Rice plant leaves showing symptoms of blast disease were collected from rice plant field located in Tanjung Karang, Selangor state, Malaysia in June 2015 after the observation of pathogen lesions, and then placed into sterilized plastic bags. The samples were transferred to the department of Plant Protection Laboratory in the Faculty of Agriculture, University Putra Malaysia. After collection, the samples were kept at 4˚C after air-drying until needed for the isolation process. The isolation of the pathogens was done using tissue plating technique described by [7] . The selected fungus A. niger was isolated from the collected soil sample by utilizing planting fold dilution method described by [8] . After air-drying, soil sample was sieved and cleaned well. Then, 1 mg of soil transferred to a sterilized tube consists of 10 mL of distilled H₂O. Next, Another 1 mL of suspension transferred from the previous tube to another one consists 9 mL of dH₂O to reach the final concentration. The procedure continued respectively to reach 10 times of dilution. Finally, 1 mL transferred and distributed on petri dish containing Potatoes Dextrose Agar (OXOID™) medium and grown for 7 - 10 days at 28˚C till use for the further experiments.

The pathogenicity test was done according to separated leaf assay technique [9] . Leaf fragments of 2 mm in size were cut from two isolated 10 days old colonies and placed onto the center of two-month-old rice plant leaf, and then incubated at room temperature (27˚C ± 2˚C). After 7 days of incubation, lesion development was observed and the length was measured.

Fungi were grown on PDA at 28˚C ± 2˚C for 3 - 4 days in order to obtain a pure fungal mycelia mats before the producing of spores that may cause contamination during DNA extraction. Fungi mycelia were cut from the colony and separated from the PDA medium using laboratory forceps in order to obtain clean fungal mycelia, then placed onto a sterilized filter paper for 30 min to be dried. Fungi mycelia transferred to a collection tube 1.5 ml and grinded using tissue grinder. DNA was extracted by the modified CTAB method by [10] . The obtained genomic DNA was incubated in TE buffer and stored at −20˚C for further use. The region (ITS 5.8S rDNA) was used to amplify the genomic DNA [11] . The universal primers (ITS1 and ITS4, synthesized by 1^st base company SdnBhd, Malaysia) were assayed to amplify the DNA [12] . PCR amplification was applied in PCR tube containing 25 µl (taq. PCR) Master Mix, 15 µl free nuclease water, 3 µl of each Forward primer ITS1 and Reverse ITS4 then 4 µl of template DNA. The PCR reaction was done due to the following cycle condition: Initial denaturation at 94˚C for 3 minutes; 30 cycles of 94˚C for 30 sec denaturation, 45 sec for annealing at 55˚C and 72˚C for 1 minute elongation 35 cycle; and a final extension step of 72˚C for 5 minutes. Electrophoresis gel technique was conducted to screening PCR products, volume of 5 µl of PCR amplified product was investigated on (1.5% Agarose gel TopVision™ eluted with 100 ml of 1X TBE buffer) using 4 µl of DNA safe dye for visualization. Sequencing technology reaction was performed at 1^st Base Company SdnBhd, Malaysia. The sequencing results were clarified by utilizing BioEdit sequence alignment editor [13] . The acquired sequence from reverse primer was transformed to reverse complement and then aligned with acquired sequence from the forward primer to obtain the consensus sequence. GenBankin formations were used to analyze and identify the species of fungal isolate sequence by BLASTn alignment. The software Mega 7 was utilized to analyze the phylogenetic tree of identified fungi.

The dual culture technique on PDA medium (Gupta et al., 2001) was used in this experiment to select the most potent fungal isolates from the soil that show better activity against P. oryzae. Disks of about 5 mm were cut from A. niger isolate 4 days-old and inoculated at 1 cm distance from the edge of the Petri dish containing PDA medium. On the other side of the plates, disks of 5 mm size cut from the pathogen isolate of 7 days old were placed at 1 cm from the other edge of the Petri dish. The disk containing the pathogen mycelium was used to prepare the controls on separate PDA plates. All the experiments were conducted in triplicates and incubated at room temperature for 7 days. After the incubation period, the radial growth of each fungus was measured and calculated using the formula below.

