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The incidence of arbuscular mycorrhiza in two submerged Isoëtes species
Aquatic Botany 94 (2011) 183–187
Contents lists available at ScienceDirect
Aquatic Botany journal homepage: www.elsevier.com/locate/aquabot
Short communication
The incidence of arbuscular mycorrhiza in two submerged Isoëtes species b,c ˇ Radka Sudová a,∗ , Jana Rydlová a , Martina Ctvrtlíková , Pavel Havránek d , Lubomír Adamec b a
Institute of Botany, Academy of Sciences of the Czech Republic, 252 43 Pr˚ uhonice, Czech Republic Institute of Botany, Academy of Sciences of the Czech Republic, 379 82 Tˇrebon, ˇ Czech Republic c ˇ Biology Centre, Academy of Sciences of the Czech Republic, Institute of Hydrobiology, 370 05 Ceské Budˇejovice, Czech Republic d Palack´ y University, Faculty of Science, Department of Botany, 783 71 Olomouc, Czech Republic b
a r t i c l e
i n f o
Article history: Received 28 September 2010 Received in revised form 2 February 2011 Accepted 8 February 2011 Available online 16 February 2011 Keywords: Arbuscular mycorrhizal fungi Aquatic macrophytes Dark septate endophytes Quillwort
a b s t r a c t We investigated the occurrence of arbuscular mycorrhizal fungi in the roots of Isoëtes lacustris and I. echinospora. These submerged lycopsids are the only macrophyte species inhabiting the bottom of two acidified glacial lakes in the Czech Republic. Arbuscular mycorrhizal (AM) fungi were detected in the roots of both species but the percentage of root colonization was both low and variable. Nevertheless, planting Littorella uniflora in the sediments from Isoëtes rhizosphere revealed high levels of viable AM propagules in both lakes. While AM colonization of Isoëtes roots did not exceed 25%, the average colonization of Littorella roots amounted to more than 80%. Although colonization of quillwort roots by AM fungi is evident, the taxonomic identity and role of these AM fungi in plant growth remain unclear. In addition to AM fungi, root-colonizing dark septate endophytic fungi were observed in both Isoëtes species. © 2011 Elsevier B.V. All rights reserved.
1. Introduction The view that the occurrence of arbuscular mycorrhiza (AM) was restricted to the terrestrial environment was revised in 1977 when AM structures were first observed in the roots of some aquatic plants from oligotrophic softwater lakes (Søndergaard and Lægaard, 1977). Since then, AM colonization has been discovered in the roots of a number of aquatic macrophytes, be they emergent, amphibious or submerged (e.g. Wigand and Stevenson, 1994; ˇ Beck-Nielsen and Madsen, 2001; Sraj-Krˇ ziˇc et al., 2006; Radhika and Rodrigues, 2007). Recently, de Maríns et al. (2009) and Baar et al. (2011) even reported AM structures in aquatic plants from the families which have non-mycorrhizal terrestrial species. The intensity of mycorrhizal colonization in water is, however, highly variable and in some plant groups (e.g. seagrasses) AM appears to be generally absent (Nielsen et al., 1999). Among the submerged aquatic macrophytes, the highest AM colonization has frequently been observed for isoetids inhabiting oligotrophic freshwater lakes (Clayton and Bagyaraj, 1984; Farmer, 1985; Wigand et al., 1998; Beck-Nielsen and Madsen, 2001). The term “isoetids” refers to a taxonomically heterogeneous group of submerged macrophytes with a rosette growth form and a welldeveloped root system which allows nutrient acquisition from the sediment. Their extensive lacunal system and permeable root surface allow isoetids to release oxygen into the rhizosphere. They
∗ Corresponding author. Tel.: +420 271015330; fax: +420 271015332. E-mail address: [email protected] (R. Sudová). 0304-3770/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.aquabot.2011.02.003
therefore have a large impact on the sediments, particularly on the redox potential and phosphorus availability (Christensen and Wigand, 1998; Smolders et al., 2002). Aerobic rhizosphere environments facilitate the formation of AM symbiosis because aerobic conditions are a prerequisite for the development of AM fungi (Beck-Nielsen and Madsen, 2001). Oxygen leakage into the sediment also contributes to phosphate limitation due to the formation of an iron hydroxide sheath around the roots which adsorbs phosphate (Christensen and Wigand, 1998). Symbiosis with AM fungi, with their far-reaching mycelium, was therefore assumed to be one of the adaptations which enabled isoetids to grow in these nutrientpoor environments. This hypothesis was supported by the results of Andersen and Andersen (2006) who recorded higher shoot biomass and tissue P and N contents in inoculated Littorella uniflora plants. The results on the mycorrhizal status of the nominate genus Isoëtes (quillwort), which are cosmopolitan heterosporous lycopsids encompassing aquatic, amphibious as well as terrestrial species, are inconsistent. Of the submerged Isoëtes species, Clayton and Bagyaraj (1984) observed AM colonization (structures nonspecified) in the roots of I. kirkii from 0.5 to 1 m lake depth and Wigand et al. (1998) reported the presence of AM structures (vesicles) in the roots of I. lacustris sampled from 4.6 m deep water. AM structures (including arbuscules) were also documented in the roots of submerged I. tuberculata (Sharma, 1998), while only hyphae and vesicles were observed in I. coromandelina (Radhika and Rodrigues, 2007). In contrast, Søndergaard and Laegaard (1977), Farmer (1985) and Nielsen et al. (2004) did not record AM colonization in I. lacustris growing at water depths of 0.3–1 m.
