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Octa Journal of Biosciences

Screening and Isolation of Salt Tolerant Bacteria from Tidal Floodplain Soils of Bangladesh 1 1,2* 1 1 3 D. Mondal , M. S. Islam , M. F. Hoque , M. Rafiq , S. M. Ahsan

1. Department of Soil Science, Patuakhali Science and Technology University, Dumki, Patuakhali-8602, Bangladesh2. Graduate School of Environment and Information Sciences, Yokohama National University, 79-7 Tokiwadai, Hodogaya-ku,

Yokohama, Kanagawa 240-8501, Japan3. Department of Horticulture, Patuakhali Science and Technology University, Dumki, Patuakhali 8602, Bangladesh

ARTICLE INFO A B S T R A C T

Received 14 2016Revised 30 May 2016Accepted 10 June 2016Available online 21 June 2016

May

Keywords: coastal soil, Rhizobium, isolation, salinity, Bangladesh Email: islam-md.saiful-nj@ynu.jp

2004). Nearly 40% of the world's land surface can be categorized as having potential salinity problems (Cordovilla et al. 1994). Salt tolerant Saline soils are defined by high concentrations of soluble

++ ++ - 4- 3- 3- rhizobia may have the potential to improve yield of legumes under salinity cations (Na+, Mg , Ca ) and anions (Cl , SO2 , CO2 , CHO ) in soil stress (El-Mokadem et al. 1991). The strategies employed in the last few solution (Rengasamy 2010). Salinity has been considered as an years to reduce the effect of salt stress on legume production have been important factor that adversely affects plant growth and cause focused on a selection of host genotypes that are tolerant to the high salt considerable loss in agricultural production (Shao et al. 2008; Shukla et conditions. Thus, an increase of tolerance to salinity of rhizobial bacteria al. 2011). Among abiotic stresses salinity is the most severe might constitute another approach to improve plant productivity under environmental stress which affecting more than 800 million hectares of symbiosis. The growth of Rhizobium strains in defined medium of land throughout the world (Munns 2005). Therefore, salinization of different salinity and pH is a valuable guide for predicting rhizobial agricultural land is steadily increasing in many parts of the world multiplication in saline soil. In saline soils, the PH and salt concentrations especially Bangladesh because of low rainfall and high potential are the important factors for soil fertility. evaporation in arid and semiarid regions (Al-Karaki 2006). It generates

Total area of Bangladesh 14.4 million hectares (ha) among this low water potential in soil which makes it difficult to obtain water and coastal area covers about 20% of the country and over 30% percent of nutrients for the plants (Porcel et al. 2012). Salinity has detrimental the net cultivable area which spreads inside up to 150 km from the coast effects on plants, microorganisms and soil properties. In soil solution, (Hoque 2013). In Bangladesh, about 1.06 million hectares of arable high concentrations of salts can change soil physical and chemical lands are affected by varying levels of salinity out of 2.85 million hectares properties by altering soil physical stability, nutrient movement and soil of the coastal and off-shore lands (FRG 2005; Asib 2011). Nitrogen input water dynamics. High amount of salts in soil solution is also a major for crop production is very important particularly in saline habitats which limiting factor for microbial diversity, abundance, composition and are N-poor. An increasing supply of N through dinitrogen fixation may functions (Kesheri et al. 2013). The high concentrations of salt in soil increase crop production in saline habitats (Zahran 1997). Salt tolerant greatly affect the growth and survival of plants by reducing symbiotic nitrogen fixers like Rhizobium increases crop productivity in evapotranspiration, photosynthesis, protein synthesis and enzyme saline soils (Zahran 1999) but the potential of free living diazotrophs is activity, which reduce growth of root and crop yield (Ravikumar et al. not well understood (Ravikumar et al. 2004; Chavada et al. 2010). 2004; Dang et al. 2008, 2010). It is well known that microorganisms have Considering the efficacy, input cost and environmental safety, use of a significant role in food chain and their existence maintains the whole chemical fertilizers for crop production in coastal saline soil is not a sound ecosystem health and functioning (Orhan and Gulluce 2015).proposition. We hypothesized that the identification and application of There are different strategies for amelioration of salt affected the most efficient microbial inoculants may help in accruing benefit to soils such as application of chemical amendments such as gypsum, agricultural crop production in the problematic saline soil. Therefore, the phosphate fertilizer, sulfuric acid, sulfur, pyrite, aluminum sulfate, and aim of this study is to isolate salt tolerant Rhizobium from saline soils in calcium chloride. Other includes leaching of excessive soluble salts from Bangladesh.upper to lower soil depths, cultivation and investigation of salt resistant

