1 Introduction

Since the early 20th Century, lichenologists have developed numerous and varied methods for isolating the different lichen photobionts. When the aim is to gain insight into the complex relationships between the lichenized algae and the mycobiont or to analyze the physiological responses of each partner to environmental factors, large scale rapid isolation methods are preferred. These methods essentially involve the homogenization of the lichen thalli, followed by separation of the photobiont from the mycobiont and fragments of thalli, using differential centrifugation (Richardson 1971), gradient centrifugation on CsCl2/KI (Ascaso 1980) or on Percoll® gradients (Calatayud et al. 2001), and/or filtering (Weissman et al. 2005). These methods are useful for studying algal metabolites or enzymatic activities of the lichenized photobionts. However, the purity of the isolated photobionts is not good enough to enable axenic cultures to be established easily.

Axenic cultures are required for algal taxonomy or when studies focus on the biochemical, molecular or physiological behaviour of microscopic algae outside the symbiosis. One of the most popular isolation methods to obtain axenic cultures was developed by Ahmadjian (1967a, b) and consists of cutting the lichen photobiont layer into thin slices, then grinding it between two glass slides and finally spreading the homogenate on a solid agar medium. Variations of this method include the micropipette method (Ahmadjian 1967a, b), the spray method (Wiedeman et al. 1964) and the cutting method (Yoshimura et al. 2002). However all are rather tedious and several months are required after isolation to obtain clones. The single-cell manipulator method used by Beck and Koop (2001) allows the straight forward attainment of clones but it involves a complex instrumental setup.

The aim of our study was to provide a simple, effective and fast method to isolate and propagate lichen photobionts, regardless of the amount of lichen thallus available or its morphology (crustose, foliose or fruticose).

2 Material and methods

2.1 Lichen material

Seven species of fruticose, foliose and crustose lichens were collected from different locations (Table 1) that represented a wide range of European bioclimates (thermo- and ombrotypes). The material was left to dry out in open air, in the shade, for one day and stored at −20°C until needed for experimentation.

Table 1 Lichens used in our study, with respective sampling site, latitude and longitude, collection data, altitude (m above sea level)
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2.2 Isolation methods–micromethod

The micromethod for isolation of lichen photobions involved starting with 15–25 mg (DW) of lichen material were washed first in tap water, and then slowly stirred in sterile distilled water in a bucket for 5 min. The fragments of thalli were homogenised in an sterile eppendorf tube with a pellet pestle and resuspended in sterile 1 ml of isotonic buffer (0.3 M sorbitol in 50 mM HEPES pH 7.5) (Good et al. 1966; Zapata et al. 2007). After filtration through sterile muslin, the filtrate was centrifuged at 490 × g in a bench-top microcentrifuge (Micro 20, Hettich, Germany) for 5 min. The pellet was resuspended in 200 μl (m) of sterile isotonic buffer and then loaded on 1.5 ml, of sterile 80 % Percoll ® in isotonic buffer. After centrifugation at 10000 × g for 10 min a clear green layer was present near the top of the eppendorf tube and some grey particles and pellet at the bottom of the tube (Fig. 1). The green layer was recovered (ca. 400 μl) avoiding any of the upper interphase. The green layer was , diluted 2-fold with sterile distilled water and centrifuged at 1000 × g for 10 min. The supernatant was discarded and the pellet resuspended in 2 ml of sterile distilled water and a drop of Tween 20 was added. The resulting suspension was sonicated at 40 KHz (Elma Transsonic Digital 470 T, 140% ultrasound power) for 1 min and again centrifuged at 490 × g for 5 min. This treatment was repeated five times. The final pellet containing the isolated algal cells was resuspended in 1 ml of sterile distilled water and the cellular density was assessed using a Neubauer hematocytometer. This micromethod was scaled up to a macromethod, which allowed preparation of large amounts of photobiont cells.