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In the Czech Republic, the genus Isoëtes is represented by two submerged species: I. lacustris and I. echinospora. Both species are critically endangered glacial relicts growing on the southern margin of their distribution range, each with only one population in ˇ Cerné Lake and Pleˇsné Lake, respectively (the Bohemian Forest). In these lakes, quillworts are the only submerged vascular plant species. Both quillwort populations were dramatically reduced due to strong acidification of the lake water caused by long-term industrial air pollution in the second half of the 20th century (Vrba et al., 2003). During the period of the strongest acidification, population recovery was not possible due to a poor survival rate of juvenile plants, the fine roots of which developed in the upper sediment layer with high concentrations of phytotoxic aluminium. The plant stands survived only due to the longevity of adult plants as their extensive root systems were preserved in deep sediment layers ˇ with circumneutral pH (Ctvrtlíková et al., 2009 and unpublished data). Recently, a progressive recovery of I. echinospora population related to a pH increase and a drop in aluminium concentration has ˇ been reported (Ctvrtlíková et al., 2009) ˇ It was proposed that quillwort survival in Pleˇsné and Cerné lakes could also be related to their potential symbiosis with naturally occurring mycorrhizal fungi. As no previous data are available in this respect, the aim of this study was to provide the primary information on AM status of I. echinospora and I. lacustris from the Bohemian Forest lakes. This is an essential prerequisite for the potential future monitoring of the development of AM symbiosis in the course of recovery of both lakes.
Table 1 Characteristics and available nutrient contents of the sediments collected from the rhizosphere (0–2 cm depth) of adult plants of I. echinospora in Pleˇsné Lake and I. ˇ lacustris in Cerné Lake in the 2004 summer season.
2. Material and methods
2.3. Sediment bioassay
2.1. Study sites
A bioassay was established to assess the relative abundance of AM propagules in the lake sediments. The sediments were collected at up to 4 cm depth directly from I. echinospora and I. lacustris stands. In each lake, the sediment was taken from ten sampling sites and mixed. The sediment mixtures in the final volume of 250 mL were then added to the respective 5.5-L plastic containers filled with Grodan rockwool cubes; a control was treated with sterile rainwater instead. L. uniflora was used as a model trap plant because of its faster growth rate and the better availability of plant material compared with Isoëtes. Each tray was planted with six AM-free rooted plantlets. The plants were grown amphibiously in a growth chamber (irradiance at the plant level 140 mol m−2 s−1 , photoperiod 16 h, 13 ◦ C/5 ◦ C day/night) and irrigated with natural sterile rainwater. After 12 months of growth, the plants were harvested and mycorrhizal colonization in their roots evaluated as described above.
ˇ Both study sites, i.e. Pleˇsné Lake and Cerné Lake, are glacial ˇ lakes situated in the Bohemian Forest (the Sumava Mountains, Czech Republic). They are in rather small and geologically sensitive ˇ catchments on a mica-schist (Pleˇsné Lake) or granitic (Cerné Lake) bedrock, both forested by Norway spruce. Pleˇsné Lake (48◦ 47 N, 13◦ 52 E, 1087 m a.s.l.) is a mesotrophic lake with an area of 7.6 ha and maximum depth of 19 m. The only submerged macrophyte species growing in the lake is I. echinospora, which inhabits an inshore area of ∼0.03 ha at depths ranging from 0.3 to 0.75 m. The stand of I. echinospora is usually ice-covered from December to April. The substrate is an aqueous sapropel with a high proportion ˇ of organic matter. Cerné Lake (49◦ 11 N, 13◦ 11 E, 1008 m a.s.l.) is oligotrophic, with an area of 18.8 ha and maximum depth of 40 m. It is again inhabited by only one submerged macrophyte species, I. lacustris, growing at depths of between 1 and 4 m, on a steep, rocky bottom covered by thin sandy (or gravel) sediment and detritus. For details on water characteristics in both lakes see Nedbalová et al. (2006). The chemical characteristics of the sediments are given in Table 1. 2.2. Field sampling of Isoëtes roots and the assessment of fungal colonization Sampling of I. echinospora and I. lacustris from Pleˇsné Lake and ˇ Cerné Lake was limited to ten adult plants per species due to conservation restrictions. I. echinospora and I. lacustris plants were sampled in September 2008 at water depths 0.4 and 2.5 m, respectively. From each plant, approximately ten roots were randomly selected from the extensive root system to avoid discrimination on the basis of root position/age. After the sediment was cleaned from the roots, they were carefully excised at the base and the plants were replanted afterwards. The roots were sectioned into pieces approximately 1 cm long and stained for subsequent microscopic
Parameter
Pleˇsné Lake
pH (H2 O) Organic matter (%) NH4 –N (mg kg−1 ) NO3 –N (mg kg−1 ) PO4 –P (mg kg−1 ) K (mg kg−1 ) Ca (mg kg−1 ) Mg (mg kg−1 )
4.94 59.6 86.5 0.22 0.06 48.6 119.0 32.0
± ± ± ± ± ± ± ±
0.11 2.1 25.7 0.04 0.01 7.7 29 8.4
ˇ Cerné Lake 5.05 8.6 15.6 0.41 0.05 15.6 11.2 5.3
± ± ± ± ± ± ± ±
0.06 1.5 3.0 0.05 0.01 2.8 3.7 0.9
All nutrients are expressed per sediment dry mass. N and P were eluted by 0.01 M CaCl2 , while cations by 0.5 M sodium acetate at pH 4.8. Means ± SE are shown; n = 6.
evaluation following the method of Koske and Gemma (1989) using 0.05% trypan blue in lactoglycerol. Root AM colonization (total, arbuscular, vesicular and hyphal) was examined using a magnified intersection method on 150 intersections at ×200 magnification according to McGonigle et al. (1990). Arbuscular and vesicular colonization was calculated as the percentage of intersections scored for the presence of arbuscules and vesicles, respectively. The hyphal colonization was calculated as the proportion of root length colonized by hyphae only. Total AM colonization provides information on the proportion of roots containing any AM structure. Similarly, the presence of microsclerocia or melanized septate hyphae of Dark Septate Endophytic fungi (DSE) was scored in each root sample.
