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In vitro growth of five Frankia isolates in the presence of four phenolic acids and juglone
SoilBid. Biochem. Vol. 18, No. 2, Printed in Great Britain. All right.s
pp. 227-231, reserved
1986 Copyright
Q
0038-0717/86$3.00+ 0.00 1986Pergamon Press Ltd
IN VITRO GROWTH OF FIVE FRANKIA ISOLATES IN THE PRESENCE OF FOUR PHENOLIC ACIDS AND JUGLONE CHRISTOPH S. VOGEL and JEFFREY 0. DAWSON Department of Forestry, University of Illinois, Urbana, IL 61801, U.S.A (Accepted 9 September 1985) Summary-The effects of four phenolic acids and juglone on the growth of five Fran/& isolates were determined using a total protein assay and light microscopy. Juglone and caffeic acid, at 100 PM and 1 mM concentrations respectively, were most inhibitory to growth of the Frunkiu isolates, although an isolate from Alnus crispa [Alms viridisssp. crispa (Ait.) Turril] nodules exhibited significantly greater growth compared to the other isolates at identical treatment levels. Gentisic, o-hydroxyphenylacetic and vanillic acids were less inhibitory to growth of the five Frankiu isolates than juglone and caffeic acid. Juglone inhibited vesicle production of two isolates that produced vesicles in controls. Juglone, caffeic acid and vanillic acid caused increased hyphal ramification, while vanillic acid also induced Frankiu to form numerous spherical structures unlike vesicles or sporangia in appearance. None of the isolates tested had the ability to utilize the phenolic acids as sole carbon and energy sources at 1 mM concentrations.
INTRODUCTION Phenolics and related compounds in soil have the potential to affect growth of microorganisms (Fisher, 1979; Li, 1974; Rice, 1974). Perradin et al. (1983) suggested that actinorhizal actinomycetes of the genus Frunkia probably exist as free-living saprophytes
in soil where they would likely encounter phenolic compounds. Perradin and co-workers also suggested that phenolics may influence Frankia growing symbiotically in host plant tissue. Nitrogen-fixing Frankia is capable of infecting the roots of certain nonleguminous trees and shrubs forming a nodular symbiotic relationship, and some actinorhizal plants have been used in forestry to increase soil nitrogen levels and the growth of associated plants. Black walnut (Jug&s nigra L.) has been coplanted with actinorhizal black alder [Alnus glutinosa L. (Gaertn.)] and autumn olive (Elueugnus umbelluta Thunb.). Walnut growth and soil nitrogen concentrations were greater in walnut-autumn olive interplantings than in plantings of walnut only (Funk et al., 1979) or in plantings of walnut and other nitrogen-fixing species (Friedrich and Dawson, 1984). Black alders interplanted with walnuts exhibited sudden decline and death 8-13 years after planting, evidently due to the release of a substance similar to phenolics, juglone (5-hydroxy-1,4_naphthoquinone), by black walnut (Rietveld et al., 1983). Juglone has also been shown to inhibit in oitro growth of some Frunkiu isolates and to inhibit nodulation of black alder seedlings in soil (Dawson and Seymour 1983; Vogel and Dawson, 1985). However, a Frankia isolate from green alder which contains high levels of the phenolic pinosylvin methyl ether was more tolerant to juglone than other isolates (Vogel and Dawson, 1985). Phenolics and polyphenols are known to occur in poplar (Populus) species (Dormaar, 1970; Olsen et at., 1971) and may have been responsible for reduced
nitrogenase activities of speckled alder [Alnus incana ssp. rugosa Clausen = A. rugosa (DuRoi) Sprengel] growing among Populus tremuloides Michx. trees in northern Wisconsin (Younger and Kaputska, 1983). Water leachates of balsam poplar (P. balsamifera L.) inhibited seed germination, growth and nodulation by Frankiu and nitrogenase activity of green alder (A. crispa var. mollis Fern.) (Jobidon and Thibault, 198 I, 1982) perhaps due to phenolics leached from poplar tissues. Since intermixtures of poplars and nitrogenfixing alders are used in forestry (Cot& and CamirC, 1984; Hansen and Dawson, 1982; Heilman and Stettler, 1983), allelopathic influence of poplars on alders and Frankia could reduce productivity of these plantations. In vitro growth of several Frankia isolates was significantly affected by the addition of some common plant phenolic compounds to the media (Perradin et al., 1983). At 1 mM concentrations of the cinnamic acids (ferulic, o-coumaric, p-coumaric, and trans-cinnamic), there was reduced growth and increased hyphal ramification of Frankia isolates from species of Ahus and Elaeagnus. However, two benzoic acids, benzoic and p-hydroxybenzoic acid, generally did not affect growth, but tended to increase vesicle numbers. Li (1974) demonstrated the presence of several phenolic compounds in the root nodules of red alder (A. rubra Bong.), the roots of another actinorhizal nitrogen-fixing species, Ceanothus velutinus Dougl. ex Hook as well as a number of understory plant species found in alder, conifer and alder-conifer stands. Evidence suggests that red alder is resistant to infection of Poriu weirii Murr. (Li et al., 1968; Wallis, 1968), a serious root rot fungus, possibly due to phenolic substances produced by red alder that have been found to inhibit in vitro growth of P. weirii (Li et al., 1969). Plant phenolics have been identified and quantified from soils of varying type, vegetative cover and location (Li et al., 1970; Shindo et al., 1978; White-
228
CHRISTOPH
S. VOGEL
