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Int.J.Curr.Microbiol.App.Sci (2013) 2(10): 406-417

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Original Research Article

Characterization of Bacillus sp. strains isolated from rhizosphere of tomato plants (Lycopersicon esculentum) for their use as potential plant growth

promoting rhizobacteria

D.Pavan Kumar Agrawal1 and Shruti Agrawal2*

1Department of Biotechnology, G. B Pant Engineering Collge, Ghurdauri, Pauri, Uttrakhand, India

2Department of Microbiology, Sai institute of paramedical and allied Sciences, 26/26A, Rajpur road, Near Meedo grand hotel, Dehradun-248001, India

*Corresponding author

A B S T R A C T

Introduction

Microbial communities are abundantly found in the rhizosphere and also in the area that is under the influence of the root and its close vicinity. The rhizosphere

gives support to many active microbial populations capable of exerting beneficial, neutral or detrimental effects on plant growth (Latour et al., 1996). In this paper

ISSN: 2319-7706 Volume 2 Number 10 (2013) pp. 406-417 http://www.ijcmas.com

K e y w o r d s

Plant growth promoting rhizobacteria; IAA production; phosphate solubilization; Seed germinationl roll towel method.

Plant growth promoting rhizobacteria (PGPR) influence the plant growth by various direct or indirect mechanisms. To look for efficient PGPR strains having multiple activities, total 28 bacterial isolates were isolated from different rhizospheric soil of tomato crop in the vicinity of Dehradun. The bacterial isolates were biochemically characterized and screened in vitro for their plant growth promoting traits like production of indole acetic acid (IAA), phosphate solubilization, hydrogen cyanide (HCN), siderophore and catalase. Total 5 bacterial isolates of Bacillus showed potential PGPR activities. The productions of indole acetic acid (IAA) by all rhizobacteria were investigated as important mechanism for plant growth stimulation. All rhizobacterial isolates produced IAA in vitro by the addition of L-tryptophan, in the culture medium. All rhizobacterial isolates in comparison with the control, also showed increment in both the shoot and the roots of tomato seedlings. PGPR were tested for seed germination and seedling vigour by using tomato seeds in roll towel method. Generally, the seed germination ranged from 90 percent (uninoculated control) to 97.5 percent. All the strains, HBS-VIII, FAR-IIIb, HBR-II, GAR-III and HBR-VII significantly improved seed germination when compared to the uninoculated control (UIC). The isolate HBRVII showed significantly increased seed germination (97.5%) and also shoot and root length as well as enhanced vigour index of 115.50, 714.35 after 6 and 16 days respectively. The rhizobacteria could exhibit more than two or three PGPR traits that promote plant growth directly or indirectly.

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we focus on the bacteria that are derived from root and exert benificial effect on the root. Such bacteria are generally designated as plant-growth-promoting rhizobacteria (PGPR). PGPR are a heterogeneous group of bacteria that are found in the rhizosphere, at root surfaces and in association with roots, which can improve the extent or quality of plant growth directly and/or indirectly. Direct effects depends on the production of plant growth regulators, improvement in plant nutrients uptake, or promote induce systemic resistance (ISR) of the plant (Chaudhary 2011).

The indirect promotion of plant growth occurs when PGPR prevent deleterious effect. The exact mechanisms by which PGPR promote plant growth are not fully understood, but are thought to include (i) the ability to produce or change the concentration of plant growth regulators like indole acetic acid (Patten and Glick, 2002), gibberellic acid , cytokinins and the ability to produce ACC deaminase to reduce the level of ethylene in the roots of the developing plants, thereby increasing the root length and growth (4. Glick,1995), (ii) asymbiotic N2 fixation (Kennedy et al., 1997), (iii) antagonism against phytopathogenic microorganisms by producing siderophores, b-1,3-glucanase, chitinases, antibiotics, fluorescent pigment and cyanide (6. Sharma et al 2003) (iv) solubilization of mineral phosphates and other nutrients (Johri et al., 2003).

In last few decades a large array of bacteria including species of Pseudomonas, Azospirillum, Azotobacter, Klebsiella, Enterobacter, Alcaligenes, Arthrobacter, Burkholderia, Bacillus and Serratia have reported to enhance plant growth (Kloepper et al., 1989; Glick, 1995).

