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Assessment of phosphate solubilization activity of Rhizobacteria in mangrove forest Article in Biocatalysis and Agricultural Biotechnology · January 2016

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Biocatalysis and Agricultural Biotechnology 5 (2016) 168–172

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Assessment of phosphate solubilization activity of Rhizobacteria in mangrove forest Manouchehr Teymouri a, Javad Akhtari b, Maryam Karkhane c, Abdolrazagh Marzban a,n a

Biotechnology Research Center, Mashhad University of Medical Sciences, Mashhad, Iran Department of Physiology and Pharmacology, Immunogenetics Research Centre, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Iran c Gastroenterology and Liver Diseases Research Center, Research Institute for Gastroenterology and Liver Diseases, Shahid Beheshti University of Medical Sciences, Tehran, Iran b

art ic l e i nf o

a b s t r a c t

Article history: Received 28 September 2015 Received in revised form 25 January 2016 Accepted 28 January 2016 Available online 29 January 2016

The current study was performed to assess the phosphate (P) solubilization potential of inhabiting bacteria from rhizosphere of Avicennia marina plants. Hence, sampling was carried out from soils near the mangrove roots in the intertidal zone. Among 13 isolates screened, four high potential solubilizing bacteria (PSB) were selected based on the formation and dimension of clear halo around the colonies. Phylogenetic analysis of 16 S rDNA of PSBs showed their close similarity to Bacillus, Pseudomonas and Acinetobacter species. Regarding phosphate solubilization index (PSI), the dimension of the clearance zone divided by that of bacterium-inoculated spot, PSB1 and PSB3 were found as the most potent isolates with 3.5 and 2.6-PSI values, respectively. However, maximum P solubilization was found in broth media for PSB10 (357 mg/l) and PSB3 (282 mg/l), respectively. The consortium experiment showed a positive effect on total P solubilized by these isolates as solubilizing efficiency increased by 373 mg/l. P solubilizing efficiency also achieved in a high percentage range for consortium culture (74% 7 8%) and PSB10 (71 7 7%). The experiments showed that P solubilization was promoted along with acidification in BSB10 and consortium culture during incubation course, while such trend was not seen for other isolates. Our investigation proposes that the isolates can be applied as biofertilizer in marine ecosystems via bioaugmentation to sustain and even restore mangrove forests. & 2016 Published by Elsevier Ltd.

Keywords: Phosphate solubilizing bacteria Mangrove Biofertilizer Rhizosphere

1. Introduction Phosphate (P) compounds act as one of the most important fertilizers in soil and promote growth of plants and algae. Amongst different P compounds, only orthophosphate can be assimilated by plants and algae (Hameeda et al., 2008). Moreover, variability in pH, humidity and cation contents of soil influences P solubilization. Considering that the use of chemical fertilizers in agriculture has created potential health hazards, the role of rhizospheric microorganisms to improve soil structure has been highly regarded (Sharma et al., 2013). Phosphate solubilizing microorganisms (PSM) play a major role in supplementing plants with P and protect plant roots against parasites (El-Hadad et al., 2011; Tallapragada and Gudimi, 2011). In addition, PSMs have been widely reported to possess many potentials, including nitrogen fixation, metal chelating, organic acid and phytohormone production, beneficial to plant health and growth (Kim et al., 2005; Pandey et al., 2006; Jiang n

Corresponding author. E-mail address: [email protected] (A. Marzban).

http://dx.doi.org/10.1016/j.bcab.2016.01.012 1878-8181/& 2016 Published by Elsevier Ltd.

et al., 2008; Hariprasad and Niranjana, 2009; Matilla et al., 2011; Kaplan et al., 2013). Up to now, a variety of microorganisms involved in P solubilization has been discovered from ectorhizospheric zone and endosymbiotic rhizobia. The most important groups belong to fungi and bacteria (known as phosphate solubilizing bacteria or PSB) such as Penicillium, Aspergillus, Pseudomonas, Azospirillum, Burkholderia, Bacillus, Enterobacter, Rhizobium, Erwinia, Serratia, Alcaligenes, Arthrobacter, Flavobacterium and Acinetobacter species (Rodriguez and Fraga, 1999; KuklinskySobral et al., 2004; Ayyadurai et al., 2006; Morales et al., 2007; Kundu et al., 2009). There are some reports concerning the isolation of several marine bacterial genera such as Pseudomonas, Bacillus, Vibrio, Alcaligenes, Micrococcus, Corynebacterium and Flavobacterium having potential for solubilizing P in seawater and marine sediments. Although high concentrations of cations in sea water cause P to precipitate in sediments, bacteria inhabited marine environments have shown the greater capability to redissolve inorganic P compounds (Mudryk, 2004). Moreover, they are capable of assimilating organic P compounds and metabolizing them to inorganic soluble compounds. The aim of this study was to assess P solubilizing potential of microorganisms inhabited sediments of

