Environmental Factors Affecting the Content of Usnic Acid in the Lichen Mycobiont of Ramalina siliquosa

Abstract

The effect of environmental factors on the content of usnic acid was examined using the cultivated lichen mycobiont of Ramalina siliquosa. There was no correlation between light intensity and the content of usnic acid or 4-O-demethylbarbatic acid. The content of usnic acid changed with variations in temperature. In our previous studies on the production of lichen substances by an isolated lichen mycobiont (Hamada & Ueno 1987, 1990), we reported that the content of the depside, 4-O-demethylbarbatic acid, in the cultured lichen mycobiont of Ramalina siliquosa was affected by temperature. Similar variations in the content of the depsidone, salazinic acid, were observed in lichens growing under natural conditions (Hamada 1989). Thus, further studies on the effect of environmental factors on the lichen mycobiont would also be valuable. As far as lichen pigments are concerned, Rundel (1969) reported that the concentration of usnic acid in Cladonia subtenuis increased with higher light intensity under natural conditions. He implied that usnic acid was not produced in the absence of sunlight. However, Ahmadjian and Jacobs (1985) reported that crystals of usnic acid were abundantly produced by synthetic lichens growing under low light intensity, and considered that the increased production of usnic acid was due to the favorable growing conditions. Moreover, I too have often found usnic acid in lichen mycobionts cultured in the dark (see Hamada 1989). Indeed earlier studies have shown that usnic acid was produced by cultured lichen mycobionts as well as by lichens grown under natural conditions (Komiya & Shibata 1969). Thus, in the present work the relationship between usnic acid content and light intensity was investigated using the cultured mycobiont from a strain ofR. siliquosa, which produces comparatively large quantities of usnic acid. MATERIALS AND METHODS A fertile thallus ofR. siliquosa was collected in Kasumicho of Hyogo Prefecture (36*N), Japan, 4 May 1988. In this thallus salazinic and usnic acids were detected by TLC (Kieselgel GF254, 0.25 mm thickness) in toluene-acetic acid (20:3), usnic acid (Rf = 0.68), 4-O-demethylbarbatic acid (Rf = 0.47), and salazinic acid (Rf = 0.05). The concentrations of these substances in the sample examined were 0.69% (usnic acid) and 0.82% (salazinic acid) as indicated by HPLC (Hamada 1988). Approximately ten spores discharged from apothecia on the thallus were inoculated into each slant, and the mycelia were cultured for 8 months in 70 test tubes containing a modified malt-yeast extract medium (malt extract 10 g, yeast extract 4 g, sucrose 4 g, agar 15 g, water 1 1, pH 7.0; see Hamada 1989). To examine the effect of light intensity, the mycobiont was cultured at 12C for 8 months in slant tubes under varied intensities of continuous fluorescent light. The surface of each slant was vertical to the light source. The light intensity was measured by photometer (Minolta T1) and adjusted from 1,100 to 4,400 lux by layers of Victoria lawn (a fine linen fabric) covering the test tubes. The fluorescent lamps (20 W x 6) were changed at intervals of 4 months. The average intensity of these fluorescent lights is shown in Table 2. In a preliminary experiment, the usnic acid content in the lichen mycobiont was found to be affected by the temperature, and is maximal at near 12*C. Therefore, in the experiments investigating the effect of time and light intensity (Tables 1-2), the temperature was maintained at 12*C. For examining the effect of temperature, the mycelia in slant tubes were kept in the phytotron at controlled temperatures of 5, 10, 12, 15, 20, and 25*C, respectively, for 8 months. This experiment was done in darkness to examine the effect of temperature without interference by light. All mycelia cultured at 15, 20, and 25*C were transplanted to fresh medium every 3 months to maintain the cultures on a slightly wet medium; those cultured at 10 and 12?C were transplanted every 4 months; while the mycelia cultured at 5?C were not transplanted at all. The mycobiont colonies coalesced and aggregated compactly on the slant. After 8 months cultivation, the mycelia were harvested, weighed, and used for the chemical determinations. (+)Usnic acid was detected by measurement of optical rotation, [a]D + 507?, in chloroform, and mass spectrometry, EI, 70eV: m/z 344 (M+, 78%), 260 (89%), 233 (100%), 217 (36%), as reported by Komiya and Shibata (1969). In colonies obtained from all test tubes other than those cultured at 25*C, 4-O-demethylbarbatic and usnic acids could be detected by HPLC in H20-methanol-acetic acid (20: 80:1); 4-O-demethylbarbatic acid (Rt = 4.6 min.); usnic acid (Rt = 17.8 min.), with a flow rate of 1.0 ml/min. using a 15 cm x 4.6 mm M & S Pack, C18 column (M & S Co.). The areas of HPLC chromatographic peaks due to usnic and 4-O-demethylbarbatic acids were used for the quantitative measurements. Each value for the acid content was an average of that observed for the colonies 0007-2745/91/57-59$0.45/0 This content downloaded from 157.55.39.78 on Thu, 23 Jun 2016 06:46:55 UTC All use subject to http://about.jstor.org/terms 58 THE BRYOLOGIST [VOL. 94