Innovative Approaches Using Lichen Enriched Media to Improve Isolation and Culturability of Lichen Associated Bacteria

Abstract

Lichens, self-supporting mutualistic associations between a fungal partner and one or more photosynthetic partners, also harbor non-photosynthetic bacteria. The diversity and contribution of these bacteria to the functioning of lichen symbiosis have recently begun to be studied, often by culture-independent techniques due to difficulties in their isolation and culture. However, culturing as yet unculturable lichenic bacteria is critical to unravel their potential functional roles in lichen symbiogenesis, to explore and exploit their biotechnological potential and for the description of new taxa. Our objective was to improve the recovery of lichen associated bacteria by developing novel isolation and culture approaches, initially using the lichen Pseudevernia furfuracea. We evaluated the effect of newly developed media enriched with novel lichen extracts, as well as the influence of thalli washing time and different disinfection and processing protocols of thalli. The developed methodology included: i) the use of lichen enriched media to mimic lichen nutrients, supplemented with the fungicide natamycin; ii) an extended washing of thalli to increase the recovery of ectolichenic bacteria, thus allowing the disinfection of thalli to be discarded, hence enhancing endolichenic bacteria recovery; and iii) the use of an antioxidant buffer to prevent or reduce oxidative stress during thalli disruption. The optimized methodology allowed significant increases in the number and diversity of culturable bacteria associated with P. furfuracea, and it was also successfully applied to the lichens Ramalina farinacea and Parmotrema pseudotinctorum. Furthermore, we provide, for the first time, data on the abundance of culturable ecto- and endolichenic bacteria that naturally colonize P. furfuracea, R. farinacea and P. pseudotinctorum, some of which were only able to grow on lichen enriched media. This innovative methodology is also applicable to other microorganisms inhabiting these and other lichen species.

Microbiome in Cladonia squamosa Is Vertically Stratified According to Microclimatic Conditions

“…In addition to the mycobiont and photobiont, diverse microorganisms, such as lichen-associated fungi (LAF) and lichen-associated bacteria, have been revealed by culturedependent and molecular approaches (Lawrey and Diederich, 2003;González et al, 2005;Liba et al, 2006;Cardinale et al, 2008;Bates et al, 2012;Hodkinson et al, 2012;Lee et al, 2014;Biosca et al, 2016;Park et al, 2016). With the dominance of Ascomycota and Basidiomycota in LAF and Alphaproteobacteria in bacterial communities across diverse lichen species, lichen-associated microbial compositions are known to be largely influenced by various environmental factors Bates et al, 2011;Cardinale et al, 2012;Hodkinson et al, 2012;U'Ren et al, 2012;Beck et al, 2014;Park et al, 2015Park et al, , 2016Wang et al, 2016).…”

Endolichenic fungi: the lesser known fungal associates of lichens

“…Although the mycobiont in most lichens is an ascomycete, a recent study shows that a basidiomycete yeast is invariably involved in this mutualism as a third partner (Spribille et al 2016). Additionally, lichens are associated with fungi lichenicolous fungi, endolichenic fungi and culturable and non-culturable non-photosynthetic bacteria (Biosca et al 2016; Muggia et al 2016). The lichens along with their associates represent a successful mode of symbiosis as they have existed for over 600 million years (Yuan et al 2005) and currently dominate nearly 10% of the Earth’s terrestrial ecosystems (Papazi et al 2015).…”

“…Chagnon et al (2016) argue that both culture-based and culture-independent studies have to be made to get a more complete picture of the diversity of endolichenic fungi. Additionally, lichen-enriched growth media could be used to isolate fastidious endolichenic fungi which fail to grow on normal growth media (Biosca et al 2016). Some lichens infect already existing lichens and develop slowly as independent forms.…”

Endolichenic fungal diversity associated with some lichens of the Western Ghats

“…By existing for more than 600 million y, lichens with their fungal partner and unicellular or filamentous algal or cyanobacterial partner, represent one of the most successful symbiotic systems [1]. A lichen represents a mini-ecosystem since apart from the obligate symbiont, the symbiome of lichen supports a basidiomycete yeast [2], nonculturable nonphotosynthetic bacteria [3], lichenicolous fungi [4], and endolichenic fungi [5]. The endolichenic fungi are nonobligate fungal component of lichen symbiome; they are similar to the endophytes of higher plants as they occur inside the lichen thallus, are nonpathogenic, and are transmitted horizontally [5][6][7].…”