Fig. 1
figure 1

Separation of Ramalina farinacea fractions after centrifugation of the extract of thalli at 10,000 × g for 10 min on 40 ml (macromethod), or 1.5 ml (micromethod) of 80 % Percoll ® in isotonic buffer. A–D, each optical micrograph refers to the corresponding Percoll layer; A and C phase contrast microscopy. Scale = 15 μm. * Not real size

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2.3 Isolation methods–macromethod

In the macromethod, one to two g DW of lichen thallus were homogenised with a mortar and pestle in 20 ml of sterile isotonic buffer. The steps following were similar to the micromethod, but the volume for resuspension of the first pellet was 1 ml. The second centrifugation step was carried out on 40 ml of sterile 80% Percoll in isotonic buffer using a fixed-angle rotor (221.22 V01/V02, Hermle, Germany) After this centrifugation step in the macromethod, four layers were visible: a) a 2–3 ml dark green supernatant at the top of the tube on the Percoll layer; b) a large and diffuse light green layer in the upper part of the Percoll gradient; c) a thick layer at the bottom of the tube and d) a grey pellet (Fig. 1). Five millilitres of the “b” layer were recovered and the subsequent isolation steps for this layer were identical to those described for the micromethod.

2.4 Culture conditions and photobiont identification

The algal suspensions were diluted with sterile distilled water to a final cell density of (5 103 cells ml−1) and 50 μl of this suspension were spread on sterile 1.5 % agar 3xN (meaning three times more nitrogen content in the form of Na NO3) Bold’s Basal Media (3NBBM) (Bischoff and Bold 1963) in each of five Petri dishes using the streak method and sterile technique. The isolated algae were cultured under 15 μmol m−2 s−1 (PPFD) with a 12 h photoperiod at 17°C. The number of algal colonies growing on each plate was counted after 45 days. Ten colonies were selected under the stereo-microscope and subcultured onto Petri dishes containing 1.5 % agar 3NBBM medium supplemented with glucose (20 g l−1) and casein (10 g l−1) (Ahmadjian 1967a) using a sterile toothpick.

DNA isolation from the algae strains and PCR was performed following the conditions of De Nova et al. (2005) with the ITS primers ITS1T and ITS4T (Kroken and Taylor 2000).

3 Results and discussion

The micromethod for lichen algae isolation was first developed employing Ramalina farinacea as starting material. It was found that after centrifugation in Percoll, fungal contamination was practically eliminated: hyphae fragments were less than 1%, most of them tightly adherant to the Trebouxia cell walls. Another important point was that only single cells remained in the Percoll® algal layer, while cell aggregates moved down (Fig. 1). This also facilitated the selection and development of monoclonal strains of photobionts on the solid agar cultures. Phase–contrast microscopy revealed a large number of very small particles (<1 μm) (around 50 times higher than the photobiont numbers) 432 106 particles ml−1 and 8.80 106 cel ml−1, respectively. However, after ultrasonication and Twin20 treatment, the number of small particles associated with the photobiont cells decreased, while, the number of algal cells remained unchanged.

The effectiveness of the “cleaning” steps in removing bacterial contamination was tested with the lichens Parmotrema perlatum and Xanthoparmelia tinctina. When Percoll gradient centrifugation was only applied to the isolation of P. perlatum and X. tinctina photobionts, a high amount of bacterial contamination (18.3 ± 2.1 and 132.8 ± 14.2 colonies per plate, respectively) was detected. However, after sonication and Tween 20 treatment of the algae isolated from Percoll gradient, the number of bacterial colonies in the cultures of algae isolated was reduced ca. 75–80%, but no differences were observed in the number of algal colonies. Non-ionic detergents can be used as surfactants in cultures of single-celled or colonial species to separate attached contaminants in association with sonication and centrifugation (Wiedeman et al. 1964; Guillard 2005).

The isolation yield obtained using the micromethod was from 14 103 cells per mg DW for Ramalina farinacea to 54 103 cells per mg DW for Acarospora hilaris (Table 2). These algal cells were successfully cultured on 1.5 % agar 3×N Bold’s Basal Media (3NBBM) (Bischoff and Bold 1963). The number of colonies developed per plate and the colony yield (colonies per 100 cells inoculated) are shown in Table 2. The number of colonies per plate varied greatly among different species, from about 20 colonies (colony yield 8.05 %) for Teloschistes chrysophthalmus, to 128 colonies (colony yield 51.54 %) for Ramalina farinacea. The algal colonies were re-cultured on Trebouxia Medium to test whether the selected colonies were free of contamination, because it is an organic nutrient medium upon which fungi, bacteria or yeasts grow quickly. Our results showed that fungal or bacterial contamination appeared in less than 3 % of the original cultures. Thus, our micromethod allows the isolation of algal cells almost free of contamination by other microorganisms.

