Purification and determination of active compounds and their antifungal activity.

Strain GP72 was grown in KMB liquid medium for 3 days at 28_C at 220 rpm in a rotary
shaker. Bulk fermentation was performed on a 6-L scale of strain GP72 in KMB medium
for 3 days. Twelve repetitions were performed under the same fermentation conditions.
The pH value of the fermentation broth was adjusted to 2.0 with 1 M HCl. Supernatant
was collected after the broth was centrifuged at 10,000 · g for 10 minutes, and then it was
extracted with equal volume of ethyl acetate with rigorous shaking. The collected organic
layer was mixed with 1/10 volume distilled water and shaken rigorously. Finally, the
organic phase was taken for evaporation under vacuum pressure. The residue was
dissolved in methanol, and major compounds were separated using preparative high-
performance liquid chromatography (HPLC) (Shimadzu LC8A) in mobile phrase of 57%
methanol plus 43% 5mM NH4AC (pH 5.0) solution with an ultraviolet (UV) light
detector monitored at 248 nm. Purified fractions were collected and used for testing
antifungal activities against a broad spectrum of phytopathogens according to the disc-
diffusion method [3]. Active fractions were further dried under decreased pressure.
Molecular structures of the active fractions were further determined by measuring mass
spectrum with a liquid chromatographer–mass spectrometer (HP1100 MSD) and 1H and
13C nuclear magnetic resonance (NMR) with AVANCE DRX400 (BRUKER,
Switzerland) spectrometer.

Detection of quorum-sensing signaling molecules produced by strain GP72.

A single colony of tested strains was inoculated into 150 ml KMB or PPM medium and
grown for 72 hours individually. For extraction of signaling molecules, 1 ml of each
broth was extracted with an equal volume of ethyl acetate, dried under vacuum pressure,
and redissolved in 10 ll methanol. A volume of 3 ll was used for inducing quorum-
sensing reporter assay for C. violaceum (cvi) C. violaceum CV026 was grown in LB
medium supplemented with 50 lg ml–1 kanamycin for 24 hours. LB agar plates were
overlaid with a semi-LB top agar layer mixed with a 24-hour culture of 200 ll ml–1 C.
violaceum CV026 indicator strain [19]. Activity of the fractions was evaluated after 24
hours of growth at 28_C by the appearance of a violet halo around the spot caused by
violascein production resulting from activation of reporter genes in C. violaceum.
Autoinducer activity on C18 reverse-phase thin-layer chromatography (TLC) (Merck,
Darmstadt, Germany), developed in methanol with water (vol:vol = 60:40), was detected
by overlaying TLC plates with a semi-LB top agar layer containing CV026 cultured cells.
The TLC plates were then incubated at 28_C for 24 hours and analyzed for the
appearance of violet spots. P. aureoginosa PAO1, P. aureofaciens 30– 84, chemically
synthesized N-butanoyl-L-homoserine lactone (C4- HSL), and N-hexanoyl-L-homoserine
lactone (C6-HSL) (Sigma) were used as standards.