head, 1964; Whitehead et al., 1983) indicating the widespread occurrence of phenolics in soils. Tannins and tannin derivatives have been implicated as possible inhibitors of nitrification by decreasing population numbers of Nitrosomonas and Nitrobacter in soils of climax forest ecosystems (Rice and Pancholy, 1973). Soils beneath red alder stands have been shown to contain phenolic compounds (Li et al., 1970), which could be partially responsible for red alder’s ability to influence soil microbe populations (Lu et al., 1968; Neal et al., 1968). It seems apparent that interactions among plant phenolics and soil microogranisms, including Frankia can have important consequences in forestry. We have quantified the interactions among previouslyuntested plant phenolics, juglone and Frankia isolates. The objectives were to determine the effects of these phenolic compounds and juglone on the growth and morphology of the Frankia isolates. MATERIALS AND METHODS
Juglone, caffeic acid, vanillic acid, gentistic acid and o-hydroxyphenylacetic acid were each dissolved in 3 ml of dimethyl sulfoxide (DMSO), brought to 100 ml volumes with distilled Hz0 and sterilized by passing through filters with 0.45 pm pores. One-ml aliquots of each phenohc solution were added to 25 x 150 mm glass culture tubes containing 9 ml of autoclaved (20 min at 1120C) broth medium. The culture tubes were then covered with plastic test tube caps. After the addition of the phenolics, the final concentrations of the broth medium components were (gl-I): casamino acids, 5.0; yeast extract. 5.0; dextrose, 10. Final concentrations of each phenolic acid in the medium was 1 and 0.5 mM each. Juglone concentrations of 100 and 50,~M were used due to juglone’s low solubility in water. In order to increase juglone solubihty for filter sterilization, the pH of the concentrate was raised to 9.4 prior to filtration by the addition of 0.1 N NaOH. Final pH of the media after the addition of the phenolics was 6.5 ( k 0.15). Two sets of controls were prepared, one containing the same concentration of DMSO found in the phenolic media (0.3% v/v) and one void of DMSO. Defined mineral media containing 1 mM concentrations of filter sterile phenohcs were employed to test the ability of Frankia to utilize the four phenolic acids as sole carbon and energy sources. The basal minimal salts used were those of the propionic acid medium developed by Shipton and Burggraaf (1982). This medium with and without propionic acid was used as a control. Five Frankia isolates, PtIl (DDB 1701 lo), Pt410 (DDB 170410), AvcIl (DDB 010110). MclO (DDB 160810) and EuIlb (DDB 130120) were used to inoculate control tubes and tubes containing phenolic broth media. Isolates PtIl and Pt410 were obtained from Purshia tridentata Pursh. nodules, AvcIl from Alnus viridis ssp. crispa nodules, MclO from nodules of Myrica cerifera L. and EuIlb from nodules of Elaeagnus umbellata. The isolates were provided by Dr Dwight Baker, Kettering Laboratory. Yellow Springs, Ohio. Added inoculum contained 4 pg total Frankia protein per tube. Inocula were prepared by harvesting 6 week old Frankia colonies grown in a
and
JEFFREY 0. DAWS~N
broth medium, macerating the colonies with a glass tissue grinder and inoculating the culture tubes with Frankia using sterile 1 ml syringes. Isolates were grown in the dark at 27°C (+ 3°C). Cultures of each isolate from each treatment were destructively sampled 3 weeks and 6 weeks after inoculation to determine growth of Frankia based on total protein per culture tube. Protein content of Frankia colonies from individual culture tubes was determined using the Coomassie Brilliant Blue (G-250, Fisher, Pittsburgh, Pennsylvania) protein-dye binding assay (Bradford, 1976). Frankia colonies were removed from the broth and washed once by centrifugation with 15 ml of pH 7 phosphate buffer. After washing, each Frankia colony was brought to a final volume of 1 ml with phosphate buffer and sonicated for 30 s. Microscopic observation of Frankia after sonication revealed total disruption of hyphae and 0.1 ml of the disrupted cells was assayed for total protein content. Standard protein curves were prepared using crystalline bovine albumin dissolved in phosphate buffer at pH 7. Phase contrast microscopy was used to make weekly observations of morphologic changes in Frankia isolates with different phenohc amendments or juglone. RESULTS AND
DISCUSSION
Growth The growth of Frankia isolate AvcI1 was less inhibited by plant phenolics than the other four isolates tested (Figs I-5). The main differences in growth of AvcIl compared with the other isolates were apparently due to AvcIl’s tolerance of 1 mM caffeic acid and its partial tolerance of 100 PM juglone (Figs 1 and 2). In an earlier experiment AvcIl was shown to have significantly greater growth relative to controls in a medium containing 100 p M juglone than four other Frankia isolates, including two alder isolates (Vogel and Dawson, 1985). The host plant from which Frankia AvcIl was isolated, A. crispa, is known to contain high concentrations of at least one phenolic compound, pinosylvin methyl ether, which discourages feeding of snowshoe hare (Bryant et al., 1983). Converselv. Purshia tridentata is commonlv known to be a preferred browse species for herbivdrous mammals in North America, suggesting that it
600
Caffeic Acid
AvcIl
Eullb
P1410
Pm
Fig. I. Frankia growth as mean total protein yields per tube (n = 3) after 6 weeks’ growth in caffeic acid. Small bars represent + 1 SD.
