Structure and function of the ectomycorrhizal association between Paxillus involutus (Batsch) Fr. and Betula pendula Roth.

Transcription

1 New Phytol. (19^5), 129, Structure and function of the ectomycorrhizal association between Paxillus involutus (Batsch) Fr. and Betula pendula Roth. I. Dynamics of mycorrhiza formation BY A. BRUN\ M. CHALOT*\ R. D. FINLAY AND B. SODERSTROM Department of Microbial Ecology, Lund University, Helgonavdgen 5, S Lund, Sweden {Received 10 May 1994 ; accepted 9 December 1994) SUMMARY Detailed examination of the structural and functional integration taking place during mycorrhiza formation necessitates rapid and aseptic synthesis of uniform mycorrhizal structures at well-defined stages of development. A system is described for formation of ectomycorrhizas between Betula peudula Roth, and Paxillus involutus (Batsch) Fr. Small sterile seedlings were placed directly on fungal colonies growing on cellophane covered agar plates. Microscopic examination during the course of development indicated an initial rapid colonization of the roots and formation of a fully developed mantle within 2-4 d. Hartig net formation was evident after 8 d of contact and involved a transition from a paraepidermal organization to a mature periepidermal Hartig net after 15 d of contact. Using a standard fungal growth medium, formation and development of mycorrhizas were not greatly affected by changes in the overall concentrations of nitrogen and phosphorus but changes in the ratio of nitrogen to phosphorus had strong effects on development and mycorrhiza formation was completely suppressed when the ratio of nitrogen to phosphorus was increased. These differences were not related to the effects of nitrogen or phosphorus on linear growth of the fungus. Key words: Betuta pendula (birch), dynamics of ectomycorrhiza formation, in vitro synthesis, Paxillus involutus. INTRODUCTION fungus can take place very rapidly. Horan, Chilvers & Lapeyrie (1988) demonstrated chemical inter- The broad host range (Duddridge, 1987) and ease of actions between Eucalyptus plants and ectoculture of Paxillus involutus have led to an increased mycorrhizal fungi within 1 d of contact and full use of the species in studies of ectomycorrhizal anatomical development within 4 d of contact. Other symbiosis (Klironomos & Kendrick, 1993). Studies studies using axenic culture techniques have shown of nitrogen metabolism in P. involutus ectomycor- similarly rapid development of ectomycorrhizas in rhizas demonstrate that the fungal symbiont plays a pine (Piche, Peterson & Acherley, 1983). In many fully integrated role in plant root metabolism and mycorrhizal associations, the exact modifications participatesactively in the assimilation and transfer of and interactions that occur between the two symbinewly absorbed nitrogen compounds (Finlay et al., onts during the first stages of the infection process 1988, 1989; Arnebrant et al., 1993). It is evident, are still not clear. Functional changes in metabolism from studies of ectomycorrhizal development, that may be closely linked to structural modifications structural and functional integration of the root and taking place during the early stages of development (Martin & Hilbert, 1991) and, for this reason, it is ' PresentaddressiUniversiteHenriPoincare, Nancy 1, Faculte important that the developmental Stages of mycordes Sciences, Laboiatoire de Biologie Forestieie, associe' INRA, l r i. BP 239, Vand,ruvre-les-Nancy Cedex. '"''"''^ formation are characterized as accurately as * To whom correspondence should be addressed. possible.

2 488 A. Brun and others Figures 1-5. For legend see opposite.