M. Teymouri et al. / Biocatalysis and Agricultural Biotechnology 5 (2016) 168–172

mangrove forests and rhizosphere of Avecinia marina at Qeshm Island, located in the Persian Gulf, south of Iran.

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3. Assessment of P solubilization efficiency 3.1. Evaluation of P solubilization activity on agar medium

2. Materials and methods 2.1. Soil sample collection Soil samples were collected from rhizosphere and sediments of mangrove forest in the intertidal coast of Qeshm Island, south of Iran. The season at the time of sampling was mid-autumn with temperature and pH variation of 30–35 °C and 7–7.7, respectively. In addition, as the location was an intertidal zone, it was difficult to assign a given amount to P and chemical oxygen demand (COD) concentrations. However, the COD varied from four to 20 mg/l and 3.5 to 10, respectively, among the samples. The samples were transferred into containers by a sterile spoon taken from the top surface of soils near the plant roots at the depth of 15 cm. To maintain aerobic microorganisms, especially bacteria, the container caps were loosed, and remained away from heat and sunlight. In the laboratory, they were kept at 4 °C in refrigerator until the start of the experiments.

2.2. Isolation of P solubilizing bacteria One gram of each collected soil sample was diluted fivefold by autoclaved sea water. One-tenth microliter of each dilution was inoculated on Pikovskaya (PVK) agar medium by an L-shape glass rod. The plates were incubated at 30 °C for one week. Then, PSBs were determined through appearing clear halo around their colonies. The isolates were purified for the study of P solubilization activity under different conditions like varying temperature, pH and salinity.

2.3. Characterization of P solubilizing bacteria Preliminary identification was performed through biochemical experiments and morphological characteristics based on macroscopic and microscopic observations. All the studies were carried out according to the experiments described in Bergey's manual of determinative bacteriology (Holt et al., 1994). To extract genomic DNA from the bacterial colonies, they were cultured on nutrient agar and incubated overnight at 35 °C. Then, DNA was isolated using the phenol/chloroform/isoamyl alcohol method as described by Sambrook et al. (2001). Polymerase chain reaction (PCR) of 16 S rDNA gene fragment was done using universal primers of Fd1 (5′AGAGTTTGATCCTGGCTCAG-3′) and Rd1 (5′-AGGAGGTGATCCAGCC-3′). PCR mixture contained reaction buffer (20 mM, Tris pH 8.4, KCl 50 mM), MgCl2 (1.2 mM), Taq polymerase (1.25 units), dNTP (10 mM) and each of primers (0.1 mM). DNA amplification was performed by a DNA thermal cycler Primus 25 with the following cycling program: initial denaturation at 94 °C for 5 min, and 30 cycles of denaturation at 95 °C for 1 min, annealing at 55 °C for 45 s, extension at 72 °C for 1 min, and final extension at 72 °C for 7 min. Phylogenetic analysis was conducted using 16 S rDNA sequencing followed by alignment with NCBI nucleotide database, from which the closest species-related sequences were retrieved and analyzed by MEGA version 4.0 software. Neighbor joining method was employed with bootstrap values generated from 1000 replicates.

To evaluate the P solubilization activity, pure colonies were spot-inoculated on PVK agar medium by a metal loop and incubated for 3 days at 30 °C. P solubilization potency was measured based on Phosphate Solubilization Index (PSI) as the following equation (Morales et al., 2011):

