CYTOKININ PRODUCTION BY ECTOMYCORRHIZAL FUNGI BY P. P. NG, A. L. J. COLE, P. E. JAMESON AND J. A. MCWHA

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1 New Phytol. (1982) 91, CYTOKININ PRODUCTION BY ECTOMYCORRHIZAL FUNGI BY P. P. NG, A. L. J. COLE, P. E. JAMESON AND J. A. MCWHA Department of Botany, University of Canterbury, Christchurch, New Zealand {Accepted 15 November 1981) SUMMARY The mycorrhizal fungi Rhizopogon luteolus Fr. and Nordh, Boletus elegans Schum. ex. Fr. and Suitlus luteus L. ex Fr., were investigated for cytokinin production in vitro. Culture filtrates in -which the fungi had been growing for at least 2 weeks showed cytokinin activity when purified and bioassayed using soybean callus tissues. The cytokinin" activity in the culture filtrate increased with longer periods of incubation. A potent inhibitor of callus growth from Boletus elegans culture filtrate which masks cytokinin activity is discussed. INTRODUCTION Ectomycorrhizal fungi infect the short-roots of woody plants. Infected roots branch repeatedly and a common growth modification is the enlargement of the cortical cells. This effect resembles that caused by growth substances such as auxins and cytokinins and which led Miller (1967) to attempt to extract cytokinins from the culture filtrates of Rhizopogon roseolus. He succeeded in extracting and identifying the cytokinins zeatin and zeatin riboside. This was the first demonstration that an organism other than a higher plant produced such compounds, although production of cytokinins by bacteria and fungi forming associations with higher plants, especially pathogenic associations, had previously been indicated (Miller, 1971). Several species of mycorrhizal fungi have been screened and found to produce cytokinins, although the screening programme adopted by Crafts and Miller (1974) showed negative results in most cases. Cytokinin production has been established in Suillus cothernatus, S. punctipes, an unidentified ectendomycorrhizal species and Rhizopogon ochraceorubens (Miller, 1971; Crafts and Miller, 1974). In this investigation an attempt was made to extract and purify cytokinins from liquid cultures of Rhizopogon luteolus. Boletus elegans and Suillus luteus, fungi commonly forming mycorrhizal associations with Pinus radiata in New Zealand. MATERIALS AND METHODS Fungal culture Young sporophores of R. luteolus and B. elegans with the annulus still intact were collected from under P. radiata at Woodend Beach, Christchurch. Small blocks of glebal material {R. luteolus) and cap material {B. elegans) were isolated and cultured in Petri dishes on Hagem's (Modess, 1941) and M40 (Stevens, 1974) media respectively containing 40 /<g ml^ streptomycin at 25 C in the dark. Isolates were subcultured weekly. S. luteus cuhures (Wills, 1978) were maintained on M40 agar at 5 C and subcultured monthly. OO28-646X/82/ I-06 S03.00/ The New Phytologist

2 58 P. P. No et al. Preliminary survey of cytokinin production The fungus under test was inoculated into the centre of a 100 ml Erlenmeyer flask containing cytokinin-free medium (basal medium) and six pieces of soybean callus tissue (approximately 10 mg per piece) were placed around it (Miller, 1971). The flasks were incubated for 1 week at 25 C in darkness before callus growth was assessed. Extraction and purification of cytokinins from liquid culture Mycelial agar discs (6 mm diameter) were taken from the growing margins of day-old cultures of the fungi and transferred to fresh medium and incubated for 2 days. After this time hyphae are seen projecting from the periphery of the agar discs (Palmer, 1969). Erlenmeyer flasks (250 ml) containing 100 ml of modified Hagem's medium were then inoculated with two such agar discs per flask (Miller, 1967) and incubated in the dark at 25 C. After 2 weeks or 50 days incubation the filtrate of 20 cultures was combined to give a volume of 2 1 which was evaporated to approximately 100 ml under reduced pressure in a rotary film evaporator and 50 ml of 70% (v/v) methanol added. The resulting extract was applied to a 25 x 300 mm column of Dowex 50W-X8 cation exchange resin (H + form, dry mesh 50 to 100) (Van Staden, 1976). The column was washed with 150 ml ice-cold 70% (v/v) methanol followed by 200 ml ice-cold water. Adsorbed cytokinins were eluted from the column with 200 ml of 2 N NH^OH followed by 500 ml of 5 N NH4OH. The effluent ammonia was removed in a rotary evaporator at 45 C and the residue taken up in 70 % (v/v) methanol and evaporated to dryness. B. elegans extracts were further taken up in 5 ml of 20% (v/v) ethanol and centrifuged through Sinta glass filters at 3000^ for 30 min. The resulting clear solution was evaporated to dryness. Bioassay and characterization of cytokinins The extracts were redissolved in 1 ml 20% (v/v) ethanol and applied to a Sephadex LH-20 column (25 x 475 mm) (Armstrong et al., 1969) and developed in an ascending manner with 20% (v/v) ethanol at a flow rate of 50 ml h"'. Ten millilitre fractions were collected. Soybean callus tissue growth was used to test the fractions for cytokinin activity (Miller, 1968). The cuhures were maintained at 28 C in darkness and the fresh wt of callus measured after 25 days. The cytokinin content was expressed as 'kinetin equivalents' by comparison with a series of reference flasks. Zeatin, zeatin riboside and isopentenyl adenine standards were also chromatographed and bioassayed. RESULTS The preliminary survey of cytokinin production using the technique involving co-culturing of fungus and callus indicated the presence of cytokinin-like activity in R. luteolus but not in B. elegans. This technique can yield useful tentative results but negative findings should not be taken to prove that cytokinins are not produced (Miller, 1971). A number of factors can interfere with this assay, e.g. the production by the fungus of compounds inhibitory to callus growth. The results of the bioassays of purified extracts of the culture filtrates are shown in Figures 1 to 4. Fourteen-day-old culture filtrates of R. luteolus, B. elegans and S. luteus showed peaks of cytokinin-like activity with elution volumes similar to

