GERMINATION OF BASIDIOSPORES OF MYCORRHIZAL FUNGI IN THE RHIZOSPHERE OF PINUS RADIATA D. DON

New Phytol. (1987) 106, 217-223 217 GERMINATION OF BASIDIOSPORES OF MYCORRHIZAL FUNGI IN THE RHIZOSPHERE OF PINUS RADIATA D. DON BY C. THEODOROU AND G. D. BOWEN* Commonwealth Scientific and Industrial Research Organization, Division of Soils, Private Bag No. 2, Glen Osmond, S.A. 5064, Australia (Accepted 3 February 1987) SUMMARY A method was developed to inoculate the surfaces of young roots of Pinus radiata D. Don uniformly with hasidiospores of mycorrhizal fungi. Germination of hasidiospores in the rhizosphere was 46 to 69 % in different experiments with Rhizopogon luteolus Fr. and Nord and 34 and 31 % with Suillus luteus (L. ex Fr.) S. F. Gray and 5. granulatus (L. ex Fr.) O. Kuntze, respectively. This compared with 0-1 % germination usually obtained for all fungi on synthetic media. Using the method, single spore cultures of R. luteolus were obtained which formed mycorrhizas on P. radiata seedlings. Germination did not respond to roots oi Eucalyptus globulus Labill, Medicago truncatula Gaertn., Trifolium subterraneum L. or Lolium perenne L., showing the germination response was specific to P. radiata, although other host tree species may produce a similar response. Specificity in such mycorrhizal associations may operate both at the germination and the infection stages. It is suggested that in vivo the necessity for a host plant to stimulate spore germination is a highly effective method of inoculum conservation in the absence of host plants. The viability of inoculum remained high during storage at freezing temperatures. High spore germination rates were also obtained when the method was used in unsterilized soil. Key words: Ectomycorrhiza, basidiospore germination, rhizosphere, Pinus radiata. INTRODUCTION Although basidiospores of ectomycorrhizal fungi are used to inoculate pines in nurseries (Theodorou, 1971; Marx & Bryan, 1975; Theodorou & Benson, 1983), the physiology of spore germination in the rhizosphere is poorly understood. Germination of basidiospores of ectomycorrhizal fungi on laboratory media is low (about 0-1 %) and requires special conditions and the presence of an activator organism (Fries, 1978). Theodorou & Bowen (1973) examined mycorrhizal production as a function of numbers of basidiospores inoculated into soil and concluded that considerably greater germination had occurred in the presence of roots than on laboratory media. Fries & Birraux (1980) placed pine seedlings on agar media seeded with basidiospores of Hebeloma species and increased spore germination to about 1 %. Germination occurred close to the root suggesting stimulation by root exudates. In studies reported here we have developed a more quantitative method to investigate the germination of basidiospores of ectomycorrhizal fungi in the rhizosphere. This is based on a method for coating roots with bacteria described * Present address: International Atomic Energy Agency, Wagramerstrasse 5, P.O. Box 100, A-1400 Vienna, Austria. 0028-646X/87/060217 + 07 $03.00/0 1987 The New Phytologist