cultivars and flushing salt-affected soils (Zhao 1993). All of these methods are often not cost effective and sustainable. Therefore, the biological amelioration method in saline soil is the planting of salt-tolerant

The soil samples used for bacterial isolation were collected plant species which improves physical and chemical structure of soil and from rhizospheric soil fractions and root-free soils from the salinity facilitates the growth of other species (Akhter et al. 2003). The other affected two districts namely Khulna and Patuakhali located at the biological approach in ameliorating salt-affected soils is the use of salt-southern part of Bangladesh (Fig. 1) in sterilized pollythene bags. The tolerant microorganisms that play several important roles in plant growth. sampling was conducted in March-April, 2014. At each sampling station, Production of exopolysaccharide is one of these roles by which they soil sample (up to 20 cm) was collected in the form of sub-samples at a restrict NaCl uptake and stimulate plant growth (Ashaf et al. 2004). distance of about 20 m each from the first sub-sample in five different Another role of microorganisms is to alleviate the antioxidant enzymes of locations. These sub-samples were thoroughly mixed to form a plant under salt stress (Han and Lee 2005). These microorganisms also composite sample. Then samples were used to investigate on salinity maintain a nutrient cycle in saline soil with their fully functional enzymes tolerant Rhizobium sp. The tests were conducted at the Central and metabolites released through soil (Dendooven et al. 2010).laboratory and Soil Science laboratory of Patuakhali Science and An environmental factor that limits crop productivity is referred Technology University, Dumki, Patuakhali, Bangladesh. The to as a stress or disturbance, salinity in soil or water is one of the major experimental procedures for the characterization and isolation ofstresses can severely limit crop production (Howieson and Ballard

INTRODUCTION

MATERIALS AND METHODSCollection and preparation of soil samples

Octa Journal of Biosciences 11

Octa Journal of Biosciences ISSN 2321 – 3663International peer-reviewed journal Jan-June 2016Octa. J. Biosci. Vol. 4(1):11-16

Use of salt tolerant bio-inoculants is better alternatives to chemical fertilizer for soil fertility enrichment in the coastal saline soils. The study was conducted to isolate and characterize salinity tolerant bacteria like Rhizobium sp. which can be used as biofertilizer. Soil samples were collected from two salinity affected districts namely Khulna and Patuakhali situated in the southern part of Bangladesh. Nine colonies were isolated from pore-plate containing yeast extract mannitol agar (YEMA) medium. All the isolates were rod-shaped, motile and gram negative. Biochemical tests indicated that they were obligate aerobes, catalase and starch hydrolysis positive. They were fast and slow growers on the YEMA medium having different pH levels. The obtained results exhibited that all the isolates tolerated at higher salt concentration (up to 4% NaCl). Among the isolates examined in this study, the KNR4 isolate was found maximum absorbance (1.265) indicating most tolerant to salinity whereas, KNR5 isolate was recorded the lowest absorbance (0.8449) resulting most sensitive. Other isolates were moderate tolerance to salinity. Therefore, KNR4 isolate may become a promising source for salinity tolerant Rhizobium sp in Bangladesh.

Octa Journal of Biosciences 12

bacterial isolates are presented in Fig. 2.