3 Mvcorrhiza formation between Paxillus involutus and Betula pendula 489 Rapid synthesis of ectomycorrhizas under aseptic conditions and in systems which produce uniform infection are therefore required for physiological and biochemical studies of the early stages of ectomycorrhizal development. Methods for rapid and sterile synthesis of ectomycorrhizas in Petri dishes have been described for species of Betula (Grellier, Letouze & Strullu, 1984), Eucalyptus (Chilvers, Douglass & Lapeyrie, 1986; Malajczuk, Lapeyrie & Garbaye, 1990), Pinus and Larix (Fortin, Piche & Lalonde, 1980; Wong & Fortin, 1989). However, as pointed out by Peterson & Chakravarty (1991), the risks of contamination are high when using the paper sandwich technique (Chilvers et al., 1986) or the nylon mesh method (Wong & Fortin, 1989) and these methods are inadequate for translocation studies since the fungus cannot be fed independently of the host plant. Similarly, the sandwich technique for aseptic synthesis between birch and P. involutus described by Simoneau et al. (1993) allowed the authors to investigate the polypeptide changes at different stages of mycorrhiza development, but is not applicable to translocation studies. The method described by Malajczuk et al. (1990) provides easy access to the eucalypt root system for external observations and for structural studies. It has been successfully used for translocation experiments using [^''CJalanine between eucalypt and Pisolithus tinctorius (Rabbani et al., 1992). In this study, a successful method for ectomycorrhizal synthesis in Petri dishes described by Malajczuk et al. (1990) has been adapted for the aseptic synthesis of ectomycorrhizas between Betula pendula Roth, and Paxillus involutus (Batsch) Fr. Mycorrhizal development was assessed by microscopy and the effects of nitrogen and phosphorus on the infection process and mycorrhiza development were evaluated. MATERIALS AND METHODS Organisms and media Paxillus involutus (Batsch) Fr. isolated from a fruit body growing under Betula pendula Roth, was maintained in Petri dishes by successive transfer on agar medium cotitaining modified Melin-Norkrans (MMN) medium from which malt extract was omitted. For the aseptic synthesis of ectomycorrhiza. P. involutus was grown on cellophane covered agar medium containing modified Melin-Norkrans medium (MMN). The complete MMN medium [NP] contained (mgr^); KH^PO, (500), (NHJ^HPO,, (250), CaCla (50), NaCl (25), MgSO,.7H2O (150), thiamine hydrochloride (0-1), FeC1.5.6H2O (1). A glucose concentration of 1 g 1~' was used. In order to optimize the production of mycorrhizal birch seedlings and produce mycorrhizas of uniform quality the effects of nitrogen and phosphorus were investigated using different media with reduced concentrations of nitrogen (N^P), phosphorus (NPj^) or both elements (NyP,)). The reduced nitrogen medium [N,,P] contained KH2PO4 (732 mg 1"') and (NH.,)2HPO4 (25 mg 1"'), giving only one tenth the amount of N but the standard P concentration. A reduced phosphorus medium [NPjJ contained KH.,PO, (50 mg 1-^, (NHJ2HPO, (25 mg T^) and NH4CI (182 mgl"') giving the standard N concentration but only one tenth of the standard P concentration. The reduced phosphorus and reduced nitrogen medium [N,;Pjj] contained KH^PO,, (50 mg r') and (NH4)2HPO4 (25 mg T'), giving one tenth of the standard concentration of both N and P. All media were adjusted to ph 5-5. Linear growth of the fungus was measured by recording the colony radius along two graduated orthogonal lines at regular time intervals, up to 22 d. Results are expressed as means of radii on six replicate plates, after subtracting the radius of the inoculum disc. Birch {Betula pendula Roth.) seeds were surfacesterilized with 3-5 % calcium hypochlorite for 30 min, rinsed in several changes of sterilized distilled water, germinated aseptically on water agar for one week and placed on top of 10 d-old colonies of P. involutus growing on cellophane covered agar medium (see above), in large round Petri dishes (150x15 mm). Plates were incubated in a growth chamber maintained at 24 C under an 18 h photoperiod (150 //mol m"^ s'^). After three weeks growth, the seedlings were removed and lateral root tips of birch were assessed for total numbers of root tips and total numbers of mycorrhizal root tips. Microscopic examination The main roots and lateral root tips of birch infected by P. involutus were sampled in a time course experiment, from 1 to 21 d and prepared for light Figure 1. Macroscopic view of a birch seedling. Three to five short lateral roots (arrowheads) have emerged per plant. The main root axis remains uninfeeted, except at the apical region (arrow), x 2-5. Figures 2, 3. Betula pendula - Paxillus involutus ectomycorrhizas 1-2 d after contact. Figure 2. Longitudinal section through the main root axis, 1 d after contact. Root hairs (arrows) are present along the root and sparse hyphae (arrowhead) are visible, x 190. Figure 3. Longitudinal section through the main root axis, 2 d after contact. The density of hyphae around the main roots has increased, x 230. Figures 4, 5. Betula pendula - Paxillus involutus ectomycorrhizas 4 d after contact. Figure 4. Longitudinal section through short lateral root showing the presence of a mantle (M) /(m thick. The epidermal cells remain tangentially elongated (arrowheads). X 90. Figure 5. Detail of a longitudinal section through a short lateral root showing the first stages of Hartig net formation (arrows) in which fungal cells start to proliferate between the root cells, x 510.