It has seen that various species of Bacillus are of interest as they are dominant in soil and are known to secrete antimicrobials and siderophores. In addition, they are also important microbial producers of biosurfactants such as rhamnolipis. These can cause lysis of zoospores of soil borne plant pathogenic fungi such as Pythium, Phytophthora and Plasmopara by interacting with and disrupting their plasma membrane (Stanghellini and Miller, 1997). Consequently, Bacillus and other rhizobacteria can facilitate a control of damping-off especially in vegetable nurseries where the disease is often prevalent by destroying zoospores of these phytopathogen. Interestingly, protection against diseases by rhizobacteria also involves quorum sensing in rhizosphere (Gray and Smith, 2005, Sharma et al., 2003) and induction of systemic resistance (ISR) wherein lipopolysaccharides have been reported to act as signal molecules (Pathak et al., 2004).

Currently our research effort is towards helping the poor farmer communities, particularly those in the state of Uttarkhand in the Central Himalayan region, that depend only on vegetable cultivation for their livelihood. In this study we focus on role the of rhizobacteria in plant growth promotion. A pool of promising rhizobacteria was screened through in-vitro and their plant growth promoting properties. To evaluate the influence of the most promising bacterial strains on plant growth, bacterized tomato seed were planted in paper towel method. The potential of these has been analyzed,that is to provide data that effect the growth parameters in tomato.

Consequently, Uttarakhand region represents a unique combination of plant and soil type which changes drastically


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with altitude. However limited efforts have been done so far to explore the available bacterial diversity. In the present study rhizospheric soil samples from tomato were collected from Dehradun region for assessment of PGPR. The objectives of this study is to isolate and characterize Bacillus sp. from the rhizosphere of tomato, and to screen their abilities and the possession of direct and indirect plant growth promoting rhizobacteria attributes. Materials and Methods

Isolation of Bacillus from the rhizosphere

Rhizospheric soil samples were collected from tomato growing fields of Dehradun. Bacillus species were isolated using dilution method with Nutrient Agar medium. Rhizosphere soil samples (10 g) were suspended in 90 mL of 0.85% normal saline (pH 7.0) and shaken vigorously at 150 rpm at 18°C for 1 h. The resulting slurry was serially diluted (100

L) to 900 L of 0.85% normal saline in each Eppendorf tube and appropriate dilution (10-4) of this suspension (0.1 mL or 100 L) was spread plated in triplicate on Nutrient Agar medium. Cultures were incubated at 37°C±2 for 2 d. For experimental use, isolates were transferred when needed to nutrient agar medium that was stored at 4°C. Each colony was assayed further for morphological and physiological characteristics including Gram reaction, endospore, motility, oxidase and catalase enzyme activity.

Phenotypic characterization of bacterial isolates

A total of 28 isolates were randomly selected morphologically from tomato

rhizosphere. The remaining bacterial isolates were phenotypically (morphotypic and physiological) characterized. Colony morphology of isolates was studied under a stereoscope microscope (Leica). This included shape, edge, elevation, surface and pigmentation.

The cellular morphology was based on cell shape and Gram staining (Leica fluorescent microscope). The Bacterial identification was carried out on the basis on Bergeys Manual of Systematic Bacteriology (Clauss and Berkeley, 1986). The Bacillus species were estimated on the basis of morphological and physiological characteristics.

Characterization of rhizobacteria for PGP traits

Siderophore production

Siderophore production was tested qualitatively using chromeazurol S medium (CAS-medium) ( Sharma and Johri, 2003). Each Bacillus isolate were streaked on the surface of CAS agar medium and incubated at room temperature for 1 to 3 days. Siderophore production was confirmed by orange halos around the colonies after the incubation, and this test was done in two replications.

Phosphate solubilization

Solubilization of tri-calcium phosphate was detected in Pikovskaya s Agar ( Johri et al., 2003). All bacterial isolates were streaked on the surface of Pikovskaya agar medium and phosphate solubilizing activity was estimated after 1 to 5 days of incubation at room temperature. Phosphate solubilization activity was determined by the development of the clear zone around bacterial colony.


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Production of HCN

All the isolates were screened for the production of hydrogen cyanide by performing the method of Lorck (1948). Briefly, nutrient broth was amended with 4.4 g glycine/l and bacteria were streaked on modified agar plate. A Whatman filter paper no. 1 soaked in 2% sodium carbonate in 0.5% picric acid solution was placed at the top of the plate. Plates were sealed with parafilm and incubated at 36±2 °C for 4 days. Development of orange to red colour indicated HCN production.