About 10g soil samples were suspended in sterilized 90 ml distilled-water, diluted and then surface plated on Yeast-Extract Mannitol Agar (YEMA) with 0.0025 % (w/v) Congo-red. After 2 days of incubation, Rhizobium colonies were obtained. Colorless and slimy colonies of Rhizobium were isolated and purified through repeated streaking method on the same medium. Nine pure isolates were slantedassigned their name as DMR1, DMR2, DMR3, NKR4, NKR5, NKR6, KKR7, KKR8 and KKR9 and used for all tests.

The characters of 9 bacterial isolates (DMR1, DMR2, DMR3, NKR4, NKR5, NKR6, KKR7, KKR8 and KKR9) were studied following the standard microbiological and molecular methods. As standard microbiological methods, the morphology and Gram staining was investigated. The physiological and biochemical characters such as pH range, salt tolerance, bromothymol blue, catalase and starch hydrolysis were investigated. The cells of Rhizobium isolates were examined under light microscope and found that the cells were rod shape and motile (Table 1). The isolates absorbed counter stain as they were Gram negative bacteria (Fig S1). Vincent et al. (1980) stated that Rhizobium was gram negative, rod shaped and generally motile which was in line with the present study. The isolates were observed to lack the ability to absorb Congo red from YEMA medium containing this dye where colonies were colorless white or very faintly pink colonies which is in agreement with the findings of Shoukry et al. (2013). Congo red is thought to form colored colloidal complex with ions on the cell surface, the colonies absorbed little dye and remain colorless or became slightly pink after 2 days of incubation.

All the tested isolates gave positive results for catalase test (Table 1). The isolates produced bubbles within a few seconds (Fig S2). Aerobic bacteria degrade hydrogen peroxide (H2O2) due to presence of catalase enzyme and produce oxygen (O2). For this, the tested isolates produced bubbles as the Rhizobium is aerobic. Positive results were obtained from the starch hydrolysis assay (Fig S3). On subjecting inoculate plates to iodine test, clear zones around the colonies were seen and the colonies turned yellow in appearance, whereas, blue color appears on no growth areas (Fig S4). This result indicated that the isolates have the potential to hydrolyze starch present in the medium (De Oliveira et al. 2007). Results in Table 1 showed that six isolates viz., DMR1, DMR2, DMR3, KNR4, KNR6 and KKR9 were fast growers where rest of the isolates viz., KNR5, KKR7 and KKR8 were slow grower. The isolates of fast growers developed yellow color (Fig S5) indicating acidic in nature where slow growers isolates developed blue color indicating alkaline in nature.

Isolation of Rhizobium

RESULTS AND DISCUSSIONMorphology and biochemical traits of Rhizobium isolates

Physiological characteristicsColony morphology

Starch hydrolysis

Bromotyhmol blue test

pH variation assay

Testing of salt tolerance

inoculated with a bacterial isolate and incubated at 30oC for 2 days. About 10 μL of the grown isolates was used for the inoculation of 50 mL YEM medium containing of known NaCl concentration ( 1, 2, 3 and 4%) and the conical flask were incubated at 30 oC for 2, 4 and 6 days. The T60 UV visible Spectrophotometer was used for the measurement of growth (in turbidity units) at 600 nm where none inoculated medium was used as a blank.

The colony morphology of isolates was examined on YEM agar plates. After an incubation of 2-3 days at 30°C, individual colonies were characterized based on their colour, shape, appearance, colony, diameter, transparency and gram stain reaction (Aneja 2003).

Starch hydrolysis test was performed to determine the ability of microorganisms to use starch as a carbon source (de Oliverira et. al. 2007). This medium was inoculated with Rhizobium and analyzed for starch utilization. Iodine test was used to determine the capability of microorganisms to use starch. Drops of iodine solution (0.1 N) were spread on a 24 h-old culture grown on Petri plates. Formation of blue color “starch iodine complex” indicated non utilization of starch and vice versa.

Bromotyhmol blue test was done to identify the isolates as fast grower or slow grower. 5 ml of 0.5% bromothymol blue solution was thoroughly mixed with one liter of YEMA and plated. Culture of different Rhizobium isolates was streaked on the plates and incubated at 30oC and observed.