229
Growth of Frankiu with phenolics and juglone
= Ii -I., =
Control 0.5mB
=l.OmM
AvcIl
Eullb
MC10
Pt410
Fig. 2. Frankia
growth as mean total protein yields per tube (n = 3) after 6 weeks’ growth in juglone. Small bars represent + 1 SD.
Fig. A. Frankia growth as mean total protein yields per tube (n = 3) after 6 week’s growth in vanillic acid. Small bars represent f 1 SD.
is low in phenolics. Perhaps strains of Frankia capable of infecting and becoming well established within a host plant high in phenolics can also tolerate phenolics in vitro and in soils. Gentisic, vanillic and o -hydroxyphenylacetic acids were less inhibitory to isolate growth than juglone or caffeic acid (Figs 3-5). Gentisic and vanillic acids are benzoic acids, and Perradin et al. (1983) found that two similar compounds, benzoic acid and phydroxybenzoic acid, were generally less inhibitory to growth of Frankia isolates than were the cinnamic acids which include caffeic acid. The o-hydroxyphenylacetic acid at 1 mM concentration stimulated growth of two of the Frankia isolates, EuI 1b and Pt410, after 6 weeks of incubation (Fig. 5). In vitro growth of P. weirii was not affected by the addition of 2 mM o-hydroxyphenylacetic acid (Li et al., 1969), and Baker and O’Keefe (1984) found that phenol incubation of soil enabled isolation of Frankia from soils, while a phenol incubation of crushed alder nodules increased both the number and robustness of developing Frankia colonies. These results demonstrate that phenolic substances can also enhance the growth of microorganisms. Addition of phenolics to the basal medium had little effect on initial pH levels of the media. After 6
weeks of incubation, however, control and phenolic media containing the isolate AvdIl had consistently higher pH values than treatments containing other Frankia isolates. The pH differences were not related to growth trends. The Frankia isolates were unable to utilize any of the four phenolic acids as sole carbon and energy sources at 1 mM concentrations. Experiments in our laboratory had shown juglone to be an unsuitable carbon and energy source for Frankia isolates at 100 and 10 PM concentrations. The addition of DMSO to the media had no detectable effects on growth or morphology of the five Frankia isolates. Morphology
Juglone inhibited vesicle production of the Frankia isolates PtIl and Pt410, the only isolates that produced vesicles in control tubes lacking phenolics. At the 100 PM juglone concentration, growth of the two Purshia isolates was completely inhibited, so vesicular status could not be determined. At the 50 PM concentration, reduced vesicle production by PtIl and Pt410 was evident. Juglone, as well as caffeic acid, caused increased hyphal ramification of all the isolates used. Struc-
u-hhvdroxvehmvl.scetic Acid
AvcIl
EuIlb
Fig. 3. Frunkia growth as mean total protein yields per tube (n = 3) after 6 weeks’ growth in gentisic acid. Small bars represent k 1 SD.
Avcll
I
EuIl b
Fig. 5. Frankia growth as mean total protein yields per tube (n = 3) after 6 weeks’ growth in o-hydroxyphenylacetic acid. Small bars represent k I SD.
CHRISTOPH S. VOGEL and JEFFREY 0.
