A technique for dual vesicular-arbuscular endomycorrhizal/ectomycorrhizal infection of Eucalyptus in vitro

Transcription

1 New Phytol. (1990), 114, A technique for dual vesicular-arbuscular endomycorrhizal/ectomycorrhizal infection of Eucalyptus in vitro BY KHADIJA BOUDARGA, FREDERIC LAPEYRIE^ AND JEAN DEXHEIMER Laboratoire de Biologie des Ligneux, JE CNRS , Universite de Nancy I, BP 239, Vandoeuvre Cedex, France ^ INRA, Centre de Recherches Forestieres de Nancy, Champenonx, Seichamps, France {Received 31 October 1988; accepted 21 September 1989) SUMMARY A few species of plant, including Eucalyptus, hear simultaneously on their root system and even on the same root apex, more than one symbiotic microorganism. The development of techniques allowing i-nixed inoculations is an essential prerequisite for the study of interactions between different symbionts. A sin-iple in vitro technique for rapid synthesis of dual vesicular-arbuscular endomycorrhiza/ectomycorrhiza on a single root apex of Eucalyptus seedlings is described. This technique was derived from the paper-sandwich technique for synthesis of ectomycorrhizas, and was modified to render it compatible with endon-iycorrhizal infection. Electron microscopy observations of dual endomycorrhiza/ectomycorrhiza showed both symbioses coexisting on the same apex, with no obvious sign of antagonisn-i. Key words: Eucalyptus, VA mycorrhizal, synthesis ectomycorrhizal synthesis, dual symbiosis, /;; vitro culture. the ectomycorrhizal fungus is restricted to the outer INTRODUCTION,,, ',.,,, no-jx T- i L- cell layer (Chilvers et al., 1987). Each symbiont can Most vascular plants form either ectomycorrhizas form physiologically active and efficient mycoror endomycorrhizas with symbiotic fungi and in a rhizas, as demonstrated with E. dumosa on calcareous few cases ectendomycorrhizas are formed. In con- soil (Lapeyrie & Chilvers, 1985), with E. camaltrast, the genus Eucalyptus forms both vesicular- dulensis seedlings infected by Glomus fasciculatum arbuscular (VA) endomycorrhizas and ectomycor- (Boudarga & Dexheimer, 1988), or with hybrid rhizas. The Eucalyptus VA-endomycorrhizas were Eiicalypttis planted after inoculation with Pisobthus first described by Asai (1934) then by Maeda (1954), tinctorius (Garbaye, Delwaulle & Diangana, 1988). but tbe first synthesis in controlled conditions was Some evidence shows that the succession between fairly recent (Malajczuk e? a/., 1981), as was tbe first VA mycorrbizas and ectomycorrhizas during host ultrastructural study (Boudarga & Dexheimer, plant ageing, could be related to competition for 1988). The anatomy, ecology and physiology of infection sites (Chilvers et al., 1987). The possible Eucalyptus ectomycorrhizas, however, have been existence, however, of a direct antagonism between extensively studied (Chilvers & Pryor, 1965; both symbionts within the plant needs further Chilvers, 1968; Ling-Lee, Ashford & Chilvers investigation. Furthermore, the fungi could also 1977; Ling-Lee, Chilvers & Ashford, 1975; Hilbert compete for a limiting substrate. A technique et al., 1987). The VA mycorrhizas seem to be more allowing controlled in vitro infection of the root prevalent on young seedlings whereas the ecto- system by botb symbionts is needed to investigate mycorrhizas appear to take over as the plant ages these possibilities. Since otber tree species, including (Chilvers, Lapeyrie & Horan, 1987). However, both Alnus and Casuarina, bear more than one type of type of symbionts can be present simultaneously on mycorrhiza (Harley & Smith, 1983), such a technique the same root apex. Here, the VA-endoinycorrhizal could be of great interest. In tbis paper a successful fungus colonizes the inner part of the cortex while method for ectomycorrhizal synthesis (Chilvers,

