Department of Biology, Faculty of Science, Ramkhamhaeng University, Bangkok, Thailand 2

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1 B_18_PF: THE INFLUENCE OF WATERING SOIL AND THALLUS ON PHYSIOLOGICAL PROCESSES AND NATURAL PRODUCTS OF THE TRANSPLANTED LICHEN Prmotrem tinctorum Mongkol Phengphech, 1 * Prichukorn Khongstr, Wetchsrt Polyim, 1 Chutim Srivioon, Knsri Boonprgo 1 1 Deprtment of Biology, Fculty of Science, Rmkhmheng University, Bngkok, Thilnd Deprtment of Chemistry, Fculty of Science, Rmkhmheng University, Bngkok, Thilnd *e-mil: mongkolpp@gmil.com Astrct: Lichen produces secondry metolites tht re different from other orgnisms nd hve gret potentil to e utilized commercilly. However, lichens hve extremely low growth rte due to their photosynthesis ctive only during high tmospheric humidity, which only exist in erly morning. The ojective of this study ws to trnsplnt the lichen Prmotrem tinctorum on mn-mde sustrte, nd provide extr wter through soil wtering, nd direct sprying on thllus. It ws hypothesized tht dditionl wter could enhnce lichen production. The trnsplnttion ws conducted t Kho Yi Ntionl Prk y using frgmented thlli. They composed of four tretments: 1) without soil wtering-without thllus wtering (), ) without soil wtering thllus wtering (), 3) soil wtering without thllus wtering (), 4) soil wtering thllus wtering (). After yer of trnsplnttion, we found tht the lichens received extr wter through soil wtering nd thllus sprying () hd the highest growth rte mesured 9.85 mm y -1, while the highest iomss production of 56.4 g g -1 ws mesured from soil wtering lone (). Contrry, photosynthesis (P N) nd Fv/Fm of oth tretments declined. More importntly, dily soil wtering lone () without thllus sprying, incresed growth, iomss nd P N of the lichens more thn only thllus sprying (), once in two weeks. This tretment should e tken into considertion for future trnsplnttion s well. Lecnoric cid ws the min nturl product tht P. tinctorum metolized. This sustnce nd methyl orsellinte, the minor sustnce, declined in ll tretments fter trnsplnttion. Contrdictory, orsellinic cid incresed in ll tretments. Wheres, trnorin, the second min sustnce, hd vrying mounts. It is most essentil to continue reserch nd development on lichen trnsplnttion to chieve the mximum production of lichens y using the informtion from this study s sis. Introduction: Lichens produce novel products tht re different from other orgnisms. They re used in phrmceuticl industries, cosmetic, foods, everges nd etc. 1 They re widely used s io-indictor of ir pollution. However, lichens grow extremely slow, which is not sufficient to utilize sustinly. Lichens depend on tmospheric moisture for cron ssimiltion. Their photosynthesis terminte in erly morning when reltive humidity drop.,3,4 Our reserch question ws, whether extr wter supplied to thllus through soil wtering, nd sprying directly over thllus cn enhnce lichen production. We hypothesized tht 1) dditionl supply of wter could increse growth rte, iomss production, photosynthesis nd vitlity of lichens ecuse wter is one of the rw mterils for orgnic mtter production ) sprying wter directly over thllus increses growth nd iomss more thn wter vpor from soil ecuse thllus sors liquid wter etter thn wter vpor or tmospheric moisture. The lichen Prmotrem tinctorum ws used s the experimentl mteril. This lichen distriutes in ll ecosystems in Thilnd, nd found undntly t Kho Yi Ntionl Prk. It hs een used successfully for silk dying nd io-indictor of tmospheric qulity in the country. It lso metolizes secondry sustnces tht hve potentil to e used phrmceuticlly nd much more. 1 Our ojectives were to improve lichen production y trnsplntion the lichen P. tinctorum on mn-mde sustrte, nd supply dditionl wter through wter evporte from soil wtering, nd sprying wter directly over thlli. 6