The ability of the Rhizobium isolates to grow in basic or acidic media was tested by streaking them on YEMagar plates with pH adjusted to 5.0, 6.0, 7.0, 8.0, and 9.0 with HCl or NaOH (Graham et. al. 1991). According to Kucuk et al. (2006), the data were noted as (-) = absence of growth, (+) = weak growth and (++) = high growth.

Salt tolerance was measured as the ability of the bacterial

cells to grow and divide under the stress of known NaCl concentration. Conical flask (250 mL) each containing 50mL YEM medium, were

Antibiotics Salmonella (%)/ N=104

R S

Amoxicillin 8 (7.7 %) 96 (92.3 %°)

Penicillin Amoxicillin + clavulanic acid 8 (7.7 %) 96 (92.3 %)

Tircacillin 48(46.15 %) 56 (53.85 %)

Cefalotin 10 (9.7 %) 98 (94.23 %)

Cephalosporin Cefoxitim 0 (0 %) 104 (100 %)

Cefotaxim 1 (0.96 %) 103 (99.04 %)

Gentamycin 11 (10. 58 %) 93 (89.42 %)

Chloramphenicol 31 (29.8 %) 73 (70.2 %)

amide

Cotrimoxazol 97 (93.37 %) 7 (6.73 %)

Nalidixic acid 37 (35.76 %) 67 (64.42 %)

Ciprofloxacin 30 (28.85 %) 74 (71.15 %)

Tetracyclin 76 (73.08 %) 28 (26.92 %)

Fig. 1. Map of the study area located at the southern part of Bangladesh

Mondal et al., 2016 / Screening and Isolation of Salt tolerant bacteria from tidal floodplain soils of bangladesh

Octa Journal of Biosciences 13

Fig. 2. Schematic diagram of experimental procedures for the present study.

Fig. 3. Effect of different salinity levels on absorbance of Rhizobium isolates at different days after culture (DAC).

Fig. 4. Effect of absorbance of Rhizobium isolates on different salinity levels at different days after culture (DAC).

Mondal et al., 2016 / Screening and Isolation of Salt tolerant bacteria from tidal floodplain soils of bangladesh

Octa Journal of Biosciences 14

Isolate

Cell shape

Motility

Gram reaction

Catalase

test

Starch Hydrolysis

test

Bromothymol blue test

Growth rate

Color

Result

DMR1

Rod

Motile

(-) ve

(+) ve

(+) ve

Fast

Yellow

Acidic

DMR2

Rod

Motile

(-) ve

(+) ve

(+) ve

Fast

Yellow

Acidic

DMR3

Rod

Motile

(-) ve

(+) ve

(+) ve

Fast

Yellow

Acidic

NKR4

Rod

Motile

(-) ve

(+) ve

(+) ve

Fast

Yellow

Acidic

NKR5

Rod

Motile

(-) ve

(+) ve

(+) ve

Slow

Blue

Alkaline

NKR6

Rod

Motile

(-) ve

(+) ve

(+) ve

Fast

Yellow

Acidic

KKR7

Rod

Motile

(-) ve

(+) ve

(+) ve

Slow

Blue

Alkaline

KKR8

Rod Motile

(-) ve

(+) ve

(+) ve

Slow

Blue

Alkaline

KKR9

Rod Motile

(-) ve

(+) ve

(+) ve

Fast

Yellow

Acidic

Isolates

pH level

5

6

7

8

9

DMR1

+

+

+

+

++

DMR2

-

+

++

++

++

DMR3

+

+

++

++

++

KNR4

-

+

++

++

++

KNR5

+

+

++

+

+

KNR6

-

+

++

++

++

KKR7

-

+

++

++

+

KKR8

+

+

++

++

+

KKR9

-

+

++

++

++

Salinity level (%)

2DAC

4 DAC

6DAC

0

0.9282a

0.9590a

0.9968a

1

0.9294a

0.9617a

1.0080a

2

0.8477ab

0.9273a

0.9740a

3

0.8048bc

0.9019ab

0.9486ab

4 0.7308c

0.8076b

0.8543b

CV

7.25%

6.81%

6.42%

SE 0.0118 0.0120 0.0118

Significance ** ** **

Table 1. Morphological and biochemical characteristics of isolated Rhizobium.