230
turally, juglone is a naphthoquinone, a double-ring compound, unlike the phenolic acids used. Li et al. (1969) found that the double-ring coumarins were more inhibitory to in vitro growth of P. weirii than were a number of other single ring phenolics. Cinnamic acids were generally found to increase hyphal ramification of Frankia isolates while not affecting vesicle production (Perradin et al., 1983), which agrees with our findings for Frankia in the presence of caffeic acid. The effects of vanillic acid on the morphology of Frankia isolates were different from those of other phenolics. Numerous spherical structures were produced, primarily in intercalary positions, by the five Frankiu isolates in the presence of vanillic acid. These structures were unlike vesicles or sporangia, because stalks and septa were lacking. The smooth, spherical structures did not mature into sporangia after 117 days incubation, but did resemble early stage sporangia described by Newcombe et al. (1979). Perhaps vanillic acid induced initial formation of large numbers of sporangia that never matured. Vanillic acid also induced high degrees of hyphal ramification by Frankia and has been shown to be present in the roots of actinorhizal A. rubra and Ceanothus velutinus and in root nodules of A. rubra (Li et al., 1974). It has been suggested that phenolics may be chemical mediators of Frankia within actinorhizal root nodules, since morphology of Frankia isolates grown in the presence of certain phenolics resembles morphology of Frankia endophytes within actinorhizal root nodules (Perradin et al., 1983). Plant phenolics and related plant compounds significantly affect the in vitro growth and morphology of Frankiu isolates and may play important roles in Frankiu growth and development in soils and within host-plant tissue. Elucidation of interactions among Frankia isolates and plant phenolics in z‘itro will aid our understanding of actinorhizal symbioses and, eventually, may lead to a better marriage of Frankia isolates, host plants and timber crops in mixed silvicultural systems. Acknowledgements-This research was supported in part by a USDA M&tire-Stennis Grant. REFERENCES Baker D. D. and O’Keefe D. (1984) A modified sucrose fractionation procedure for the isolation of frankiae from actinorhizal root nodules and soil samples. Plum and Soil,
Special Volume 78, 23-28. Bradford M. M. (1976) A rapid and sensitive method for the quantification of microgram quantitites of protein utilizing the principal of protein-dye binding Annuls of’ Biochemistry 72, 2488254. Bryant J. P., Wieland G. D., Reichardt P. B., Lewis V. E. and McCarthy M. C. (1983) Pinosylvin methyl ether deters snowshoe hare feeding on green alder. Science 222, 1023-1025. Cote B. and Camire C. (1984) Growth, nitrogen accumulation, and symbiotic dinitrogen fixation in pure and mixed plantings of hybrid poplar and black alder. Plant and Soil, Special Volume 78, 2099220. Dawson J. 0. and Seymour P. E. (1983) Effects of juglone concentration on growth in-vitro of Frankia Ar13 and Rhizobium japonicum Strain 7 I. Journal of Chemical Ecol-
ogy 9, 1175-I 183.
DAW~.ON
Dormaar J. F. (1970) Seasonal
pattern of water-soluble constituents from leaves of Populus X ‘Northwest’ (Hort.). Journal of Soil Science 21. 105-l IO. Friedrich J. M. and Dawson J. 0. (1984) Soil nitrogen concentration and Juglans nigra growth in mixed plots with nitrogen-fixing Alnus, Elaeagnus, Lespedeza. and Robinia species, Canadian Journal of Forest Research 14, 864-868. Fisher R. F. (1979) Allelopathy. In Plant Disease (J. G. Horsfall and E. B. Cowling, Eds), Vol. 4, pp. 313-330. Academic Press, New York. Funk D. T.. Schlesinger R. C. and Ponder F. (1979) Autumn-olive as a nurse plant for black walnut. Botanical Gazette 140 (Suppl.), 110~114. Hansen E. A. and Dawson J. 0. (1982) Effect of Alnus glurinosa on hybrid Populus height growth in a shortrotation intensively cultured plantation. Fores/ Science 28, 49-59. Heilman P. and Stettler R. F. (I 983) Phytomass production in young mixed plantations of Alnus rubra (Bong.) and cottonwood in western Washington. Canadian Journal of Microbiology 29, 1007-1013. _ Jobidon R. and Thibault J. R. (1981) Allelonathic effects of balsam poplar on green alder germination. Bulletin of the Torrey Botanical Club 108, 413418. Jobidon R. and Thibault J. R. (1982) Allelopathic growth inhibition of nodulated and unnodulated Alnus crispa seedlings by Populus balsamtfera. American Journal of Borany 69, 1213-1223. Li C. Y. (1974) Phenolic compounds in understory species of alder, conifer. and mixed alder-conifer stands of coastal Oregon. Lloyd& 37, 6033607. Li C. Y., Lu K. C., Trappe J. M. and Bollen W. B. (1968) Enzyme systems of red alder and Douglas-fir in relation to infection by Poriu weirii. In Biology of Alder (J. M. Trappe, J. F. Franklin, R. F. Tarrant and G. M. Hansen, Eds), Northwest Scientific Association 40th Annual Meet ing, Symposium Proceedings, 1967, pp. 