سویه‌های جدید Bacillus cereus Wah1 وEnterobacter cloacae Wkh با پتانسیل بالا برای تولید سیدروفورها

Introduction

Iron is the fourth most abundant element in the soil, but a small amount of it is available for the microorganisms and plants due to the very low solubility of iron minerals, especially in the arid areas, (1- 4). Plants have the ability to absorb iron under iron deficiency conditions by using two different strategies, lowering pH of rhizospher by the release of the protons or production of the siderophores (5, 6). Nevertheless, iron deficiency in calcareus and dry soils is a common phenomenon leading to a significant reduction in the crop yields (5, 7).

This deficiency is compensated by using the chemical fertilizers (chelated iron) that are not eco-friendly and have high cost. In addition, the production or selection of the tolerant cultivares to Fe deficiency are extremely complex due to the non-uniform nature of soils and environmental conditions. Thus, plant breeding programs have slow progress (5).

Therfore, the use of microorganisms such as PGPR to improve the availability of nutrients for plants is an important and necessary factor for agriculture (8) to reduce risks for the human health and the environment (9, 10).

PGPRs are useful for the plant growth. These rhizobacteria stimulate the plant growth with both direct and indirect mechanisms (11, 12). Direct mechanisms include production of the phytohormons like auxin, ACC deaminase, siderophores and improvment of the solubilization of insoluble phosphate (13- 16). Indirect mechanisms also will begin when PGPR acts like biocontrol agents and reduce the disease (11).

Various works have indicated that many PGPRs have the ability for siderophore production (17- 22). Siderophores are low-weight molecules (500 to 1000 Dalton) that could solubilize Fe⁺³ by chelating them. Then, palnts and microorganisms can absorb Fe⁺³-siderophore combination and improve their growth (23- 26). The growth factors of plants would be increased using siderophore-producing rhizobacteria (27).

The purpose of this study was the isolation and identification of the best siderophore-producing rhizobacteria originated from Iran.

Materials and methods

General Isolation of rhizobacteria: The soil samples collected from the rhizospher of different plants from different provinces of Iran. Soil samples were placed in the plastic bags, and transported to the laboratory. Soil was crushed with a mortar thoroughly, then 1 gram of the crushed soils was used for preparation of the serial dilution of 10^-1 – 10^-6 in the physiological serum. After that 100 µl from 10^-6 dilution was added to the nutrient agar plates. Inoculated cultures were incubated at 30ºC during 72 hours. After appearance of colonies, single colony was isolated and prepared for more analysis (13, 28).

Specific isolation of bacteria containing siderophore

Cultivation of rhizobacteria:In this stage, siderophore producing rhizobacteria were screened using the specific medium of liquid CAS assay (29). Bacterial strains were inoculated into M9 minimal medium. The M9 medium was prepared as follow: KH₂PO₄ 3 gr/L, NaCl 0.5 gr/L, CaCl₂ 0.025 gr/L, MgSO₄ 0.088 gr/L, Glucose 2gr/L and NH4Cl 1gr/L. After inoculation, the samples were incubated four days at 30° C and 100 rpm.  After four days, 1 ml of liquid medium centrifuged for 10 minutes at 5000 rpm and after centrifugation, 0.5 ml of each sample was transferred to the new vial.

Chrome Azurol S Liquid Assay: Reagents were prepared as follow:

CAS assay solution:

2 mM CAS stock solution: 0.121g CAS in 100 ml water,

1 mM Fe stock solution: 1 mM FeCl3.6H2O in 10 mM HCL,

Piperazine buffer: dissolved 4.307 g piperazine in 30 ml water. Add 6.75 ml concentrated HCl to bring the pH to 5.6,

Hexadecyltrimetyl ammonium bromide (HDTMA): dissolved 0.0219 g HDTMA in 50 ml water in a 100 ml mixing cylinder.

We mixed 1.5 ml Fe solution with 7.5 ml CAS solution and add to the HDTMA in the mixing cylinder. Piperazine-1, 4-bis(2-ethanesulfonic acid) (PIPES) solution was added to the mixing cylinder and brought volume up to 100 ml with water.

Shuttle solution was 0.2 M 5-Sulfosalicylic acid. We added 0.5 ml CAS assay solution to 0.5 ml culture supernatant and mixed. Then 10 µl of the shuttle solution was added and mixed. After a few minutes, Siderophores, if present, will remove iron from the dye complex, resulting in a reduction in blue color of the solution. The absorbance (A₆₃₀) for the loss of blue color was measured. For A₆₃₀ measurements, we used the minimal medium as a blank, and used the minimal medium plus CAS assay solution plus shuttle as a reference (r). The sample (s) should have a lower reading than the reference. Siderophore units are defined as

Statistical analysis: All experiments were done based on completely randomized design (CRD) and all data were analyzed using excel and SAS softwares.

O-CAS assay

Cultivation of rhizobacteria: First, all bacteria were grown in the nutrient broth medium for 24 hours. After that 1 ml of the bacterial suspensions was centrifuged and bacterial pellet were washed by NaCl (8% w/v) solution. Then, the washed pellet was used for inoculation of solid M9 medium. It should be mentioned that solid M9 medium has the same combination like M9 medium discussed above plus agar 15 gr/L.

