Screening of Compounds Secreted by Local Isolates of Phosphate Solubilizing Fungi (PSF) by GC-MS Analysis

The study conducted to isolate and characterise PFS from rhizosphere soil of plants that grown in Wasit province, south central Iraq (32.5086651°N, 45.790801°E) and some organic wastes. Five (g) of soil from rhizosphere and plants residue were collected, then suspended in 100 ml of sterile distilled water and shaken on a rotary shaker for 5 min on to separate the microorganisms from the sample. All sampling procedures were repeated in triplicate.
 
The samples were serially diluted to 10^-3, 10^-5 and 10^-7 and spread on petri dish contain potato dextrose agar (PDA). The plates were incubated at 27°C for 5 days or until the colonies developed and then transferred to fresh medium and purified. The pure strains were maintained on PDA slants at 4°C. The isolates were identified by their colony characteristics, spores morphology and microscopic observations. The fungi frequency ratio was determined according to the following equation:   
For investigation of phosphate-solubilizing isolates activity, laboratory study conducted at mycology laboratory, plant protection department, college of Agriculture, university of Wasit in September 2016. The cultures were spot inoculated on solid phosphate solubilization media: PVK¹ (Pikovaskya, 1948) and NBRIP² (Mehta and Nautiyal, 2001). After 5 days of incubation at 27°C, the plates were examined for the presence of clear zones around colonies. The experiment was repeated in triplicate for each isolates. For indicating P solubilization activity, colonies forming a clear halo were counted and further used to determine the P-solubilization index. Solubilization index (SI) was measured using following formula (Edi-Premono et al., 1996) :  
Fungal culture of P solubilization activity were inoculated in to 100 ml in broth of phosphate solubilization media (PVK and NBRIP) in conical flasks 250 ml and incubated on a shaker incubator 120 rpm at 27°C for 14 days. Un-inoculated Sterile medium served as control. Initial pH and changing in pH was noted after 14 days of inoculation using pH digital meter (Nenwani et al., 2010), in addition to weight of mycelia dry masses of each isolates at the end of incubation periods by drying the biomass colony in oven at 90°C till their weight are to be constant. The dry biomass was calculated in g/l of fermentation medium. The experiment was performed in triplicate for each isolates.
 
Secondary (Organic) compounds formed by PSF with GC-MS
 
For organic compounds determination, on the 14^th day of incubation, 10 ml of each inoculated flasks was removed and filtered, then pooled to be extracted and also used for GC-MS analysis. The extracted and analysis of GC-MS conducted in laboratories of ecology department, ministry of science and technology, Iraq.
 
Sample extraction
 
Organic compounds in PSF culture broth, samples were extracted into 10 ml tube contain 5 ml of ethyl acetate using mechanically shaking for 10 min, then the tube was immediately centrifuged at 800 g for 5 min. The organic layer was transferred to a second tube and solvent was evaporated under reduced pressure. The residue was dried in vacuum desiccators over P₂O₅-KOH.
 
GC-MS analysis
 
GC-MS QP 2010 plus shimadzu, A fused-silica capillary column coated with Optima-5 m scroos-linked 5% phenylmethyl silicone (SE-30 m, 0.25 mm i.d., 0.32 mm film thickness used). The GC temperature program was as follows: initial temperature was 50°C, held for 2 min) increased to 200°C at a rate of 5°C/min, then to 280°C rate of 10°C/min and finally to 280°C held for 10 min. The split ratio was 1:10 injection temperature was 280°C, transfer line temperature) was 250°C and ion source temperature was 200°C). The mass spectrometer was operated at 70 eV in the electron impact mode with SCAN or selected ion monitoring, The selected ion groups for the identification of organic acid and organic compounds.
 
Statistical analysis
 
All experiments were performed in triplicate. Genstat statistical program was used to analyse the results that conducted in completely randomized design-CRD in experiments, a one and two-way ANOVA were used and the averages were compared using the LSD test (p<0.01) to compare between means.

The isolation results revealed that Aspergillus genus was the highest frequency in the Rhizosphere soil and some organic residue by using dilution method (Table 1), sixth fungal strains were identified on the basis of colonies microscopic characters as Aspergillus niger, A. terrus and Trichoderma hamatum based upon their colony morphological characteristics. The A. niger was found in all replicates and ranged by 15.3% in the rhizosphere soil of Alfa-Alfa plant to 100% in the garlic Rhizosphere soil, followed by 60.7% in the sample containing Date palm residue, While the lowest frequency was T. hamatum with 22.5 % in the sample containing cucumber.
 

 
Kalaf et al., (2018) reported that numbers of different microorganisms were isolated from two samples of soil with and without plant residues and the Aspergillus niger was record about 25.5 to 41.6% respectively, While the lowest frequency was T. harzianum with 8.7% in the soil sample containing plant residues.
 
Fungal culture of phosphate solubilizing activity
 
In solid media
 
The study results showed that all fungal isolates formed a clear halo zone indicate of phosphate solubilisation activity. after 5 days of incubation a clear halo zone was formed around isolate colonies on solid PVK and NBRIP medium which supplemented with calcium phosphate, indicating fungal isolates ability of phosphate-solubilizing (Table 2). Aspergillus niger isolates AD-A2 and AG-B1 showed strong phosphate solubilization with average colony diameter 7.20 and 6.73 cm in PVK and NBRIP medium respectively, while other fungi was less effective solubilization by making smaller clear zones around their colonies.
 