241-250. USDA Forest Service, Portland, OR. Li C. Y., Lu K. C., Nelson E. E., Bollen W. B. and Trappe J. M. (1969) Effect of phenolic and other related compounds on growth of Poria weirii in-vitro. Microbios 3, 30553 1I. Li C. Y., Lu K. C., Trappe J. M. and Bollen W. B. (1970) Separation of phenolic compounds in alkali hydrolysates of a forest soil by thin-layer chromatography. Cunadiun Journul of Soil Science 50, 458460. Lu K. C.. Chen C. S. and Bollen W. B. (1968) Comparison of microbial populations between red alder and conifer soils. In Biology of Alder (J. M. Trappe, J. F. Franklin, R. F. Tarrant and G. M. Hansen, Eds), Northwesr Scien@c Association 4Oih Annual Meeting. Symposium Proceedings, 1967, pp. 173-178. USDA Forest Service, Portland, OR. Neal J. L., Lu K. C., Bollen W. B. and Trappe J. M. (I 968) A comparison of rhizosphere microfloras associated with mycorihizae of red alder and Douglas-fir. In Biology of Alder (J. M. Tranoe. J. F. Franklin, R. F. Tarrant and G. M. Hansen, ‘Eds), Northwest 3cienfiJic Associbfion 40th Annual Meeting, Symposium Proceedings, 1967, pp. 57-72. USDA Forest Service, Portland, OR. Newcombe W., Callaham D., Torrey J. G. and Peterson R. L. (1979) Morphogenesis and fine structure of the actinomycetous endophyte of nitrogen-fixing root nodules of Comptonia peregrina. Bolanical Gazette I40 (Suppl.). 22234. Olsen R. A., Odham G. and Lindberg G. (1971) Aromatic substances in leaves of Populus tremulr as inhibitors of mvcorrhizal fungi. Physiology of Plants 25, 122-129. Per&din Y., Mot& M. J, and%alonde M. (1983) Influence of phenolics on in vitro growth of Frankia strains. Canadian Journal of‘ Botany 6, 2807.--2814. S. K. (1973) Inhibition of Rice E. L. and Pancholy
Growth
of Frankia with phenol&
nitrification by climax ecosystems. II. Additional evidence and possible role of tannins. American Journal of Botany 60, 691-702. Rice E. L. (1974) Allelopafhy. Academic Press, New York. Rietveld W. J., Schlesinger R. C. and Kessler K. J. (1983) Allelopathic effects of black walnut on European black alder coplanted as a nurse species. Journal of Chemical Ecology 9, I1 19-l 133. Shindo H., Ohta S. and Kuwatsuka S. (1978) Behavior of phenolic substances in the decaying process of plants. IX. Distribution of phenolic acids in soils of paddy fields and forests. Soil Science and Plant Nutrition 24, 2333243. Shipton W. A. and Burggraaf A. J. P. (1982) A comparison of the requirements for various carbon and nitrogen sources and vitamins in some Frankia isolates. Planr and Soil 69, 149-161. Vogel C. S. and Dawson J. 0. (1985) Effect of juglone on growth in vitro of Frankiu isolates and nodulation of
and juglone
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Alnus glutinosa in soil. Plant and Soil, Special Volume 87, 79-89. Wallis G. (1968) Resistance of Alnus rubra to infection by the root rot fungus Poriu weirii. In Biology ofAlder (J. M. Trappe, J. F. Franklin, R. F. Tarrant and G. M. Hansen, Eds) Northwest Scientific Association 40th Annual Meeting, Symposium Proceedings, 1967, p. 195. USDA Forest Service, Portland, OR. Whitehead D. C. (1964)Identification of p-hydroxybenzoic, vanillic, p-coumaric, and ferulic acid in soils. Nature, London 202, 417419. Whitehead D. C., Dibb H. and Hartley R. D. (1983) Bound phenolic compounds in water extracts of soils. plant roots and leaf litter. Soil Biology & Biochemistry 15, 133-136. Younger P. D. and Kaputska L. A. (1983) N2(C,H,)ase activity by Alnus incana ssp. rugosa (Betulaceae) in the northern hardwood forest. American Journal qf Botany 70, 30-39.
pp. 227-231, reserved
1986 Copyright
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0038-0717/86$3.00+ 0.00 1986Pergamon Press Ltd
IN VITRO GROWTH OF FIVE FRANKIA ISOLATES IN THE PRESENCE OF FOUR PHENOLIC ACIDS AND JUGLONE CHRISTOPH S. VOGEL and JEFFREY 0. DAWSON Department of Forestry, University of Illinois, Urbana, IL 61801, U.S.A (Accepted 9 September 1985) Summary-The effects of four phenolic acids and juglone on the growth of five Fran/& isolates were determined using a total protein assay and light microscopy. Juglone and caffeic acid, at 100 PM and 1 mM concentrations respectively, were most inhibitory to growth of the Frunkiu isolates, although an isolate from Alnus crispa [Alms viridisssp. crispa (Ait.) Turril] nodules exhibited significantly greater growth compared to the other isolates at identical treatment levels. Gentisic, o-hydroxyphenylacetic and vanillic acids were less inhibitory to growth of the five Frankiu isolates than juglone and caffeic acid. Juglone inhibited vesicle production of two isolates that produced vesicles in controls. Juglone, caffeic acid and vanillic acid caused increased hyphal ramification, while vanillic acid also induced Frankiu to form numerous spherical structures unlike vesicles or sporangia in appearance. None of the isolates tested had the ability to utilize the phenolic acids as sole carbon and energy sources at 1 mM concentrations.