2.4. Statistical analysis Data on the percentage of AM and DSE colonization in plant roots were arcsin transformed and assessed using one-way analysis of variance using Statistica 8.0 software. In the first series of observations, the difference in the level of root colonization between the two Isoëtes species was tested, while the sediments from the two lakes were compared in the bioassay. 3. Results The microscopic analyses revealed the presence of AM structures in the roots of both Isoëtes species (Table 2). However, the percentage of root length colonized was generally low (on average, 14% in I. echinospora and 4% in I. lacustris) and highly varied between individual samples. When both species were compared, I. echinospora showed significantly higher mycorrhizal colonization than I. lacustris (F1,18 = 9.1, P < 0.01). While all sampled I. echinospora
R. Sudová et al. / Aquatic Botany 94 (2011) 183–187 Table 2 The percentage of colonization by AM fungi and DSE in the roots of I. echinospora and ˇ I. lacustris from Pleˇsné Lake and Cerné Lake (field samples) and L. uniflora planted in the sterile substrate treated with lake sediments (sediment bioassay). Field samples AM colonization
I. echinospora
Total (%) Arbuscular (%) Vesicular (%) Hyphal (%) DSE colonization (%)
13.3 0.4 1.7 11.2 9.6
± ± ± ± ±
1.9 0.2 0.5 1.7 1.2
Sediment bioassay I. lacustris
Pleˇsné Lake
4.0 ± 2.4 0.0 0.5 ± 0.4 3.5 ± 0.9 3.2 ± 1.0
82.1 66.8 27.3 8.9 8.0
± ± ± ± ±
7.0 5.4 6.1 1.7 1.2
ˇ Cerné Lake 89.9 76.7 31.9 5.2 8.3
± ± ± ± ±
2.6 4.5 2.2 1.4 1.7
Field samples, n = 10; sediment bioassay, n = 6. Means ± SE are presented.
plants showed the presence of AM structures, 6 of the 10 specimens of I. lacustris were free of AM colonization. In both species, AM colonization was mostly formed by dichotomously branched non-septate hyphae, whereas vesicles and, in particular, arbuscules were very infrequent (Table 2). Globose and oval vesicles (diameter 25–65 m), which are characteristic of Glomus species, as well as lobed and rectangular vesicles (up to 100 m in length) typical of Acaulospora species were observed (Fig. 1). There was no obvious relation between host plant identity and the proportion of different vesicle types. On the other hand, the arbuscules were recorded only in the roots of I. echinospora. In addition to AM colonization, microsclerotia and hyphae of DSE were observed in the roots of
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both Isoëtes species (Table 2; Fig. 1), with I. echinospora showing significantly higher DSE colonization than the other species (F = 16.9, P < 0.001). DSE and AM colonization were often observed in the same roots. While AM colonization of Isoëtes roots from the field reached no more than 25%, a very high percentage of AM colonization (on average 86%) with frequent arbuscules and vesicles was observed in the roots of L. uniflora grown in a sterile substrate treated with lake sediments from Isoëtes rhizosphere (Table 2, Fig. 1). No traces of AM colonization were observed in the control plants. There were no significant differences observed in the total percentage root length colonized or arbuscular or vesicular colonizations between samples from the two lakes. As with field root samples, DSE colonization was also observed in the sediment bioassay, with a mean colonization reaching 8% (Table 2). 4. Discussion The present study demonstrated AM colonization in the roots of two submerged Isoëtes species, I. lacustris and I. echinospora. To the best of our knowledge, this is the first report on AM colonization in I. echinospora. With I. lacustris, no AM colonization was observed in three previous studies (Søndergaard and Lægaard, 1977; Farmer, 1985; Nielsen et al., 2004) but our results correspond with those of (Wigand et al., 1998) who reported the presence of small vesicles in the roots of I. lacustris from a Danish oligotrophic lake. Inconsistent
Fig. 1. Root colonization by arbuscular mycorrhizal fungi (A–E) and dark septate endophytes (F). A, B, C, F–I. echinospora, D–I. lacustris, E–L. uniflora.
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results on the AM status of I. lacustris may be partially caused by different abiotic conditions of sampling sites, such as trophic status, water depth, organic matter content and redox potential. The level of AM colonization in the present study does not seem to be related to the trophic status of the lake, with a higher colonization observed for I. echinospora from the mesotrophic lake than for I. lacustris from the oligotrophic lake. Nevertheless, we are aware that the relation between AM colonization and the lake trophic status cannot be interpreted unambiguosly, as differences other than trophic status exist between both lakes. No relationship between AM colonization of aquatic plants and lake trophic status was reported by Clayton and Bagyaraj (1984), Farmer (1985) or Wigand et al. (1998). On the other hand, Beck-Nielsen and Madsen (2001) recorded higher AM colonization in root samples from oligotrophic compared to eutrophic lakes. Lower AM colonization of I. lacustris in the present study may be related to its deeper stands. A decrease of AM colonization with an increase in water depth has previously been reported by Clayton and Bagyaraj (1984) and Wigand et al. (1998), likely due to lower redox potential, higher oxygenconsuming sediment organic content and higher phosphate levels in the pore water at deeper sites. Although this study showed AM colonization of both Isoëtes species, the levels of colonization were very low and the actual impact of AM symbiosis on plant growth and fitness remains questionable. It could be hypothesized that low colonization levels were caused by impairment of AM symbiosis by the adverse conditions of acidified lakes with pH ∼4.9 and mean concentration of ionic Al ∼200 g L−1 at the sampling time (Nedbalová et al., 2006). Nevertheless, this hypothesis does not seem plausible in the light of the results of the sediment bioassay. In contrast to low colonization levels in the Isoëtes field samples, very high AM colonization were observed in the roots of Littorella plants treated with lake sediments from the Isoëtes rhizosphere. As both lake bottoms are inhabited solely by Isoëtes as the only submerged macrophyte species, there is no doubt that the pool of viable AM propagules is maintained due to symbiosis with Isoëtes