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

نوع مقاله: پژوهشی- انگلیسی

نویسندگان

1 استادیار اصلاح نباتات، دانشگاه تحصیلات تکمیلی صنعتی و فناوری پیشرفته، کرمان، ایران

2 استادیار علوم دامی، دانشگاه تحصیلات تکمیلی صنعتی و فناوری پیشرفته، کرمان، ایران

3 دانشیار بیوتکنولوژی، دانشگاه اصفهان، ایران

چکیده

مقدمه: سیدروفورها مولکول‌هایی با وزن مولکولی نسبتا پایین (500 تا 1000 دالتون) هستند که به عنوان لیگاندهای کلاته کننده آهن در شرایط کمبود آهن توسط بسیاری از میکروارگانیزم‌ها تولید می شوند. گیاهان می‌توانند ترکیب آهن-سیدروفور را به منظور رفع نیازشان به آهن جذب کنند. هدف از پژوهش حاضر جداسازی و شناسایی باکتری‌های تولید کننده سیدروفور است.
مواد و روش‏‏ها: در این پژوهش از روش سنجش مایع CAS، برای جداسازی باکتری‌های محرک رشد گیاهی (PGPR) تولید کننده سیدروفوراستفاده شد. سپس، انواع مختلف سیدروفورهای تولید شده توسط بهترین سویه‌ها، با استفاده از روش سنجش O-CAS تعیین شدند. سرانجام دو سویه برتر تولید کننده سیدروفور با استفاده از آنالیز توالی ژن 16S rDNAشناسایی شدند.
نتایج: نتایج نشان داد که 3/69 درصد از باکتری‌های جداسازی شده توانایی تولید سیدروفور را دارند. پایین‌ترین و بالاترین میزان سیدروفورها به ترتیب به سویه های Cke1 (58/17 درصد واحد سیدروفوری) و Wah1 (76/97 درصد واحد سیدروفوری) تعلق داشتند. بر پایه روش سنجش O-CAS این سویه‌ها سیدروفورهایی از نوع کاتکول و هیدروکسامات را تولید کردند. دو سویه Wah1 و Wkh با استفاده از آنالیز توالی‌یابی ژن 16S rDNA به ترتیب به عنوان Bacillus cereus و Enterobacter cloacae شناسایی شدند.
بحث و نتیجهگیری: Wah1 و Wkh سیدروفورهای بیشتری (به صورت واحد سیدروفوری) نسبت به سایر سویه‌هایی که تا کنون با روش مشابهی مطالعه شده بودند، تولید کردند. بنابراین، آن‌ها سویه‌های با ارزشی برای مطالعه پتانسیل‌شان به عنوان کودهای زیستی و حفاظت گیاه در برابر پاتوژن‌های خاکزاد خواهند بود.

کلیدواژه‌ها

موضوعات


عنوان مقاله [English]

Novel strains of Bacillus cereus Wah1 and Enterobacter cloacae Wkh with high potential for production of siderophores

نویسندگان [English]

  • Mahmood Maleki 1
  • Sedigheh Norouzpour 2
  • Elham Rezvannejad 3
  • Shahryar Shakeri 1
1 Assistant Professor of Plant Breeding, Department of Biotechnology, Institute of Science and High Technology and Environmental Science, Graduate University of Advanced Technology, Kerman, Iran
2 MSc student of Plant Breeding, Graduate University of Advanced Technology, Kerman, Iran
3 Assistant Professor of Animal Breeding, Department of Biotechnology, Institute of Science and High Technology and Environmental Science, Graduate University of Advanced Technology, Kerman, Iran
چکیده [English]