 
The results also reflex on Solubilization Index (SI) of Phosphate, were it ranged 1.03 to 1.29. Data recorded high SI for Aspergillus niger isolate AD-A2 and AG-B1 with average 1.29 and 1.28 respectively than other isolates especially Trichoderma hamatum which showed least phosphate-solubilizing activity with SI 1.03 in both PVK and NBRIP medium respectively. 
 
The results also showed that Aspergillus niger have significant ability to solubilizing phosphorus and make it available for absorbs by plants and improves plant growth more than other fungi. However, solubilization of various forms of precipitated calcium phosphate in solid agar plates has been widely used as an initial isolation criterion for Phosphorus solubilizing microorganisms (Alikhani et al., 2006). Whitelaw, (2000) and Al-Taie and Alwan, (2014) indicates that precipitated phosphorus agar techniques are useful for isolating and selecting microorganisms for further investigations but have limited sensitivity.
 
Results of the present study are supported by the observation of Al-Taie, (2014) and Saber et al., (2009) who studied ability of isolate Aspergillus niger to produce a clear halo zone around their colony in NBRIP solid media have similar results, this indicates its ability to precipitated phosphorus due to produce of organic acids. 
  
In broth media
 
After fungal isolates phosphorus solubilizing ability was confirmed on solid medium, this ability was carried out in liquid (broth) medium. The PSF isolates ability was accompanied by a decreasing pH of the medium. The results (Table 3) showed that PSF more significant of decreasing pH as compare with control treatment in both PVK and NBRIP broth medium at 14 days of shaking incubation 120 rpm at 27°C.
 

 
Aspergillus niger isolate AD-11 was dropped pH of final product from fermentation significantly 3.52 and 4.71 followed by the isolate AD-A2 with 5.05 and 5.30 in both PVK and NBRIP broth medium compared with control treatment where it remained constant around pH 5.75 and 6.61 respectively, while Trichoderma hamatum which showed least decreasing of pH compared with other fungal isolates with 5.32 and 5.39 in both PVK and NBRIP medium respectively. 
 
The study indicated that mycelia dry masses production by Aspergillus niger isolate correlated with acidity (reducing pH). The results (Table 3) showed that isolates AD-11 were recorded high amounts of fungal biomass 0.30 and 0.35 g and 190.8 and AD-A2 were recorded 0.28 and 0.34 g at both PVK and NBRIP broth medium respectively after 14 days of shaking incubation at 27°C. Such results revealed that the fungal strains have ability to solubilizing P and change it to available form and consume enough P for its own growth.
 
Yuan et al., (2005) reported that the significant decrease in pH broth medium as a result from organic acids production, that effect comes from consequence consumption of sugar. Barroso and Nahas (2008) revealed that rhizosphere microorganisms also associated the acid producing activity by decrease in the pH of the culture medium. This effect were explain how organic acids can release soluble phosphate by chelating the cations complex to inorganic phosphate (Whitelaw et al., 1999), which sutable to plant because of P availability to plants. Also, releasing of organic acids by non-vesicular mycorrhizal fungi can sequester cations beside create a microenvironment acidifying near the roots, this effect is considered to be a major mechanism of P-solubilization, as well as Mn, Fe and Zn by plants (El-Azouni, 2008).
       
Due to the fact that many different mechanisms effects were involved in organic compound in the fungal filtrate (Zeng et al., 2001). The organic-chemical of PSF effects on cucumber seedling were demonstrated with Aspergillus sp. (14) and Trichoderma hammatum (Zayed and Motaal, 2005).   
 
Identification of organic Allelochemical compounds formed by PSF by GC-MS
 
It was observed that, ethyl acetate  extract of PSF culture filtrate that analysis by GC-MS, resulted different compounds, in this study, Eleven major compounds were detected in the crude extract of PSF culture filtrate of Aspergillus sp. isolates and Trichoderma hamatum (T-113) as PSF (Table 4).
 

       
Many compounds are formed by submerged fermentation of Aspergillus isolates, were identified in as following: 1,3-Dioxan-5-ol (96.47%); Octanoic acid, 4-chlorobenzyl ester (0.14%); 1,3,5-Cycloheptatriene (0.58%); Crotonic acid (2.64%); 2-Propanone, 1,1-diethoxy (0.18%); 2-Butanone, 4-hydroxy (13.11%); 1-Tetrazol-2-ylethanone (86.4%); Propanethioamide (0.49%); Acetic acid, ethyl ester; Acetidin (100%); Methylolacetone (100%) and Trichoderma hamatum (T-113) were identified: Ethane, 1,1-diethoxy (100%) and 2-Butanone, 4-hydroxy-(100%). Most of these compounds have not been reported earlier, this screening of LF of PSF is recorded for the first time in Iraq. 
       
Used organic solvents are reported to be more efficacious in extraction of antimicrobial compounds (Parekh et al., 2005). The organic solvent ethyl acetate was thought to enhance the solubility of the compound. In addition to, that ethyl acetate extracts have higher concentration of active components and thus have greater potency against the pathogen and soluble phosphorous (Bibi et al., 2011).

The secondary compounds of PSF play an important role in Phosphate Solubilizing by decrease pH of rhizosphere and inhibited the growth of pathogenic microorganisms. We report the isolation of PSF from A. niger have been governing organic compounds producing which carried out to identify by GC-MS analysis for the first time in Iraq. More trails need to be conducted to determine which of compounds will be affected by A. niger isolation. Details about the mode of action of organic compounds will also be required.

None.