plants. In the presence of a more mycotrophic plant species, the initial amount of AM propagules was likely readily multiplied, resulting in higher root colonization in Littorella. The contrast in the percentage of AM colonization in Isoëtes and Littorella roots is likely caused by genus-specific differences, although the release from the stress conditions of the acidified environment might have contributed to higher colonization rates in the bioassay. While other isoetids such as Lobelia dortmanna or L. uniflora showed high AM colonization in all previously published studies (Søndergaard and Lægaard, 1977; Farmer, 1985; Wigand et al., 1998; Beck-Nielsen and Madsen, 2001; Nielsen et al., 2004; Baar et al., 2011), the results on AM status of Isoëtes species are inconsistent, possibly indicating lower significance of AM symbiosis for Isoëtes spp. growth and survival. Both L. uniflora and L. dortmanna are characterised by faster growth and smaller root systems when compared with I. lacustris and I. echinospora and, therefore, may be more dependent on nutrients supplied by the mycorrhizal fungi. Different levels of AM colonization in Isoëtes vs. Littorella plants that were observed in the present study may also be related to the difference in the presence of root hairs. While Littorella plants do not possess root hairs, both Isoëtes species, particularly juvenile plants, bear abundant and relatively long root ˇ hairs (Ctvrtlíková et al., 2009 and unpublished results). A trade-off between the investment into root hairs and AM symbiosis could be suggested based on the results of Beck-Nielsen and Madsen (2001). Our observation of dark septate endophytes in Isoëtes roots provides additional evidence that these root-colonizing endophytic fungi are native to aquatic environments. Although DSE were reported from many habitats worldwide, little is known about their ecological role, even in terrestrial ecosystems (Jumpponen and
Trappe, 1998; Mandyam and Jumpponen, 2005). Due to common occurrence of DSE under extreme environmental conditions, their important role in stress alleviation has been suggested (Mandyam and Jumpponen, 2005; Yuan et al., 2010). The attention that has been given to the occurrence of DSE in aquatic ecosystems was even lower than that afforded to AM fungi. Kai and Zhiwei (2006) were the first to record DSE colonization in a limited number of aquatic ˇ plants involved in their study. Sraj-Krˇ ziˇc et al. (2006) and de Maríns et al. (2009) reported the presence of microsclerotia in the roots of different submerged and emergent aquatic plants. As with previous observations (Kai and Zhiwei, 2006; de Maríns et al., 2009), co-colonization of the same roots by AM and DSE was observed in our study. To conlude, this study provides evidence for colonization of two submerged Isoëtes species, I. echinospora and I. lacustris, by arbuscular mycorrhizal fungi as well as by dark septate endophytes. The taxonomic identity of these fungal colonizers, together with their impact on host plant growth and fitness, still remain to be elucidated. Acknowledgements ´ The authors thank to Zuzana Sykorová, Petr Kohout and two anonymous reviewers for their valuable comments on the manuscript. Sincere thanks are due to Dr. Brian G. McMillan (Glasgow, Scotland) for correction of the language. Financial support of the Czech Science Foundation (projects P504/10/0781 and 206/07/1200), and the Research Programme of the Institute of Botany (AV0Z 60050516) is gratefully acknowledged. This study of Isoëtes populations in the Bohemian Forest was made under the Act No. 114/1992 Gazette on the Protection of Nature and the Landscape and the statutory exception Nr. 1677/04-620/308/04 given for the specially protected wild plants. References Andersen, F.Q., Andersen, T., 2006. Effects of arbuscular mycorrhizae on biomass and nutrients in the aquatic plant Littorella uniflora. Freshwater Biol. 51, 1623–1633. Baar, J., Paradi, I., Lucassen, E.C.H.E.T., Hudson-Edwards, K.A., Redecker, D., Roelofs, J.G.M., Smolders, A.J.P., 2011. Molecular analysis of AMF diversity in aquatic macrophytes: A comparison of oligotrophic and utra-oligotrophic lakes. Aquat. Bot. 94, 53–61. Beck-Nielsen, D., Madsen, T.V., 2001. Occurrence of vesicular–arbuscular mycorrhiza in aquatic macrophytes from lakes and streams. Aquat. Bot. 71, 141–148. Christensen, K.K., Wigand, C., 1998. Formation of root plaques and their influence on tissue phosphorus content in Lobelia dortmanna. Aquat. Bot. 61, 111–122. Clayton, J.S., Bagyaraj, D.J., 1984. Vesicular–arbuscular mycorrhizas in submerged aquatic plants of New Zealand. Aquat. Bot. 19, 251–262. ˇ Ctvrtlíková, M., Vrba, J., Znachor, P., Hekera, P., 2009. The effects of aluminium toxicity and low pH on the early development of Isoëtes echinospora. Preslia 81, 135–149. de Maríns, J.F., Carrenho, R., Thomaz, S.M., 2009. Occurrence and coexistence of arbuscular mycorrhizal fungi and dark septate fungi in aquatic macrophytes in a tropical river–floodplain system. Aquat. Bot. 91, 13–19. Farmer, A.M., 1985. The occurrence of vesicular–arbuscular mykorrhiza in isoetidtype submerged aquatic macrophytes under naturally varying conditions. Aquat. Bot. 21, 245–249. Jumpponen, A., Trappe, J.M., 1998. Dark septate endophytes: a review of facultative biotrophic root-colonizing fungi. New Phytol. 140, 295–310. Kai, W., Zhiwei, Z., 2006. Occurrence of arbuscular mycorrhizas and dark septate endophytes in hydrophytes from lakes and streams in southwest China. Int. Rev. Hydrobiol. 91, 29–37. Koske, R.E., Gemma, J.N., 1989. A modified procedure for staining roots to detect VA mycorrhizas. Mycol. Res. 92, 486–505. Mandyam, K., Jumpponen, A., 2005. Seeking the elusive function of the root colonising dark septate endophytic fungi. Stud. Mycol. 53, 173–189. McGonigle, T.P., Miller, M.H., Evans, D.G., Fairchild, G.L., Swan, J.A., 1990. A new method which gives an objective measure of colonization of roots by vesiculararbuscular mycorrhizal fungi. New Phytol. 115, 495–501. Nedbalová, L., Vrba, J., Fott, J., Kohout, L., Kopáˇcek, J., Macek, M., Soldán, T., 2006. Biological recovery of the Bohemina Forest lakes from acidification. Biologia 61, S453–S465. Nielsen, S.L., Thingstrup, I., Wigand, C., 1999. Apparent lack of vesicular–arbuscular mycorrhiza (VAM) in the seagrasses Zostera marina L. and Thalassia testudinum Banks ex König. Aquat. Bot. 63, 261–266.