Introduction: Siderophores are relatively low-weight molecules (500 to 1000 Dalton), which are produced by many microorganisms as the chelating ligands of iron under iron deficiency. Plants can absorb the siderophore-iron complex to meet their needs for iron. The aim of this study was isolation and identification of siderophore producing bacteria.
Materials and methods: In this study, we used liquid chrome azurol S (CAS) assay for the isolation of siderophore producing plant growth promoting rhizobacteria (PGPRs). Then, different kinds of the produced siderophores by the best strains were determined using overlaid chrome azurol S (O-CAS) assay. Finally, two best siderophores producing strains were identified using the 16S rDNA gene sequencing analysis.
Results: The results showed that 69.3 percent of the isolated bacteria could produce siderophores. The lowest and highst levels of siderophores belonged to the strains Cke1 (17.58 percent siderophore unit) and Wah1 (97.76 percent siderophore unit) respectively. Based on the O-CAS assay method, these strains produced catchol and hydroxamat types of siderophores. Two strains Wah1 and Wkh were identified using 16S rDNA gene sequencing analysis as Bacillus cereus and Enterobacter cloacae, respectively.
Discussion and conclusion: Wah1 and Wkh produced more siderophores (as siderophore unit) than other strains, which were studied using the same method until now. Therefore, they could be valuable strains to study their potential as biological fertilizers and plant protection against the soil born pathogens.

کلیدواژه‌ها [English]

  • Bacteria
  • Liquid CAS Assay
  • O-CAS Assay
  • Siderophore

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.

Mean Square

Sum of Squares

DF

Sources of Variation

0.16**

8.11

51

Treatment

0.00056

0.058

104

Error

 

8.17

155

total

** Significant differences at 1% probability level

 

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

No.

Strains

Siderophore unit

No.

Strains

Siderophore unit

No.

Strains

Siderophore unit

1

Wah1

97.76 a

19

Ake4

63.35 jk

37

Hke5

42.52 rst

2

Wkh

96.99 ab

20

Gke3

63.19 jk

38

Ver19

41.84 st

3

Ver4

93.69 abc

21

Mho

62.47 k

39

Mke2

39.17 tu

4

Hke2

92.00 bc

22

Hke6

61.20 k

40

Hke1

36.21 uv

5

Hke3

90.59 c

23

Aes1

58.47 kl

41

Ake3

36.06 uv

6

Aes2

90.04 cd

24

Ama3

57.76 klm

42

Ver11

33.88 uvw

7

Wta2

88.74 cde

25

Ver2

55.74 lmn

43

Ake1

33.71 uvw

8

Lka4

88.70 edf

26

Sho4

54.09 lmno

44

Aes4

32.47 vwx

9

Hke8

84.88 def

27

Wha

53.58 lmno

45

Ama1

29.06 wxy

10

Ake7

83.56 efg

28

Lka7

52.59 mnop

46

Lka5

27.88 xyz

11

Hke4

81.98 fg

29

Ver6

52.03 nop

47

Sho1

27.78 xyz

12

Ake9

81.32 fg

30

Aes3

50.46 nopq

48

Ver15

26.35 yza

13

Hke7

78.47 gh

31

Ver13

49.47 opq

49

Cke2

23.41 zab

14

Wah2

73.53 hi

32

Ver17

49.44 opq

50

Min2

21.77 abc

15

Ver9

73.52 hi

33

Ver18

48.93 opq

51

Wta1

19.74 bc

16

Ver10

72.36 i

34

Ver16

47.80 pqr

52

Cke1

17.58 c

17

Gke4

68.46 ij

35

Ver14

46.12 qrs

 

 

 

18

Ake2

68.03 ij

36

Cke3

45.74 qrs

 

 

 

 

 

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.

No

Strains

color

Siderophore type

 

Strains

color

Siderophore type

1

Wah1

Orange-purple

 

Catechol and hydroxamate

9

Hke8

Orange-purple

 

Catechol and hydroxamate

2

Wkh

Purple

catechol

10

Ake7

Purple

catechol

3

Ver4

Purple

catechol

11

Hke4

Orange

hydroxamate

4

Hke2

Purple

catechol

12

Ake9

Purple

catechol

5

Hke3

Orange

hydroxamate

13

Hke7

Orange

hydroxamate

6

Aes2

Purple

catechol

14

Wah2

Purple

catechol

7

Wta2

Purple

catechol

15

Ver9

Purple

catechol

8

Lka4

Purple

catechol

16

Ver10

Orange

hydroxamate

 

 

 

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.