4 490 A. Brun and others Figures For legend see opposite.

5 Mvcorrhisa formation betiveen Paxillus involutus and Betula pendula microscopy, as described by Peterson (1991). The tissues were fixed in 2-5 "' glutaraldehyde in loottimhepes buffer (ph 6-8) for 3 h at room temperature atid then overnight at 4 C, rinsed three times for 15 min each iti the same buffer and dehydrated in a graded ethanol series (30, 50, 70, 90 and 100%). Tissues were then infiltrated with LR White resin gradually over one day in the follovvitig sequence: loo",, ethanol-lr White (1:1); 100% ethanol-lr White (1:2); 100% ethanol-lr White (1 :3) and finally pure LR White. Tissues were left in a second change of LR White resin overnight, at room temperature, then embedded in gelatine capsules and the resiti polymerized at 60 C for 48 h. Sectiotis were cut with glass knives at a thickness of 2'0 /im and stained with methyletie blue/azure B (1/1) prepared in Borax buffer. The experiment was repeated three times. For each treatment/time interval at least three replicate plates were used and 10 sections from each of five plants were examitied, making a total of 150 sections per treatment. RESULTS Mycorrhizal development Paxillus itwolutus mycelium colotiized roots oi Betula pendula within h, otice hyphae had cotitacted the root surface. At this stage, microscopic examination of longitudinal sections revealed the presence of root hairs and sparse hyphae along the root (Fig. 2). Two days after inoculation, the hyphae remained sparse and unorganized and the presence of root hairs was still evident. However, the density of hyphae around the main roots had inereased and the fungal mycelium remained attached to the root when the plant was harvested (Fig. 3). In particular, there was an accumulation of poorly aggregated hyphae at the apieal region of the main root axis (not shown). In eontrast, the newly-emerging lateral roots are completely enveloped by the fungal hyphae which form two distinct layers, one inner layer of closely aggregated hyphae, and an outer layer consisting of sparse, less well organized hyphae (not shown). Macroscopic examinations of the root system at day four showed that the fungus totally enveloped the root system (Fig. 1) and that there were 3-5 newly-emerging roots per plant. A-Iycorrhiza were 491 white when young, slowly developing a beige/brown colour with age. The main root axis remained uninfected, except at the apical region. Microscopic observations at this stage showed the presence of a mantle //m thick and differentiated into two distinct layers, an inner layer consisting of highly stained and closely aggregated hyphae, and an outer region consisting of sparse and poorly stained hyphae (Fig. 4). Details of longitudinal sections (Fig. 5) revealed the first stages of Hartig net formation where fungal cells start to proliferate between the root cells. The epidermal cells remain tangentially elongated (Fig. 4). Within 8-10 d of contact, a thick mantle (95-150/mi) had developed and there was a recognizable Hartig net (Fig. 6). The epidermal root cells had elongated radially during Hartig net formation, a feature characteristic of angiosperms (Agerer, 1991). At this stage of ectomycorrhiza formation, the Hartig net was restricted to the anticlinal walls of the epidermal cells as shown in a detailed longitudinal section (Fig. 7). This type of structure corresponds to the paraepidermal Hartig net described by Agerer (1991). Longitudinal sections through the Hartig net (not shown) revealed a single layer of hyphae between the epidermal cells, but occasionally several hyphal rows were seen. Longitudinal sections through the mantle (not shown), revealed net-like hyphal bundles with frequent clamp connections, and a plectenchymatous structure, according to the classification of Agerer (1991). Within 