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Contents lists available at ScienceDirect
Aquatic Botany journal homepage: www.elsevier.com/locate/aquabot
Short communication
The incidence of arbuscular mycorrhiza in two submerged Isoëtes species b,c ˇ Radka Sudová a,∗ , Jana Rydlová a , Martina Ctvrtlíková , Pavel Havránek d , Lubomír Adamec b a
Institute of Botany, Academy of Sciences of the Czech Republic, 252 43 Pr˚ uhonice, Czech Republic Institute of Botany, Academy of Sciences of the Czech Republic, 379 82 Tˇrebon, ˇ Czech Republic c ˇ Biology Centre, Academy of Sciences of the Czech Republic, Institute of Hydrobiology, 370 05 Ceské Budˇejovice, Czech Republic d Palack´ y University, Faculty of Science, Department of Botany, 783 71 Olomouc, Czech Republic b
a r t i c l e
i n f o
Article history: Received 28 September 2010 Received in revised form 2 February 2011 Accepted 8 February 2011 Available online 16 February 2011 Keywords: Arbuscular mycorrhizal fungi Aquatic macrophytes Dark septate endophytes Quillwort
a b s t r a c t We investigated the occurrence of arbuscular mycorrhizal fungi in the roots of Isoëtes lacustris and I. echinospora. These submerged lycopsids are the only macrophyte species inhabiting the bottom of two acidified glacial lakes in the Czech Republic. Arbuscular mycorrhizal (AM) fungi were detected in the roots of both species but the percentage of root colonization was both low and variable. Nevertheless, planting Littorella uniflora in the sediments from Isoëtes rhizosphere revealed high levels of viable AM propagules in both lakes. While AM colonization of Isoëtes roots did not exceed 25%, the average colonization of Littorella roots amounted to more than 80%. Although colonization of quillwort roots by AM fungi is evident, the taxonomic identity and role of these AM fungi in plant growth remain unclear. In addition to AM fungi, root-colonizing dark septate endophytic fungi were observed in both Isoëtes species. © 2011 Elsevier B.V. All rights reserved.
1. Introduction The view that the occurrence of arbuscular mycorrhiza (AM) was restricted to the terrestrial environment was revised in 1977 when AM structures were first observed in the roots of some aquatic plants from oligotrophic softwater lakes (Søndergaard and Lægaard, 1977). Since then, AM colonization has been discovered in the roots of a number of aquatic macrophytes, be they emergent, amphibious or submerged (e.g. Wigand and Stevenson, 1994; ˇ Beck-Nielsen and Madsen, 2001; Sraj-Krˇ ziˇc et al., 2006; Radhika and Rodrigues, 2007). Recently, de Maríns et al. (2009) and Baar et al. (2011) even reported AM structures in aquatic plants from the families which have non-mycorrhizal terrestrial species. The intensity of mycorrhizal colonization in water is, however, highly variable and in some plant groups (e.g. seagrasses) AM appears to be generally absent (Nielsen et al., 1999). Among the submerged aquatic macrophytes, the highest AM colonization has frequently been observed for isoetids inhabiting oligotrophic freshwater lakes (Clayton and Bagyaraj, 1984; Farmer, 1985; Wigand et al., 1998; Beck-Nielsen and Madsen, 2001). The term “isoetids” refers to a taxonomically heterogeneous group of submerged macrophytes with a rosette growth form and a welldeveloped root system which allows nutrient acquisition from the sediment. Their extensive lacunal system and permeable root surface allow isoetids to release oxygen into the rhizosphere. They
∗ Corresponding author. Tel.: +420 271015330; fax: +420 271015332. E-mail address: [email protected] (R. Sudová). 0304-3770/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.aquabot.2011.02.003
therefore have a large impact on the sediments, particularly on the redox potential and phosphorus availability (Christensen and Wigand, 1998; Smolders et al., 2002). Aerobic rhizosphere environments facilitate the formation of AM symbiosis because aerobic conditions are a prerequisite for the development of AM fungi (Beck-Nielsen and Madsen, 2001). Oxygen leakage into the sediment also contributes to phosphate limitation due to the formation of an iron hydroxide sheath around the roots which adsorbs phosphate (Christensen and Wigand, 1998). Symbiosis with AM fungi, with their far-reaching mycelium, was therefore assumed to be one of the adaptations which enabled isoetids to grow in these nutrientpoor environments. This hypothesis was supported by the results of Andersen and Andersen (2006) who recorded higher shoot biomass and tissue P and N contents in inoculated Littorella uniflora plants. The results on the mycorrhizal status of the nominate genus Isoëtes (quillwort), which are cosmopolitan heterosporous lycopsids encompassing aquatic, amphibious as well as terrestrial species, are inconsistent. Of the submerged Isoëtes species, Clayton and Bagyaraj (1984) observed AM colonization (structures nonspecified) in the roots of I. kirkii from 0.5 to 1 m lake depth and Wigand et al. (1998) reported the presence of AM structures (vesicles) in the roots of I. lacustris sampled from 4.6 m deep water. AM structures (including arbuscules) were also documented in the roots of submerged I. tuberculata (Sharma, 1998), while only hyphae and vesicles were observed in I. coromandelina (Radhika and Rodrigues, 2007). In contrast, Søndergaard and Laegaard (1977), Farmer (1985) and Nielsen et al. (2004) did not record AM colonization in I. lacustris growing at water depths of 0.3–1 m.