3 Cytokinin production by ectomycorrhizal fungi 59 0 Kinetin Fig. 1. Soybean bioassay of 14-day-old culture filtrate of Rhizopogon luteolus after partial purification on Dowex 50 H+ followed by fractionation on Sephadex LH-20, eluted with 20% ethanol. ZR and Z indicate elution zones of authentic zeatin riboside and zeatin respectively. Broken line represents the void volume baseline. 160 ZR i /xg r' kinetin 6 80 Fig. 2. Soybean bioassay of 50-day-old culture filtrate of Rhizopogon luteolus after partial purification on Dowex 50 H+ followed by fractionation on Sephadex LH-20, eluted with 20% ethanol. ZR indicates elution zones of authentic zeatin riboside. The lowest broken line represents the void volume baseline. Error bars indicate ± standard error for at least three replicates.

4 6o P. P. NG et al ZR Z 2iP 15 ^i.q C kinefin = 20 - [ } ^ _r n LJ LJ L-i 0 IXQ L T kinetin fmiooo CvJCijCvJW'OrOfO^ ^ ^ I I I I I I I I I I I I I I I I I I I I I I I f I I I I I I T Fig. 3. Soybean bioassay of 14-day-old culture filtrate of Boletus etegans after partial purification on Dowex 50 H^ followed by fractionation on Sephadex LH-20, eluted witb 20'^,, etbanol. ZR, Z and 2iP indicate elution zones of authentic zeatin riboside, zeatin and isopentenyl adenine respectively. Tbe lowest broken line represents the void volume baseline. Error bars indicate + standard error for at least three replicates. 350 ZR ^g r' kinetin S 250 e 200 g f fj.qc' kinetin I I I 1 I I I I I I I I I I I 1 I I I 1 I I I I I I I I I I I Fig. 4. Soybean bioassay of 14-day-oId culture filtrate of SuiHus luteus after partial purification on Dowex 50 H"^ fouowed by fractionation on Sephadex Iv}i-20, eluted with 20% ethanol. ZR and Z indicate elution zones of authentic zeatin rihoside and zeatin respectively. The lowest broken line represents the void volume baseline. Error bars indicate i standard error for at least three replicates.

5 Cytokinin production hy ectomycorrhizal fungi 6i zeatin riboside (fractions 17 to 38) and zeatin (fractions 42 to 53) standards. Several small peaks of activity were observed in fractions eluting later than the zeatin standard (Figs 1 and 3). The isopentenyl adenine standard eluted in fractions 78 to 89. An isopentenyl adenosine standard was not available but this would be expected to elute after zeatin and before isopentenyl adenine (Van Staden and Dimalla, 1980). Culture filrates of R. luteolus cultured for 50 days showed a peak of activity in fractions 33 to 35 (Fig. 2). The activity which corresponded with zeatin riboside was significantly greater than that obtained from the bioassay ofthe 14-day-old culture (Fig. 1). Miller (1967) also reported an increase in cytokinin activity with longer periods of incubation. However, no comparable accumulation of activity was recorded in the fractions corresponding with zeatin. Cytokinin-like activity eluting from the column earlier than the zeatin riboside standards shown in Figures 3 and 4 could be attributed to zeatin glucoside, zeatin riboside glucoside or zeatin ribotide which elute soon after the void volume (Davey and Van Staden, 1978, 1979; Van Staden and Dimalla, 1980). Activity eluting later than the zeatin (Figs 1 and 3) may indicate cytokinins or merely experimental variation. Further work will be needed to clarify this. Using a line of soybean callus which grows independently of added cytokinin, it was possible to demonstrate a potent inhibitor of callus growth in B. elegans culture filtrate (Fig. 5). S Fig. 5. Bioassay of 14-day-old culture filtrate of Botetus etegans in which the soybean callus tissues became habituated on cytokinin. Fractions 15 to 20 indicate presence of soybean callus inhibitor. Broken line represents the void volume baseline.