Qualitative assay of IAA Production:

The qualitative assay of IAA produced by Rhizobacterial strains were conducted on the basis of Bric s et al. (1991) proposed method. In this method sterilized Petri dishes (9-cm diameter) were used. Each Petri dish was inoculated with toothpicks and three repeats (3 squares) were considered for any rhizobacterial isolate. Then each inoculated Petri dish was overlaid with an 82-mm-diameter disk of nitrocellulose membrane. Petri dishes were inversely incubated into incubator (at 27°C) for 2 to 4 days. Finally when the diameter of appeared colonies on nutrient agar media was about 2 mm, IAA production assay was conducted as following exposition: The disks of nitrocellulose membrane including rhizobacterial grown colonies (about 2 mm) were treated with Salkowski reagent within other Petri dish including a reagent

saturated filter paper (whatman no. 2). The reaction was allowed to proceed until adequate colour developed. All the reagent incubations was conducted at room temperature. Bacteria producing IAA were identified by the formation of a characteristic red halo within the

membrane immediately surrounding the colony (after time of 0.5 to 2 hours) that the color and size of the colonies varied in different isolates (depending on the amount of produced IAA by isolates). Generally, the tested isolates were group of IAA hormone production ability as follows. The halo diameter (HD) and the colony diameter in strains were measured and the mean ratio of halo diameter to colony diameter were calculated for each isolate and considered as the qualitative gradation base of IAA production ability.

Quantitative assay for Production of Indole acetic acid

Indole acetic acid (IAA) production was detected as described by Agrawal et al., (2011). Bacterial cultures were grown for 48hours on the nutrient broth at 37±2 °C. Bacteria were grown overnight in five ml of nutrient broth supplemented with L-tryptophan to achieve a final concentration of 0, 50, 100, 200 and 500 g ml-1. After incubation for 42 h, bacterial growth was measured spectrophotometrically at 600 nm; the cells were removed from culture medium by centrifugation at 7,500 rpm for 10 min. 1 ml aliquot of supernatant was mixed with 4 ml of Salkowski s reagent (150 ml of concentrated H2SO4, 250 ml of D.W, 7.5 ml of 0.5 M FeCl3, 6H2O). Samples were left at 28 20C for 25 min and absorbance was read at 535 nm. The concentration of IAA was determined by referring to a standard curve.

Evaluation of effective PGPR strains on seed germination and seedling vigour

Seed germination assay:

The experiment was conducted to assess the influence of 5 selected efficient isolates on seed germination and tested for their plant growth promotion ability by the


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standard roll towel method (ISTA, 1985) in growth chamber. Bacteria were grown in nutrient broth medium on a shaker (150 rpm) for 2 days and centrifuged at 10,000 rpm for 5 minutes. The pellet was mixed with sterile carboxy methyl cellulose (CMC) (HiMedia) suspension (1%). Tomato seeds were surface sterilized with 0.1% mercuric chloride for 5 min, rinsed with sterilized distilled water (SDW) and soaked in bacterial suspension (3×108 cfu ml-1) using 1% carboxymethyl cellulose (CMC) for 24h and sterile blank nutrient broth served as control. Then the seeds were blot dried, placed in wet blotters and incubated in growth chamber maintained at 25±20C and 95±3% RH. Each treatment was replicated four times with 40 seed per each. The percentage of germination was recorded at fifth day. Ten seedlings were taken at random from each replication and length of root and shoot measured separately at 6 and 16 days (Abdul Baki and Anderson, 1973). Plant growth promotion of tomato seedling was assessed using Vigour Index (VI). VI = per cent germination x mean total length of seedling (root length + shoot length)

Results and Discussion

A total three rhizospheric and three soil samples were collected from tomato growing fields, home and garden in Dehradun. These samples were plated on Nutrient agar medium. The total cfu obtained in these cases were seen to be maximum in Rhizosphere layer of Garden soil (3.41 X106 CFU/gram soil) as compared to Field and Home samples (table1).

Bacterial morphotypes were selected on the basis of colour, morphological characteristics viz., colony morphology

(shape, margin, elevation and surface) and cell morphology (Gram s reaction, cell shape and arrangement) and were studied in detail (table2). Bacteria exhibited wide morphological variation. Maximum variation in colony shape viz.,irregular, circular. Maximum of the obtained bacterial from different soil samples were Gram Positive Bacilli with Mucoid texture, entirely edged and creamy white in appearance as tabulated in table 2.

