Secondary metabolites from the Aspergillus sp. in the rhizosphere soil of Phoenix dactylifera (Palm tree)

The soil-derived fungus Aspergillus sp. isolated from the rhizospheric soil of Phoenix dactylifera (Date palm tree) and cultured on the large scale solid rice medium yielded a novel compound 1-(4-hydroxy-2,6-dimethoxy-3,5-dimethylphenyl)-2-methyl-1-butanone (1) and four known compounds; citricin (2), dihydrocitrinone (3), 2, 3, 4-trimethyl-5, 7-dihydroxy-2, 3-dihydrobenzofuran (4) and oricinol (5). The structures of the isolated compounds were elucidated by MS, 1H, 13C and 2D NMR spectra. Compound (1) exhibited potent antimicrobial activities against Staphylococcus aureus with MIC values of 2.3 μg mL−1 and significant growth inhibitions of 82.3 ± 3.3 against Candida albicans and of 79.2 ± 2.6 against Candida parapsilosis. This is the first report to isolate metabolites from the fungus Aspergillus found in temperate region date plant rhizospheres. Electronic supplementary material The online version of this article (10.1186/s13065-019-0624-5) contains supplementary material, which is available to authorized users.


Introduction
The rhizosphere is the portion of the soil which is surrounding the plant root [1,2]. This soil inhabited a great microbial diversity than nonrhizosphere soil [3].The microorganisms in the rhizosphere play a great biological role in the growth of host plant. This occurs through the defense mechanism provided by the rhizosphere microbial communities against pathogens or through providing nutrition to the plant by their role in mineralization of different organic compounds [4,5]. Fungi for instance, provide the plant with phosphorous while asymbiotic and symbiotic bacteria play an important role in nitrogen fixation and instantly increase of the available nitrogen in the rhizosphere region [6]. However, the diversity of microbial strains varies from one rhizosphere to another according the species of the plant and the environmental factors [7,8].
Recent reports show that the rhizosphere region of soil hills is untapped source of clinically important microorganisms, especially fungi [9][10][11][12][13][14] which produce a large number of bioactive metabolites. However, the attention for isolation of novel compounds with great pharmaceutical value from this fungal habitat still limited comparing to endophytes and marine niches.
Phoenix dactylifera, usually known as a date palm tree, it is globally valued for its health and nutritional-promoting fruit [15]. This tree grown in the arid and semi-arid regions especially areas which have long, dry summer and mild winter are best for date palm cultivation [16]. Kingdom of Saudi Arabia is the second top producer and exporter of dates since this tree covers more than 170 thousand hectares [17].
The filamentous fungi Aspergillus are ubiquitous opportunistic moulds that are pathologically and therapeutically important [18]. Many literatures reported numerous bioactive metabolites isolated from Aspergillus sp. [19][20][21]. These metabolites showed significance therapeutic importance such as anticancer and antimicrobial activities. The biological value of this fungal species, make it of considerable interest to the scientific research community for discovering further novel bioactive compounds [22].
As a part of our ongoing search on bioactive fungal secondary metabolites from unexplored niches [23,24], in this study, a fungal strain RO-17-3-2-4-1, identified as Aspergillus sp., was isolated from the rhizosphere soil of P. dactylifera, Wadi Hanifa, 15 km Northwest of Riyadh, Saudi Arabia. To the best of our knowledge, it is the first research report on the isolation of secondary metabolites from the rizosphere soil of temperate region plants P. dactylifera.

Isolation and structural identification
Disease suppressive soils offer effective protection to plants against infection by soil borne pathogens. Therefore, suppressive soils are considered as a rich source for the discovery of microorganisms which provides novel secondary metabolites on large scale culture. To date, a plethora of work has been done on the fungal culture of the obtained microorganism from these soils which led to the isolation of novel biologically active constituents. In our ongoing research on the findings of soil based microorganism and its culture for the identification of secondary metabolites, we worked on the crude ethyl acetate extract of the interrhizospheric fungus (Aspergillus sp.). It exhibited considerable antimicrobial activity against the tested bacterial and fungal strains. Bioactivity-guided fractionation led to the isolation of one new compound 1-(4-hydroxy-2,6-dimethoxy-3,5-dimethylphenyl)-2methyl-1-butanone 1, together with four known compounds; citricin 2, dihydrocitrinone 3, 2, 3, 4-trimethyl-5, 7-dihydroxy-2, 3-dihydrobenzofuran 4, and oricinol 5 ( Fig. 1). Herein, we report the structure elucidation and biological evaluation of the isolated compounds.
of the NMR data with literature values.

