Molecular characterization and identification of plant

Molecular characterization and identification of plant growth promoting endophytic bacteria isolated from the root nodules of pea (Pisum sativum L.) Mohsin Tariq, Sohail Hameed, Tahira Yasmeen, Mehwish Zahid & Marriam Zafar World Journal of Microbiology and Biotechnology ISSN 0959-3993 Volume 30 Number 2 World J Microbiol Biotechnol (2014) 30:719-725 DOI 10.1007/s11274-013-1488-9

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Author's personal copy World J Microbiol Biotechnol (2014) 30:719–725 DOI 10.1007/s11274-013-1488-9

ORIGINAL PAPER

Molecular characterization and identification of plant growth promoting endophytic bacteria isolated from the root nodules of pea (Pisum sativum L.) Mohsin Tariq • Sohail Hameed • Tahira Yasmeen Mehwish Zahid • Marriam Zafar

•

Received: 20 May 2012 / Accepted: 30 January 2013 / Published online: 26 September 2013 Ó Springer Science+Business Media Dordrecht 2013

Abstract Root nodule accommodates various non-nodulating bacteria at varying densities. Present study was planned to identify and characterize the non-nodulating bacteria from the pea plant. Ten fast growing bacteria were isolated from the root nodules of cultivated pea plants. These bacterial isolates were unable to nodulate pea plants in nodulation assay, which indicate the non-rhizobial nature of these bacteria. Bacterial isolates were tested in vitro for plant growth promoting properties including indole acetic acid (IAA) production, nitrogen fixation, phosphate solubilization, root colonization and biofilm formation. Six isolates were able to produce IAA at varying level from 0.86 to 16.16 lg ml-1, with the isolate MSP9 being most efficient. Only two isolates, MSP2 and MSP10, were able to fix nitrogen. All isolates were able to solubilize inorganic phosphorus ranging from 5.57 to 11.73 lg ml-1, except MSP4. Bacterial isolates showed considerably better potential for colonization on pea roots. Isolates MSP9 and MSP10 were most efficient in biofilm formation on polyvinyl chloride, which indicated their potential to withstand various biotic and abiotic stresses, whereas the remaining isolates showed a very poor biofilm formation ability. The

M. Tariq S. Hameed T. Yasmeen M. Zahid M. Zafar Microbial Physiology Laboratory, National Institute for Biotechnology and Genetic Engineering (NIBGE)/ PAEC, Islamabad, Pakistan M. Tariq (&) T. Yasmeen Government College University Faisalabad (GCUF), Allama Iqbal Road, Faisalabad, Pakistan e-mail: [email protected]; [email protected] M. Zafar Centre of Agricultural Biochemistry and Biotechnology (CABB), University of Agriculture Faisalabad, Faisalabad, Pakistan

most efficient plant growth promoting agents, MSP9 and MSP10, were phylogenetically identified by 16S rRNA gene sequence analysis as Ochrobactrum and Enterobacter, respectively, with 99 % similarity. It is suggested the potential endophytic bacterial strains, Ochrobactrum sp. MSP9 and Enterobacter sp. MSP10, can be used as biofertilizers for various legume and non-legume crops after studying their interaction with the host crop and field evaluation. Keywords Pisum sativum L. Plant growth promoting bacteria 16S rRNA sequencing Nodules

Introduction Rhizobia are soil bacteria, recognized by the character of infecting roots of legume (Fabaceae) plants and developing an outgrowth on root, called nodule, which are the small factories of atmospheric nitrogen fixation. In the past, rhizobia were considered to include the bacteria belonging to genera Rhizobium, Azorhizobium, Bradyrhizobium, Ensifer and Mesorhizobium. Recently, it is found that rhizobia is a paraphyletic group, fall into three classes of proteobacteria-alpha, beta and gamma-proteobacteria (Angus and Hirsch 2010; Benhizia et al. 2004). Root nodules also accommodate various non-nodulating bacteria having definite influence on the survival, nodulation and grain yield of crop and their densities are reported to be very high (Mishra et al. 2009; Tariq et al. 2012). These endophytic bacteria live inside the nodule tissues without substantially harming or gaining benefit other than shelter (Kobayashi and Palumbo 2000). Such bacteria are relatively protected from the competitive, high-stress environment of soil and promote plant growth by producing plant growth promoting substances (Aravind et al. 2012; Patel et al. 2012).

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Furthermore, these bacteria act synergistically with rhizobia to improve nodulation and nitrogen fixation (Duangpaeng et al. 2012). Non-nodulating bacteria for the first time isolated from nodules of legume plant were identified as Agrobacterium radiobacter (Beijerinck and Delden 1902). Non-nodulating bacteria isolated from nodules of various legume plants include Inquilinus, Pantoea, Escherichia, Bosea, Phyllobacterium, Sphingomonas, Pseudomonas, Agromyces, Microbacterium, Paenibacillus, Aerobacter, Agrobacterium, Chryseomonas, Curtobacterium, Erwinia, Flavimonas, Sphingomonas, Methylobacterium, Blastobacter, Devosia, Rhodopseudomonas, Paracraurococcus, Phyllobacterium, Ochrobactrum, Cupriavidus, Herbaspirillum, Pseudomonas, Enterobacter, Leclercia, Ochrobactrum, Starkeya, Azotobacter, Azospirillum, Ornithinicoccus, Bacillus (Sturz et al. 1997; Selvakumar et al. 2008; Tariq et al. 2012). The present study was aimed to isolate the non-nodulating endophytic bacteria from the root nodules of pea plants and characterize in vitro for plant growth promoting properties. Using 16S rRNA gene sequence analysis, we also studied the taxonomic position of these non-nodulating endophytic bacteria.