2 Methodology: Trnsplnttion site: The secondry forest, previously tropicl rin forest, t Kho Yi Ntionl Prk (KYPN), Thilnd ws used s the experiment ground. Trnsplnted method nd smple collection: The trnsplnted thlli included few peripherl loes of out 3-4 cm from P. tinctorum inhited the secondry forest. Ech trnsplnted thllus ws fixed on 5x5 cm lck polyethylene net mesh size x mm y.5 mm monofilment fishing line (Figure 1.). Forty trnsplnted thlli were fixed on trnsplnted frme mde from PVC pipe lining with lck polyethylene net. They stnd on the ground t 45-degree inclintion fcing the Est under % lck shding net (Figure 1). The trnsplnted thlli received extr moisture from vpor evported from soil wtering nd sprying distilled wter directly over thlli. They composed of four tretments s show in tle 1. Tle 1. The four wter tretments on the trnsplnted lichen P. tinctorum conducted in the secondry forest t KYNP. Thllus Soil Dry soil (SD) Soil wtering (SW) Dry thllus (TD) Thllus wtering (TW) These tretments were orgnized on ten trnsplnted frmes, which divided into ech 5 wet soils nd 5 dry soils. A trnsplnted frme hd 8x5 thlli (column x row), of which columns lterntely treted y TD nd TW, (Figure 1), mking up (4x5) x 5 x = thlli. In ddition, two thlli were trnsplnted to ech trnsplnted frmes for mesuring the initil physiologicl prmeters. All together mking up totl of trnsplnted thlli. The experiment lsted from Novemer, 15 to Novemer, 16. Growth nd Biomss mesurement:twenty thlli of ech tretment from the trnsplnted frmes were rndomly selected for growth mesurement. The photogrmmetry method ws used to clculted growth rte ccording to Hooker nd Brown (1977) 5 nd Sncho et l., (11) 6. Photogrphs of the thlli on the trnsplnted frmes were tken y mm lens Cnon SH6. Are nd dimeter were clculted y AxioVision softwre LE Rel. 4.1 from Crl Zeiss Industrielle Messtechnik GmH, Germny. Twenty thlli from the trnsplnted frmes were weighed efore nd fter trnsplnttion for iomss mesurement. The growth rte nd iomss ws clculted sed on Hill (). 7 Photosynthesis nd Chlorophyll fluorescence: Five thllus of ech tretment were mesured for net photosynthetic rte (P N) nd Chlorophyll fluorescence under 3 mol m - s -1 light intensity, % thllus wter content nd 5 o C room temperture t lortory of Rmkmheng University. P N ws mesured y LI64 (Licor inc. USA) using the conifer chmer. Chlorophyll fluorescence prmeter, Fv/Fm, ws mesured y mini-pam (Wlz inc. Germny) under sme condition. Fv/Fm ws used to evlute thllus vitlity. Secondry metolites nlysis: Lichen smples were ir-dried t room temperture for two dys nd ground into powder with liquid nitrogen, nd were then sieved through μm filter. A ten milligrm of smples ws ccurtely weighted nd extrcted for lichen sustnces y cetone fter soking overnight. The extrcts were filtered nd evported to dryness. The residues were then dissolved y smll mount of methnol nd diluted to exct volume 7:3 of methnol: DI wter using enzoic cid s n internl stndrd. The solution smples were filtered through.45 μm syringe memrne efore injection to HPLC. 8 Dt nlysis: The differences of growth rte nd secondry metolites mong the four tretments (,, nd ) were exmined y one-wy ANOVA, nd ll pirwise multiple comprison verified y Tukey Test (P<.5). 7

3 Wet soils (SW) Dry soils (SD) - - Figure 1. Trnsplnttion of the lichen P. tinctorum t KYNP, () the trnsplnted frmes stnd on the ground t 45-degree inclintion fcing the Est () the trnsplnted thlli on lck polyethylene nets were fixed on the trnsplnted frme. The tretment ws: soil wtering (SW), without soil wtering or dry soil (SD), thlli spryed with distill wter (TW) nd thlli without wter (TD). Results nd Discussion: Figure demonstrted growth, iomss production, photosynthesis nd chlorophyll fluorescence (Fv/Fm) of the trnsplnted lichens. Growth rte (mm y -1 ) Growth rte Biomss Net Photosynthesis (P N ) c. d. c c Biomss (mg g -1 ) P N ( mol CO m - s -1 ) Fv/Fm Fv/Fm 1.. Figure. Men vlues of growth rte (), iomss (), Net photosynthesis (c) nd Fv/Fm (d) of lichen P. tinctorum fter trnsplnttion under =control-dry soil without TW, =dry soil with TW, =Wet soil without TW nd =Wet soil with TW. Different letters over rs refer to sttisticlly significnt difference (Tukey test, p <.5). Evportive moisture from soil wtering without thllus sprying () rised up iomss nd growth over the thlli in the nturl condition, without soil nd thllus moistening, (). Regrdless, net photosynthesis (P N) nd Fv/Fm seem to e lower etween such tretments, ut t non sttisticl different (Figure ). Thllus sprying () resulted in the higher iomss, ut lower growth thn the tretment without extr wter on thllus (). Whilst, P N nd Fv/Fm were not enefited from thllus wtering s these vlues were not significntly different etween the two tretments. Remrkly, y given extr wter to the trnsplnted lichens oth through soil wtering nd direct sprying the thlli () resulted in the highest growth rte mong ll tretments. Nevertheless, iomss production ws lower thn the tretment tht received only soil wtering without thllus wetting (). 8