I n h i b i t i o n   G r o w t h   R e d i a l ( % ) = R a d i a l   g r o w t h   o f   c o n t r o l − R a d i a l   g r o w t h   o f   t r e t m e n t R a d i a l   g r o w t h   o f   c o n t r o l × 100 %

The culture filtrate method was used to determine the secondary metabolites efficiency of the selected isolates against blast pathogen. The experiment was conducted according to the method described by [14] with some modification. Firstly, disks of about 5 mm containing the A. niger isolate was inoculated into 250 mL of PDB (OXOID™) prepared in sterilized 500 mL conical flasks and incubated at 26˚C ± 2˚C on an orbital rotary incubator shaker at 120 rpm for 14 days. After every 7 days of incubation, three volumes (50, 25 and 12.5 mL) of fungal supernatant were taken from A. niger culture. The collected supernatants were transferred to 50 mL test tubes and centrifuged at 5000 rpm for 5 min to remove remaining mycelial fragments that might be present in the supernatant. After centrifugation, the supernatants were transferred to a new set of tubes and then filtered twice using sterilized Whatman No. 1 filter paper; followed by filtering through 0.45 and 0.22 µm pore biological membrane filters. Molten PDA medium was used to make up the final concentration of the filtrates to 100% (v/v). The filtrates were poured into Petri plates and inoculated with 5 mm disks of P. oryzae placed at the center of the plates. Finally, for the positive control, sterilized dH₂O with the same concentration and volume was mixed with molten PDA to obtain a final concentration of 100% (v/v). All the treatments were performed in triplicates and incubated at 26˚C ± 2˚C for 10 days. The growth inhibition percentage was measured at the end of the incubation period.

Minimum inhibitory concentrations (MICs) were conducted to determine the lowest concentration of secondary metabolites of A. niger in which no growth of P. oryzae can be detected in the experiment. Concentrations less than 12.5% (v/v) were performed with following of fold serial dilutions. A. niger was incubated for 14 days in incubator shaker at 26˚C ± 2˚C. After incubation, volumes (6.25, 3.125 and 1.562 ml) were collected and filtered using (Watman No.1) filter paper then 0.45 µm followed by 0.22 µm millix syringe filter. Agar diffusion method was assayed to all filtered volumes and adjusted to the final concentration 100% (v/v) with molted PDA (OXIOD™). Each plate received a 5 mm disk of P. oryzae, and then incubated at 26˚C ± 2˚C for 10 days. The inhibition percentage was measured.

The production of soil fungal secondary metabolites is affected by different factors such as the temperature and pH [15] . This experiment was conducted to determine the optimum circumstances in producing of secondary metabolites of A. niger. Therefore, A. niger was grown on PDB distributed in 4 conical flasks and pH medium adjusted to (3, 5, 7 and 9) respectively by adding HCL and NaOH [4] . The measurement of pH was by pH meter. Flasks were kept at 26˚C ± 2˚C for 14 days to let A. niger grows in different level of pH. Besides, PDB was added to other 4 conical flasks inoculated with A. niger then incubated under various temperature (21˚C, 25˚C, 29˚C and 34˚C) for 14 days to find the optimum temperature for producing of metabolites. Volume 12.5 ml of each treatment (pH and temperature) was filtered then added to 85.5 ml of molten PDA to provide the final concentration 100% (v/v). Lastly, volumes poured in petri dishes then inoculated by 5 mm mycelial disks of 5 days old colony of P. oryzae. Treatments incubated for 10 days then percentage of radial growth was measured.

About 5 mL of fungal secondary metabolites of A. niger aged 14 days on PDB was filtered and sent to the Department of Chemistry, Faculty of Sciences, University Putra Malaysia for GC-MS dissection analysis. The GC-MS analysis of the sample was done using SHIMADZU GC-MS QP 2010 plus with EI electron impact ion source of 70 eV employing a BPX5 Fused Silica Capillary Column (30 m × 0.25 m ID × 0.25 FILM THICKNESS). The temperature program was at the range of 100˚C for 5 minutes to 250˚C for 10 minutes. The purge flow program was 3.0 mL/min. The retention time and peak of areas were recorded by integrating electronically. The obtained peaks were compared and matched with the institute of National Standards and Technology (NIST 08 and NIST 08s) library and correlated with the distributed data that have been published.