2 74 K. Boudarga, F. Lapeyrie and J. Dexheimer Douglass & Lapeyrie, 1986), has been adapted for double inoculation of Eucalyptus seedlings. The resulting dual endo-ectomycorrhizas were observed by transmission electron microscopy. MATERIALS AND METHODS Host plant Seeds of Fucalyptus camaldulensis (Denhardt) were surface sterilized by shaking them gently for 20 min in chlorine solution (prepared from 5 g of calcium hypochlorite in 100 ml distilled water and filtered). They were then rinsed in three changes of sterile distilled water for 5 min each, plated on 1 % agar, 2 % sucrose medium in Petri dishes and incubated upside down for 9 d at 25 C for germination. Endomycorrhizal fungus inoculum A strain of Gigaspora margarita Becker & Hall was maintained on onion growing in pot culture. The spores were collected by sieving the substrate between 100 and 350//m. They were surface sterilized by soaking them for 20 min in an antibiotic solution (chloramine T 2%, streptomycin 0-04%), and were then rinsed in three changes for 5 min each, of sterile di.stilled water. The spores were then plated on sterile 1 % water agar, the ph having been adjusted to with HCl before autoclaving. After 6 d at 25 C, 80% of the spores had a germ tube between 0'3 and 8 mm long. Ectomycorrhizal fungus inoculum Pisolithus tinctorius (Mich, ex Pers.) Ckr. & Couch, was grown, from multiple mycelial plugs, on absorbant cards of stiff paper placed on potato dextrose agar in 90 mm plates, at 25 C for 10 d. Eucalyptus axenic culture for dual mycorrhizal infection The technique used was very similar to that of Chilvers et al. (1986). The Eucalyptus seedlings were grown on agar in a large Petri dish kept at an angle of 75, but no filter paper separated the roots from the medium, the germinated seedlings were placed at the top of the plate to let the root system spread over the surface of the agar. The agar medium was based on that of Crush & Hay (1981). It was supplemented with active charcoal (5 g T') and the ph was adjusted to 6-8 with HCl (1 N). The seedlings were raised in controlled environment cabinets at 22 C (under a regime of 1 2 h light (12 tubes Sylvania 'Grolux', 30 w), the root system being protected from light by a dark plastic sheet over the base of the plate. Dual inoculation for mycorrhizal synthesis The germinated spores were placed in contact with the young lateral roots 6 d after disinfection and 9 d after the seed germination. Fifty four days later, after abundant growth of the endophyte, the card bearing the inoculum of the ectomycorrhizal fungus was laid over the roots. Microscopic observations Fourteen days after ectomycorrhizal inoculation lateral roots were sampled. Root pieces were fixed in 2-5 % glutaraldehyde in phosphate buffer ph 7-2 for 7 h at 0 C. The samples were then rinsed in phosphate buffer and postfixed with 2 % osmium solution in distilled water for 1 h, before being dehydrated by acetone and impregnated with Fpon (Dexheimer, Gianinazzi & Gianinazzi-Pearson, 1979). Semi-thin sections were stained with Toluidine Blue ph 11, for light microscope observations, whereas ultrathin sections were contrasted with lead citrate and uranyle acetate for electron microscopy. RESULTS Newly formed ectomycorrhizas could be seen in the vicinity of Gigaspora spores (Fig. 1 a). Light microscope observations of these mycorrhizas showed that Pisolithus tinctorius was restricted to the external cell layer, forming a Hartig net between the cells and a thin sheath around the root. Gigaspora margarita was present as well but was restricted to the inner cortex where most of the cells were infected. The endophyte formed arbuscules in the deeper layers of the inner cortex, but hyphal coils were observed in the more superficial ones (Fig. 1 b). The percentage of lateral roots including both fungal symbionts varied between 60 and 70%. In the ectomycorrhiza, the Hartig net was occasionally made up of multiple layers. The fungal cytoplasm was dense with numerous organelles, indicating that it was probably metabolically active (Fig. 1 c). The host cortical cells were vacuolated, with only a thin layer of cytoplasm around the plasmalemma. Some dense granules, probably tannin deposits, were accumulated in the vacuole but never filled it, contrary to usual observation. In the endomycorrhiza, the endophyte formed either arbuscules, or coils in the more superficial layers. The arbuscule was always surrounded by the host plasmalemma (Fig. \d). The cytoplasm of an infected host cell was dense, with numerous organelles, especially mitochondria. DISCUSSION The anatomical structure of dual mycorrhizas synthesised in vitro resembled that of dual symbioses found in pot grown Eucalyptus dumosa on unsterile soil (Chilvers et al., 1987).

3 75 Dual mycorrhizal infection of Eucalyptus Figure 1. Dual ectomycorrhizal/va endomycorrhizal infection of Eucalypt root apex, (a) Eucalyptus root in contact with a spore of Gigaspora rnargarita, and mycelium of Pisolithus tinctorius ( x 25). (ft) Transverse section through a root with dual infection showing Hartig net between external cortical cells and arbuscules or hyphal coils inside the deeper cortical cells (x 160). (c) Ultrastructural localization of both symbionts inside the root showing multiple layers Hartig net, hyphal coils and arbuscules, as well as tannin deposits in vacuoles of cortical cells ( X 3000). (d) Ultrastructure of tbe arbuscule, totally surrounded by host plant plasmalema (x 23000). Key: Sp, spore of Gigaspora margarita; hn, hartig net: ar, arbuscule: he, hyphal coil; p, plasmalema: P, host plant cell wall; t, tannin. Both types of mycorrhiza could be found in an active state, even when they were involved in a dual endo-ectomycorrhiza association. In such dual endoectomycorrhiza the ultrastructure of each individual symbiotic association was indistinguishable from that found in single VA endomycorrhiza (Boudarga & Dexheimer, 1988) or in single ectomycorrhiza (Lei, 1988). The VA endophyte was still functional under the ectomvcorrhiza as indicated bv the occurrence of a continuous host cell plasma membrane around the arbuscule. Our technique was \'ery suitable for the synthesis of endomycorrhizas and ectomycorrhizas on the same root system or on the same root apex. Interactions between three symbiotic organisms could henceforth be studied in detail. Many parameters may be controlled, such as medium composition or time sequence of the infection, to