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In the Czech Republic, the genus Isoëtes is represented by two submerged species: I. lacustris and I. echinospora. Both species are critically endangered glacial relicts growing on the southern margin of their distribution range, each with only one population in ˇ Cerné Lake and Pleˇsné Lake, respectively (the Bohemian Forest). In these lakes, quillworts are the only submerged vascular plant species. Both quillwort populations were dramatically reduced due to strong acidification of the lake water caused by long-term industrial air pollution in the second half of the 20th century (Vrba et al., 2003). During the period of the strongest acidification, population recovery was not possible due to a poor survival rate of juvenile plants, the fine roots of which developed in the upper sediment layer with high concentrations of phytotoxic aluminium. The plant stands survived only due to the longevity of adult plants as their extensive root systems were preserved in deep sediment layers ˇ with circumneutral pH (Ctvrtlíková et al., 2009 and unpublished data). Recently, a progressive recovery of I. echinospora population related to a pH increase and a drop in aluminium concentration has ˇ been reported (Ctvrtlíková et al., 2009) ˇ It was proposed that quillwort survival in Pleˇsné and Cerné lakes could also be related to their potential symbiosis with naturally occurring mycorrhizal fungi. As no previous data are available in this respect, the aim of this study was to provide the primary information on AM status of I. echinospora and I. lacustris from the Bohemian Forest lakes. This is an essential prerequisite for the potential future monitoring of the development of AM symbiosis in the course of recovery of both lakes.
Table 1 Characteristics and available nutrient contents of the sediments collected from the rhizosphere (0–2 cm depth) of adult plants of I. echinospora in Pleˇsné Lake and I. ˇ lacustris in Cerné Lake in the 2004 summer season.
2. Material and methods
2.3. Sediment bioassay
2.1. Study sites
A bioassay was established to assess the relative abundance of AM propagules in the lake sediments. The sediments were collected at up to 4 cm depth directly from I. echinospora and I. lacustris stands. In each lake, the sediment was taken from ten sampling sites and mixed. The sediment mixtures in the final volume of 250 mL were then added to the respective 5.5-L plastic containers filled with Grodan rockwool cubes; a control was treated with sterile rainwater instead. L. uniflora was used as a model trap plant because of its faster growth rate and the better availability of plant material compared with Isoëtes. Each tray was planted with six AM-free rooted plantlets. The plants were grown amphibiously in a growth chamber (irradiance at the plant level 140 mol m−2 s−1 , photoperiod 16 h, 13 ◦ C/5 ◦ C day/night) and irrigated with natural sterile rainwater. After 12 months of growth, the plants were harvested and mycorrhizal colonization in their roots evaluated as described above.
ˇ Both study sites, i.e. Pleˇsné Lake and Cerné Lake, are glacial ˇ lakes situated in the Bohemian Forest (the Sumava Mountains, Czech Republic). They are in rather small and geologically sensitive ˇ catchments on a mica-schist (Pleˇsné Lake) or granitic (Cerné Lake) bedrock, both forested by Norway spruce. Pleˇsné Lake (48◦ 47 N, 13◦ 52 E, 1087 m a.s.l.) is a mesotrophic lake with an area of 7.6 ha and maximum depth of 19 m. The only submerged macrophyte species growing in the lake is I. echinospora, which inhabits an inshore area of ∼0.03 ha at depths ranging from 0.3 to 0.75 m. The stand of I. echinospora is usually ice-covered from December to April. The substrate is an aqueous sapropel with a high proportion ˇ of organic matter. Cerné Lake (49◦ 11 N, 13◦ 11 E, 1008 m a.s.l.) is oligotrophic, with an area of 18.8 ha and maximum depth of 40 m. It is again inhabited by only one submerged macrophyte species, I. lacustris, growing at depths of between 1 and 4 m, on a steep, rocky bottom covered by thin sandy (or gravel) sediment and detritus. For details on water characteristics in both lakes see Nedbalová et al. (2006). The chemical characteristics of the sediments are given in Table 1. 2.2. Field sampling of Isoëtes roots and the assessment of fungal colonization Sampling of I. echinospora and I. lacustris from Pleˇsné Lake and ˇ Cerné Lake was limited to ten adult plants per species due to conservation restrictions. I. echinospora and I. lacustris plants were sampled in September 2008 at water depths 0.4 and 2.5 m, respectively. From each plant, approximately ten roots were randomly selected from the extensive root system to avoid discrimination on the basis of root position/age. After the sediment was cleaned from the roots, they were carefully excised at the base and the plants were replanted afterwards. The roots were sectioned into pieces approximately 1 cm long and stained for subsequent microscopic
Parameter
Pleˇsné Lake
pH (H2 O) Organic matter (%) NH4 –N (mg kg−1 ) NO3 –N (mg kg−1 ) PO4 –P (mg kg−1 ) K (mg kg−1 ) Ca (mg kg−1 ) Mg (mg kg−1 )
4.94 59.6 86.5 0.22 0.06 48.6 119.0 32.0
± ± ± ± ± ± ± ±
0.11 2.1 25.7 0.04 0.01 7.7 29 8.4
ˇ Cerné Lake 5.05 8.6 15.6 0.41 0.05 15.6 11.2 5.3
± ± ± ± ± ± ± ±
0.06 1.5 3.0 0.05 0.01 2.8 3.7 0.9
All nutrients are expressed per sediment dry mass. N and P were eluted by 0.01 M CaCl2 , while cations by 0.5 M sodium acetate at pH 4.8. Means ± SE are shown; n = 6.
evaluation following the method of Koske and Gemma (1989) using 0.05% trypan blue in lactoglycerol. Root AM colonization (total, arbuscular, vesicular and hyphal) was examined using a magnified intersection method on 150 intersections at ×200 magnification according to McGonigle et al. (1990). Arbuscular and vesicular colonization was calculated as the percentage of intersections scored for the presence of arbuscules and vesicles, respectively. The hyphal colonization was calculated as the proportion of root length colonized by hyphae only. Total AM colonization provides information on the proportion of roots containing any AM structure. Similarly, the presence of microsclerocia or melanized septate hyphae of Dark Septate Endophytic fungi (DSE) was scored in each root sample.
