ISOLATION AND IDENTIFICATION OF CYTRID FROM MANGROVE

AREA
FAIQ, I. S.1, ASMAT, A.1*
1

School of Biosciences and Biotechnology,

Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor
*
Corresponding author email: drasmart@gmail.com
ABSTRACT
Chytrids are very important components of freshwater ecosystems. In this study, chytrids were
successfully isolated using baiting method from water sample taken from mangrove area of
Teluk Kemang Port Dickson. Pollen grains were used to attract zoospores. Molecular
identification using BLASTn showed isolated chytrid has 98% similarity with Thrautochytrium
sp. FJN -10 in Gent Bank data. Lipid analysis has been done to one of isolated chytrid showed
that isolated chytrid has potential in docosahexanoic acid (DHA) production.
INTRODUCTION
Thraustochytrids are a widespread group of marine fungi, belonging to the Kingdom Chromista
(Cavalier-Smith et al. 1994) or Straminipila (Raghukumar 2002). Chytrid is considered
euryhaline organisms, which are able to adapt to a wide range of salinities (Jones and Harrison
1976) and has been found in estuarine, littoral, mangrove and oceanic water and sediments
around the world (Raghukumar 1987). Isolation also has been made from hyper saline
environment, such as evaporated rock pools and salt pan. Schneider (1981) has described growth
and sporulation of an isolate of Thrautochytrium pachydermum in salinities up to 60%.
Thraustochytrids contain the genera Thraustochytrium and Schizochytrium, which has been
known to produce lipids rich in polyunsaturated fatty acids such as eicosapentaenoic acid
(C20:5n-3), docosapentaenoic acid (C22:5n-6), and docosahexaenoic acid (C22:6n-3; DHA)
(Yongmanitchai & Ward 1989). In addition, Aki et al. (2003) previously reported that the DHA
producing Schizochytrium sp. strain KH105 also accumulates a significant amount of
xanthophylls such as canthaxanthin and astaxanthin.
METHOD
Isolation Of Chytrid Using Baiting Method
Baiting method was conducted according to Barr (1987). A total of 10ml of each sample was
pour into the petri dish. Then a few of pollen grains were spread out on the surface of the sample.
Ampicillin 500mg/L and novobiocin 500mg/L were added to the water sample to suppress
bacteria growth. Each sample was incubated for two weeks at 29 C with present of sunlight.
After 24 hours, the pollen grains were observed under the microscope for the present of chytrid
zoospores or thalli. If no zoospore present, samples were incubated again and observed every day
until 14 days. After zoospore colonized the pollen grain, the pollen grains were transferred into

B1 agar medium containing ampicillin 500mg/L and novobiocin 500mg/L. Cultures were
incubated at 29C for 4 days.
Characterization And Identification
Characterization and identification of chytrid were done based on colony morphology
observation, cell morphology observation and molecular identification.
Lipid Extraction
Lipid extraction was conducted according to the method by Sasser (1990) with slight
modification. A total of 3.0ml of Reagent 1 was added to each tube containing cells. The tubes
were vortexed briefly and heated in a boiling water bath for 5 minutes, at which time the tubes
were vigorously vortexed for 5-10 seconds and returned to the water bath to complete the 30
minute heating. 6ml of Reagent 2 was added. The tubes briefly vortexed. After vortexing, the
tubes were heated for 10 minutes at 80C. Addition of 4 ml of Reagent 3 to the cooled tubes was
followed by gentle tumbling for about 10 minutes. The tubes were uncapped and the aqueous
(lower) phase was pipetted out and discarded. The remaining organic phase in the tubes was
transfer into vial and dried for one day.
RESULTS AND DISCUSSION
Isolation
Sample from Teluk Kemang 1 (Wtk1) and Teluk Kemang 2 (Wtk2) show the appearance of
round structure fill with granular attached to pollen grain surface after 48 hours. Chytrid
zoospores are motile and attract to pollen grain because it contain cellulose. Zoosporic fungi feed
on chitin, keratin and cellulose they obtain from their environment or their hosts including algae,
fungi, plants, invertebrates and, as recently discovered, vertebrates (Gleason et al. 2008). This is
because chytrid also has the ability to efficiently digest particulate organic matter. After
incubation of chytrid on B1 agar in 29C, colonies were observed. Colony of both Wtk1 and
WTK2 are orange in color. Some colonies appear pale yellow. After 24- 48 hour, both grow as
small circular shape colonies.
Molecular Identification
Polymerase chain reaction (PCR) produced 1800bp single band. Result of sequencing for Wtk1
and Wtk2 is shown on Appendix A. DNA sequence extract from Wtk1 isolate produced
significant alignments with Thraustochytrium sp. FJN-10 with 97% similarity with Gene Bank
database. Accesion number from Gene Bank is FJ821482.1. DNA sequence extracted from Wtk2
isolate also produced significant alignments with Thraustochytrium sp. FJN-10 with 98%
similarity with Gene Bank database. Accesion number from Gene Bank is FJ821482.1
By using neighbour-joining method, phylogenetic tree were derived from DNA sequences
of Wtk1 and Wtk2 isolate formed one group . Wtk1 and Wtk2 isolate form clad with
Thraustochytrium sp. FJN-10.

Lipid Profile
Result showed that C16 were the highest lipid produced by chytrid isolate Wtk1 with 42.859%.
C16 is palmitic acid, which one of the most common saturated fatty acids found in animals and
plants. Chytrid isolate Wtk1 produced 18.636% of C22:6n3. C22:6n3 is DHA, which a highly
unsaturated long chain fatty acid.
CONCLUSION
Chytrid were isolated from Teluk Kemang 1 and Teluk Kemang 2 sites of Port Dickson
mangrove. The thalli of chytrid attached to pollen grain contained spherical and globose
zoosporangium. The Colony grows circular in the beginning and then became irregular as the
colonies grow. Color of the colony is orange. The cytoplasm appeared granular or filled with
multiple oil globules. DNA sequences of chytrid isolate Wtk1 produced significant alignments
with Thraustochytrium sp. FJN-10 with 98% similarity while chytrid isolate Wtk2 significant
alignments with Thraustochytrium sp. FJN-10 with 97% similarity. Lipid analysis showed
chytrid isolate Wtk1 produced palmitic acid with 42.859% and DHA with 25%.
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