15 d, the first leaves had appeared and there were 3-6 mycorrhizal short roots ( > 2 mm in length) per plant. Microscopic examinations of these short roots indieated that no change in the structure or width ( //m) of the mantle had occurred whereas the Hartig net had eontinued to develop and become fully established along the length of the root (Fig. 8). Detail of a longitudinal section (Fig. 9) clearly showed that the fungal cells had fully enveloped the cells of the epidermal layer forming a periepidermal Hartig net (Agerer, 1991). Several hyphal rows were present between the epidermal cells (Fig. 9). Three weeks after inoculation some of the root apiees had produced side branches (not shown) but no major structural changes occurred in the third week. Figures 6,7. Bettila peitdttla - Paxillus itwolutus ectomycorrhizas 8 d after contact. Figure 6. Longitudinal section through a lateriil root. A typical paraepidl'rmal Hartig net (HN) has formed. The epidermal cells have elongated radially (arrowheads). x210. Figure 7. Detail of a longitudinal section showing the Hartig net restricted to the anticlinal walls of the epidermal cells (arrows), x 420. Figures 8, 9. Bettila pendula - Paxillus invohttus ectomycorrhizas \S d after contact. Figure 8. Longitudinal section through a mature ectomyeorrhiza showing the fully estahlished mantle (M) and Hartig net (HN). x 200. Figure 9. Detail of the same longitudinal section. The fungal cells have fully enveloped the cells of the epidermal layer (arrows) forming a periepidermal Hartig net. x 480. Figures 10, 11. Betula pendula - Paxilhis involutus eetomycorrhizas 15 d after contact on M M N containing reduced nitrogen (N,P). Figure 10. The mantle (M) is thinner (50-60/mi) and the penetration by the Hartig net (HN) between the cells of the epidermis is severely restricted to a single row of cells, never extending heyond the outer half of the epidermal layer (arrowheads), x 210. Figure 11. Detail of a longitudinal section showing restricted Hartig net de\-elopment (arrows), x 470.

6 492 A. Brun and others treatment but less extensively distributed (not shown). Microscopic examinations of mycorrhizal roots developed on reduced nitrogen medium (N,jP) at day 15 showed that a high proportion of roots did not possess a complete Hartig net and had a thinner mantle (50-60//.m) (Fig. 10). Penetration by the Hartig net between the cells of the epidermis was restricted to a single row of cells, never extending beyond the outer half of the epidermal layer (Fig. 11). This incomplete structure was never found in NP or NjjPji treatments at this stage of development. However, the effects of the different nutrient regimes on mycorrhizal infection were not the same as those on the radial growth of the fungus in pure culture (Fig. 12). Indeed, radial growth of P. involutus on reduced phosphorus (NPjj) or reduced phosphorus and nitrogen (N,^P () medium was faster than that on complete (NP) or reduced nitrogen (Nj^P) medium Time (days) Figure 12. Effect of nitrogen and phosphorus concentration on radial growth of Paxitlus involutus. The fungus was grown on cellophane covered agar medium containing complete MMN meditam ( ), reduced nitrogen MMN medium ( ), reduced phosphorus MMN medium (O) or reduced nitrogen and reduced phosphorus MMN medium (#) as described in the Materials and Methods section. Data are expressed as means of five replicates. Vertical bars indicate SE. Effects of nitrogen and phosphorus on the infection process The first steps of colonization, involving the presence of a sparse layer of hyphae around the roots within 1-2 d, as shown in Figures 2 and 3, took place in all treatments. However, the percentage of root tips forming fully developed mycorrhizas with P. involutus after 15 d differed greatly between the treatments. Mycorrhiza