6 62 p. p. NG et al. DISCUSSION Evidence of cytokinin production by R. luteolus, B. elegans and S. luteus in vitro was recorded in this study. A number of roles for cytokinins in mycorrhizal associations have been suggested including the provision by the fungus of cytokinins that may occur in the tips of uninfected roots (Kende, 1971; Short and Torrey, 1972), supplementing their production by the root (Crafts and Miller, 1974), controlling the infection and colonization of the fungal symbiont or other invading fungi (Mothes, Engelbrecht and Kulajewa, 1959; Miller, 1969) and affecting seed and spore germination if secreted into the soil (Van Staden and Dimalla, 1976). None of these has, as yet, been substantiated but the production of cytokinins by these fungi which cause growth modifications of roots they invade tempts such explanations. In a preliminary survey of mycorrhizal fungi for cytokinin production the screening technique used by Miller (1971) gave negative results with B. elegans. This fungus was shown to produce a callus inhibitor which masked cytokinin activity. Some limitation of this technique for screening such fungi is evident. ACKNOWLEDGEMENTS We wish to thank Mrs M. M. Stevens and Mrs A. P. Luney for their assistance. REFERENCES ARMSTRONG, D. J., BURROWS, W, J., EVANS, P. K. & SKOOG, F, (1969), Isolation of cytokinins from trna. Biochemical and Biophysical Research Communications, 37, , CRAFTS, C, B, & MILLER, C, O. (1974), Deteetion and identification of cytokinins produced by mycorrhizal fungi. Plant Physiology, 54, , DAVEY, J, E. & VAN STADEN, J. (1978). Cytokinin activity in Lupinus albus III, Distribution in fruits. Physiohgia Plantarum, 43, DAVEY, J, E, & VAN STADEN, J. (1979), Cytokinin activity in Lupinus albus L, IV, Distribution in seeds. Plant Physiology, 63, KENDE, H. (1971). The cytokinins. International Review of Cytology, 31, MILLER, C. O, (1967), Zeatin and zeatin riboside from a mycorrhizal fungus. Science, 157, MILLER, C. O. (1968). Naturally occurring cytokinins. In: Biochemistry and Physiology of Plant Growth Substances (Ed, by F, Wightman & G, Setterfield), pp, 33-45, Runge Press, Ottawa, MILLER, C, O, (1969), Control of deoxyisoflavone in soybean tissue, Planta, 87, 26-35, MILLER, CO, (1971), Cytokinin production by mycorrhizal fungi. In; Ectomycorrhizae (Ed, by E. Hacskaylo), pp, Proceedings of the 1st North American Conference on Mycorrhizae, U.S. Department of Agriculture, Washington, MODESS, O, (1941), Zur Kenntnis der mykorrhizabildner von Kiefer und fichte Symbolae Botanicae Upsalienses, 5, 1-147, MOTHES, K,, ENGELBRECHT, L. & KULAJEWA, O, (1959), tjber die Wirkung des Kinetins auf Stickstoffverteilung und Eiweiss-synthese in isolierten Blattern, Flora, 147, 445^64. PALMER,J. G,(1969), Techniques and Proceduresfor Culturing Ectomycorrhizal Fungi,pp , lstnorth American Conference on Mycorrhizae. U.S. Department of Agriculture, Washington, SHORT, K. C, & TORREY, J, G, (1972), Cytokinins in seedling roots of pea. Plant Physiology, 49, STEVENS, R, B, (1974). Mycology Guidebook, 700 pp. Washington, University of Washington Press. VAN STADEN, J. (1976), The identification of zeatin glucoside from coconut milk, Physiohgia Plantarum, 36, , VAN STADEN, J, & DIMALLA, G. G. (1976). Cytokinins from soil, Planta, 130, 85-87, VAN STADEN, J. & DIMALLA, G, G. (1980), Endogenous cytokinins in Bougainvillea 'San Diego Red'. I. Occurence of cytokinin glucosides in the root sap. Plant Physiology, 65, , WILLS, B, (1978), Mycorrhiza development in trees for revegetation of eroded mountain slopes. Ph,D, thes University of Canterbury, New Zealand.

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