4 76 K. Boudarga, F. Lapeyrie and J. Dexheimer determine how they influence competition between the symbiotic associations. Presently, antagonism does not seem to exist between the two fungal symbionts. This supports the hypothesis of Chilvers et al. (1987), that endomycorrhizal ectomycorrhizal succession is simply related to spatial competition for infection sites. Some complementary work to characterize in detail the metabolic activities of tbe three symbionts during the establishment, maturation and ageing of this dual association could give valuable information. ACKNOWLEDGEMENTS The authors wish to thank D. Vairelles for valuable technical assistance. REFERENCES ASM, T. (1934). Uber das Vorkommen und die Bedeutung der Wurzelpilze in den Landpflanzen. Japanese Journal of Botany 1, BOUDARGA, K. & DEXHEIMER, J. (1988). Etude ultrastructurale des endomycorhizes a vesieules et arbuscules de jeunes plants d'eucalyptus camaldulensis (Denhardt) Myrtacees. Bulletin de la Societe Botanique de France 135, CHILVERS, G. A. (1968). Low power electron microscopy of the root cap region of Eucalypt mycorrhizas. New Phytologist 67, CHILVERS, G. A., DOUGLASS, P. A. & LAPEYRIE, F. F. (1986). A paper-sandwich technique for rapid synthesis of ectomycorrhizas. New Phytologist 103, 397^02. CHILVERS, G. A., LAPEYRIE, F. F. & HORAN, D. P. (1987). Ectomycorrhizal vs. endomycorrhizal fungi within the same root system. New Phytologist 107, CHILVERS, G. A. & PRYOU, L. D. (1965). The structure of Eucalypt mycorrhizas. Australian Journal of Botany 13, CRUSH, J. R. & HAY, M. J. M. (1981). A technique for growing mycorrhizal clover in solution culture. New Zealand Journal Agricultural Research 24, DEXHEIMER, J., GIANINAZZI, S. & GIANINAZZI-PEARSON, V. (1979). Ultrastructural cytochemistry of the host-fungus interfaces in the endomycorrhizal association Glomus mosseae/allium cepa. Zeitschrift fiir Pflanzen physiologie 92, GARBAYE, J., DELWAULLE, J. C. & DIANGANA D. (1988). Growth response of Eucalypts in the Congo to ectomycorrhizal inoculation. Forest Research and Management 24, HARLEY, ]. J. & SMITH, S. E. (1983). Mycorrhizal symbio.ns. Academic Press, London. HiLRERT, J. L., GAUDIN, V., MARTIN, F. & LAPEYRIE, F. (1987). Identification of 'ectromycorrhiza-specific' proteins (ectomycorthizins) involved in the development of Pisolithus- Eucalyptus symbiosis. Proceedings of the 7th North American Conference on Mycorrhizae, Gainesville, Florida. LAPEYRIE, F. F. & CHILVERS, G. A. (1985). an endomycorrhiza-ectomycorrhiza succession associated with enhanced growth hy Eucalyptus dumosa seedlings planted in a calcareous soil. New Phytologist 100, LEI, J. (1988). Etude experimentale des systhnes symbiotiques mvcorhiziens de quelques essences ligneuses, application pratique a la mycorhization de vitroplants. Ph. D. thesis, Llnivetsity of Nancy. LING LEE, M., ASHFORD, A. E. & CHILVERS, G. A. (1977). A histochemical study of polysaccharide distribution in Eucalypt mycorrhizas. New Phytologist 78, LING LEE, M., CHILVERS, G. A. & ASHEORD, A. E. (1975). Polyphosphate granules in three different kinds of tree mycorrhiza. New Phytologist 75, MAEDA, M. (1954). The meaning of myeorrhiza in regard to systematic botany. Kumamoto Journal of Science, series B3, MAI.AJCZUK, N., LINDERMAN, R. G., KOUGH, J. & TRAPPE, J. M. (1981). Presence of vesicular-arbuscular mycorrhizae in Eucalyptus sp. and Acacia sp., and their ahsence in Banksia sp. after inoculation with Glomus fasciculatus. New Phytologist 87,

5