2i8 C. T H E O D O R O U A N D G. D. B O W E N by Bowen (1979). The method was used to examine the specificity of the rhizosphere germination response, to produce fungal colonies from single germinating spores, and as an assay for the viability of stored basidiospore inoculum. MATERIALS AND METHODS Inoculation with basidiospores Spore suspensions in distilled water were made from surface-sterilized fruit bodies of Rhizopogon luteolus Fr. and Nord, Suillus luteus (L. ex Fr.) S. F. Gray and S. granulatus (L. ex Fr.) O. Kuntze as previously reported (Theodorou, 1971). Twenty millilitres of 1 % distilled water agar was allowed to cool to 36 C and 1 ml of basidiospore suspension containing about 10^ spores ml~^ was added and shaken to distribute the spore suspension evenly. Sterile Pinus radiata D. Don seedlings, 4 or 21 d old with radicles 25 and 6 cm long, respectively, were dipped into the spore suspension momentarily and excess agar allowed to drain from the root. This inoculated the roots uniformly with a layer, a few microns thick, of spores in agar. Growth conditions Inoculated seedlings were placed on sterile 1*5 % distilled water agar in 20 cm diameter Petri dishes and the root was covered with sterile moistened perlite. Dishes were covered with thin polyethylene film ('Gladwrap', 12 5 jltm thick) to minimize loss of moisture and placed in a growth cabinet at 25/15 C day/night temperature and a daytime irradiance of approximately 24 W m"^. In other studies, the inoculated seedlings were placed in an unsterilized forest soil [Mt Burr sand, an orthic podzol (FAO-Unesco, 1978)], which had been moistened to field capacity. Assessment of germination Roots were gently washed to remove adhering perlite or soil and stained with Jones & Mollison (1948) stain for 3 min. The epidermis and attached cell layers of the roots together with the spore film were then teased off and mounted in glycerol. Germinated and ungerminated spores in 40 fields (10 fields from each of four seedlings) were counted under a microscope at 400 x magnification. RESULTS Germination with time and position on root The germination of i^. luteolus basidiospores on 4 and 21 d old seedlings raised on a glucose-yeast-peptone agar (Rovira, 1959) or in perlite, respectively, was examined at 7, 14, 18, 28 and 35 d from inoculation and at different positions (base, 1-5 cm from junction of stem and root; mid-root, mid-position between base and apex; and apex, apical 10 cm) on the root. Four replicates were examined. At all sampling times, the germination of basidiospores on 4 d old seedlings was slower than that on the 21 d old seedlings, with the final germination on 4 d old seedlings being 53-7% [Table l(a)]. Germination on 4 d old seedlings increased significantly with time over the course of the experiment. No germination occurred when the spore suspension was plated on either distilled water agar or Melin Norkrans medium (Melin, 1959). As observed on the stained sections under the microscope, bacterial and fungal colonies developed in the rhizosphere of the inoculated seedlings but when suspensions of these were plated on media seeded with basidiospores they did not induce germination in the absence of roots.

Germination of basidiospores in the rhizosphere 219 Table 1. Germination of Rhizopogon luteolus basidiospores in the rhizosphere of Pinus radiata Days from inoculation Germination at different positions on the roots (%) Base Mid-root Apex LSD between positions P = 0-05 < 0-01 Germ tube length {ji)f (a) Seedlings 4d old when inoculated* 0 20-2 35-2 43-0 53-7 7 14 18 28 35 LSD P = 0-05 0-01 4-9 6-6 (b) Seedlings 21 d old when inoculated* 7 14 18 28 35 LSD between times P == 0-05 0-01 9-8 46-5 64-5 67-2 68-8 11-3 15-7 5-2 35-0 38-5 48-2 48-8 17-2 23-8 4-0 7-7 14-0 26-2 29-8 11-8 16-4 3-0 17-8 21-8 10-2 12-2 4-3 25-6 31-4 14-8 17-5 21 3-4 3-9 81 11-7 0-4 06 * Mean of 40 fields; 10 fields per seedling, f Mean of 80 measurements,, not determined. Germination on 21 d old seedlings commenced within 7 d from inoculation (9-8%) and reached its maximum at about 18 d (64-5%); there was no further significant increase in the germination between 18 and 35 d (68 8%) after inoculation [Table l(b)]. A greater percentage of spores germinated on the basal part of the root (maximum 68-8 %) than on the mid-section (max. 48-8 %) or the apex (max. 29-8 %). Growth of the spore germ tubes was slow; between 7 and 35 d the mean increase in germ tube length was only 9-6 ptm. (from 21 to 11-6 /im). Single spore cultures At harvest on day 18 in the experiment above, 0 5 cm long pieces from the basal part of the inoculated root were shaken for 5 min with beads in 10 ml sterile distilled water and 01 ml was plated on Melin-Norkrans medium which contained the antibiotics tetracycline-hcl and streptomycin-sulphate (50/*g ml~^). Colonies (42 per plate, 4200 colonies cm"^ root) of a fungus with cultural characteristics similar to those of R. luteolus grew from the plated suspension of germinated single spores within 5 d. These colonies were isolated on fresh Melin-Norkrans medium and two pieces, 1 cm^ each, of actively growing mycelium were inoculated aseptically onto the roots of 15 d old sterile P. radiata seedlings growing in sterile Mt Burr sand in preserving jars plugged with cotton wool. The inoculated seedlings formed mycorrhizas within 8 weeks. Thirty-two per cent of the short roots became mycorrhizal; non-inoculated seedlings did not produce any mycorrhizas.

