetin, is a widespread and frequently occurring flavonol in plants. Quercetin, and other flavonoid aglycones, occur rarely [8], but have been isolated from soil [4]. Because of this, and their suspected antimicrobial properties, these compounds were utilized in this study. Results
Zones of inhibition of microbial growth were observed with flavonoid-soaked discs. How-
ever, when the flavonoid solutions were adjusted to pH 7.0, no microbial inhibition was observed. Discs soaked with distilled water adjusted to pH 5.7-5.8 with 1N HCI produced zones of inhibition similar to the flavonoid solutions which were not adjusted to neutral pH. In addition, the number or relative distribution of microbes isolated from soil did not differ when results from NA or NA plus rutin, quercetin, or rutin and quercetin plates were compared. Furthermore, the growth curve of A. tumefaciens was not inhibited by the flavonoids (Fig. 1 ). A. tumefaciens grew in the vicinity of the flavonoid-soaked discs (pH 7.0) on MS agar plates. Bacterial growth was moderate and did not differ in the vicinities of discs soaked in either of the two or a mixture of the two flavonoids. These discs provided the sole source of carbon for these plates. When cultured in liquid MS medium with rutin as the only carbon source, A. tumefaciens Bom exhibited a normal growth pattern (Fig. 1 ). MS medium containing 100 I~g/ml rutin and MS medium containing 0.1% glucose (without flavonoids) supported growth of A. tumefaciens equally well under the conditions assayed. Much lower growth occurred with rutin at 10 IJg/ml. Thus, bacterial growth appeared dependent upon the concentration of rutin when the flavonoid provided the sole carbon source. E. coil E-26 grew in MS medium containing rutin or quercetin as the sole carbon source.
(Received 20 November 1976; received for publication 18 July 1977)
2
JOHN E. AVERETT, WILLIAM A, BANKS AND DANIEL E, BOEHME
2.0 /
1.0
08
//
.5 0.6
~o4 m.
I
0 2 ~'~AmAmAmA~A~AmA j
OI
t 5
I Time,
h
FIG. 1. BACTERIAL GROWTH CURVES OF A. TUMEFACIENS STRAIN B6m IN LIQUID MS MEDIA CONTAINING 0,1% D-GLUCOSE (w/v) A - - A AND 100 i~g/ml RUTIN WITHOUT GLUCOSE i - - i . All media were adjusted to pH 7.0. Absorbance was monitored with a Klett Summerson photoelectric colorimeter (green filter No. 54).
A loop-sized inoculum from an overnight MS plus 0.1% glucose culture produced turbid cultures within 24 h in glucose-free media containing rutin or quercetin. Bacterial growth was moderate and was apparently dependent upon the flavonoid concentration when these compounds provided the sole carbon/energy source. The addition of rutin or quercetin (pH 7.0) to exponentially growing E. coil E-26 (bacterial generation time 38.3 4- 0.8; avg. 4- s.e., N=12) produced only an expected dilution effect. Exponential growth was not significantly altered (bacterial generation times: 100 I~g/ml rutin 38.5 4- 0.9, N=6 and 100 pg/ml quercetin 38.1 4- 1.5, N=6, avg. 4- s.e.). Discussion Growth in minimal salts medium necessitates the de novo synthesis of all organic molecules required for cellular growth from exogenous supplements. Presumably, cells cultured in such medium would have a large number of biosynthetic processes available for inhibition by rutin or quercetin. However, it is conceivable that rutin or quercetin may have inhibited a variety of nonessential metabolic processes. In any case, the bacteria were not pre-adapted to flavonoid containing media nor were flavonoid-resistant colonies isolated prior to these studies. These data indicate that the flavonoids
rutin and quercetin have no apparent antimicrobial activity at neutral pH. In fact, the only antimicrobial feature of these compounds was their acidic properties. The ability of A. tumefaciens and E. coil to grow in minimal salts medium where rutin and quercetin provided the only source of carbon indicates bacteria can even metabolize flavonoids if no alternative carbon source is available. However, flavonoids may have a role as contributors to soil acidity, along with a variety of other plant constituents. Crude leaf extracts (acidic pH) are known to be bacteriocidal and it would appear that plants contain several compounds including flavoholds, which display synergistic antimicrobial acidosis. Hence, flavonoids may have an allelopathic effect as suggested by Rice and Pancholy [4] and others; but the effect is from the acidic properties of the compounds. The allelopathic role of flavonoids is probably not major, especially compared to such compounds as tannins. Flavonoids are widespread in plants and their occurrence does not appear to be correlated with successional stages. If this is true, their impact on inhibition of nitrification would be as great in pioneer as in climax communities. This study did not examine the process of nitrification or the possible involvement of flavonoids in this process. Likewise, a physiological influence of flavonoids on nitrifying bacteria was not examined. Rather, a general antimicrobial effect of rutin or quercetin on soil microflora was analyzed. While the microflora from open field and forest floor samples differed, the addition of rutin or quercetin or both flavonoids to the culture medium did not alter these microbial populations. Caution is required when interpreting inhibition of soil microbes by plant and soil constituents. These microbial populations may have evolved a variety of resistance mechanisms to protect against any adverse effects of flavonoids, The absence of flavonoid-induced growth inhibition did not warrant identification of these microbes. However, the ability of rutin and quercetin to support the growth of A. tumefaciens and E. coil suggests that the ability to utilize these compounds could confer a selective advantage for soil microbes by providing an additional nutrient source and it is conceivable that some flavonoids under certain cultural conditions could exert an adverse effect on microbial growth or survival. However, we do not believe that selection stemming from such
NEGATIVEALLELOPATHICEFFECTSOF RUTINAND QUERCETINON FOURTEENSOILAND ENTERICMICROBES a c t i v i t y has played a major e v o l u t i o n a r y role in the p r o d u c t i o n of f l a v o n o i d s in plants.
