30 Foxing or fox spots Florian,1994 Foxing Foxbrown Arai, 1992 Foxed Cain, 1984; Mori and Tachibana, Foxing-causing fungi Arai, Asper

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1 24 (4) : 2944, Foxing Aspergillus Penicillium Aspergillus niger, Aspergillus flavus, Aspergillus ochraceus, Aspergillus ustus, Aspergillus spp.,penicillium citrinum, Penicillium chrysogenum, Penicillium spp.,chaetomium globosum, Trichoderma viride,fusarium solani, Cladosporium sp., Rhizopus sp., Phoma sp.,umbelopsis sp., Metarrhizium sp., Eurotium sp. 70 % 60 % Research paper Separation and Identification of Foxed Spots on Paperbased Cultural Relics Tsang-Chyi Shiah 1 Feng-Jyi Chang 2 AbstractFoxing of paper-based cultural properties could be caused by the biodegradations. Brown stains not only apparently decrease the life of rare books, paintings and art crafts, but also affect the preservation of cultural relics. The aim of this study is to explore the contributing factors of foxed spots showed on paper-based materials. The experimental results are summarized as follows Foxed spots usually showed yellowish color in the serface of paper artifacts. Hyphae and conidia of microorganisms were observed on the foxed area in SEM observation. Among 17 species of fungi isolated from paper-based materials of which the main species, were identified as the genera of Aspergillus and Penicillium. The foxing-causing fungi of Pineapple xuan paper were inhibited by less than 60% R.H. at room temperature, and over 70% R.H.,the fungal growth was very vigorous. 1. Assistant scientist, Division of Forest Chemistry, Taiwan Forestry Research Institute, Council of Agriculture. 2. Professor, Department of Forestry, NCHU.

2 30 Foxing or fox spots Florian,1994 Foxing Foxbrown Arai, 1992 Foxed Cain, 1984; Mori and Tachibana, Foxing-causing fungi Arai, Aspergillus sp.,alternaria sp.,penicillium sp.,stachybotrys sp.,stemphylium sp.,tricoderma sp.,chaetomium sp.,chrysosporium sp.,eurotium sp Szczepanowska, ,

3 24 (4) : 2944, NBKP ; Tzean et al., 1990; 1994 (1)39 g PDAMerck 1000 ml (2)Mineral salt agar NH4NO33 g K2HPO4 1.4 gkcl 0.25 gmgso4 7H2O 0.25 g 10 g 1000 ml (3)CZ Czapek's agar NaNO3 3 g K2HPO41 gmgso47h2o 0.5 gkcl0.5 g FeSO47H2O0.01 gsucrose 30 g 10 g 1000 ml Aspergillus (4)CYA Czapek's yeast extract arar Czapek's extract 20 g Yest extract 5 gk2hpo4 1 gsucrose30 g 10 g 1000 mlpenicillium (5)MEA Malt extract agar Malt extract 20 g Peptone 1 gdextrose 20 g10 g 1000 ml Aspergillus Penicillium 3. PDA 25 (1)Aspergillus niger var. niger (2)Aspergillus flavus (3)Penicillium citrinum (4)Trichoderma sp. (5)Rhizopus sp. (6)Mucor sp. (7)Umbelopsis sp. (8)Metarrhizium sp. (9)Chaetomium globosum ml,, 10 l ml 10 4~ Arai,1984; 1987; Szczepanowska, ph (1) ph 67 8 ph ADVENTEC, TOYO, FILTER PAPER, No.1 PDA 100l L (2)

4 ph (1) 66 cm 200 l L (2) (3) 80 Cupriethylene diamine % R.H Aspergillus ochraceus, Aspergillus ustus, Penicillium chrysogenum, Chaetomium globosum, Trichoderma viride, Fusarium solani Cellulolytic microorganisms 200 Dhawan, 1986; Gallo, 1994 Penicillium, Aspergillus, Chaetomium, Mucor, Rhizopus, Fusarium, Stemphylium, Cladosporium,Stachibotrys, Alternaria, Trichoderma Czerwinska, ; Hsieh et al., 1987; Tzean et al., 1990; Aspergillus flavus Link 1 Aspergillus flavus MEA Czapek's cm PDA

24 (4) : 2944, 2002 33 1. Table 1. Identification of fungi from PDA cultivation of foxed spots found on paper-based cultural relics. Samples 1. 2. 3. 4. 5. 6.-1 7.-2 8. 9. 10. 11. 12. 13. 14. 15. 16.