formation occurred on almost all (> 95 %) roots when both P and N were lowered to one-tenth of the concentration in standard MMN (NRPR), giving an average of 3-8 mycorrhizal root tips per plant (40-60 plants were analyzed). Only 70 % of the lateral roots became mycorrhizal within 15 d when seedlings were placed on colonies grown on complete MMN (NP), giving an average of 2-1 mycorrhizal root tips per plant. Mycorrhiza formation by P. involutus was totally suppressed on reduced phosphorus medium (NPj^) and partially suppressed on reduced nitrogen medium (N,jP), where the number of mycorrhizal root tips per plant was 1-4. Mycorrhizal roots formed on NP, Nj^Pj^ or N jp media had similar macroscopic appearances. The Hartig net of roots from the NP treatments was structurally similar to that of roots in the Nj^P,^ DISCUSSION The in vitro technique described in this study provides a convenient method for direct observation of the dynamics of root infection and mycorrhizal development. The rapid and aseptic synthesis of ectomycorrhizas between birch and Paxillus involutus in Petri dishes allows production of large numbers of plants with a uniform level of infection which can be used in physiological experiments requiring material at a well-defined stage of development. Seedlings produced in this way and subsequently transferred to a peat substrate rapidly developed an extensive mycelial phase, indicating that the synthesized mycorrhizas were fully functional. Microscopic examination during the course of integration indicates rapid colonization of the roots by the mycelium. The present study indicates that the process of mycorrhiza formation in Betula pendula is characterized by tbe formation of a fungal mantle prior to the development of the Hartig net and supports earlier studies on various angiosperms (Grellier et al., 1984; Horan et al., 1988). These results contrast with those of Nylund & Unestam (1982), who showed that the Hartig net appears before the fungal mantle in Picea ectomycorrhizas. The first stages of Hartig net formation are evident 4 d after contact. The maturation of the Hartig net is characterized by a transition from a paraepidermal structure (day 8) to a periepidermal structure (day 15), thus differing from the organization described by Grellier et al. (1984) and Agerer (1991) for Betula ectomycorrhizas, in which the Hartig net does not completely envelop the epidermal cells. The process of mycorrhizal infection observed in the present study also involved the same sequence of events as described by Horan et al. (1988) for eucalypt ectomycorrhizas, except that the typical periepidermal Hartig net was formed earlier in the eucalypt

7 Mycorrhiza formation between Paxillus involutus and Betula pendula system. The two types of mycorrhizal roots which developed in the Betula pendula - Paxillus involutus system correspond to those described in the Eucalyptus pilularis Hydnangium carneum association (Moore, Massicotte & Peterson, 1989). Roots emerging after contact between the main root axis and hyphae possess a mantle along the whole length of the root, while others have a mantle confined to the root apex. Although Grellier et al. (1984) used micropropagated platitlets their line drawings suggest that the dynamics of infection are similar to those in seedlings. Simultaneous reduction in the supply of both P and N caused a marked increase in the development of mycorrhizas. Mycorrhiza formation was reduced when nitrogen alone was decreased and completely suppressed when only P was reduced. These differences are not related to the effects of mineral nutrients on linear growth of the fungus. Gibson & Deacon (1990) also reported that development of mycorrhizas on birch using different fungi was markedly reduced at a reduced nitrogen level compared with that on the complete