Experimental This study was conducted with strains of Agrobacter/um tumefaciens, Bacillus subti/is, Erwinia amylovora, Escherichia coil, Pseudomonas aeruginosa, Rhizobium leguminosarum, R. meliloti, R. trifoli, Salmonella typhimurium, Staphylococcus aureus, Saccharomyces cereviciae, Penicillium sp. and two unidentified fungi isolated from soil. The organisms were grown on nutrient agar and minimal salts media. Nutrient agar (NA) contained 23g Difco nutrient agar, 5g D-glucose, and l g Difco yeast extract per 1 H20, adjusted to pH 7.0. Minimal salts (MS) medium contained 5g NH4CI, l g NH4NO3, 2g Na2SO3 (anhydrous), 3g K2HPO4 (anhydrous), l g KH2PO4 and O.lg MgSO4 .7H20 per 1 H20, pH 7.0. Where indicated, MS medium was solidified with Difco purified agar, 1.5%. K phosphate buffer. 0.1 M pH 7.0 was used for all dilutions and cell suspensions. Distilled water suspensions (100 pg/ml) were pH 5.8 for quercetin and pH 5.7 for rutin. Where indicated, these solutions were adjusted to pH 7.0 with 1 N NaOH. Flavonoid solutions were filter sterilized before use (0.45 #m Millipore filter). Antimicrobial A cdvity Assay The sensitivity disc method as described in the "Manual of Microbiological Methods" (Society of American Bacteriologists, 1957 [9]) was utilized. Before use, discs were soaked in water saturated with rutin and/or quercetin (100 IJg/ml). A. tumefaciens B6m and E. coil E-26 growth curves were assayed. Overnight bacterial cultures in MS media plus l g / I glucose were inoculated into MS medium plus l g/I glucose and supplemented with 100 l~g/ml rutin and/or quercetin and compared to
3
growth curves obtained in MS medium plus l g / I glucose. Soil microflora was isolated from open field and forest floor soil samples, l g of soil was suspended in 10 ml potassium phosphate buffer. The suspension was centrifuged and the supernatant was collected and diluted to obtain between 100 and 300 colonies per plate. The soil isolates were plated on NA containing 100 t~g/ml rutin, quercetin or rutin and quercetin (100 I~g/ml each) and on NA without flavonoids. Flavonoid Metabolism Flavonoid-soaked discs were placed on MS agar and covered with a lawn of bacteria or fungi. Flavonoids provided the only source of carbon for growth. In addition, 10-100 t~g/ml rutin was added to MS cultures of A. tumefaciens B6m and 100 pg/ml quercetin or rutin to E. coil E-26 as the only carbon source.
References 1. Levin, D. A. (1971) Am. Natur. 105, 157. 2. McClure, J. W. (1976) Physiology and function of flavonoids, in The Flavonoids (Harborneo J. B., Mabry, T. J. and Mabry, H., eds.), Chapman ~ Hall, London. 3. Rice, E. L. and Pancholy, S. K. (1973) Am. J. Botany 60, 691. 4. Rice, E. L. and Pancholy, S. K. (1974) Am. J. Botany 61, 1095. 5. Muller, C. H. (1966) Bull. Torrey Dot. Club 95, 432. 6. Rice, E. L. (1967) Bios 38, 67. 7. Lodhi, M. A. K. and Rice, E. L. (1967) Bull. Torrey Dot. Club 98, 83. 8. Harborne, J. B. (1967) Comparative Biochemistry of the Flavonoids, Academic Press, London. 9. Society of American Bacteriologists, (1957) Manual of Microbiological Methods, McGraw-Hill, New York.