The occurrence of anastomosis formation and nuclear. exchange in intact arbuscular mycorrhizal networks

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1 Research The occurrence of anastomosis formation and nuclear Blackwell Science Ltd exchange in intact arbuscular mycorrhizal networks Manuela Giovannetti 1, Paola Fortuna 2, Anna Silvia Citernesi 1, Stefano Morini 2 and Marco Paolo Nuti 1 1 Dipartimento di Chimica e Biotecnologie Agrarie, Centro di Studio per la Microbiologia del Suolo, C. N. R., Università di Pisa, Via del Borghetto 80, Pisa, Italy; 2 Dipartimento di Coltivazione e Difesa Specie Legnose, Università di Pisa, Via del Borghetto 80, Pisa, Italy Summary Author for correspondence: Manuela Giovannetti Tel: Fax: mgiova@agr.unipi.it Received: 4 January 2001 Accepted: 29 March 2001 The widespread occurrence of anastomoses and nuclear migration in intact extraradical arbuscular mycorrhizal (AM) networks is reported here. Visualization and quantification of intact extramatrical hyphae spreading from colonized roots into the surrounding environment was obtained by using a twodimensional experimental model system. After 7 d the length of extraradical mycelium in the AM symbiont Glomus mosseae ranged from 5169 mm in Thymus vulgaris to 7096 mm in Prunus cerasifera and 7471 mm in Allium porrum, corresponding to 10, 16 and 40 mm mm 1 root length, respectively. In mycelium spreading from colonized roots of P. cerasifera and T. vulgaris, contacts leading to hyphal fusion were 64% and 78%, with 0.46 and 0.51 anastomoses mm 1 of hypha, respectively. Histochemical localization of succinate dehydrogenase activity in hyphal bridges demonstrated protoplasmic continuity, while the detection of nuclei in the hyphal bridges confirmed the viability of anastomosed hyphae. The ability of AM extraradical mycelium to form anastomosis and to exchange nuclei suggests that, beyond the nutritional flow, an information flow might also be active in the network. Key words: arbuscular mycorrhizas, Glomus mosseae, extraradical mycelium, anastomosis, nuclear exchange, mycorrhizal networks, wood wide web. New Phytologist (2001) 151: Introduction Most terrestrial plant species live in symbiosis with mycorrhizal fungi, which develop extraradical hyphal systems fundamental for the uptake of nutrients from soil and their translocation to the host plant (Smith & Read, 1997). Many studies have demonstrated the major role played by the extramatrical mycelium in the mobilization and transfer of soil nutrients such as phosphorus, nitrogen, Cu, Fe, K, Zn, Ca and S (Smith & Read, 1997). Moreover, works with experimental microcosms and field-based studies reported transfer of carbon, nitrogen and phosphorus between plant species interconnected by a common mycorrhizal mycelium, highlighting the importance of the mycorrhizal web for the exploitation and redistribution of resources within plant communities (Simard et al., 1997). Investigations on the structure and function of the vegetative mycelium of ectomycorrhizal fungi have utilized microcosm systems and two-dimensional transparent observation chambers, which allowed nondestructive visualization of the fungal networks (Finlay & Read, 1986; Bending & Read, 1995a, 1995b; Perez-Moreno & Read, 2000). On the other hand, most studies of the extramatrical mycelium of arbuscular mycorrhizas involved destructive extraction from soil (Jakobsen & Rosendhal, 1990; Jakobsen et al., 1992; Jones et al., 1998). A few nondestructive observations of arbuscular mycorrhizal (AM) extraradical mycelium have been carried out by using root observation chambers (Friese & Allen, 1991) and in vitro dual systems (Bago et al., 1998). Such studies have provided qualitative information on the architecture of AM mycelium, and on its development before and after symbiosis establishment. Nevertheless, virtually nothing is known New Phytologist (2001) 151:

2 718 Research of the dynamics of hyphal growth during the extramatrical phase and of the mechanisms allowing the formation of extensive extraradical mycelial networks through which nutrients are proposed to flow. One of these mechanisms could be represented by the formation of anastomoses, which have been described to occur widely between vegetative hyphae of other mycorrhizal fungi (Fries, 1987; Dahlberg & Stenlid, 1994; Sen et al., 1999). So far, only one report has visualized and quantified hyphal fusions in AM mycelium, and detected cytoplasmic flow and nuclear exchange between anastomosing hyphae during presymbiotic growth (Giovannetti et al., 1999). We describe an experimental in vivo system which allowed