PSI=

Total diameter of halo zone Colony diameter

PSIs were calculated in triplicate for the various colonies. The most potent bacteria were selected for further studies. 3.2. Evaluation of P solubilization activity in broth medium The selected bacterial strains were evaluated for P solubilization activity in 100 ml broth medium containing 1 g glucose, 0.5 g MgCl2 6H2O, 0.025 g MgSO4 7H2O, 0.02 g KCl, 0.01 g (NH4)2SO4, 2.0 g NaCl as well as 0.5 g tricalcium phosphate amended as insoluble P source. All experiments were carried out in 250 ml Erlenmeyer flasks containing 100 ml liquid medium. The flasks were autoclaved at 121 °C under 15-atmosphere pressure for 15 min prior to adding tricalcium phosphate. Culture conditions for the isolates were initial pH 7.0 and temperature 30 °C with rotary shaking at 100 rpm for seven days. P solubilization activity was assayed as pure isolates and a consortium of all isolates. Growth rate and P solubilization were measured at daily intervals. Two milliliters of media was centrifuged at 6000 rpm for 15 min, and then the supernatant was taken to measure the P concentration using ascorbic acid method as described by Murphy and Riley (1962). Briefly, 1 ml of the supernatant was added to 5 ml ammonium heptamolybdate solution (2.5 g/ml) in 0.2 M H2SO4. Then, 1 ml fresh-made ascorbic acid solution (0.4% w/v) was added and mixed. The solution was incubated in dark for half an hour and the absorbance was measured via spectrophotometry at 587 nm wavelength. Using KH2PO4 standard calibration line, the P content of the supernatants was determined. Control flasks without bacterial inoculation were also incubated at the same condition used for samples. This control showed non-bacterial P solubilization at the end of the incubation period and their values were subtracted from experimental values. 3.3. Statistical data analysis Statistical analyses were performed using GraphPad Prism version 5 (GraphPad Software, San Diego, CA). All experiments were conducted in triplicate and the values expressed as mean along with standard deviation. One-way ANOVA and Tukey's multiple comparison were employed at the significance level of 5%.

4. Results and discussion From the total soil samples, 86 colonies were isolated in the primary screening, of which 13 colonies exhibited P solubilizing activity. Among these colonies, four colonies (PSB1, PSB3, PSB10 and PSB12) showed maximum P solubilizing potential on agar medium (Fig. 1). As seen in Table 1, maximum PSIs were determined for PSB1 and PSB3 strains, which were 3.5 and 2.6, respectively. PSB1 was the main prominent P solubilizer on agar media, while the highest P solubilization occurred in the broth

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Fig. 1. Growth profile of phosphate solubilizing bacteria on Pikovskaya agar medium after 7 days incubation at 30 °C and clear zone formed from solubilization of P in medium containing tricalcium phosphate. Table 1 Isolates related phosphate solubilizing potential parameters. PSIa

PS in broth P solubilizing efficiency (%)

Isolate

Microorganism

PSB1 PSB3 PSB10 PSB12 Consortium

Bacillus sp. 3.5 7 0.2b 2217 14 Pseudomonas sp. 2.6 7 0.2 3567 20c Pseudomonas sp. 2.3 7 0.4 280 7 29 Acinetobacter sp. 2.0 7 0.1 2157 14 – – 3717 16

45 76 72 75c 57 77 43 77 74 78

a Phosphate solubilization index. The values are presented as means7 standard deviations (n¼ 3). b indicates the significant difference compared to all other isolates. c indicates the non-significant difference compared to consortium.

media for PSB3 (357 mg/l) and then PSB10 (282 mg/l). This is consistent with the report of Sarkar et al. (2012) that showed contradictory results between clear zone diameter and P solubilization activity in liquid media. Many studies have been carried out on several PSBs, which some of them were similar to our isolates. For example, Vyas and Gulati (2009) isolated a number of Pseudomonas sp. that exhibited high P solubilization activity in broth media, while giving a small halo zone on agar media. In this regard, Qian et al. (2010) explained that the P solubilization activity of PSMs may be influenced by media compositions and culture conditions when they are transferred to different media. Comparing the consortium activity with those of pure isolates, although it seemed that the main P solubilizing activity could be attributed to PSB3, the four isolates in combination could collaborate and improve P solubilization efficiency by 373 mg/l. Maximum P solubilizing efficiency also achieved in a high percentage range for consortium and PSB3 inoculated cultures. There are some reasons illustrating as to why consortium behaves better than single isolates. May be one or some products of metabolism of an isolate can provide improved conditions for another isolate to grow (Dipak and Sankar, 2015). Another justification is that other isolates could still contribute to increasing P solubilization when PSB3 is no longer capable of dissolving P due to unfavorable changes in culture condition. It has been reported that bacteria could solubilize P in the range of 30 to 900 mg/l depending on insoluble P composition, media components and initial pH (Ma et al., 2009). Another parameter for such diversity in P solubilization is the types of microorganisms involved in the process (Saxena et al., 2015). Based on 16 S rDNA sequences and alignment with database deposited in NCBI GenBank, the isolates were found to belong to Bacillus, Pseudomonas and Acinetobacter species (Table 1). Fig. 2 also shows the relationship of the isolates with their closest relatives retrieved from NCBI GenBank. Up to now, many different bacterial strains have been isolated from various environments shown to be involved in P solubilization. The bacterial genera, identified in the study, have already been known as potent P solubilizing bacteria, especially Bacillus and Pseudomonas sp. (Igual et al., 2001; Islama et al., 2007; Misra et al., 2012). It was noteworthy that these bacterial strains were also determined to be vital to P recycling in the studied locals owing to lack of efficient