INTRODUCTION Phenolics and related compounds in soil have the potential to affect growth of microorganisms (Fisher, 1979; Li, 1974; Rice, 1974). Perradin et al. (1983) suggested that actinorhizal actinomycetes of the genus Frunkia probably exist as free-living saprophytes
in soil where they would likely encounter phenolic compounds. Perradin and co-workers also suggested that phenolics may influence Frankia growing symbiotically in host plant tissue. Nitrogen-fixing Frankia is capable of infecting the roots of certain nonleguminous trees and shrubs forming a nodular symbiotic relationship, and some actinorhizal plants have been used in forestry to increase soil nitrogen levels and the growth of associated plants. Black walnut (Jug&s nigra L.) has been coplanted with actinorhizal black alder [Alnus glutinosa L. (Gaertn.)] and autumn olive (Elueugnus umbelluta Thunb.). Walnut growth and soil nitrogen concentrations were greater in walnut-autumn olive interplantings than in plantings of walnut only (Funk et al., 1979) or in plantings of walnut and other nitrogen-fixing species (Friedrich and Dawson, 1984). Black alders interplanted with walnuts exhibited sudden decline and death 8-13 years after planting, evidently due to the release of a substance similar to phenolics, juglone (5-hydroxy-1,4_naphthoquinone), by black walnut (Rietveld et al., 1983). Juglone has also been shown to inhibit in oitro growth of some Frunkiu isolates and to inhibit nodulation of black alder seedlings in soil (Dawson and Seymour 1983; Vogel and Dawson, 1985). However, a Frankia isolate from green alder which contains high levels of the phenolic pinosylvin methyl ether was more tolerant to juglone than other isolates (Vogel and Dawson, 1985). Phenolics and polyphenols are known to occur in poplar (Populus) species (Dormaar, 1970; Olsen et at., 1971) and may have been responsible for reduced
nitrogenase activities of speckled alder [Alnus incana ssp. rugosa Clausen = A. rugosa (DuRoi) Sprengel] growing among Populus tremuloides Michx. trees in northern Wisconsin (Younger and Kaputska, 1983). Water leachates of balsam poplar (P. balsamifera L.) inhibited seed germination, growth and nodulation by Frankiu and nitrogenase activity of green alder (A. crispa var. mollis Fern.) (Jobidon and Thibault, 198 I, 1982) perhaps due to phenolics leached from poplar tissues. Since intermixtures of poplars and nitrogenfixing alders are used in forestry (Cot& and CamirC, 1984; Hansen and Dawson, 1982; Heilman and Stettler, 1983), allelopathic influence of poplars on alders and Frankia could reduce productivity of these plantations. In vitro growth of several Frankia isolates was significantly affected by the addition of some common plant phenolic compounds to the media (Perradin et al., 1983). At 1 mM concentrations of the cinnamic acids (ferulic, o-coumaric, p-coumaric, and trans-cinnamic), there was reduced growth and increased hyphal ramification of Frankia isolates from species of Ahus and Elaeagnus. However, two benzoic acids, benzoic and p-hydroxybenzoic acid, generally did not affect growth, but tended to increase vesicle numbers. Li (1974) demonstrated the presence of several phenolic compounds in the root nodules of red alder (A. rubra Bong.), the roots of another actinorhizal nitrogen-fixing species, Ceanothus velutinus Dougl. ex Hook as well as a number of understory plant species found in alder, conifer and alder-conifer stands. Evidence suggests that red alder is resistant to infection of Poriu weirii Murr. (Li et al., 1968; Wallis, 1968), a serious root rot fungus, possibly due to phenolic substances produced by red alder that have been found to inhibit in vitro growth of P. weirii (Li et al., 1969). Plant phenolics have been identified and quantified from soils of varying type, vegetative cover and location (Li et al., 1970; Shindo et al., 1978; White-
228
CHRISTOPH
S. VOGEL
head, 1964; Whitehead et al., 1983) indicating the widespread occurrence of phenolics in soils. Tannins and tannin derivatives have been implicated as possible inhibitors of nitrification by decreasing population numbers of Nitrosomonas and Nitrobacter in soils of climax forest ecosystems (Rice and Pancholy, 1973). Soils beneath red alder stands have been shown to contain phenolic compounds (Li et al., 1970), which could be partially responsible for red alder’s ability to influence soil microbe populations (Lu et al., 1968; Neal et al., 1968). It seems apparent that interactions among plant phenolics and soil microogranisms, including Frankia can have important consequences in forestry. We have quantified the interactions among previouslyuntested plant phenolics, juglone and Frankia isolates. The objectives were to determine the effects of these phenolic compounds and juglone on the growth and morphology of the Frankia isolates. MATERIALS AND METHODS