the visualization of intact extraradical mycorrhizal networks produced by the AM fungus Glomus mosseae living in symbiosis with Allium porrum, Thymus vulgaris and Prunus cerasifera. By using this system we investigated: the extension of extraradical mycelium originating from infected roots; anastomosis formation and structure of the mycorrhizal network; and viability and nuclear occurrence in the anastomosing mycelium. Materials and Methods Fungal material The AM fungus Glomus mosseae (Nicol. & Gerd.) Gerdemann & Trappe (Kent isolate) (Banque Européenne des Glomales code 12) was obtained from pot-cultures maintained in the collection of the Department of Chemistry and Agricultural Biotechnology, University of Pisa, Italy. Sporocarps of G. mosseae were rinsed five times in sterile distilled water and germinated in the dark at 24 C between two Millipore membranes (0.45 µm diameter pores), which were placed on moist sterile quartz grit in 14 cm diameter Petri dishes. Ten sporocarps were used for each membrane sandwich. Plant material Sterile seeds of Allium porrum and Thymus vulgaris were germinated in moist sterile grit. After 15 d the root system of each seedling was sandwiched between the Millipore membranes containing G. mosseae germinated sporocarps (Giovannetti et al., 1993b). Micropropagated plantlets of a selection of Prunus cerasifera Ehrh (MrS. 2/5) were obtained as described by Fortuna et al. (1996). After 2 wk in the rooting medium, rooted shoots were removed from in vitro cultures, washed in distilled water and the root systems were sandwiched as described above. Plants were individually placed in 10 cm-diameter pots and the sandwiched root systems were buried in sterile quartz grit. Pots were closed in Sun Transparent Bags (Sigma, St. Louis, MO, USA) and maintained in a growth chamber with 24 C day and 21 C night temperatures and 16/8 light/dark cycle. Experimental model system After 20 d growth in the sandwich system, plants were harvested; roots were gently removed from sandwiches by immersion in water and checked for the occurrence of extramatrical mycelium, which was carefully plucked with forceps under a Wild dissecting microscope (Leica, Milano, Italy). Mycorrhizal infection was confirmed on sample plants by clearing and staining, using lactic acid instead of phenol (Phillips & Hayman, 1970). The roots of each plant were then placed between two 8 cm 8 cm Millipore membranes, transferred into pots containing sterile quartz grit and maintained in a growth chamber as described. At intervals (3, 5, 7, 9, 11 d), after removing the external mycelium and transplanting, three replicate plants of A. porrum, T. vulgaris and P. cerasifera were harvested, the root sandwiches were carefully opened and the roots and the extramatrical mycelium growing from the roots on the membranes were stained with Trypan blue in lactic acid (0.01%). Extension and structure of the mycorrhizal network Experiments were carried out as described above, using eight replicate plants of A. porrum, T. vulgaris and P. cerasifera, which were harvested 7 d after removing the external mycelium and transplanting into the sandwich system. The membranes were gently opened and the extramatrical mycelium was stained with Trypan blue in lactic acid (0.01%), and observed under the Wild dissecting microscope (Phillips & Hayman, 1970). Hyphal density (hyphal length mm 2 ) was estimated with the gridline intersect method (Giovannetti & Mosse, 1980) by counting the length of hyphae in four areas of 64 mm 2, using a grid eyepiece. Numbers of hyphal contacts, anastomoses and hyphal branches were counted in four areas of 16 mm 2. Frequency of anastomosis was calculated by determining the proportion of hyphal contacts that had anastomosed. Total hyphal length was calculated multiplying hyphal density by the area covered by the mycelial network, which was measured with an area meter. Finally, the root systems were removed from the membranes, cleared and stained to assess the percentage of AM infection and the total length of the whole root system (Giovannetti & Mosse, 1980). Data were subjected to ANOVA and differences in treatment means were evaluated by Duncan s multiple range test. Percentage data were subjected to arcsine transformation before analysis. Viability of the mycorrhizal network To assess viability of the extramatrical mycorrhizal mycelium and the establishment of protoplasmic continuity in anastomosed hyphae, succinate dehydrogenase (SDH) activity (MacDonald & Lewis, 1980; Saito et al., 1993) was New Phytologist (2001) 151:

3 Research 719 assessed on the mycelium produced by five plants for each species and grown for seven days in Millipore membrane sandwiches as described above. After SDH staining, the extraradical mycelium was observed under the dissecting microscope, selected areas of membranes were cut, mounted on microscope slides and observed under a Polyvar light microscope (Reichert-Jung, Vienna, Austria) for the presence of formazan salt depositions in hyphal bridges. Nuclear occurence in the mycorrhizal network Occurence and localization of nuclei in hyphae was observed by diaminophenylindone (DAPI) (Sigma, St. Louis, USA) staining (Logi et al., 1998). The root systems of five G. mosseae infected plants of A. porrum were sandwiched between two polycarbonate black 47 mm membranes (Costar Nucleopore), placed between two 8 cm 8 cm Millipore membranes and transferred into pots containing sterile quartz grit as described above. The polycarbonate black membranes were used to obtain a better visualization of nuclei under epifluorescence. On these membranes fungal growth was slow, and an extensive mycorrhizal network could be detected only after 18 d. After opening the root sandwiches, selected areas of the membranes bearing extraradical mycelium were mounted on microscope slides in DAPI (5 µg ml 1 in 1 : 1 water: glycerol) and observed under epifluorescence, by using the filter combination U1(BP , LP 418, DS 420). Results Experimental model system The experimental model system allowed the detection and visualization of intact extraradical AM mycelium. After transplanting (3 d), the sandwiched root systems of plants of A. porrum, T. vulgaris and P. cerasifera colonized by G. mosseae showed poor production of extraradical fungal hyphae. The extramatrical mycelium growing from the roots on the membranes was detectable 2 d later and after 7 d an extensive AM extramatrical mycelial network, evenly spreading around the whole root systems was visible to the naked eye (Fig. 1a,b). At the following harvests, the extraradical mycelium had overgrown the membranes in several sandwiched root systems in the three plant species. Following this feasibility experiment, further quantitative and qualitative assessments were performed after growing the plants in the sandwich system for 7 d. Extension of the mycorrhizal network The extension and architecture of the root system of A. porrum, T. vulgaris and P. cerasifera appeared different in our bidimensional experimental system: P. cerasifera and T. vulgaris showed a branched root system, whereas in A. porrum no lateral roots were present and total root length was lower than in the other two plant species. However, total hyphal length and the extension of the mycelial networks were similar in the three plant species (Table 1). Since total root length of A. porrum was lower than that of P. cerasifera and T. vulgaris, the length of external fungal hyphae per mm of root was highest in A. porrum, but this difference was not related to a higher AM root infection. The mean growth rate of the extraradical hyphal network during the first 7 d, calculated by dividing total hyphal length by the number of days growth, ranged from 738 to 1067 mm d 1 (Table 1). Structure and viability of the mycorrhizal network The AM extraradical mycelium appeared as an anastomosing mycelial network surrounding the root system (Fig. 1a,b). Hyphal density was higher in A. porrum than in T. vulgaris and P. cerasifera (Table 2). Fungal hyphae growing from mycorrhizal roots were highly branched ( branches mm 1 ) and showed many anastomoses (Fig. 1c,d). The number of anastomoses per mm of hypha ranged from 0.46 in A. porrum to 0.51 in T. vulgaris and P. cerasifera. The frequency of anastomoses was 75% in A. porrum, 78% in T. vulgaris and 64% in P. cerasifera. Viability of extraradical mycelium determined by SDH activity was 100% in the whole mycelial network in the three plant species. Protoplasmic continuity, the characteristic feature of true anastomosis, and viability of anastomosed hyphae were demonstrated by the localization of formazan salts depositions in the hyphal bridges (Fig. 2a,b). Table 1 Root length, arbuscular mycorrhizal colonization and extension of Glomus mosseae extraradical mycelial networks in three different mycorrhizal plants Allium porrum Thymus vulgaris Prunus cerasifera Root length (mm) ± 29.4b ± 35.6a ± 58.8a AM colonization (% root length) 54.4 ± 3.6b 46.7 ± 2.6b 64.4 ± 3.1a Total hyphal length (mm) 7471 ± 1356a 5169 ± 441a 7096 ± 897a Area covered by the extraradical mycelium (mm 2 ) 2755 ± 453a 2525 ± 161a 2898 ± 254a Hyphal length per total root length (mm mm 1 ) 40.2 ± 7.2a 10.1 ± 0.7b 15.9 ± 1.7b Hyphal length per mycorrhizal root length (mm mm 1 ) 73.7 ± 12.2a 21.9 ± 1.4b ± 2.9b Values, means ± SE; n = 8. Values followed by the same letters within the same row are not significantly different (P < 0.05; Duncan s test). New Phytologist (2001) 151:

4 720 Research Fig. 1 Visualization of the development of intact extraradical mycelial networks produced by Glomus mosseae, which grow from mycorrhizal roots of Prunus cerasifera (a) and Allium porrum (b) and uniformly colonize the surrounding environment. (c) Micrograph showing densely branched hyphae spreading from A. porrum mycorrhizal roots. (d) Micrograph of G. mosseae mycelium spreading out from root-based mycorrhizal hyphae. Bars, (c) 300 µm; (d) 200 µm. New Phytologist (2001) 151:

5 Research 721 Table 2 Structure and anastomosis formation ability of intactextraradical mycelial networks produced by Glomus mosseae living in symbiosis with three different mycorrhizal plants Allium porrum Thymus vulgaris Prunus cerasifera Hyphal density (mm mm 2 ) 2.72 ± 0.21a 2.07 ± 0.17b 2.61 ± 0.25ab No. of anastomoses mm ± 0.18a 1.09 ± 0.15a 1.34 ± 0.17a No. of anastomoses mm 1 (length) of hypha 0.46 ± 0.04a 0.51 ± 0.04a 0.51 ± 0.03a No. of hyphal branches mm 1 (length) of hypha 0.97 ± 0.06a 0.86 ± 0.05a 0.92 ± 0.08a No. of anastomoses per hyphal contact 0.75 ± 0.02a 0.78 ± 0.02a 0.64 ± 0.03b Values, means ± SE; n = 8. Values followed by the same letters within the same row are not significantly different (P < 0.05; Duncan s test). Fig. 2 Light micrographs showing complete fusions of hyphal walls and the establishment of protoplasmic continuity in anastomosing extramatrical hyphae of Glomus mosseae. (a, b) Visualization of succinate dehydrogenase (SDH) activity evidenced by formazan salt depositions in hyphal bridges. (c) Localization of nuclei (stained with diaminophenylindone (DAPI)) the middle of a fusion bridge. Bars, (a) 7 µm; (b) 10 µm; (c) 10 µm. Nuclear occurence in the mycorrhizal network DAPI staining and epifluorescence microscopy showed that nuclei were evenly distributed in extraradical hyphae, confirming that the whole mycorrhizal network was viable and actively growing (Fig. 3a). Nuclei were detected in hyphal bridges between anastomosing hyphae, showing complete fusion of hyphal walls and the establishment of protoplasmic continuity (Fig. 2c). Interestingly, a high number of nuclei were consistently detected in the middle of most fusion New Phytologist (2001) 151:

6 722 Research Fig. 3 Epifluorescence microscopy of extramatrical mycelium of Glomus mosseae showing the consistent occurrence of nuclei (visualized by diaminophenylindone (DAPI) staining) within the anastomosing hyphal network. (b) Arrows indicate many nuclei in the middle of fusion bridges. Bars, (a) 25 µm; (b) 10 µm. bridges, suggesting nuclear migration and exchange during hyphal anastomosis (Fig. 3b). Discussion In this study we visualized and quantified the development of intact extraradical AM mycelium growing in vivo, by using a two-dimensional experimental model system, and we showed the large occurrence of anastomoses in the mycorrhizal network. The results provide data on: the extension of intact AM extraradical mycelium; the formation of anastomosis within the hyphal network; and protoplasmic continuity and nuclear exchange in anastomosing hyphae. To our knowledge this is the first visualization and quantification of the whole intact AM network in vivo. The experimental model system allowed us to monitor the growth of extramatrical hyphae spreading from colonized roots into the surrounding environment, and the rate of development of the mycorrhizal network, since a fixed starting point for data collection was represented by the transfer of plantlets into the sandwich system after removal of all the mycelium, both presymbiotic and symbiotic, around mycorrhizal roots. Although the bidimensional sandwich environment may influence network extension, this technique can be used in further spatio-temporal studies aimed at monitoring the development of the mycorrhizal network and the process of formation of hyphal interconnections between different plants. Moreover, the clearcut detection of AM extraradical mycelium was due to the absence of background nonmycorrhizal hyphae, which represented a severe limitation to the assessment of hyphal growth in previous works (Abbott & Robson, 1985; Sylvia, 1988). The hyphal densities we found after 7 d growth ranged from 10 to 40 mm mm 1 root length, which compare to figures ranging from 1.6 to 1420 mm mm 1 root in studies of mycelium extracted from soil (Sanders & Tinker, 