Raman spectroscopic analysis of the effect of the lichenicolous fungus Xanthoriicola physciae on its lichen host

Symbiosis (2017) 71:57–63 DOI 10.1007/s13199-016-0447-2 Raman spectroscopic analysis of the effect of the lichenicolous fungus Xanthoriicola physciae on its lichen host Howell G.M. Edwards 1 & Mark R.D. Seaward 2 & Tom F. Preece 3 & Susana E. Jorge-Villar 4 & David L. Hawksworth 5,6,7 Received: 16 August 2016 / Accepted: 6 September 2016 / Published online: 5 October 2016 # The Author(s) 2016. This article is published with open access at Springerlink.com Abstract Lichenicolous (lichen-dwelling) fungi have been extensively researched taxonomically over many years, and phylogenetically in recent years, but the biology of the relationship between the invading fungus and the lichen host has received limited attention, as has the effects on the chemistry of the host, being difficult to examine in situ. Raman spectroscopy is an established method for the characterization of chemicals in situ, and this technique is applied to a lichenicolous fungus here for the first time. Xanthoriicola physciae occurs in the apothecia of Xanthoria parietina, producing conidia at the hymenium surface. Raman spectroscopy of apothecial sections revealed that parietin and carotenoids were destroyed in infected apothecia. Those compounds protect healthy tissues of the lichen from extreme insolation and * Mark R.D. Seaward 1 Centre for Astrobiology & Extremophiles Research, Division of Chemical &Forensic Sciences, School of Life Sciences, University of Bradford, Bradford BD7 1DP, UK 2 School of Archaeological Sciences, University of Bradford, Bradford BD7 1DP, UK 3 34 Meadowbrook, Twmpath Lane, Gobowen, Oswestry, Shropshire SY10 7HD, UK 4 Area Geodinamica Interna, Facultad de Humanidades y Educacion, Universidad de Burgos, Calle Villadiego s/n, 09001 Burgos, Spain 5 Departamento de Biología Vegetal II, Facultad de Farmacia, Universidad Complutense de Madrid, Plaza Ramón y Cajal, 28040 Madrid, Spain 6 Department of Life Sciences, The Natural History Museum, Cromwell Road, London SW7 5BD, UK 7 Comparative Plant and Fungal Biology, Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3DS, UK their removal may contribute to the deterioration of the apothecia. Scytonemin was also detected, but was most probably derived from associated cyanobacteria. This work shows that Raman spectroscopy has potential for investigating changes in the chemistry of a lichen by an invading lichenicolous fungus. Keywords Parasitism . Parietin . Pathogenicity . Protective biochemicals . Scytonemin . Xanthoria parietina 1 Introduction Lichenicolous (lichen-dwelling) fungi have proved to be a major ecological group of fungi, with around 2000 species already described. Many of the genera consist only of lichenicolous species, and most are restricted to particular lichen hosts, commonly single host genera or single lichenized species. The biological interactions vary from saprobes or commensals, to gall-formers or necrotizing pathogens, but in many cases the relationships are unclear and some may even be mutualistic. Some start as pathogens, kill the host lichen, and then utilize the algal partner of the host to form an independent lichen. For more information on the variety of relationships involved see Richardson (1999), Hawksworth (2003), Lawrey and Diederich (2003), and Divakar et al. (2015). To date, our knowledge of the interactions that take place at the biochemical or cellular level in these associations is somewhat limited (cf. Lawrey 1995, 2000; Lawrey et al. 1999; Merinero et al. 2015; Asplund et al. 2016). Microscopy has provided information on whether the fungal or the algal partner in a lichen is parasitized (de los Rios and Grube 2000) and thin-layer chromatography suggests compounds not detected in the host lichen may be produced, and perhaps originate from the invading fungus (Hawksworth et al. 1993). More 58 sensitive methods able to examine changes in situ are needed to better explore these relationships in depth. Here we use Raman spectroscopy, which has proved particularly suitable for the molecular analysis of the protective compounds produced by lichens and cyanobacteria in stressed environments (e.g. Seaward and Edwards 1995, 1997; Russell et al. 1998; Wynn-Williams and Edwards 2000a; Edwards et al. 2004), but not previously in investigations of lichenicolous fungi. Surface-dwelling organisms require photosynthetically active radiation in the visible region of the electromagnetic spectrum for survival, but insolation by low wavelength high energy ultraviolet radiation and exposure to high intensities of visible radiation (e.g. Solhaug and Gauslaa 2012) can be damaging unless these organisms have developed a photoprotective screening strategy (Cockell and Knowland 1999; Wynn-Williams and Edwards 2000, 2002); however, it must be noted that some authors (Robson et al. 2015; Hideg et al. 2013) have emphasised UV as a regulatory factor rather than as a stressor in such circumstances. In the case of Xanthoria (e.g. Gauslaa and Ustvedt 2003), photoprotection is provided by bright yellow to orange anthraquinone pigments, of which parietin predominates (Culberson et al. 1977). The amount of pigment produced varies according to the light regime of the habitat (Edwards et al. 2003a) and Xanthoria parietina protected under perspex cloches in an Antarctic habitat produced less parietin than similar colonies outside; specimens can vary from white through grey, yellowish-grey, yellow, orange, to orange-red with increasing light intensity, and when suddenly put in the dark they become greenish within a few days (Hawksworth and Wiltshire 2011). Earlier Raman spectroscopic studies of X. parietina (Edwards et al. 2003b, 2004) identified the characteristic spectral biomarkers (19 bands) of parietin, along with associated accessory carotenoids, on several substrata in different environments. The dualistic role of important photoprotective pigments has been recognised (Cockell and Knowland 1999) and their production, along with other key lichen chemicals, in response to stressed habitats have been monitored by means of Raman spectroscopy (Edwards et al. 2003a, 2004; WynnWilliams and Edwards 2000), its discriminatory sensitivity being used for the recognition of key biological signatures of the protective chemicals. Here we report for the first time a Raman spectroscopic analysis of X. parietina parasitized by the lichenicolous asexual fungus Xanthoriicola physciae which appears to be largely confined to Europe and is particularly frequent in the British Isles (Hawksworth and Punithalingam 1973; Hawksworth 1979; Preece 2013). In the case of Xanthoria parietina, the invading fungus is parasitic, the hyphae growing through the host hymenium and forming conidiogenous cells just below the surface, with conidia at the surface (Fig. 1). Extensive sooty black discoloration occurs. The association is specialized, the fungus evidently being restricted to a single host H.G.M. Edwards et al. Fig. 1 Xanthoria pari (...truncated)