2.4. Statistical analysis Data on the percentage of AM and DSE colonization in plant roots were arcsin transformed and assessed using one-way analysis of variance using Statistica 8.0 software. In the first series of observations, the difference in the level of root colonization between the two Isoëtes species was tested, while the sediments from the two lakes were compared in the bioassay. 3. Results The microscopic analyses revealed the presence of AM structures in the roots of both Isoëtes species (Table 2). However, the percentage of root length colonized was generally low (on average, 14% in I. echinospora and 4% in I. lacustris) and highly varied between individual samples. When both species were compared, I. echinospora showed significantly higher mycorrhizal colonization than I. lacustris (F1,18 = 9.1, P < 0.01). While all sampled I. echinospora
R. Sudová et al. / Aquatic Botany 94 (2011) 183–187 Table 2 The percentage of colonization by AM fungi and DSE in the roots of I. echinospora and ˇ I. lacustris from Pleˇsné Lake and Cerné Lake (field samples) and L. uniflora planted in the sterile substrate treated with lake sediments (sediment bioassay). Field samples AM colonization
I. echinospora
Total (%) Arbuscular (%) Vesicular (%) Hyphal (%) DSE colonization (%)
13.3 0.4 1.7 11.2 9.6
± ± ± ± ±
1.9 0.2 0.5 1.7 1.2
Sediment bioassay I. lacustris
Pleˇsné Lake
4.0 ± 2.4 0.0 0.5 ± 0.4 3.5 ± 0.9 3.2 ± 1.0
82.1 66.8 27.3 8.9 8.0
± ± ± ± ±
7.0 5.4 6.1 1.7 1.2
ˇ Cerné Lake 89.9 76.7 31.9 5.2 8.3
± ± ± ± ±
2.6 4.5 2.2 1.4 1.7
Field samples, n = 10; sediment bioassay, n = 6. Means ± SE are presented.
plants showed the presence of AM structures, 6 of the 10 specimens of I. lacustris were free of AM colonization. In both species, AM colonization was mostly formed by dichotomously branched non-septate hyphae, whereas vesicles and, in particular, arbuscules were very infrequent (Table 2). Globose and oval vesicles (diameter 25–65 m), which are characteristic of Glomus species, as well as lobed and rectangular vesicles (up to 100 m in length) typical of Acaulospora species were observed (Fig. 1). There was no obvious relation between host plant identity and the proportion of different vesicle types. On the other hand, the arbuscules were recorded only in the roots of I. echinospora. In addition to AM colonization, microsclerotia and hyphae of DSE were observed in the roots of
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both Isoëtes species (Table 2; Fig. 1), with I. echinospora showing significantly higher DSE colonization than the other species (F = 16.9, P < 0.001). DSE and AM colonization were often observed in the same roots. While AM colonization of Isoëtes roots from the field reached no more than 25%, a very high percentage of AM colonization (on average 86%) with frequent arbuscules and vesicles was observed in the roots of L. uniflora grown in a sterile substrate treated with lake sediments from Isoëtes rhizosphere (Table 2, Fig. 1). No traces of AM colonization were observed in the control plants. There were no significant differences observed in the total percentage root length colonized or arbuscular or vesicular colonizations between samples from the two lakes. As with field root samples, DSE colonization was also observed in the sediment bioassay, with a mean colonization reaching 8% (Table 2). 4. Discussion The present study demonstrated AM colonization in the roots of two submerged Isoëtes species, I. lacustris and I. echinospora. To the best of our knowledge, this is the first report on AM colonization in I. echinospora. With I. lacustris, no AM colonization was observed in three previous studies (Søndergaard and Lægaard, 1977; Farmer, 1985; Nielsen et al., 2004) but our results correspond with those of (Wigand et al., 1998) who reported the presence of small vesicles in the roots of I. lacustris from a Danish oligotrophic lake. Inconsistent
Fig. 1. Root colonization by arbuscular mycorrhizal fungi (A–E) and dark septate endophytes (F). A, B, C, F–I. echinospora, D–I. lacustris, E–L. uniflora.
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results on the AM status of I. lacustris may be partially caused by different abiotic conditions of sampling sites, such as trophic status, water depth, organic matter content and redox potential. The level of AM colonization in the present study does not seem to be related to the trophic status of the lake, with a higher colonization observed for I. echinospora from the mesotrophic lake than for I. lacustris from the oligotrophic lake. Nevertheless, we are aware that the relation between AM colonization and the lake trophic status cannot be interpreted unambiguosly, as differences other than trophic status exist between both lakes. No relationship between AM colonization of aquatic plants and lake trophic status was reported by Clayton and Bagyaraj (1984), Farmer (1985) or Wigand et al. (1998). On the other hand, Beck-Nielsen and Madsen (2001) recorded higher AM colonization in root samples from oligotrophic compared to eutrophic lakes. Lower AM colonization of I. lacustris in the present study may be related to its deeper stands. A decrease of AM colonization with an increase in water depth has previously been reported by Clayton and Bagyaraj (1984) and Wigand et al. (1998), likely due to lower redox potential, higher oxygenconsuming sediment organic content and higher phosphate levels in the pore water at deeper sites. Although this study showed AM colonization of both Isoëtes species, the levels of colonization were very low and the actual impact of AM symbiosis on plant growth and fitness remains questionable. It could be hypothesized that low colonization levels were caused by impairment of AM symbiosis by the adverse conditions of acidified lakes with pH ∼4.9 and mean concentration of ionic Al ∼200 g L−1 at the sampling time (Nedbalová et al., 2006). Nevertheless, this hypothesis does not seem plausible in the light of the results of the sediment bioassay. In contrast to low colonization levels in the Isoëtes field samples, very high AM colonization were observed in the roots of Littorella plants treated with lake sediments from the Isoëtes rhizosphere. As both lake bottoms are inhabited solely by Isoëtes as the only submerged macrophyte species, there is no doubt that the pool of viable AM propagules is maintained due to symbiosis with Isoëtes plants. In the presence