22O C. THEODOROU AND G. D. BOWEN Specificity in germination stimulation The specificity of the germination response of the basidiospores of R. luteolus was examined by coating sterile glass fibres and roots of 3 d old seedlings of P. radiata. Eucalyptus globulus Labill, Medicago truncatula Gaertn., Trifolium subterraneum L. and Lolium perenne L. and incubating on agar/perlite as described above. Germination was measured 4 weeks after inoculation. No germination occurred on glass fibres, or in the rhizosphere of L. perenne, E. globulus, T. subterraneum and M. truncatula, but in the rhizosphere of P. radiata 54+10% germination occurred. Viability of stored inoculum The method was used to assay the viability of basidiospore suspensions either after storage deep frozen ( 15 C) for three or seven months or freshly collected from fruiting bodies frozen for three months. Freshly extracted spore suspensions from immature and mature fruiting bodies served as controls. Roots of 3 d old P. radiata seedlings were coated with the spores and germination was assessed 4 weeks after inoculation. Basidiospore inoculum stored deep frozen for up to seven months remained viable. Spores from the immature fruiting bodies did not germinate. Although the fresh inoculum from mature fruiting bodies showed 49 % germination and the stored inoculum 22 to 27 % (even after seven months storage), a valid comparison of the stored against the fresh inoculum could not be made, since inoculum collected at different times might have attained a different degree of maturation. However, indirect evidence (Theodorou & Bowen, 1973) suggests that basidiospores may lose some viability on storage. Table 2. Germination of basidiospore inoculum o/rhizopogon luteolus after storage in different forms and for different lengths of time {Germination was measured 4 weeks after inoculation of roots of Pinus radiata) Source and history of inoculum Spore suspension freshly prepared from mature fruiting bodies Spore suspension freshly prepared from immature fruiting bodies Spore suspension deep frozen ( 15 C) for three months Spore suspension deep frozen ( 15 C) for seven months Fruiting bodies deep frozen ( 15 C) for three months LSD P = 005 001 Germination (%)* 49 0 23 27 22 7 10 * Mean of 40 fields: 10 fields per seedling. Germination in soil systems Basidiospores were coated on roots of 4 d old P. radiata seedlings which were planted into pots of unsterilized Mt Burr sand at field capacity. Forty-two percent of spores had germinated 4 weeks after inoculation.

Germination of basidiospores in the rhizosphere 221 Stimulation of Suillus species Basidiospores of Suillus luteus, S. granulatus and R. luteolus were coated individually on 4 d old seedlings of P. radiata and germination was examined by the standard method described above. Four weeks after inoculation 34 and 31 % basidiospores of S. luteus and S. granulatus had germinated, respectively, while 46 % R. luteolus spores germinated under the same conditions. DISCUSSION Fries & Birraux (1980) and Birraux & Fries (1981) showed that when pine roots were placed on plates of nutrient agar seeded with basidiospores of the ectomycorrhizal fungi Hebeloma spp. and Thelephora terrestris Ehrh. ex Fr., root exudates stimulated germination of the basidiospores from 0-1 % usually obtained on synthetic media to approximately 1 %. Our studies show that even higher percentage germination can be obtained with basidiospores oi R. luteolus, S. luteus and»s. granulatus when they are coated on roots of P. radiata seedlings in a thin film of agar and in the absence of any external energy source in the medium. Since very sparse spore germination was obtained on synthetic media in the absence of roots, it is evident that the stimulation of germination was due to root exudates. They were probably effective in inducing germination because of the close contact of spores with the source of the stimulus. No germination was obtained in the rhizosphere of non-host plants. Our results indicate, therefore, that the germination response of R. luteolus spores is specific to host roots, although host species other than P. radiata may induce a similar response. This is in agreement with the results of Birraux & Fries (1981) with spores of T. terrestris. The results explain the specificity of infection shown naturally by ectomycorrhizal fungi and the conservation of their spores in 'alien' environments. R. luteolus and some other Rhizopogon species show specificity in that they infect pines but not eucalypts (Theodorou & Bowen, 1971; Malajczuk, Molina & Trappe, 1982). The present studies show that incompatibility between fungus and roots of non-host species can occur at the germination stage as well as at the infection stage. It is likely that germination responds to specific compounds in the exudate, rather than to energy sources for growth. Theodorou & Bowen (1971) showed that JR. luteolus mycelium can grow in the rhizosphere of non-host plants (eucalypts, grasses and subterranean clover), indicating that germination and mycelial growth respond to different exudate compounds. Factors other than plant species, such as the age of the root at time of inoculation, the position of the spores on the root, the maturity of the spores and the nutrition of the seedlings before and after inoculation, as well as other soil factors, may also influence the rate of spore germination. Germination was faster on older parts of the roots. This is consistent with the finding of Theodorou (1980) that mycorrhizal infection from basidiospores and mycelium of R. luteolus was faster on older than on younger roots. Although it is thought that exudation is greater near the apex of roots (McDougall, 1970), Bowen (1969) and Bowen & Theodorou (1979) showed that microbial growth in the rhizosphere of P. radiata was equal or greater at the basal part of 14 and 28 d old roots. The similar effect for spore germination may be due to greater loss of substrate (exudate plus lysates) (Rovira, Foster & Martin, 1979) from the older part of the root, or to a change in the composition