medium. The results of our investigations indicated a negative effect of P under conditiot-is of reduced N availability (i.e. NjjP treatment), supporting previous results (Newton & Pigott, 1991). It appears to be essential to maintain a P: N ratio of about 3 to optimize the infection rate it-i our axenic system. Further studies are now in progress in our labs using the system described in this paper to investigate the basis of functional changes in N-assimilation occurring during the early stages of integration between fungus and host. ACKNO-WLEDGEMENTS This work was supported hy a grant (FRB-EV5V-CT ) from the Commission of the Furopean Comn-iuiiities to M. C. Financial support from the Swedish Natural Science Research Council, the S-wedish Council for Forestry and Agricultural Research and the Swedish Fnvironmental Protection Agency is gratefully acknowledged. REFERENCES Agerer R Characterization of ectomycorrhizae. In; Norris JR, Read DJ, Varma AK, eds. Methods in Mierobiology, vol. 23, Teehiri(iues for the study of mycorrhiza. London: Academic Press, Arnebrant K, Ek H, Finlay RD, Soderstrom B Nitrogen translocation between Atnus glutinosa (L.) Gaertn. seedlings inoeuiated witii Frankia sp. and Pinus eontorta Doug, ex Loud seedlings connected by a eommon ectoniycorrl-iizai n-iycelium. New Phytotogist 124: Chilvers GA, Douglass PA, Lapeyrie F A paper- 493 sandwich teci-inique for rapid synthesis of ectomycorrhizas. Neiv Phytologist 103: Duddridge JA Specificity and recognition in eetomycorrhizai associations. In: Pegg G F, Ayres PG, eds. Fungal infection of plants. Cambridge: Cambridge University Press, Finlay RD, Ek H, Odham G, Soderstrom B Myeelial uptake, translocation and assimilation of nitrogen from N labelied ammonium by Pinus sylvestris piants infected with four different ectomycorrhizal fungi. Neiv Ptiytotogist 110: Finlay RD, Ek H, Odham G, Soderstriim B Uptake, translocation and assimiiation of nitrogen from '-''N-labelled ammonium and nitrate sources by intact ectomycorrhiza systems infected with Paxillus involutus. New Phytologist 113: Fortin JA, Piche Y, Lalonde M Teehnique for the observation of early morphological elianges during ectomycorrhiza formation. Canadian Journal of Botany 58: Gibson F, Deacon JW Establishment of ectomycorrhizas in aseptic eulture: efteets of glucose, nitrogen and phosphorus in relation to suecessions. Myeotogieal Research 94: Grellier B, Letouze R, Strullu DG Micropropagation of birch and mycorrhizal forn-iation ;'/; 'nitro. 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New Phytotogist 111: Nylund J, Unestam T Strueture and physiology of ectomycorrhizae. I. The process of mycorrhiza formation in Norway spruce in vitro. New Phytotogist 91: Peterson RL Histochemistry of ectomycorrhiza. In: Norris JR, Read DJ, Varma.\K, eds. Methods in mierobiotogy, vol. 23, Teehniqties for the study of myeorrhiza. London: Academic Press, Peterson RL, Chakravarty P Techniques in synthesizing ectomyeorrhiza. In: Norris JR, Read DJ, Varma AK, eds. Methods in mierobiotogy, vot. 23, Teehniques for the study of myeorrhiza. London:.^eademic Press, Piche Y, Peterson RL, Acherley CA Early development of ectomyeorrhizal short roots of pine. Seamiing Electron Mieroseopy3: Rabbani S, Chalot M, Botton B, Martin F Utilisation de l'alanine par les plantules d'eucalyptus associees an champignon ectot-nycorhizien Pisolithus tinetorius. Butletin de I'Aeademie et de la Soeiete' Lorraine des Seienees 31, Simoneau P, Viemont JD, Moreau JC, Strullu DG Symbiosis-related polypeptides associated with the early stages of ectomyeorrhiza organogenesis in bircli {Betuta pendula Roth.). New Phytologist 124: Wong KK, Fortin JA A Petri dish technique for the aseptic synthesis of ectomycorrhizae. Canadian Journal of Botany 67:

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