Indian Journal of Marine Sciences Vol. 31(1), March 2002, pp. 73-74

Short Communication

Endophytic fungi associated with the tropical ovalis ()

P. T. Devarajan & T. S. Suryanarayanan* Department of Botany, Ramakrishna Mission Vivekananda College, Chennai 600 004, Tamil Nadu, India and V. Geetha Department of Botany, Bharathi Women’s College, Chennai 600 108, Tamil Nadu, India Received 14 May 2001, revised 6 November 2001

Leaf blade, petiole and rhizome of the seagrass Halophila ovalis were examined for the presence of endophytic fungi. Although the seagrass harboured endophytic fungi, their number and colonization densities in seagrass tissues were low. This is presumed to be due to a combination of physical and chemical factors.

[ Key words : Seagrass, Halophila, fungal endophyte, tropical endophytes ]

Fungal endophytes colonize healthy tissues and damaged shoot and root system were carefully either remain dormant in the tissue or produce more washed to remove sediments and used. The leaf blade, extensive but symptomless infections1. The contem- petiole and rhizome of 500 were screened for porary interest in the study of fungal endophytes is endophytes. From each plant, a tissue segment partly due to the recognition that some of them pro- (0.5cm) was obtained from its rhizome, middle part of duce novel metabolites that are of agricultural and the petiole and the leaf blade. The tissue segments pharmaceutical importance2. There appears to be no from each organ were separately surface sterilized. published report on fungal endophytes of marine an- Ethanol and sodium hypochlorite were used for sur- giosperms although mangrove trees3 and some terres- face sterilization6. The segments were immersed in trial halophytes4 have been studied for their endophyte 70% ethanol for 5 sec and then treated with sodium assemblages. Therefore, we examined Halophila hypochlorite (4%) for 1 minute. They were then ovalis (R.Br.) Hook. f. the most common seagrass of rinsed in sterile water for 10 sec and plated on potato the Coromandel coast of India, for the presence of dextrose agar (PDA) medium amended with chloram- endophytes. phenicol (150 mg l−1). Surface sterililzed segments of Vegetative plants of seagrass Halophila ovalis (Hy- rhizome, petiole and leafblade were plated separately. drocharitaceae) were collected from Muttukadu estu- About 15 segments were plated on 20 ml of PDA me- ary (13°N latitude and 80o E longitude) in Chengal- dium contained in a 9 cm dia. petridish and incubated7 pattu district of Tamil Nadu, southeast coast of India. in a light chamber at 26°C for 30 days. The light was It is the most common seagrass distributed along the provided by three cool white fluorescent tubes. The entire Coromandel coast; it forms large, dense, almost light regimen was 12 h dark: 12 h light cycles. The monospecific meadows5. The leaf paste of this sea- tissue segments received about 2200 lux of light grass is used to cure skin diseases; the plant is also through the petridish lid. The petridishes were ob- used as manure in coconut plantations5. Plants grow- served periodically and the fungi that grew out from ing on the surface of estuary bed present at a depth of the tissues were isolated and identified. The coloniza- 1 m from the water level were collected and brought tion density (CD) of each endophyte species was cal- to the laboratory in sterile polythene bags. They were culated as follows: processed within 5 h of collection. Plants with un- ______No. of tissue segments colonized CD (%) = ×100 *For correspondence Total no. of tissue segments observed E-mail : [email protected] 74 Indian J. Mar. Sci., Vol. 31, No. 1, March 2002