of a more mycotrophic plant species, the initial amount of AM propagules was likely readily multiplied, resulting in higher root colonization in Littorella. The contrast in the percentage of AM colonization in Isoëtes and Littorella roots is likely caused by genus-specific differences, although the release from the stress conditions of the acidified environment might have contributed to higher colonization rates in the bioassay. While other isoetids such as Lobelia dortmanna or L. uniflora showed high AM colonization in all previously published studies (Søndergaard and Lægaard, 1977; Farmer, 1985; Wigand et al., 1998; Beck-Nielsen and Madsen, 2001; Nielsen et al., 2004; Baar et al., 2011), the results on AM status of Isoëtes species are inconsistent, possibly indicating lower significance of AM symbiosis for Isoëtes spp. growth and survival. Both L. uniflora and L. dortmanna are characterised by faster growth and smaller root systems when compared with I. lacustris and I. echinospora and, therefore, may be more dependent on nutrients supplied by the mycorrhizal fungi. Different levels of AM colonization in Isoëtes vs. Littorella plants that were observed in the present study may also be related to the difference in the presence of root hairs. While Littorella plants do not possess root hairs, both Isoëtes species, particularly juvenile plants, bear abundant and relatively long root ˇ hairs (Ctvrtlíková et al., 2009 and unpublished results). A trade-off between the investment into root hairs and AM symbiosis could be suggested based on the results of Beck-Nielsen and Madsen (2001). Our observation of dark septate endophytes in Isoëtes roots provides additional evidence that these root-colonizing endophytic fungi are native to aquatic environments. Although DSE were reported from many habitats worldwide, little is known about their ecological role, even in terrestrial ecosystems (Jumpponen and
Trappe, 1998; Mandyam and Jumpponen, 2005). Due to common occurrence of DSE under extreme environmental conditions, their important role in stress alleviation has been suggested (Mandyam and Jumpponen, 2005; Yuan et al., 2010). The attention that has been given to the occurrence of DSE in aquatic ecosystems was even lower than that afforded to AM fungi. Kai and Zhiwei (2006) were the first to record DSE colonization in a limited number of aquatic ˇ plants involved in their study. Sraj-Krˇ ziˇc et al. (2006) and de Maríns et al. (2009) reported the presence of microsclerotia in the roots of different submerged and emergent aquatic plants. As with previous observations (Kai and Zhiwei, 2006; de Maríns et al., 2009), co-colonization of the same roots by AM and DSE was observed in our study. To conlude, this study provides evidence for colonization of two submerged Isoëtes species, I. echinospora and I. lacustris, by arbuscular mycorrhizal fungi as well as by dark septate endophytes. The taxonomic identity of these fungal colonizers, together with their impact on host plant growth and fitness, still remain to be elucidated. Acknowledgements ´ The authors thank to Zuzana Sykorová, Petr Kohout and two anonymous reviewers for their valuable comments on the manuscript. Sincere thanks are due to Dr. Brian G. McMillan (Glasgow, Scotland) for correction of the language. Financial support of the Czech Science Foundation (projects P504/10/0781 and 206/07/1200), and the Research Programme of the Institute of Botany (AV0Z 60050516) is gratefully acknowledged. This study of Isoëtes populations in the Bohemian Forest was made under the Act No. 114/1992 Gazette on the Protection of Nature and the Landscape and the statutory exception Nr. 1677/04-620/308/04 given for the specially protected wild plants. References Andersen, F.Q., Andersen, T., 2006. Effects of arbuscular mycorrhizae on biomass and nutrients in the aquatic plant Littorella uniflora. Freshwater Biol. 51, 1623–1633. Baar, J., Paradi, I., Lucassen, E.C.H.E.T., Hudson-Edwards, K.A., Redecker, D., Roelofs, J.G.M., Smolders, A.J.P., 2011. Molecular analysis of AMF diversity in aquatic macrophytes: A comparison of oligotrophic and utra-oligotrophic lakes. Aquat. Bot. 94, 53–61. Beck-Nielsen, D., Madsen, T.V., 2001. Occurrence of vesicular–arbuscular mycorrhiza in aquatic macrophytes from lakes and streams. Aquat. Bot. 71, 141–148. Christensen, K.K., Wigand, C., 1998. Formation of root plaques and their influence on tissue phosphorus content in Lobelia dortmanna. Aquat. Bot. 61, 111–122. Clayton, J.S., Bagyaraj, D.J., 1984. Vesicular–arbuscular mycorrhizas in submerged aquatic plants of New Zealand. Aquat. Bot. 19, 251–262. ˇ Ctvrtlíková, M., Vrba, J., Znachor, P., Hekera, P., 2009. The effects of aluminium toxicity and low pH on the early development of Isoëtes echinospora. Preslia 81, 135–149. de Maríns, J.F., Carrenho, R., Thomaz, S.M., 2009. Occurrence and coexistence of arbuscular mycorrhizal fungi and dark septate fungi in aquatic macrophytes in a tropical river–floodplain system. Aquat. Bot. 91, 13–19. Farmer, A.M., 1985. The occurrence of vesicular–arbuscular mykorrhiza in isoetidtype submerged aquatic macrophytes under naturally varying conditions. Aquat. Bot. 21, 245–249. Jumpponen, A., Trappe, J.M., 1998. Dark septate endophytes: a review of facultative biotrophic root-colonizing fungi. New Phytol. 140, 295–310. Kai, W., Zhiwei, Z., 2006. Occurrence of arbuscular mycorrhizas and dark septate endophytes in hydrophytes from lakes and streams in southwest China. Int. Rev. Hydrobiol. 91, 29–37. Koske, R.E., Gemma, J.N., 1989. A modified procedure for staining roots to detect VA mycorrhizas. Mycol. Res. 92, 486–505. Mandyam, K., Jumpponen, A., 2005. Seeking the elusive function of the root colonising dark septate endophytic fungi. Stud. Mycol. 53, 173–189. McGonigle, T.P., Miller, M.H., Evans, D.G., Fairchild, G.L., Swan, J.A., 1990. A new method which gives an objective measure of colonization of roots by vesiculararbuscular mycorrhizal fungi. New Phytol. 115, 495–501. Nedbalová, L., Vrba, J., Fott, J., Kohout, L., Kopáˇcek, J., Macek, M., Soldán, T., 2006. Biological recovery of the Bohemina Forest lakes from acidification. Biologia 61, S453–S465. Nielsen, S.L., Thingstrup, I., Wigand, C., 1999. Apparent lack of vesicular–arbuscular mycorrhiza (VAM) in the seagrasses Zostera marina L. and Thalassia testudinum Banks ex König. Aquat. Bot. 63, 261–266.
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