(-) ve = Gram negative, (+) ve = Positive

Table 2. Tolerance of Rhizobium at different pH levels in YEMA medium.

Note: (-) = Absence of growth, (+) = weak growth and (++) = High growth

Table 3. Effect of different salinity levels on absorbance of Rhizobium isolates at different days after culture (DAC).

Vertically different letters a,b and c indicates significant difference at 5% level by DMRT

Mondal et al., 2016 / Screening and Isolation of Salt tolerant bacteria from tidal floodplain soils of bangladesh

Octa Journal of Biosciences 15

Isolates

2DAC

4DAC

6DAC

DMR1

0.8496b

0.8348bc

0.9451bc

DMR2

0.8666b

0.8868bc

0.9578bc

DMR3

0.8611b

0.9279b

0.9705b

KNR4

1.1000a

1.2220a

1.265a

KNR5

0.7276c

0.7823c

0.8449c

KNR6

0.8301bc

0.8703bc

0.9295bc

KKR7

0.7961bc

0.8618bc

0.9130bc

KKR8

0.8415bc

0.9151b

0.9045bc

KKR9

0.7607bc

0.9025b

0.8775bc

CV

7.25%

6.81%

6.42%

SE

0.0159

0.0160

0.0159

Significance

**

**

**

Table 4. Effect of absorbance of Rhizobium isolates under different salinity levels at different days after culture (DAC).

Vertically different letters a,b and c indicates significant difference at 5% level by DMRT

2 DAC

4 DAC

6 DAC

Isolates

% Salinity level

0

1

2

3

4

0

1

2

3

4

0

1

2

3

4

DMR1

1

0.872

0.821

0.79

0.766

1.021

0.912

0.887

0.853

0.839

1.048

0.959

0.934

0.899

0.886

DMR2

1.016

0.844

0.902

0.847

0.724

1.034

0.9

0.9

0.907

0.835

1.061

0.946

0.947

0.954

0.882

DMR3

1.028

0.876

0.887

0.77

0.745

1.053

0.912

0.904

0.934

0.837

1.079

0.958

0.951

0.981

0.884

KNR4

1.184

1.208

1.017

1.116

0.977

1.31

1.259

1.245

1.243

1.054

1.337

1.305

1.292

1.29

1.101

KNR5

0.802

0.83

0.706

0.734

0.566

0.822

0.847

0.834

0.822

0.586

0.949

0.893

0.881

0.869

0.633

KNR6

0.808

1.018

0.858

0.801

0.666

0.828

1.014

0.922

0.942

0.728

0.855

1.061

0.969

0.989

0.775

KKR7

0.93

0.889

0.789

0.721

0.652

0.953

0.933

0.908

0.84

0.718

0.98

0.979

0.955

0.887

0.765

KKR8

0.857

0.908

0.903

0.747

0.793

0.856

0.935

0.876

0.812

0.83

0.883

0.982

0.923

0.859

0.877

KKR9

0.73

0.92

0.749

0.717

0.688

0.754

0.945

0.87

0.764

0.841

0.78

0.992

0.917

0.811

0.888

CV

7.25%

6.81%

6.42%

SE

0.0355

0.0359

0.0355

Significance

**

**

**

Table 5. Interaction effect of growth of Rhizobium isolates and different salinity levels at different days after culture (DAC).

