SUMMARY
Pecteilis
susannae is a wild types of terrestrial orchid grown on
Forest-floor of Wonosadi Gunung Kidul. P.
susannae naturally occurring orchid was grown associated with soil fungi; Fusarium and Epuloriza. These fungi promote their seeds germination (Chutima et al., 2010 ). Besides that, The
capability of P. susannae to grow in
nature was assisted by phytohormone IAA derived from plant itself or
environment. Indole-3-acetic acid (IAA) is the most active auxin hormone group responsible
for stimulating plant growth and roots formation. The IAA synthesis in plants was
affected by tryptophan. Root exudates are known to be the source of
tryptophan in rhizosphere soil (Tsavkelova et
al., 2005). The previous research had proven that IAA
producing yeasts had been found from rhizosphere especially Williopsis (Nassar et al., 2005), Pichia (Nakamura
et al., 1991), Rhodotorula (Azeredo et al.,
1998), Candida, Cryptococcus
(Slavikova dan Vadkertiova, 2000) dan Saccharomyces
(Spencer dan Gorin, 1971). Therefore, the study was carried in order to
obtain indole-3-acetic acid producing yeasts from rhizosphere soil of
terrestrial orchid Pecteilis susannae
from several location in Wonosadi Forest Gunung kidul and to investigate the capability
of yeast isolates to produce IAA on medium supplemented with L-tryptophan as
precursor.
This study was commenced with direct isolation of
yeast from rhizosphere soil of orchids P.
susannae, using Yeast Malt Agar (YMA) medium. Yeast isolates were selected
based on their capability to produce IAA on Glucose Peptone Broth (GPB) medium
supplemented with 200 μg/ml L-tryptophan. The concentration of IAA was
determined colorimetrically on UV-1601PC spectrophotometer (OD530
nm) with Salkowsky’s reagent added to supernatant of yeast culture. Uninoculated
medium with the reagent added was used as the control. The ability of selected
yeasts to produce IAA was elucidated through IAA production experiment on
Glucose Peptone broth (GPB) and Salt Basal Medium (SBM) supplemented with and
without L-tryptophan (200 μg/ml), incubated on a shaker (120 rpm, 30oC).
Uninoculated medium was used as the control. Biomass (OD600 nm) and
IAA production (OD530 nm) were determined per 24 hours for 4 days. The
IAA compound on culture of selected yeast isolates was extracted with ethyl
acetate. The IAA was identified through Thin-Layer Chromatography (TLC) by
spotted the yeast culture extract on silica gel covered-plates. The plate was
observed under UV illumination (λ254 nm and λ365). Selected isolates of IAA
producing yeasts were characterized and identified using standard methods for
yeast.
The results of the study revealed that twenty nine yeast
isolates had been successfully isolated from rhizosphere of P. susannae, only 4 isolates (K2, U5,
A3, and A1) were able to produce high concentration of IAA on medium GPB supplemented
with L-tryptophan 200 μg/ml. These selected yeasts showed the higher production
of IAA up to 5 times on GPB and SBM supplemented with 200 μg/ml L-tryptophan than
non-supplemented medium. IAA production of selected yeasts (K2, U5, A3, and A1)
on GPB medium without L-tryptophan were 11.048, 10.690, 10.595, and 9.738 μg/ml
respectively, and 2.428, 1.167, 1.119, and 1.452 μg/ml respectively on SBM. On the
other hand, IAA production on medium supplemented with 200 μg/ml L-tryptophan
were 31.191, 51.524, 56.262, and 13.286 μg/ml respectively on GPB medium, and
2.789, 5.095, 2.619, and 2.619 μg/ml respectively on SBM. These result
indicated that L-tryptophan is the main precursor in IAA synthesis by yeast and
enhances the production of IAA, and also that the synthetic medium
(SBM) lacked the nutrients required for optimal growth and development of yeast
cultures and for IAA biosynthesis (Tsavkelova et al., 2005). The IAA identification on silica gel-plate showed
that U5 and A3 isolates produce compound with the same Rf value compared to IAA standard.
Identification of selected yeast isolates based on
their morphology and physiological-biochemical characteristics, K2 and U5 isolates
share the same character with member genera of Saccharomyces, so that these isolates presumably identified as strain
Saccharomyces sp.1 (K2) and Sachharomyces sp.2 (U5). The character
of A3 isolate share the same character with member genera of Cryptococcus, these isolate presumably identified
as strain Cryptococcus sp. 1. The
characters of A1 isolate share the same characters with the member genera of Candida, so that this isolate was presumably
identified as strain Candida sp.1.
Based on the result,
concluded that at the rhizosphere of terrestrial orchid Pecteilis susannae (L.) Rafin can be found the IAA producing
yeasts. Those yeast produce IAA 5 times higher on medium containing 200 μg/ml
L-tryptophan than medium without L-tryptophan.
Those IAA producing yeasts from rhizosphere of orchid Pecteilis
susannae (L.) Rafin presumably identified as strain Saccharomyces sp.1 (K2), Saccharomyces
sp.2 (U5), Cryptococcus sp. (A3), and
Candida sp.1 (A1).
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