Fig. 2. Phylogenetic tree of isolates as well as their neighboring relatives retrieved from NCBI (BLASTn) based on the 16 S rRNA sequences. Gene Bank accession numbers are listed with species names. The PSBs were labeled as black diamonds. Bootstrap values were generated from 1000 replicates and are shown as percentages at nodes.


10

A

B b

8

300

c

6

pH

P Concentration (mg/l)

400

171

200

4 100 0

a 0

PSB1

1

2

2

3

4

5

Day PSB3

6

7

0

8

0

1

2

3

4

5

6

7

8

Day PSB10

PSB12

Consortium

Fig. 3. Phosphate solubilization (A) and pH (B) follow ups within seven days incubation in liquid media. Data are presented as means 7 standard deviations (n¼ 3), a, b and c indicate significant difference compared to consortium after first, third and seventh day of incubation, respectively.

knowledge about marine ecosystems, especially mangrove rhizobia in Qeshm Island. Some literatures noted that difference in P solubilization of liquid and solid media cultivation is related to the type and amount of organic acids produced such as oxalic, malonic, fumaric, tartaric, alpha ketobutyric, citric, 2-ketogluconic, and gluconic and succinic acid that PSBs release in media (Vyas and Gulati, 2009; Zhu et al., 2011; Panhwar et al., 2013). The secretion of organic acids could be decreased when the bacteria are inoculated on solid media compared to liquid media cultivation. However, the role of organic acids in P solubilization has been questioned because some reports have mentioned other mechanisms to be responsible for P solubilization such as chelating agents produced by bacteria from different metabolic pathways (Rodriguez and Fraga, 1999; Park et al., 2011). Fig. 3 presents the P solubilization activity of the isolates within seven days incubation as well as pH changes in broth media. The pH value decreased with growth promotion of the bacteria in consortium culture. However, pH variation had no consistent correlation with P solubilization activity. For example, in PSB1, PSB3, PSB10, pH did not drop in the culture media. Although the consortium culture showed a remarkable higher P solubilization within four days of incubation than each isolate, pH value decreased significantly compared to PSB12 after sixth day of incubation. In this regard, some authors attributed the ability of PSMs concerning P solubilization to the drop of pH and protonating the media during growth period. Other literatures have suggested other mechanisms involved in P solubilization activity, such as P adsorption onto bacteria, production of chelating compounds by bacteria, Fe absorption into microorganisms, which indirectly results in improved P solubilization (Holmboe and Kristensen, 2002). Fe ions precipitate along with inorganic P, therefore, their reduction in environment leads to P solubilization (Reef etal., 2010). Finding the mechanisms and interactions between such bacteria and the root of plants might provide opportunity for human contribution to forestation like in situ augmentation of these bacteria. Therefore, in depth studies still is necessitated about how bacterial activities can play a critical role in P solubilization and following absorption into plant rhizosphere. On the other hand, what impacts plants could have on microorganisms and which mechanisms involve in the cycle of P among plants, bacteria and others are other issues that their examinations expand our knowledge about mangrove system and help us to preserve them.

5. Conclusion P solubilization in saline alkali soils, especially marine and coastal ecosystems only performed by P solubilizing microorganisms. Therefore, it is a rational hypothesis that increasing bacterial number and also their diversity enhances P mineralization facilitating P assimilation by plant roots. Our study suggests that employment of the isolates in fertilizing soils and sediments in mangrove area is a promising interference that contributes to nutrient availability and promoting growth of mangrove plants.

Acknowledgment The authors are thankful for kindly help of Dr. Kavyani during research steps, especially bacterial isolation from the soil samples.

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