5 24 (4) : 2944, Table 1. Identification of fungi from PDA cultivation of foxed spots found on paper-based cultural relics. Samples Species of fungi Phoma sp. Aspergillus niger var. niger Aspergillus niger var. niger Aspergillus niger var. niger Aspergillus niger var. niger Penicillium citrinum Penicillium citrinum Trichoderrma viride Aspergillus flavus Cladosporium sp. Aspergillus niger var. niger Aspergillus niger var. niger Aspergillus niger var. niger Umbelopsis sp. Metarrhizium sp. Aspergillus sp. Rhizopus sp. Aspergillus niger var. niger Eurotium sp. Aspergillus niger var. niger Remark NBKP Aspergillus flavus Fig. 1. Colony morphology and SEM photographs of Aspergillus flavus. Topon MEA, at 2 for 14 daysleftaspergillumrightconidia

34 110780 5.423 m 11 60m Aspergilla 4.021.42.89.1 m 5.613.2.45.4 m 3.0 5.6 2.45.1 m 2. Aspergillus niger var. niger 2 Aspergillus niger MEA 25 Czapek's 14 5.2 cm Vesicle 170 m 22024604.

6 m 11 60m Aspergilla m m m 2. Aspergillus niger var. niger 2 Aspergillus niger MEA 25 Czapek's cm Vesicle 170 m m Aspergilla Biseriate Metulae mPhialidely m m 3. Aspergillus niger var. awamori 25 Czapek's cm 1 70 m m Aspergilla 2. Aspergillus niger Fig. 2. Colony morphology and SEM photographs of Aspergillus niger. Topon MEA, at 2 for 14 daysleftaspergillumrightconidia

7 24 (4) : 2944, m m m 4. Aspergillus ustus Thom et Church Czapek's cm 4080 m m 1015 m m m 5. Aspergillus ochraceus Czapek's cm m m 1015 m m 33.2 m 6. Pencillium chrysogenum Thom Czapek's cm 12 mm m m m 7. Pencillium citrinum Thom 3 Pencillium citrinum CYA 25 Czapek's cm m m 22.5 m 8. Trichoderma viride Pers. ex Gray 4 Trichoderma viride PDA MA 4.5 m m m 9. Chaetomum globosum Kunze mm

4. Colony morphology and SEM photographs of Trichoderma viride.

8 36 3. Pencillium citrinum Fig. 3. Colony morphology and SEM photographs of Penicillium citrinum. Topon CYA, at 25 for 14 daysleftpenicillusright conidia 4. Trichoderma viride Fig. 4. Colony morphology and SEM photographs of Trichoderma viride. Topon PDA, at 25 for 14 daysleftphialides showing formation of conidiarightconidia

9 24 (4) : 2944, m m 10. Cladosporium sp PDA Fusarium solani 24 PDA cm m m m ph Trichoderma viride, Penicillium citrinum Cladosporium sp Szczepanowska, CZCYA MEA Aspergillus niger Aspergillus flavus Penicillium citrinum ph Szczepanowska ph Szczepanowska, ph ph 5 ph % ph 7 ph 8 30 Trichoderma virvide % Chaetomium globosum 75.2 % Metarrhiziumn sp % 10 % % Trichoderma viride415.1metarrhizium sp Aspergillus niger Chaetomiun globosum510.7 Thakre

10 38 2. Table 2. Effect of temperature on the growth of fungi and stain formation on inoculated filter paper. Fungi Penicillium citrinum Umbelopsis sp Metarrhizium sp Aspergillus flavus Aspergillus niger Trichoderma sp Cladosporium sp Chaetomium globosum Growth Stain 1+ yellowish 1 1 yellowish 1 2 Growth 3 4 Stain 1 greenish 3 reddish 3 blackish 3 1 yellowish 3 yellowish 3 yellowish Growth Stain 3 reddish 3 blackish 1 yellowish 3 3 brownish Fungi were grown for 7 days on filter paper added with mineral salt agar at 12,24,36 in dark incubator respectively. Growthon paper surface no fungi colony growth 1paper was covered with 20 % of fungi colonies 2paper was covered with 40 % of fungi colonies 3paper was covered with 60 % of fungi colonies 4paper was covered with 80 % of fungi colonies paper was covered with 100 % of fungi colonies Stainon paper surface colorless 1stained noticeably 2stained appreciably 3stained apparently Cellulolytic fungi Thakre and Bhajbhuje, 1991Thakre 7 60 % R.H % nm 60 % R.H. UV SEM 70 %

11 24 (4) : 2944, Aspergillus niger,aspergillus flavus Penicillium citrinum Fig. 5. Effect of temperature on the mycelia growth of Aspergillus niger,aspergillus flavus on CZ and MEA mediaand Penicillium citrinum on CYA and MEA media. 3. ph Table 3. Effect of ph on the growth of fungi and stain formation on inoculated filter paper. Fungi ph 5 ph 6 ph 7 ph 8 Penicillium citrinum Umbelopsis sp Metarrhizium sp Aspergillus flavus Aspergillus niger Mucor sp Cladosporium sp Chaetomium globosum Trichoderma sp Growth Stain 2 brownish 1 brownish Growth 4 4 Stain 2 brownish 1 brownish yellowish Growth Stain 1 brownish brownish Growth 4 Stain brownish Symbols see Table 2 shown.