1973; Tisdall & Oades, 1979; Abbott & Robson, 1985; Sylvia, 1988). The very high values occasionally reported could be set in the context of the average rate of hyphal growth obtained in our experimental system, which ranged from 738 to 1067 mm d 1 in the first 7 d of growth. These data compare very favourably with those reported for the presymbiotic mycelium, 2.8 mm d 1 after 7 d (Logi et al., 1998). We detected and quantified anastomoses in the extraradical mycelium. The occurrence of hyphal fusions in AM fungi had been previously mentioned by some authors (Mosse, 1959; Francis & Read, 1984; Tommerup, 1988; Friese & Allen, 1991; Giovannetti et al., 1993a), and a recent study determined the occurrence and frequency of anastomosis between hyphae originating from different spores growing in vitro, in the absence of the host, in three Glomus species (Giovannetti et al., 1999). In our experimental model system the frequencies of anastomoses per hyphal contact, ranging from 64 to 78%, are higher than those found within self-anastomosing isolates of Rhizoctonia solani (> 50%: (Hyakumuchi & Hui, 1987)). They are also higher than those obtained in G. mosseae presymbiotic mycelium growing in vitro, originating from the same (57%) and from different (40%) germinating spores (Giovannetti et al., 1999). The number of anastomoses per mm (length) of hyphae ranged from 0.46 in A. porrum to 0.51 in T. vulgaris and P. cerasifera (Table 2). Interestingly, such values were times higher than those observed in presymbiotic mycelium produced by G. mosseae germinated spores (Giovannetti et al., 1999). These data confirm the ability to form hyphal anastomosis by AM fungi, though lower than values for saprophytic fungi: on an area basis, anastomosis frequency was between 1.1 and 1.3 fusions mm 2, compared with fusions mm 2 for Gibberella fujikuroi (Correll et al., 1989). New Phytologist (2001) 151:

7 Research 723 Histochemical localization of SDH activity in hyphal bridges showed that protoplasmic continuity, the characteristic feature of true anastomosis, was established between fusing hyphae of the symbiotic mycelium, in accordance with observations on presymbiotic mycelium (Giovannetti et al., 1999). SDH activity was detected in all the extraradical hyphae after 7 d growth, showing 100% viability of the mycorrhizal network. Previous works reported that metabolic activity of extraradical mycelium in soil, measured by using different vital stains, ranged from 63% in 6-wk-old G. intraradices hyphae to 100% in 3-wk-old G. clarum hyphae (Schubert et al., 1987; Hamel et al., 1990). These authors maintained that the viability of the extraradical mycelium decreased significantly with increasing hyphal age. Other data showed that the activity of extraradical mycelium ranged from 0 to 32% in hyphae extracted from soil, increasing greatly in hyphae attached to colonized roots 96% in 6, 9 and 13-wk-old G. mosseae and G. intraradices hyphae (Sylvia, 1988). A recent work reported that the length of vital extraradical hyphae ranged from 20 to 40 m m 1 colonized root in Eucalyptus coccifera seedlings inoculated with three different AM fungi (Jones et al., 1998). These data are comparable with the values obtained in our study, which ranged from 22 to 74 m m 1 colonized root length (Table 1). DAPI staining and epifluorescence microscopy allowed the localization of nuclei in hyphal bridges and confirmed the viability of anastomosed hyphae and the establishment of protoplasmic continuity during hyphal fusions, in agreement with previous findings (Giovannetti et al., 1999). The large occurrence of nuclei in extraradical mycelium and in anastomosing hyphae suggests that, beyond the nutritional flow, an information flow may also be active. The high frequency of hyphal fusions in symbiotic mycelium of AM fungi confirms that the phenomenon may represent an important event in the biology of these obligate biotrophs (Giovannetti et al., 1999). Moreover, anastomosis formation may represent a fundamental mechanism for the development of the AM network, since in natural situations such ability, together with the wide host range of AM fungi, could lead to the establishment of nonfinite hyphal webs, linking together many different plant species (Perry et al., 1989; Molina et al., 1992; Read, 1997). The ability of AM extraradical mycelium developing from different plants to form anastomosis, to exchange nuclei and to establish interconnections should be further investigated, to improve our understanding of the functional significance of the wood wide web. 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