4 Most interestingly, we found tht soil wtering every dy without thllus sprying (SW- TD) incresed growth, iomss nd P N of the trnsplnted lichens more thn thllus wtering (once in two weeks) without soil wtering (), lthough Fv/Fm did not follow this pttern. Wtering oth soil nd thllus enriched growth to the highest rte mong ll tretments. However, iomss nd P N ws lower thn soil wtering lone, whilst Fv/Fm lmost equl to soil wtering, nd seems to e the lowest mong ll. Therefore, soil wtering should e used s sic guideline for future lichen trnsplnttion in order to stimulte lichen production for commercil uses under sustinle utiliztion of nturl resources nd conservtion policy. Notwithstnding, dditionl reserches should e conducted to pplied these informtion in different environment, long with pplying growth stimulting fctor, fertilizer nd hormone, to chieve the supreme condition for iomss production of lichens. Secondry metolites: Lecnoric cid, the mjor sustnce of this lichen, in ll tretments declined fter trnsplnttion (Figure 3). The highest vlues were 357 g g -1 in, while the lowest vlues were recorded in (1877 g g -1 ). Atrnorin of dry soil tretments ( nd ) incresed slightly. While wet soils ( nd ) declined s much s 8% of the initil. The highest mount ws 185 g g -1 from (Figure 3), with significntly difference etween nd (p<.5). Secondry metolites ( g g -1 ) Lecnoric cid Atrnorin Orsellinic cid Methyl orsellinte c. d c 4 15 c Initil Initil Initil Initil Figure 3. Men vlues of the secondry metolites in the lichen P. tinctorum fter trnsplnttion in different wter tretments. Lecnoric cid (), Atrnorin (), Orsellinic cid (c) nd Methyl orsellinte (d) =Dry soil without TW (control), =Dry soil with TW, =Wet soil without TW nd TH, =Wet soil with TW. Different letters over rs refer to sttisticlly significnt difference (Tukey test, p <.5). Orsellinic cid of ll tretments incresed fter trnsplnttion (Figure 3c). Interestingly, dry stte thlli ( nd ) hd higher concentrtion of this metolite thn the wetted thlli ( nd ), lthough they were not different sttisticlly. Methyl orsellinte in ll tretments oviously declined more thn other lichen sustnces fter trnsplnttion (Figure 3d). Prticulrly, under the wet condition,, the mount reduced s much s 37%. Whilst lichen in dry soil (SD) nd wet soil tretments (SW) fell 3 nd 31% respectively. Lichens produce secondry metolites for protection ginst environmentl stress nd defend from iologicl thretening. 1,9 Overll reduction of secondry metolites fter trnsplnttion on the mn-mde sustrte, the lck polyethylene net, could possily e low stress condition nd less threten y other orgnisms. 9

5 Conclusion: 1. Evportive moisture from soil wtering lone without thllus sprying, elevted growth, iomss nd P N of the trnsplnted lichens more thn only thllus wtering.. Thllus sprying lone without soil wtering, lthough improved iomss production, ut reduced growth of the lichens grew nturlly, neither soil nor thllus wtering. Whilst, P N nd Fv/Fm were not enefited from thllus wtering. 3. Wtering oth soil nd thllus enriched growth to the highest rte mong ll tretments, lthough iomss nd P N ws lower thn soil wtering lone. 4. Both wter tretments, soil nd thllus, lthough stimulted growth nd iomss, ut seems to reduce nturl products of the lichens. Prticulrly, lecnoric cid, which is the mjor metolite of this lichen. Only orsellinic cid, the minor product, incresed s consequence of wter tretments. References: 1. Rnkovic B, Studzińsk-Srok E, FŁtynowicz W. 15; 16: Phengphech M, Fungkew P, Polyim W, Wtthn S, Boonprgo K. 16;4: Welch AR, Gillmn MP, John EA. 6; 38: Plmqvist K, Sunderg B. ; 3: Hooker TN, Brown DH. 1977; 9: Sncho L, Dvid P, Green TGA, Mercedes V, Pintdo A. 11; 34(6): Hill DJ.. Protocols in Lichenology; Thepnun P, Srivioon C, Rukchisirikul T, nd Boonprgo K. 13; 38: 1 9. Deduke C, Timsin B, Piercey-Normore MD. 1. Interntionl Perspectives on Glol Environmentl Chnge; Acknowledgements: This study ws supported y the Ntionl Reserch Council of Thilnd (NRCT). We wish to thnk Mr. Sumrit Senglek for their ssistnce in fieldwork nd the officers nd stffs t Kho Yi Ntionl Prk for their kind coopertion. Our grtitude lso goes collegues t Lichen Reserch Unit, Rmkhmheng University. 3