Isolation and culture of lichen bacteriobionts

Lichens are increasingly regarded as a complicated microcosm formed by a large number of lichen-associated fungi and nonphotoautotrophic bacteria, in addition to the photobionts and mycobionts (Grube et al. 2012). Some non-photoautotrophic bacteria harboured in lichen microbiomes are considered by some as integral players in the lichen symbiosis and have been referred to as bacteriobionts (Grube & Berg 2009). The first studies on lichen-associated bacteria using culture-independent molecular methods were conducted by Cardinale et al. (2006). Subsequent research with molecular and microscopic methods clearly showed that Alphaproteobacteria is the predominant bacterial group in growing parts of lichens (Cardinale et al. 2008, 2012; Grube et al. 2009; Bates et al. 2011; Mushegian et al. 2011; Schneider et al. 2011; Hodkinson et al. 2012). In some cases, host species specificity of the lichen-associated bacteria has been shown (Grube et al. 2009; Bates et al. 2011; Bjelland et al. 2011). These studies provide evidence that bacteriobionts are integral components in lichen symbioses. Rhizobiales (Alphaproteobacteria) was shown to be the predominant order among lichen bacteriobionts (Hodkinson et al. 2012) and includes many nitrogen-fixing taxa forming plant-associated root nodules (Sy et al. 2001; Ngom et al. 2004). A recently discovered and uncultured lineage of this order, LAR1, has been found in many lichens (Hodkinson & Lutzoni 2009; Bates et al. 2011; Hodkinson 2011; Hodkinson et al. 2012). Furthermore, the bacterial nitrogenfixing genes were also detected in chlorolichens using PCR (Grube et al. 2009). These studies suggest that bacteriobionts are potential nitrogen-fixers in the lichen symbioses. Despite living in nutrient-poor conditions, most lichen mycobionts produce high levels of secondary compounds. For example, the usnic acid content in Alectoria ochroleuca (Hoffm.) A. Massal. is 8% of the dry weight of the lichen thallus, while lecanoric acid may constitute up to 23·5% of the thallus dry weight of Parmotrema tinctorum (Despr. ex Nyl.) Hale (Wei 1998). More than 700 lichen compounds have been reported, almost all of which are insoluble in water (Huneck & Yoshimura 1996; Dembitsky & Tolstikov 2005). Most lichen compounds are aromatics (containing one or more benzene rings), while terpenoids are also very common in lichens (Huneck & Yoshimura 1996; Dembitsky & Tolstikov 2005). Considering the high content of secondary compounds in lichen thalli and the enormous diversity of carbon sources utilized by bacteria, we first investigated the culturable bacteria from the chlorolichens Umbilicaria esculenta (Miyoshi) Minks and Parmelia omphalodes (L.) Ach. , and the cyanolichen Lobaria retigera (Bory) Trevis. , on media with acetone extracts of corresponding lichens as the nutrient source. However. some bacteria can use acetone as a carbon source (Platen & Schink 1989). Therefore, we selected dimethyl sulfoxide (DMSO) to dissolve the acetone extracts, and DMSO media were used to clarify whether or not culturable bacteriobionts utilize DMSO as a substrate. All the lichen compounds extracted from these species are water-insoluble, but soluble in acetone and DMSO.