Biological activities
All isolated compounds (1-5) were evaluated for their antimicrobial activity against pathogenic bacteria and fungi by disc diffusion method by measuring the inhibition zones and for the active compounds (MIC) minimum inhibitory concentration values were also determined. Interesting antimicrobial properties were observed ( For human pathogenic fungi, the simple aromatic compound 5 disclosed the most significant growth inhibitions of 92 ± 3.9 and 90 ± 2.8 at 50 μg mL −1 against Candida albicans and Candida parapsilosis, respectively. Followed by compounds 1, 2, and 4 with higher inhibition value than the positive control Itraconazole a broad-spectrum antifungal drug. Compounds 3 neither showed antifungal nor antibacterial activity at 25 μg mL −1 . These result suggested that the aromatic ring in polyketides may strengthen the antibacterial and antifungal activities of this class of compounds.

General experimental procedures
The experimental procedure has written in Additional file 1.

Plant and fungal strain materials
The fungal strain was isolated from rhizosphere soil of P. dactylifera, Wadi Hanifa, 15 km Northwest of Riyadh, KSA, in October 2017 and deposited in the laboratory of Pharmacognosy department, KSU. The fungus was identified as Aspergulis sp. (GenBank accession No. MK028999) according to DNA amplification sequencing of the fungal ITS region as reported in literature [28,29].

Antibacterial assay
The antibacterial activity was determined according the reported method [20]. The Gram-positive, Staphylococcus aureus (CP011526.1) and Bacillus licheniformis (KX785171.1) and the Gramnegative, Enterobacter xiangfangensis (CP017183.1), Escherichia fergusonii (CU928158.2) and Pseudomonas aeruginosa (NR-117678.1) bacteria were suspended in a nutrient broth for 24 h then spread on Muller Hinton agar plate. 10 µL of the sample solution were loaded in wells using Amikacin as positive control. The clear area which was free of microbial growth was measured triplicate to detect the diameter of zone of inhibition and the mean were recorded. The lowest concentration of the tested isolated compounds that will inhibit the visible bacterial growth, minimal inhibitory concentration (MIC, μg mL −1 ) was determined as well [28].

Antifungal assay
The antifungal activity of isolated compounds was assessed using well diffusion and broth microdilution techniques with positive control, Itraconazole. The tested pathogenic fungi were Candida albicans and C. parapsilosis. According to Gong and Guo [29], in SDA plate the sample solutions (100 µl), approximately 3 × 10 6 colonyforming units (CFU) mL −1 was smeared. Wells were created in SDA plates and loaded with the 10 µg of the tested compounds. The plates were then incubated at 37 °C for 1 day. The diameters (in mm) of zone of inhibition were measured and the rates of growth inhibition were obtained according the following formula taking on consideration ± SD as means: where d c : Diameter of the untreated control fungus, d s : Diameter of the sample-treated fungus and d 0 : Diameter of the fungus cut.

Conclusions
Polyketides possess a wide range of significant biological activities, such as anti-tumor, antimicrobial and antiinflammatory. In our study, one new and four known metabolites were obtained from the large scale fermentation of the interrhizospheric fungus Aspergillus sp., and their antimicrobial activity was evaluated. The isolation of compounds 1-5 suggested that this Aspergillus strain is a powerful producer of polyketides with diverse structures. Compounds 1 showed significant antimicrobial activity against two pathogenic fungal strains Candida albicans and C. parapsilosis and a pathogenic strain of bacteria Staphylococcus aureus with MIC 2.3 μg mL −1 . This study shows the importance of rhizospheric soil inhibited fungi as untapped source for novel secondary metabolites.

Additional file
Addit ional file 1 NMR, Mass spectrum & chromatogram of extracts.