Table 2 Different amounts of lichen thalli were used: micromethod (15 or 25 mg) or macromethod (1 g). At the end of the isolation protocol the cells were resuspended in distilled water and adjusted to a cell density of 5.103 cells ml−1. 50 μl of the cell suspensions were spread on Petri dishes with 3NBBM culture medium and kept at 15 μmol m−2 s−1 (PPFD) in a 12/12 h light-dark cycle and 17°C; algal colonies were counted after 45 days
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The yield of algal cells isolated by the macromethod varied widely depending on the lichen species. The yield was from 10.70 103 per mg DW for Lobaria pulmonaria to 1.86 103 cells per mg DW for Xanthoparmelia tinctina (Table 2). Curiously, both the isolation yield and the viability of algal cells were higher for Ramalina farinacea after the micromethod than the macromethod, but for Seirophora villosa the two methods gave similar results. This may be explained, on one hand, by the differences in the thalli homogenisation procedure between the macro and micromethod. We have found that the employment of different homogenisation devices in macromethods (mortar, polytron, waring blendor) influenced dramatically the final yield of algal cells isolated from different lichens. For instance, in previous works we observed that there was a ten-fold reduction in the yield for Parmelia quercina (Willd.) Hale when a polytron was used instead of mortar and pestle (Calatayud et al. 2001). Similarly, the homogeneization with a pellet pestle in an eppendorff at microscale seemed to be more efficient for algae extraction than with a mortar and pestle at macroscale. On the other hand, only 5 ml were recovered from the algal layer in the macromethod and some cells were left in the centrifugation tube, which will also decreased the final yield.

Viability differences observed in R. farinacea comparing the macro and micromethods may be due to age diversity in the starting biological material. In many lichens, photosynthetic and metabolic activity is not homogeneous and seems to be age-dependent along the thallus (Moser et al. 1983, Barták et al.2000; Gasulla 2009). Whereas in the macromethod algal cells were isolated from whole thalli, in the micromethod only a small piece from a young area of a single healthy thallus was used. This may explain why the number of algal colonies that grow per 102 cells recovered by the micromethod was higher than when algae were isolated from the whole thallus. Physiological diversity could also explain the apparent differences in viability of algae isolated from different lichen species.

The micromethod described here has shown to be useful for obtaining hundreds of axenic colonies of photobionts from fruticose, foliose and crustose lichens (Table 1), allowing the isolation of algal cells from very small amounts of lichen thallus (15–25 mg). Furthermore, it can be scaled up if more photobiont material should be required. Our macro method provides advantages to other similar methods, such as differential centrifugation (Drew and Smith 1967), centrifugation on CsC2, KI (Ascaso 1980) or Percoll® gradient (Calatayud et al. 2001). A disadvantage of these previously reported methods is that the final algal isolates still contain a relatively high proportion of fungal or bacterial contamination. With our isolation method we were able to reduce contamination to very low levels. Hyphal fragments and spores were practically eliminated after centrifugation on Percoll® and with sonication and Twin 20 treatment, bacterial contamination was reduced to very low levels, which improves the use of these algae for biochemical or physiological studies. In fact, after detergent/sonication treatment, the number of bacterial colonies growing in our cultures was reduced by around 80%, more than enough to obtain dozens of algal colonies without contamination, facilitating the development of axenic cultures.

The absence of significant external contamination and the purity of the obtained algal strains was partially confirmed after amplification and sequencing of the ITS region including the 5.8 S gene of the nuclear rDNA from the algal strains and the corresponding lichens. Amplification of algal DNA gave clear single bands in agarose gels and sequences were unambiguous without peak superposition. Sequences were deposited in National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov). Accession numbers of the GenBank: FJ418565, FJ418566, FJ792797, FJ792798, FJ792800–FJ792803, and HM064495.

A final advantage with respect to other methods such as the micropipette method (Ahmadjian 1967a, b), the spray method (Wiedeman et al. 1964), the cutting method (Yoshimura et al. 2002) or other centrifugation methods (Richardson 1971), is that our new method is less time-consuming. We can obtain millions of clean algal cells in less than 2 h and furthermore fewer post- isolation subcultures are needed to obtain axenic and monoclonal strains. In conclusion, this isolation method provides a simple, effective and fast method for isolating and growing lichen algae, regardless of lichen morphology or amount of thallus available.