Plant growth promoting activity of bacterial isolates

The total isolates (28) obtained in the present study were mostly gram positive rods and were subjected to PGPR properties determining tests (Kloepper et al., 1980) i.e. siderophore production and phosphate solublization test. Appearance of clear zone indicated the positive result i.e., phosphate solubilization and Siderophore production was indicated by orange halos around the colonies. Out of 28 only 5 isolates showed positive test namely FAR-IIIb, HBR-II, GAR-III, HBR-VII and HBS-VIII which were tabulated. All the isolates were then subjected to HCN production test (Rezzonico et al., 2007). Appearance of reddish brown zone around the inoculated colony shows positive result. Out of 28 isolates some showed strong results while other gave moderate and low results. Isolates which gave negative result had PGPR property because HCN is a lethal byproduct which is harmful for the plant growth promotion. HCN, produced by many soil microorganisms and it is postulated to play a role in biological control of pathogens (Defago et al., 1990). Production of HCN by certain strains of fluorescent pseudomonads has been involved in the suppression of soil borne pathogens (Siddiqui et al., 2006). The isolates were also subjected to siderophore


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Table.1 Functionally active bacterial population associated with tomato crop recorded on

various selective media (CFU g-1 soil)

Sample Type King s B Media Nutrient Agar Media

Garden Soil Sample ( I ) 1.15X106 2.44 X106

Garden Rhizosphere Sample (I) 3.41 X106 1.98 X106

Home Soil Sample ( II ) 0.49 X106 1.80 X106

Home Rhizosphere Soil Sample (II) 0.89 X106 1.40 X106

Field soil sample 0.6 X106 0.4 X106

Field rhizosphere 2.38 X106 0.79 X106

Table.2 Morphotypic characterization of rhizobacteria associated from Tomato

Sample Name of Isolate

Grams Reaction Colour of Colony

Margin Elevation

Surface Form

Morphotypic characterization of rhizobacteria associated from Tomato of Garden Soil Sample GASIa Gram Ve, Coccus

Off White Repand Flat Dry Irregular

GASIb Gram +Ve,Shorts thin Rods

Transparent

Entire Convex Mucoid Round

GASIIa Gram Ve, Coccus

Creamy White

Entire Raised Mucoid Round

GASIIb Gram +Ve, Coccus

Creamy White

Entire Umbonate

Mucoid Round

GASIII Gram +Ve, Short Rods

Creamy,Translucent

Undulate

Flat Dry Irregular

GASIV Gram +Ve, Baccillus

Creamy White

Erose Umbonate

Glisining Irregular

GASVI Gram +Ve, Short Rods

Yellow Entire Slightly Raised

Mucoid Irregular

GASVII Gram +Ve, Coccus

Off White Entire Convex Mucoid Round

GASIX Gram Ve, Coccus Orange Entire Raised Mucoid Irregular

Morphotypic characterization of rhizobacteria associated from Tomato of Garden Rhizo-sphere Sample

GARI Gram +Ve, ShortRods (Streptococci)

Off White Cilliated

Flat Dry Irregular

GARII Gram Ve, Cocco baccillus

White Cilliated

Flat Dry Irregular

GARIII Gram +Ve, Baccillus

Off White Cilliated

Flat Mucoid Irregular

GARIV Gram +Ve, Baccillus

White Entire Flat Mucoid Fusiform


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Morphotypic characterization of rhizobacteria associated from Tomato of Home Soil Sample

HBSIII Gram +Ve, Thin Rods

White Lobate Flat Dry Irregular

HBSV Gram Ve,

Spindle Shape Off White Entire Flat Mucoid Irregular

HBSVII Gram +Ve,

Rods White Entire Raised Mucoid Circular

HBSVIII Gram+Ve,Rods (Streptococcus)

White Entire Raised Dry Irregular

Morphotypic characterization of rhizobacteria associated from Tomato of Home Rhizos-phere Sample

HBRI Gram +Ve, Baccillus

Creamy Entire Umbonate

Dry Irregular

HBRII Gram +Ve, Coccus

Off White Entire Umbonate

Dry Round

HBRIII Gram +Ve, Coccobaccillus

Off White Lobate Convex Dry Round

HBRVII Gram +Ve, Baccillus

Off White Entire Flat Mucoid Irregular

Morphotypic characterization of rhizobacteria associated from Tomato of Field Soil Sample

Table.3 Plant growth promoting attributes of the Bacterial isolates from the rhizosphere of tomato plants