Note: CV= Coefficient of Variance, SE= Standard Error, ** indicates significant difference at 1% level

The pH is an important parameter for the growth of the organism. Slight variations in pH of medium might have enormous effect on the growth of organism. All isolates were grown in YEM medium with pH values of 7 and 8, but differences were detected at pH 5 and 6 (Table 2). Of all the isolates even grew at a basic condition as high as pH 9. Superior growth of Rhizobium has been reported at neutral to alkaline pH. Results showed that cells were able to grow well at pH 6.0, 7.0, 8.0 and 9.0 at 28 ºC temperature. Only growth of four isolates (DMR1, DMR3, KNR5 and KKR8) was observed in medium with pH 5.0. Similar observation was found by Kucuk, et al. (2006) who earlier reported that Rhizobium can grow on YEMA medium having different levels of pH.

Isolation of salt tolerant bacteria

CONCLUSION

Acknowledgments

salinity levels at different days after culture (DAC) was highly significant (Fig. 4). At all days after culture (DAC), the highest absorbance was recorded by the growth of KNR4 isolate and the lowest absorbance was found by the growth of KNR5 isolate (Table 4).

The interaction effect of salinity levels and isolates of Rhizobiumon absorbance at different days after culture (DAC) was highly significant. The interaction of KNR4 isolate with different salinity levels was highest at all DAC (Table 5). At 2 DAC and 6 DAC, the lowest interaction was found by KKR9 isolate with control and 3% (w/v) NaCl where at 4 DAC, lowest in control. The lowest value of remaining interaction was observed by KNR5 isolate at all DAC (Table 5). There is variation in salt resistance among Rhizobium strains, e.g., Rhizobium strains from Vignaun guiculata were tolerant to NaCl up to 5.5%, which is

It is known that salt stress significantly reduces nitrogen equivalent to about 450 mM NaCl (Mpepereki et al. 1997). Harashit fixation and nodulation in legumes. Hashem et al. (1998) have proposed Kumar Mandal (2014) found that Rhizobium trifolii grew at 3% NaCl that salt stress may decrease the efficiency of the Rhizobium-legume where Kucuk et al. (2006) found that rhizobia grew at 5% NaCl.symbiosis by reducing plant growth and photosynthesis survival and proliferation of rhizobia in the soil and rhizosphere or by inhibiting very early symbiotic events, such as chemotaxis and root hair colonization,

In this study the strains of Rhizobium sp. isolated were rod thus, directly interfering with root nodule function. Some rhizobial

shaped, Gram negative first growing in nature. One isolate (NKR4) in the isolates have been shown to grow under high salt conditions 4-5%

present study was salt tolerant which can be used as a desirable (Kucuk et al. 2006). Results indicated that the effect of salinity levels on

biofertilizer in the salt affected regions in Bangladesh. The increasing the absorbance of Rhizobium at different days after culture (DAC) was

levels of salinity had significant effect on the growth of Rhizobium. The highly significant (Fig. 3 and Table 3). At 2 DAC, the highest absorbance

present study suggested that more advance research is needed in this (0.9294) was found at 1% (w/v) NaCl which was statistically similar with

area, particularly on field evaluation and application of potential control (0.9282) and the lowest absorbance (0.7308) was recorded at

organisms especially salt tolerant Rhizobium as biofertilizer in the salt 4% (w/v) NaCl (Table 3). At 4 DAC, the highest absorbance (0.9617) was

affected soil.found at 1% (w/v) NaCl which was statistically similar with control (0.9590) and 2% (w/v) NaCl (0.9273) and the lowest absorbance (0.8076) was recorded at 4% (w/v) NaCl which was similar at 6 DAC

The authors thank to the authority of Patuakhali Science and(Table 3).The effect of absorbance of Rhizobium isolates on different

Mondal et al., 2016 / Screening and Isolation of Salt tolerant bacteria from tidal floodplain soils of bangladesh

Octa Journal of Biosciences 16

alfalfa inoculated with salt tolerant strains of Rhizobium. Ain Shams Science Bulletin, 28B: 441-Technology University (PSTU), Bangladesh for providing laboratory 468.

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Mondal et al., 2016 / Screening and Isolation of Salt tolerant bacteria from tidal floodplain soils of bangladesh