Table 1 ⎯ Occurrence (CD %) of fungal endophytes in different plants with antifungal compounds in their leaves are tissues of the seagrass H. ovalis known to harbour lesser number of foliar endo- 15 Fungus Leaf blade Petiole Rhizome phytes . Thus, it is possible that a combination of physical and chemical factors is responsible for the Acremonium sp.1 0.2 0.2 ⎯ A. strictum ⎯ 0.2 ⎯ low recovery of endophytes from such as Aspergillu flavus ⎯ ⎯ 0.2 H. ovalis. Studies on other seagrasses, especially A. niger ⎯ ⎯ 0.2 those with sturdier tissue than H. ovalis would throw Chaetomium globosum 0.2 ⎯ 0.2 more light on endophyte-seagrass associations and Chaetomella sp. ⎯ ⎯ 0.2 also on the possible occurrence of antifungal com- Cladosporium cladosporioides 0.2 ⎯ 0.6 pounds in these marine angiosperms. Curvularia lunata 0.2 ⎯ ⎯ Fusarium sp. 0.2 ⎯ ⎯ References Monodictys sp. ⎯ ⎯ 0.2 1 Carroll G C, Fungal endophytes in vascular plants: myco- Penicillium sp. 0.2 ⎯ ⎯ logical opportunities in Japan, Trans Mycol Soc Japan, Phialophora sp. 0.2 0.4 ⎯ 31 (1990) 103-106. Phoma sp. ⎯ ⎯ 0.2 2 Strobel G A, Yang X, Sears J, Kramer R, Sidhu R S & Hess Talaromyces sp. ⎯ 0.6 ⎯ W M, Taxol from Pestalotiopsis microspora, an endophytic Sterile mycelium ⎯ 0.2 ⎯ fungus of Taxus wallachiana, Microbiology, 142 (1996) 435-440. Almost all the terrestrial angiosperms studied so far 3 Suryanarayanan T S, Kumaresan V & Johnson J A, Foliar have mitosporic fungi, ascomycete fungi and some fungal endophytes from two species of the mangrove Rhizo- 8 phora, Can J Microbiol, 44 (1998) 1003-1006. sterile forms as endophytes . The leaf blade, petiole 4 Suryanarayanan T S & Kumaresan V, Endophytic fungi of and rhizome of H. ovalis also harboured such fungi as some halophytes from an estuarine mangrove forest, Mycol endophytes (Table 1). Normally, a large number of Res, 104 (2000) 1465-1467. fungal taxa were isolated as endophytes from the leaf 5 Ramamurthy K, Balakrishnan N P, Ravikumar K & Ganesan 9 R, Seagrasses of Coromandel coast of India, Flora of India- tissue of a single plant species . This is especially true Series 4, (Botanical Survey of India), 1992, pp 80. of tropical plants. Suryanarayanan et al.3 isolated 28 6 Dobranic J K, Johnson J A, & Alikhan Q R, Isolation of and 25 endophytes respectively from leaves of man- endophytic fungi from eastern larch (Larix laricina) leaves from New Brunswick, Canada, Can J Microbiol, 41 (1995) groves Rhizophora apiculata and R. mucronata. In the 194-198. case of H. ovalis however, we isolated only six endo- 7 Bills G F & Polishook J D, Recovery of endophytic fungi phyte species from tissue segments derived from 500 from Chamaecyparis thyoides, Sydowia, 44 (1992) 1-12. leaves (Table 1). Not only was the number of endo- 8 Bills G F, Isolation and analysis of endophytic fungal com- munities from woody plants, in Endophytic fungi in grasses phyte taxa less in this seagrass but their CD% was and woody plants: Systematics, ecology and evolution, edited also meager (Table 1). It is interesting to note that by Redlin S C & Carris L M, (APS Press, Minnesota), 1996, genera such as Phoma, Cladosporium and Fusarium pp. 31-65. that are frequently reported to occur as endophytes in 9 Petrini O, Fungal endophytes of tree leaves, in Microbial 10 ecology of leaves, edited by Andrews J A & Hirano S S, terrestrial plants of the tropics also occur in the tis- (Springer-Verlag, New York), 1991, pp 179-197. sues of seagrass. 10 Brown K B, Hyde K D & Guest D I, Preliminary studies on Leaves of healthy seagrasses are commonly colo- endophytic fungal communities of Musa acuminata species complex in Hong Kong and Australia, Fungal Diversity, 1 nized externally by algae, bacteria and protozoa. It is (1998) 27-51. now clear that endophytic fungi also colonize seagrass 11 Nielsen S L, Thingstrup I & Wigand C, Apparent lack of ve- leaves albeit in low frequencies. The restricted fre- sicular-arbuscular mycorrhiza (VAM) in the seagrasses Zos- quency of occurrence and diversity of endophytes in tera marina L. and Thalassia testudinum Banks ex König., Aquat Bot, 63 (1999) 261-266. H. ovalis could be due to the environment where it is 12 Zapata O & McMillan C, Phenolic acids in seagrasses, Aquat 11 found. Nielsen et al. attributed the lack of mycorrhi- Bot, 7 (1979) 307-317. zal fungi in the roots of seagrasses to the salinity and 13 Cariello L & Zanetti L, Effect of Posidonia oceanica extracts on the growth of Staphylococcus aureus, Bot Mar, 22 (1979) low oxygen level that prevail in the marine environ- 129-131. ment where seagrasses grow. In addition, the leaves 14 Jensen P R, Jenkins K M, Porter D & Fenical W, Evidence of seagrasses contain phenolic acids12 and other com- that a new antibiotic flavone glycoside chemically defends pounds that are antibiotic in nature13. Recently, Jensen the sea grass Thalassia testudinum against zoosporic fungi, 14 Appl Environ Microbiol, 64 (1998) 1490-1496. et al. reported the presence of a novel flavone glyco- 15 Rajagopal K & Suryanarayanan T S, Isolation of endophytic side antibiotic Thalassia testudinum that defends this fungi from leaves of neem (Azadirachta indica A. Juss.), seagrass from infection by zoosporic fungi. Terrestrial Curr Sci, 78 (2000) 1375-1378.