Isolate code

Qualitative assay for IAA PRODUCTION

HCN production

Siderophore production

P-solublization activity

HBS-VIII +++ - + + FAR-IIIb +++ - + + HBR-II ++ - + + GAR-III +++ ++ + + HBR-VII +++ - + +

FASIV Gram +Ve, Strepto Baccillus

White Entire Raised Mucoid Irregular

FARIa Gram Ve, Coccobaccillus

Creamy White

Entire Raised Mucoid Round

FARIb Gram +Ve, Coccus

Cream Translucent

Entire Raised Mucoid Irregular

FARII Gram +Ve, Coccus (Cluster)

White Entire Convex Mucoid Round

FARIIIa

Gram +Ve, Coccus

Off White Entire Raised Mucoid Irregular

FARIIIb

Gram +Ve, Coccus

Off White Entire Raised Mucoid Irregular

FARIV Gram Ve, Coccus

Creamy Entire Raised Mucoid Round


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Fig.1 Production of Indole Acetic Acid ( IAA)

Table.3 Biochemical characterizations of bacterial isolates recovered from Tomato rhizosphere

Isolate Grams reaction

Endospore staining

Cata-lase

Citrate utilization

Starch hydrolysis

Gelatin utilization

Oxi-dase

Cellulose utilization

Cellular morphology

Motility

HBS-VIII

+ + + - + + + - Long Rods

+

HBR-II + + + + + + - - Bacillus + FAR-IIIb + + + + + + + - Long rods + GAR-III + + + + + + + - Bacillus + HBR-VII + + + - + + - - Rods +

Fig.2 Efficacy of rhizobacterial isolates on seed germination


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Fig.3 Efficacy of rhizobacterial isolates on seedling vigour by roll towel method.

Fig.4 Efficacy of rhizobacterial isolates on root length and shoot length by roll towel method


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production test. Siderophores are low molecular weight, extracellular compounds with a high affinity for ferric iron, that are secreted by microorganisms to take up iron from the environment ( Sharma and Johri, 2003) and their mode of action in suppression of disease were thought to be solely based on competition for iron with the pathogen (Bakker et al., 1987;Duijff et al., 1997). On the basis of above three test screening of isolates were done.

A total five isolates were further selected and subjected for IAA production test. (fig1 ). Indole acetic acid (IAA) production was detected as described by Brick et al., (1991).The ability of bacteria to produce IAA in the rhizosphere depends on the availability of precursors and uptake of microbial IAA by plant. Growth

promotion may be attributed to other mechanisms such as production of plant growth promoting hormones in the rhizosphere and other PGP activities (Glick, 1995). Production of IAA by Bacillus is a general characteristic of our test isolates. Higher level of IAA production by Pseudomonas was recorded by other workers (Xie et al 1996). Production of IAA was indicated by appearance of pink colour. After qualitative analysis these isolates were subjected for quantitative production of IAA which were tabulated. Isolates GARIII showed maximum IAA production and least by isolates FARIIIb which was obtained manually from standard graph as shown in fig1. These isolates (HBS-VIII, FAR-IIIb, HBR-II,

GAR-III and HBR-VII) were then subjected to biochemical characterization for the determination of probable microorganism which were tabulated. Bacillus species were estimated by morphologies and physiology characteristics based techniques table3.

These isolates were then assessed to know their influence on the seed germination by measuring the shoot and root length. The growth promoting activity of 5 isolates of PGPR was tested for seed germination and seedling vigour by using tomato seeds in roll towel methods. The data on seed germination and seedling vigour of tomato as influenced by seed bacterization with different rhizobacterial isolates by roll towel method are given in Fig 2,3,4 In general, the seed germination ranged from 90 per cent (uninoculated control) to 97.5 percent (HBRVII). All the strains,HBS-VIII, FAR-IIIb, HBR-II, GAR-III and HBR-VII significantly improved seed germination when compared to the uninoculated control (UIC). The isolate

HBRVII showed significantly increased seed germination (97.5%) and also shoot and root length as well as enhanced vigour index of 115.50, 714.35 after 6 and 16 days respectively.

Acknowledgement

The author wish to thank to chairman Mr Harish Arora and Vice chairperson Mrs. Rani arora of SIPAS, Dehradun. We acknowledge the sincere thanks to Dean life science Prof V D Sharma and Principal SIPAS Dr. Sandhya Dogra for providing us research facilities.

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