222 C. THEODOROU AND G. D. B o W E N of the exudates. The nutrition of the host may also affect the physiology of the root and its exudation (Bowen, 1969). Preliminary findings (Theodorou & Bowen, unpublished data) indicated that when seedlings were raised in Hoagland & Arnon (1950) nutrient solution (with or without phosphorus), germination was lower than when seedlings were raised in distilled water. Germination in distilled water was 4 5 %, whilst in plant nutrient solution with phosphorus it was 16% and without phosphorus 19% (there was no significant difference between the phosphorus treatments). As phosphorus did not affect the spore germination it is probable that the effect of high phosphorus in decreasing mycorrhizal infection (Marx, Hatch & Mendicino, 1977) is on the growth of the fungus and on the infection phase. This is in agreement with the findings of Sanders (1975) with vesicular-arbuscular mycorrhizas. The method developed in these studies provides a means of studying spore germination in unsterilized soil and thus for ecological studies on the influence of soil factors on germination of spores in a range of natural ecosystems. Growth of mycelium from the germ tube was extremely slow in the rhizosphere of young seedlings (9 6 /*m over a period of 28 d). Since germinated spores grew into colonies visible to the naked eye within 5 d from plating on Melin-Norkrans medium, it is probable that the supply of growth substances in the rhizosphere limited fungal growth. Although the rhizosphere is considerably richer in energy sources than is soil, the much longer generation time of bacteria in the rhizosphere than on laboratory media (Bowen & Rovira, 1976) indicates that it is certainly not an optimal medium. Fries & Birraux (1980) observed that certain young myeelia from germinated spores in synthetic media died for unknown reasons. However, the percentage germination obtained in these studies guarantees a good level of infection even if some germ tubes die off before they grow into infective mycelium. This was shown to be the case by Theodorou & Bowen (1973) in their study of spore dose mycorrhizal infection. REFERENCES & FRIES, N. (1981). Germination of Thelephora terrestris basidiospores. Canadian Journal of Botany, 59, 2062-2064. BOWEN, G. D. (1969). Nutrient status eftects on loss of amides and amino acids from pine roots. Plant and Soil, 30, 139-142. BOWEN, G. D. (1979). Integrated and experimental approaches to the study of organisms around roots. In: Soil Borne Plant Pathogens (Ed. by B. Schippers & W. Gams) pp. 209 227. Academic Press, New York & London. BOWEN, G. D. & ROVIRA, A. D. (1976). Microbial colonization of plant roots. Annual Review of Phytopathology, 14, 121-144. BOWEN, G. D. & THEODOROU, C. (1979). Interactions between bacteria and ectomycorrhizal fungi. Soil Biology and Biochemistry, 11, 119-126. FAO-UNESCO (1978). Soil Map of the World, vol. 10, Australasia. Unesco, Paris. FRIES, N. (1978). Basidiospore germination in some mycorrhiza-forming Hymenomycetes. Transactions of the British Mycological Society, 70, 319^324. FRIES, N. & BIRRAUX, D. (1980). Spore germination in Hebeloma stimulated by living plant roots. Experimentia, 36, 1056-1057. HOAGLAND, D. R. & ARNON, D. I. (1950). The water culture method for growing plants without soil. California Agricultural Experimental Station Circular, 347, 1-39. JONES, P. C. T. & MOLLISON, J. E. (1948). A technique for the quantitative estimation of soil microbirraux, D. organisms. Journal of General Microbiology, 2, 54 69. & TRAPPE, J. M. (1982). Ectomycorrhiza formation in Eucalyptus. I. Pure culture synthesis, host specificity and mycorrhizal compatibility with Pinus radiata. New Phytologist 91, 467-482. MALAJCZUK, N., MOLINA, R.

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