The two obtained isolates of P. oryzae were named as UPMZPO and UPMZPO1. These strains identified morphologically as a simple whitish mycelium toward the colony center, moderately and become greyish brown to dark looking for the rest of the colony, and single (or fasciculate) conidiophores [16] . These two isolates were revealed typical characteristics of blast pathogen. Besides, pure isolates of A. niger colony aged 7 days was observed as grayish black to black colony with white mycelia at the edge of the fungus colony [17] .

The two obtained isolates were shown virulence pathogenicity on rice plant leaf (Figure 1). The isolate UPMZPO was selected for the further testes according to lesion length on the surface of rice leaf. The disease symptoms appear as brown lesions while on the sensitive leaves, diamond-form or spindle-form spots.

The DNA purity and quantity were identified by NanoDrop 2000 c spectrophotometer (Thermo Fisher Scientific, USA). Based on screening of gel electrophoresis, DNA fragments for the isolate UPMZ01 and UPMZPO were amplified at range (582 and 527 bp) respectively. Measurement was according to molecular weight marker (100 bp ladder) as in (Figure 3). Investigations of DNA sequencing were obtained by NCBI Blast. The first isolates identified as Aspergillus niger named as UPMZ01. The partial sequence of 18s rDNA of UPMZ01 isolate was shown up to 100% similarity to A. niger with an accession (KU729033). While the pathogen isolate identified as Magnaportha oryzea (Pyriculariaoryzae) were shown 100% similarity to the accession (AB274418) P. oryzae. Lastly, phylogenetic tree for the identified fungi was made by using Mega 7 software (Figure 2). The identified fungi were recorded in GenBank with accessions (KY698415 and KY698417).

The dual culture result was shown a clear inhibition zone of the pathogen P. oryzae. The percentage inhibition of redial growth is up to 81.326% compared with the control plate (Chart 1 & Figure 3).

The results were observed from the agar diffusion method of filtered metabolites of UPMZ01 strain at the concentrations (12.5, 25 and 50 mL) shown (100%, 100% and 53.32%) percent inhibition growth respectively. These results were at 7 days of metabolites age dissolved in broth medium. While, the observations was done at 14 days of metabolites age (100%, 100% and 100%) of inhibition growth to all treatments. All were obtained results, compared with negative control plates (0% inhibition) which were had the similar concentration volume of sterilized distilled water as showing in Figure 4 and Figure 5.

The observed results were shown mycelium growth of P. oryzae in the plate with concentration 6.25% (v/v) of A. niger metabolites with inhibition percentage 42.85%. The inhibition percentage reduced respectively based on the decreasing of aqueous metabolites solution in Table 1 and Figure 6.

The treatments were shown significantly different results. Treatment with pH 5.0 was observed with 100% PIGR to the mycelia of tested pathogen while the treatment with PH 3.0 was given 83.803% PIGR. The pH 7.0 treatment was given 42.33% PIGR and pH 9.0 was shown inhibition percentage 13.26%. The Figure 7 and Figure 8 showed the PIGR mean of treatments.

The temperature test showed that the metabolites antimicrobial product of A. niger resulted under temperature range between 21˚C to 29˚C have been revealed inhibitory percentage up to 100% against P. oryzae (Figure 9 and Figure 10), while the treated metabolites under temperature 34˚C has been given inhibition 55.66˚C. As a result were observed, the range between 21˚C and 29˚C is the most efficient temperature for the production of A. niger antimicrobial metabolites.

The analysis of aqueous secondary metabolites was performed at the Department of Chemistry, Faculty of Science at University Putra Malaysia. The

Means of PIGR with the same letter are not significantly different.

obtained results indicated of 15 compounds were detected and identified by using mass spectrometry linked with gas chromatography. Most of them are fatty acids. The area and retention time of each presented compound was showed in Table 2.