Preparation of CAS medium: The medium for 100 ml was as follows: CAS 6.05 mg, HDTMA 7.29 mg, PIPES 3.024 g, and 1 mM FeCl₃·6H₂O in 10 mM HCl 1 mL. Agarose (0.9%, w/v) was used as gelling agent (30).

Determination of produced siderophores: Ten ml of the CAS medium was applied over those mediums containing cultivated rhizobacteria to test the siderophore production. After 30 min, a change in the color will be appeared in the overlaid medium from blue to purple (for siderophores of the catechol type) or from blue to orange (for hydroxamates type) (30).

PCR amplification of bacterial 16S rDNA genes and electrophoretic analysis: Genomic DNA of isolated bacteria was extracted according to the method of Ausubel et al. (13, 31). The 16S rDNA gene was PCR amplified from genomic DNA isolated from the best siderophore producing rhizobacteria following standard PCR protocols (32).  Primers 8F (5′-AGA GTT TGA TCC TGG CTC AG-3′) and 1541R (5′-AAG GAG GTG ATC CAG CCG CA-3′) were used to amplify the 16S ribosomal gene. The PCR products were separated by their electrophoresis in a 1% agarose gel for 2 h. After that, staining was done using the ethidium bromide. Amplification products (1531 bp in size) were stored at -20°C. The clean PCR product was subjected to cycle sequencing in forward directions.

Molecular identification and phylogenetic analysis: Sequence analysis was performed by using the algorithms BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Multiple-sequence alignment was carried out using a freely available alignment program, Mega4.1. Bacterial identifications were based on the 16S rDNA gene sequence similarity. Neighbors joining phylogenetic trees were generated using the top alignment matches (13, 32).

Result

Isolation of PGPR: Soil samples were collected from different plant rhizosphere in different provinces of Iran. In the present work, we conducted an extensive screening procedure for the isolation of PGPRs. PGPRs were isolated on the nutrient agar medium. Out of large number of bacterial colonies appeared in the primary culture, seventy five colonies were selected based on their shapes and colors.

Siderophore Production:As mentioned above, liquid CAS assay was used based on completely random design with three replications to screen siderophore-producing PGPRs. The primary results showed that 52 strains had the ability to produce siderophores.

Data analysis indicated that there was a significant difference at 1% level between treatments (Table 1). It means that at least one strain has a significant difference with others.

In order to identify the best siderophore-producing strain, Duncan’s Multiple Range Test was conducted between all the siderophore-producing PGPRs (Table 2). Based on the mean comparison, Wah1 and Wkh were the best strains for production of siderophores. These strains could produce 97.76 and 96.99 percent of siderophore unit, respectively. Cke1 had the lowest ability to produce siderophore (Table 2).

Table 1- Variance analysis of siderophore producing bacteria.

** Significant differences at 1% probability level

Table 2- Mean comparison between the siderophore producing PGPRs based on Duncan’s Multiple Range Tests.

Among all the strains that were able to produce siderophore, 8 strains (15.38 %) produced less than 30 percent of siderophore unit, 14 (26.9 %) between 30 and 50%, 14 strains (26.9 %) between 50 and 70%, 10 strains (19.23 %) between 70 and 90 and 6 strains (11.5 %) more than 90 percent (Figure 1).

CAS Detection Method: We used O-CAS method (30) to determine the type of siderophores produced by the best siderophore-producing rhizobacteria. 16 strains were analyzed for the determination of the type of siderophores. The results showed that 10 strains changed the blue color of medium to purple (catechol-type siderophores), 4 strains changed the blue color to orange (hydroxamate-type siderophores) and 2 strains changed the blue color to orange-purple (catechol and hydroxamate) (Table 3, Figures 2 and 3).

Fig. 1- Different capabilities of siderophore producing rhizobacteria based on their siderophore unit.

Table 3- Determination of siderophore types by using O-CAS method.

Fig. 2- O-CAS assay carried out with the best siderophore producing Bacteria.

Fig. 3- Distribution of different siderophore types between siderophore producing rhizobacteria.

Phylogenetic Analysis:The phylogenetic affiliation of the best siderophore-producing strains (Bacillus cereus strain Wah1 and Enterobacter cloacae strain Wkh) was obtained by 16S rDNA gene sequence analysis. The blast search showed that the strains Wah1 and Wkh were more similar to Bacillus cereus and Enterobacter cloacae, respectively. The 16S rDNA sequence of Wah1 and Wkh reported in this paper had been deposited in the GenBank database under accession numbers: KU744631 and KU744630, respectively.

In order to find the most similar available sequences, a BLAST search was conducted in NCBI database. 16S rDNA sequence data of the most closely related species of Bacillus and Enterobacter were extracted and used in a tree construction to demonstrate the taxonomy of these strains (Figures 4 and 5). The phylogenetic relationships derived from the neighbor-joining analysis of the16S rDNA gene sequence of the Wah1 and Wkh with the most validly described species of the genus Bacillus and Enterobacter were shown in Figures 4 and 5.

Fig. 4- Neighbor-joining tree based on 16S rRNA gene sequences, showing relationships of Bacillus cereus strain Wah1 with closely related members of the genus Bacillus.

Fig. 5- Neighbor-joining tree based on 16S rRNA gene sequences, showing relationships of Enterobacter cloacae strain Wkh with closely related members of the genus Enterobacter.