A Streptomyces isolate having antifungal activity against Pyricularia oryzae , the causal agent of rice blast disease, was isolated from soil collected in rice fields of Tanjung Karang Selangor, peninsula Malaysia. The aim of the study was to determine the antifungal activity of Streptomyces sp. isolate UPMRS4 extracts against P. oryzae and to identify bioactive antifungal compounds produced by UPMRS4. Various solvents were used for extraction of antifungal compounds and well diffusion method was used to determine the antifungal activity of the extracts. The ethyl acetate extract demonstrated the highest activity against mycelial growth of P. oryzae, with an effective inhibitory concentration (EIC) of 1.562 μg/ml significantly higher compared to that of chloroform, diethyl ether, methanol, acetone, ethanol and water. Based on GC-MS and LC-MS/MSanalyses, compounds with antifungal activity were detected such as (Pyrrolo[1,2-a] pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl); Pyrrolo[1,2-a] pyrazine-1,4-dione, hexahydro-3-(phenylmethyl); ergotamine; amicomacin ; fungichromin; rapamycin and N-Acetyl-D, L-phenylalanine . These compounds had good general antifungal activity and might have potential future agricultural applications.
Rice (Oryza sativa L.) is among the most important crops all over the world and it is consumed as a staple as well as primary source of energy and protein. Rice blast disease, caused by the fungus Pyricularia oryzae, is among the most significant diseases affecting rice cultivation, since it is prevalent in most rice growing regions and causes serious yield losses [
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 (ALBERTR) 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. [
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 [
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
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 (
The result related to GC-MS dissection showed the presence of 22 different volatile compounds in the ethyl acetate crude extract (
The presence of peaks that appeared in the chromatogram showed the presence of 35 different compounds from ethyl acetate crude extract (
The use of antagonistic microorganisms like Streptomyces spp. is an appropriate approach to effectively control
diseases in plants [
In the present study, the result demonstrates the presence of 22 different volatile compounds in the ethyl acetate crude extract. Recent studies have illustrated that a number of these compounds can lead to direct inhibition of fungal and bacterial pathogens. Pyrrolo 1,2-a pyrazine group is a group of potent naturally occurring antibiotics from various Streptomyces species, which are among the most promising types of lead compounds [
Based on the chemical constituents identified using LC-MS/MS, Azumi [
Our findings revealed that rapamycin (sirolimus), it was initially found to be an antimicrobial compound to bust activity against Candida albicans [
In conclusion, the findings of the current study showed that Streptomyces sp. isolate UPMRS4 had significant antifungal activity against rice blast pathogen, P. oryzae. Ethyl acetate crude extract showed the highest inhibition on the mycelial growth of P. oryzae and the EIC was determined as 1.562 μg/ml. GC-MS analysis showed that ethyl acetate crude extract of isolate UPMRS4 contained macrolides compounds, Pyrrolo[1,2-a] pyrazine-1,4-dione group while LC-MS/MS demonstrated the presence of fungicidal compounds such as amicoumacin, fungichromin, N-acetyl-D, L-phenylalanine and rapamycin. These results indicate that Streptomyces sp. isolate UPMRS4 had the potential to be developed as a biocontrol agent to control rice blast pathogen, P. oryzae.
Hayman Kakakhan Awla,Jugah Kadir,Radziah Othman,Tavga Sulaiman Rashid,Mui-Yun Wong, (2016) Bioactive Compounds Produced by Streptomyces sp. Isolate UPMRS4 and Antifungal Activity against Pyricularia oryzae. American Journal of Plant Sciences,07,1077-1085. doi: 10.4236/ajps.2016.77103