Juglone, caffeic acid, vanillic acid, gentistic acid and o-hydroxyphenylacetic acid were each dissolved in 3 ml of dimethyl sulfoxide (DMSO), brought to 100 ml volumes with distilled Hz0 and sterilized by passing through filters with 0.45 pm pores. One-ml aliquots of each phenohc solution were added to 25 x 150 mm glass culture tubes containing 9 ml of autoclaved (20 min at 1120C) broth medium. The culture tubes were then covered with plastic test tube caps. After the addition of the phenolics, the final concentrations of the broth medium components were (gl-I): casamino acids, 5.0; yeast extract. 5.0; dextrose, 10. Final concentrations of each phenolic acid in the medium was 1 and 0.5 mM each. Juglone concentrations of 100 and 50,~M were used due to juglone’s low solubility in water. In order to increase juglone solubihty for filter sterilization, the pH of the concentrate was raised to 9.4 prior to filtration by the addition of 0.1 N NaOH. Final pH of the media after the addition of the phenolics was 6.5 ( k 0.15). Two sets of controls were prepared, one containing the same concentration of DMSO found in the phenolic media (0.3% v/v) and one void of DMSO. Defined mineral media containing 1 mM concentrations of filter sterile phenohcs were employed to test the ability of Frankia to utilize the four phenolic acids as sole carbon and energy sources. The basal minimal salts used were those of the propionic acid medium developed by Shipton and Burggraaf (1982). This medium with and without propionic acid was used as a control. Five Frankia isolates, PtIl (DDB 1701 lo), Pt410 (DDB 170410), AvcIl (DDB 010110). MclO (DDB 160810) and EuIlb (DDB 130120) were used to inoculate control tubes and tubes containing phenolic broth media. Isolates PtIl and Pt410 were obtained from Purshia tridentata Pursh. nodules, AvcIl from Alnus viridis ssp. crispa nodules, MclO from nodules of Myrica cerifera L. and EuIlb from nodules of Elaeagnus umbellata. The isolates were provided by Dr Dwight Baker, Kettering Laboratory. Yellow Springs, Ohio. Added inoculum contained 4 pg total Frankia protein per tube. Inocula were prepared by harvesting 6 week old Frankia colonies grown in a
and
JEFFREY 0. DAWS~N
broth medium, macerating the colonies with a glass tissue grinder and inoculating the culture tubes with Frankia using sterile 1 ml syringes. Isolates were grown in the dark at 27°C (+ 3°C). Cultures of each isolate from each treatment were destructively sampled 3 weeks and 6 weeks after inoculation to determine growth of Frankia based on total protein per culture tube. Protein content of Frankia colonies from individual culture tubes was determined using the Coomassie Brilliant Blue (G-250, Fisher, Pittsburgh, Pennsylvania) protein-dye binding assay (Bradford, 1976). Frankia colonies were removed from the broth and washed once by centrifugation with 15 ml of pH 7 phosphate buffer. After washing, each Frankia colony was brought to a final volume of 1 ml with phosphate buffer and sonicated for 30 s. Microscopic observation of Frankia after sonication revealed total disruption of hyphae and 0.1 ml of the disrupted cells was assayed for total protein content. Standard protein curves were prepared using crystalline bovine albumin dissolved in phosphate buffer at pH 7. Phase contrast microscopy was used to make weekly observations of morphologic changes in Frankia isolates with different phenohc amendments or juglone. RESULTS AND
DISCUSSION
Growth The growth of Frankia isolate AvcI1 was less inhibited by plant phenolics than the other four isolates tested (Figs I-5). The main differences in growth of AvcIl compared with the other isolates were apparently due to AvcIl’s tolerance of 1 mM caffeic acid and its partial tolerance of 100 PM juglone (Figs 1 and 2). In an earlier experiment AvcIl was shown to have significantly greater growth relative to controls in a medium containing 100 p M juglone than four other Frankia isolates, including two alder isolates (Vogel and Dawson, 1985). The host plant from which Frankia AvcIl was isolated, A. crispa, is known to contain high concentrations of at least one phenolic compound, pinosylvin methyl ether, which discourages feeding of snowshoe hare (Bryant et al., 1983). Converselv. Purshia tridentata is commonlv known to be a preferred browse species for herbivdrous mammals in North America, suggesting that it
600
Caffeic Acid
AvcIl
Eullb
P1410
Pm
Fig. I. Frankia growth as mean total protein yields per tube (n = 3) after 6 weeks’ growth in caffeic acid. Small bars represent + 1 SD.
229
Growth of Frankiu with phenolics and juglone
= Ii -I., =
Control 0.5mB
=l.OmM
AvcIl
Eullb
MC10
Pt410
Fig. 2. Frankia
growth as mean total protein yields per tube (n = 3) after 6 weeks’ growth in juglone. Small bars represent + 1 SD.
