Streptomyces sp. isolate UPMRS4 (KT693185), identified from preliminary screening, was cultivated on potato dextrose agar (PDA) (Difco™ Company USA) and incubated at 28˚C for seven days. The agar was cut into small pieces (about 1 cm) to be used for extraction with solvents of different polarity (chloroform, ethyl acetate, methanol, acetone, ethanol, diethyl ether and water) and placed into 500 ml top capped bottles with 250 ml of each solvent. The mixtures of agar and each solvent were shaken in a rotating shaker at 150 rpm overnight at 30˚C. This was followed by centrifugation of the mixtures at 10,000 rpm (Avanti J-26 XPI centrifuge, Beckman Coulter, USA) for 10 min to separate from supernatants. A vacuum filtration pump was used to filter the extracts with two layers of Whatman No.1 filter papers (ALBERT^R) before drying with a rotary evaporator at 40˚C. The concentrated powder of each extract was regarded as crude sample and stored at −20˚C for further use. Crude extracts were dissolved in 50% methanol for antifungal, bioassay and chromatography analysis.

Agar well diffusion method was used to screen the crude extracts for any antifungal activity following Patel et al. [12] with some modifications. Antifungal bioassay was done on 9 cm Petri plate with 20 ml of PDA. The fungal pathogen used was P. oryzae (KT693184), obtained from the Department of Plant Protection, Universiti Putra Malaysia. A 5 mm fungal plug was cut from the leading edges of a seven-day-old pure culture of P. oryzae, and was put in the center of the plate. A 5 mm diameter well was made at 2.5 cm from the fungal plug and another well at the opposite side, followed by pipetting 30 µl aliquot of each crude extract into the two wells on each plate. Control plates contained wells of filter-sterilized methanol 50% and sterile distilled water. Radial growth was recorded after incubation for 10 days. The radial growth of fungal colony was recorded with a meter ruler along two diagonal lines drawn on the reverse side of each plate. Three plates with two radial measurements per plate were conducted and the experiment was repeated twice. The percent inhibition of radial growth (PIRG) of each treatment compared to control was computed utilizing the formula below:

Crude extract which showed the highest antifungal activity was selected to determine the effective inhibitory concentration (EIC) at 50% inhibition of mycelial growth. The EIC was determined using the antifungal bioassay described in section 2.2 above with concentrations of 1.56, 3.125, 6.25, 12.5, 50 and 100 µg/ml.

Crude extract which showed the highest antifungal activity was subjected to aShimadzu GC-17A attached to a Shimadzu GC-MS-QP5050A system. The column used was a Phenomenex Zebron ZBFFAP ultra-low-bleed Bonded Polyethylene Glycol fused capillary column of 30 ml × 0.25 mm I.D × 0.25 µm film thickness. Split ratio 20 injection was performed. Helium was the transporter carrier gas with a stream flow rate of 0.7 ml/ min. The column temperature was kept at 70˚C for 3 min, then modified at 10˚C/min to 90˚C via programming and finally modified at 5˚C/min to 230˚C. The inlet and detector temperatures were 230˚C and 250˚C, respectively, while the dissolvable deferral (solvent delay) was 5.75 min [13] .

An AB Sciex 5500QTrap (Linear Quadrupole Hybrid Ion ‎Trap Mass Spectrometer, AB Sciex, Toronto, Canada) mass spectrometer operating in ‎electrospray ionization (ESI) negative mode and hyphenated with an Agilent 1290 ultra-high ‎performance liquid chromatography system was used. The high purity nitrogen gas for the ‎mass spectrometer was set at 40 psi for source gas, 40 psi for the heating gas and HIGH for collision ‎gas with a source temperature of 500˚C. ‎The setting for electrospray ionization voltage was set to 4500 kV. ‎The collision energy to attain fragmentation was set at 35 eV with a spread of ±15 eV.‎ Mass range for MS/MS scan was set from ‎‎50 - 1000 m/z while mass range for full scan was set from 100 - 1000 m/z while scan speed was set at 1000 m/z per second.‎A Phenomenex Synergi Fusion RP (100 mm × 2.1 mm i.d., 3 um ‎particle size, Phenomenex, CA, USA) was used to obtain separation. The mobile phase was made up of aqueous ammonium ‎formate (5 mmol/L) with 0.1% formic acid (solvent A) and acetonitrile with ammonium formate‎‎ (5 mmol/L) with 0.1% formic acid (solvent B). The compounds were separated with the ‎following linear-programmed solvent gradient: 0 min (10% B), 10 min (95% B), 2 min (95% B) ‎then equilibrating back to 10% B for 3 min.‎ The flow rate for the column was set at 0.25 mL/min while the column temperature was set at 40˚C and injection ‎volume at 10 uL.‎

Statistical analysis was done using one way Analysis of Variance (ANOVA) at confidence level 95% using SAS, 2003. Comparison of means was conducted with Duncan.

The inhibition rates of different solvent extracts of Streptomyces sp. isolate UPMRS4 on the mycelia growth of P. oryzae are shown in Figure 1. All extracts showed some effectiveness on the tested pathogen ranging from 55.3% to 98.33%. Ethyl acetate extract showed the highest inhibition significantly different from other solvent extracts. Based on this result, ethyl acetate was chosen for the rest of the experiments. Figure 2 shows the 98.33% inhibition of mycelial growth of P. oryzae compared to the control.

All the tested concentrations of ethyl acetate crude extract exhibited inhibition on mycelial growth of P. oryzae. The suppressive effect increased with the ‎increase in concentration at 1.56 to 100 μg/ml (Figure 3). Concentration of 100 µg/ml showed significantly higher inhibition of the pathogen (98.33%) followed by 50 µg/ml (78.33%) and 25 µg/ml (75.66%). There was no significant difference between the three of them but there was significant difference compared to that of 12.5 µg/lm (64%). Lower concentrations of 6.25, 3.125 and 1.562 µg/ml were also able to inhibit mycelia growth at 56.66%, 56.16% and 55.3%, respectively.

The result related to GC-MS dissection showed the presence of 22 different volatile compounds in the ethyl acetate crude extract (Figure 4). The major compounds present in the extract as indicated by some highest peaks were identified as 1) Pyrrolo[1,2-a] pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl) with the retention time 16.748; 2) Pyrrolo[1,2-a] pyrazine-1,4-dione, hexahydro-3-(phenylmethyl) with the retention time 22.148; 3) ergotamine with the retention time 21.783.

The presence of peaks that appeared in the chromatogram showed the presence of 35 different compounds from ethyl acetate crude extract (Figure 5). Four of the compounds in the extract indicated by highest peaks were identified as 1) Amicomacin(C₂₀H₂₉N₃O₇), with peak at retention time of 3.25 and molecule weight 421.68 (Figure 6); 2) Fungichromin (C₃₅H₅₈O₁₂), with peak at retention time of 11.85 and molecule weight 670.49 (Figure 7); 3) N-Acetyl-D, L-phenylalanine (C₁₁H₁₃NO₃), with peak at retention time of 2.98 and molecule weight 206.38 (Figure 8) and (4) Rapamycin, (C₅₁H₇₉NO₁₃), with peak at retention time of 6.61 and molecule weight 912.85 (Figure 9).