12 40 6. Fig. 6. Reduction of filter paper DP after 30 days foxing-induced fungi inoculation. A B C D E % R.H. 2.5 Fig. 7. Observation of paper samples after 2.5 years storage at 60 % R.H. Anaked eyes, Bunder UV light, C ESEM photographs

13 24 (4) : 2944, % 75 % 5.79 % 90 % 8.96 % 80 % 7.33 % 75 % 5.79 % 70 % 5.59 % UV SEM 8 90 % R.H % Arai 7 94 % 235 Arai, 1987Dhawan 75 % R.H. 235 Dhawan, 1986 Gallo 65 % 10 % 20 Gallo, % Craig % Craig, Aspergillus niger, Aspergillus flavus, Aspergillus ochraceus, Aspergillus ustus, Aspergillus spp., Penicillium citrinum, Penicillium chrysogenum, Penicillium spp., Chaetomium globosum, Trichoderma viride, Fusarium solani, Cladosporium sp., Rhizopus sp., Phoma sp., Umbelopsis sp., Metarrhizium sp., Eurotium sp., Aspergillus Penicillium ph ph 7, 8 3. Trichoderma viride Chaetomium globosum, Metarrhizium sp. Aspergillus niger % UV SEM 70 % 90 %80 %75 % 70 % R.H %

42 A B C D E F 8. 90 % R.H. 2.5 Fig. 8. Observation of paper samples after 2.

14 42 A B C D E F % R.H. 2.5 Fig. 8. Observation of paper samples after 2.5 years storage at 90 % R.H. Anaked eyes, Bunder UV light, C FSEM photographs

15 24 (4) : 2944, Trans. mycol. Soc. R.O.C., 1(2) (3) (4) Arai, H. (1984) Microbiological studies on the conservation of paper and related cultural properties, (part1) Isolation of fungi from the foxing on paper. Science for Conservation (in Japanese) Arai, H. (1987a) Microbiological studies on the conservation of paper and related cultural properties. (part 5) Physiological and morphological characteristics of fungi isolated from foxing, foxing formation mechanisms and countermeasures.science for Conservation (in Japanese) Arai,H. (1987b) Control of biodeterioration on the foxing-causing fungi. ICOM Committee for Conservation, working group Arai,H. (1992) Relationship between fungi and brown spots found in various materials. 2nd Int.Conference on Biodeteriation of Cultural Property, Yokohama 5-8 October pp Barnett, H.L. and B.B. Hunter (1987) Illustrated Genera of Imperfect Fungi. Fourth Edition, Macmillan publishing company, New York. Czerwinska, E. and R. Kowalik (1979) Microbiodeterioration of audiovisual collections an art Protection of audiovisual records against destructive microflora. Part. Microbial problems in photographic print collections. RESTAURATOR Cain,C.E., and B.A.Miller (1984) Proposed classification of foxing. AIC 10th annual meeting pp Craig, R. (1986) Alternative approaches to the treatment of mold biodeterioration -an International problem. The paper conservation: Journal of the institute of paper conservation Coleman, M.D., C.S. Bledsoe and W. Lopushinsky (1989) Pure culture response of ectomycorhizal fungi to imposed water stress

16 44. J. Can. Bot Dhawan, H Microbial deterioration of paper material (A literature review). Publication ManagementKhan, M. M. India. pp Florian, M-L. E. (1994) Conidia fungi mould, mildewbiologya basis for logical preservation, eradication and treatment for museum and archival collections. Leather Conservation News Hsieh, H.M., H.J. Su and S.S. Tzean (1987) The genus Penicillium in Taiwn. Two new taxa of synnematous Penicillium. Trans. Mycol. Soc. R.O.C.. 2 (2) Mori,W.and Tachibana,N., (1992) Foxing found in books and other paper materials. 2 nd Int. Conference on Biodeterioration of Cultural Properties p.77. Szczepanowska, H. (1986) Biodeterioration of art objects on paper. The Paper Conservator Tzean, S.S., J.L. Chen, G.Y. Liou, C.C. Chen and W.H. Hsu (1990) Aspergillus and related teleomorphs from Taiwan. Food industry Research Development Institute, Taiwan. Tzean, S.S., S.C. Chiu, J.L. Chen, S.H. Hseu, G.H. Lin, G.Y. Liou, C.C. Chen and W.H. Hsu (1994) Penicillium and related teleomorphs from Taiwan. Food industry Research Development Institute, Taiwan.

36(4) (2004) Journal of Soil and Water Conservation, 36(4) (2004) earthworms dig soil on surface and prevent plants to grow. But until D

36(4) (2004) Journal of Soil and Water Conservation, 36(4) (2004) earthworms dig soil on surface and prevent plants to grow. But until D (1) (2) (3) 16 20 35 Effect of Vegetation and Soil Properties on Earthworm Population Li-Ling Lin Professor, Department of Soil and Water Conservation, National Chung Hsing University, Taichung, Taiwan,