Fig. A. Frankia growth as mean total protein yields per tube (n = 3) after 6 week’s growth in vanillic acid. Small bars represent f 1 SD.
is low in phenolics. Perhaps strains of Frankia capable of infecting and becoming well established within a host plant high in phenolics can also tolerate phenolics in vitro and in soils. Gentisic, vanillic and o -hydroxyphenylacetic acids were less inhibitory to isolate growth than juglone or caffeic acid (Figs 3-5). Gentisic and vanillic acids are benzoic acids, and Perradin et al. (1983) found that two similar compounds, benzoic acid and phydroxybenzoic acid, were generally less inhibitory to growth of Frankia isolates than were the cinnamic acids which include caffeic acid. The o-hydroxyphenylacetic acid at 1 mM concentration stimulated growth of two of the Frankia isolates, EuI 1b and Pt410, after 6 weeks of incubation (Fig. 5). In vitro growth of P. weirii was not affected by the addition of 2 mM o-hydroxyphenylacetic acid (Li et al., 1969), and Baker and O’Keefe (1984) found that phenol incubation of soil enabled isolation of Frankia from soils, while a phenol incubation of crushed alder nodules increased both the number and robustness of developing Frankia colonies. These results demonstrate that phenolic substances can also enhance the growth of microorganisms. Addition of phenolics to the basal medium had little effect on initial pH levels of the media. After 6
weeks of incubation, however, control and phenolic media containing the isolate AvdIl had consistently higher pH values than treatments containing other Frankia isolates. The pH differences were not related to growth trends. The Frankia isolates were unable to utilize any of the four phenolic acids as sole carbon and energy sources at 1 mM concentrations. Experiments in our laboratory had shown juglone to be an unsuitable carbon and energy source for Frankia isolates at 100 and 10 PM concentrations. The addition of DMSO to the media had no detectable effects on growth or morphology of the five Frankia isolates. Morphology
Juglone inhibited vesicle production of the Frankia isolates PtIl and Pt410, the only isolates that produced vesicles in control tubes lacking phenolics. At the 100 PM juglone concentration, growth of the two Purshia isolates was completely inhibited, so vesicular status could not be determined. At the 50 PM concentration, reduced vesicle production by PtIl and Pt410 was evident. Juglone, as well as caffeic acid, caused increased hyphal ramification of all the isolates used. Struc-
u-hhvdroxvehmvl.scetic Acid
AvcIl
EuIlb
Fig. 3. Frunkia growth as mean total protein yields per tube (n = 3) after 6 weeks’ growth in gentisic acid. Small bars represent k 1 SD.
Avcll
I
EuIl b
Fig. 5. Frankia growth as mean total protein yields per tube (n = 3) after 6 weeks’ growth in o-hydroxyphenylacetic acid. Small bars represent k I SD.
CHRISTOPH S. VOGEL and JEFFREY 0.
230
turally, juglone is a naphthoquinone, a double-ring compound, unlike the phenolic acids used. Li et al. (1969) found that the double-ring coumarins were more inhibitory to in vitro growth of P. weirii than were a number of other single ring phenolics. Cinnamic acids were generally found to increase hyphal ramification of Frankia isolates while not affecting vesicle production (Perradin et al., 1983), which agrees with our findings for Frankia in the presence of caffeic acid. The effects of vanillic acid on the morphology of Frankia isolates were different from those of other phenolics. Numerous spherical structures were produced, primarily in intercalary positions, by the five Frankiu isolates in the presence of vanillic acid. These structures were unlike vesicles or sporangia, because stalks and septa were lacking. The smooth, spherical structures did not mature into sporangia after 117 days incubation, but did resemble early stage sporangia described by Newcombe et al. (1979). Perhaps vanillic acid induced initial formation of large numbers of sporangia that never matured. Vanillic acid also induced high degrees of hyphal ramification by Frankia and has been shown to be present in the roots of actinorhizal A. rubra and Ceanothus velutinus and in root nodules of A. rubra (Li et al., 1974). It has been suggested that phenolics may be chemical mediators of Frankia within actinorhizal root nodules, since morphology of Frankia isolates grown in the presence of certain phenolics resembles morphology of Frankia endophytes within actinorhizal root nodules (Perradin et al., 1983). Plant phenolics and related plant compounds significantly affect the in vitro growth and morphology of Frankiu isolates and may play important roles in Frankiu growth and development in soils and within host-plant tissue. Elucidation of interactions among Frankia isolates and plant phenolics in z‘itro will aid our understanding of actinorhizal symbioses and, eventually, may lead to a better marriage of Frankia isolates, host plants and timber crops in mixed silvicultural systems. Acknowledgements-This research was supported in part by a USDA M&tire-Stennis Grant. REFERENCES Baker D. D. and O’Keefe D. (1984) A modified sucrose fractionation procedure for the isolation of frankiae from actinorhizal root nodules and soil samples. Plum and Soil,
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