Discussion and conclusion

The ability of siderophore production or absorption is one of the traits that cause the superiority of microorganisms in different environments (33) and facilitate the relationship between plant and bacteria and create a community around the roots, stems and leaves (34). This mechanism involves producing siderophore with high affinity to Fe3+ and absorption of siderophore loaded with iron (35).

Heretofore, several studies have been conducted to isolate the siderophore-producing rhizobacteria, all of whom confirmed that many strains can produce siderophores (36-39). Tian et al. could isolate 229 siderophore-producing bacteria (20). Their results showed that seventy five percent of siderophore-producing strains could produce siderophore unit between 20 and 40 percent and less than 25 percent between 40 and 60 percent of siderophore unit. Sayyed et al., optimized best conditions for the production of siderophores by two pseudomonas strains (40). These strains could produce approximately 85 percent of siderophore unit. Tailor and Joshi assessed 63 strains of pseudomonas for siderophore production (19). Their results showed that only 12 strains were able to produce siderophore and among these 12 strains, 7 strains were accounted for more than 85 percent of the siderophore unit. The best strain in the optimal conditions was accounted for 96% of the siderophore unit. Zhu and Yang investigated the ability of different strains for the production of siderophores (22). Their results showed that 19 strains could produce siderophores and strain MB8 produced a relatively high level of siderophore (70.38 % siderophore unit). By studying different researches on the capability of different bacteria for producing bacteria, it seems that the best strains in our study could produce more siderophores. In our study, results showed that more than 21 percent of siderophore-producing strains could produce siderophore unit more than 70 percent and the best one was accounted for more than 97 percent of siderophore unit.

Pérez-Miranda et al. introduced O-CAS assay method (30). Their results showed that color changes from blue to purple correspond to catechol-type siderophores and changes from blue to orange are consistent with hydroxamate-type siderophores. Sullivan et al. screened siderophore-producing bacteria from African dust using O-CAS method (18). Their results showed that 53 percent of bacteria could produce siderophores. Among siderophore-producing bacteria, two strains produced catechol siderophores and 10 strains produced hydroxamate siderophores, but most of them (approximately 90 percent) produced carboxylate siderophores (18). In our study, we used O-CAS assay to determine the type of siderophores in the best strains (having more than 70 percent siderophore unit). Most of the strains could produce catechol siderophores.

In our study, two best siderophore-producing strains belonged to the Bacillus and Enterobacter genus. These results indicated the importance of these genera for the production of siderophores. Different studies have shown the capability of both genera of Enterobacter and Bacillus to produce siderophores (17, 21, 41, 42, 43). For example, Van Tiel-Menkveld et al., extracted two types of siderophores from the iron-starved cultures of Enterobacter cloacae (43). Ramesh et al. showed that Enterobacter cloacae subsp. MDSR9 can produce the hydroxamate type of siderophore (17). Yaish et al. isolated PGPRs from the rhizospher of the date palm tree. Most isolated bacteria belonged to the Bacillus and Enterobacter genera. Some of these strains were able to chelate the ferric iron and solubilize phosphorus, zinc and potassium (21). In another work, Kumar et al. (42) and Kaki et al. (41) demonstrated different capabilities of Bacillus strains such as production of siderophores and IAA.

Iron in the soil is in the form of insoluble Fe3+ under aerobic conditions that it is not available for plants or microorganisms. Iron is essential for the main physiological processes such as nitrogen fixation, photosynthesis, and respiration (44). Microorganisms and plants have developed special mechanisms to chelate insoluble iron through the release of siderophores and absorption of iron-siderophore complexes through receptor proteins from the outer membrane to meet their needs for iron (45-48). Here, we isolated strains with high capabilities to produce siderophores. The best strains, Bacillus cereus strain Wah1 and Enterobacter cloacae strain Wkh, produce siderophores more than the most identified strains in other researches until now. They could be potential biologic fertilizers and biological enemies of soil born pathogens.

 

Acknowledgment

We gratefully acknowledge for the research funding provided for this project (No. 94/3854) by Institute of Science and High Technology and Environmental Sciences, Graduate University of Advanced Technology, Kerman, Iran.

 

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