Materials and methods Isolation of nodule endophytic bacteria Pea plants grown in the field of National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan, were sampled for bacterial isolation at nodulation stage after 4 weeks. Roots were washed thoroughly using sterile distilled water to remove adhering soil. About 10 nodules were detached from the roots with a sterilized forceps. The intact and undamaged nodules were immersed in ethanol for 30 s and transferred to a 3 % solution of calcium hypochlorite for 5 min under aseptic conditions. Nodules were rinsed in five changes of sterile water. Surface sterilization of nodules was ensured by incubating nodules on yeast extract mannitol (YEM) agar (Vincent 1970) plate for 72 h at 28 ± 2 °C. Nodules were crushed to prepare a suspension in 20 ll sterile water in a Petri dish. One loop full of nodule suspension was streaked on YEM plates containing Congo red and incubated at 28 ± 2 °C until the appearance of single colonies, which were selected after 24 h of showing different morphotypes and subcultured until the purity of cultures was confirmed (Marsudi et al. 1999). Pure cultures were stored in 20 % glycerol at -80 °C. Cell shape and size were observed under light microscope by taking a drop of bacterial culture suspension in saline. Gram’s reaction was performed according to Vincent (1970).

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Nodulation assay Nodulation assays were performed according to Ma et al. (2003) with some modifications. All the potential nodule endophytic bacteria were grown individually in YEM broth, and maintained at 106 c.f.u. ml-1 in sterile water. Rhizobium leguminosarum bv. viciae PS-I and PS-II were used as positive control for nodulation. Seeds of pea cv. Meteore were surface sterilized with 0.1 % mercuric chloride for 5 min and washed extensively with sterile water. Surface sterilized seeds were allowed to germinate in dark room at 25 ± 2 °C on moist filter paper kept in sterile Petri plates containing 15 seed. Germinated seedlings (1 cm) were treated with inocula of each endophytic bacteria, separately, and transplanted in the sterilized magenta boxes containing vermiculite-perlite (ratio 1:1 v/v) and incubated at 30 ± 2 °C (day) and 20 ±2 °C (night) for a light photoperiod of 16 h per day (Fraile et al. 1988). Plants were watered with quarter strength nitrogen free Hoagland solution (Arnon and Hoagland 1940). After 4 weeks, plants were observed for nodulation. Characterization of endophytic bacteria for plant growth promoting attributes Bacterial isolates were also studied in vitro for plant growth promoting properties including indole acetic acid (IAA) production, nitrogen fixation, solubilization of phosphate, root colonization and biofilm formation. Detection of IAA production by bacterial strains was carried out by growing cultures in YEM broth supplemented with tryptophan (100 mg l-1) for 1 week. Qualitative estimation of IAA was assessed by Fe-HClO4 and Fe-H2SO4 reagents producing pink color (Gordon and Weber 1951). IAA was quantified by ethyl acetic acid oxidation method (Tien et al. 1979) using HPLC with Turbochom software (Perkin Elmer USA). Nitrogenase activity was examined by acetylene reduction assay (Hardy et al. 1968) on a gas chromatograph (Thermoquest, Trace G.C. Italy) using Porapak Q column and H2-flame ionization detector. Nitrogen fixation ability of bacterial strains was assessed by inoculating single colony in 5 ml semisolid nitrogen free media (NFM) (Okan et al. 1977) in 15 ml vials and incubated at 28 ± 2 °C for 48 h. Acetylene (10 % v/v) was injected to the vials and incubated for 16 h at room temperature. Gas samples (100 ll) from these vials were analyzed on a gas chromatograph as described by Hameed et al. (2004). For phosphate solubilization study, a single colony of each bacterial culture grown on YEM plate was streaked on to Pikovskaya’s agar plates containing tricalcium phosphate (Pikovskaya 1948) and incubated at 25 ± 2 °C for 7–10 days. The plates were analyzed for clear phosphate solubilization zone around the colonies. Phosphate

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solubilization was quantified by Phospho-molybdate blue color method (Nair et al. 2007) using spectrophotometer (Campec M350 Double Beam). Root colonization assay Potential of bacterial isolates to colonize pea roots was also studied. Ten-day-old plants were harvested and the roots were cut into 1.5 cm segments. Pieces of uniform shape and size were placed into the 96-wells of a microtiter plate. 200 ll of bacterial culture maintained at OD600 = 0.2 were added to the wells and the plates were incubated at 28 °C for 24 h for fast growing and 72 h for slow growing bacteria. After the incubation period, the root pieces were removed from the cultures, washed with sterile water, and then added to 1 ml sterile water. Bacterial biofilms were removed from the root surface and dispersed in sterile water by vigorous shaking. An aliquot (100 ll) of the dispersed preparation was plated on YEM agar and counted after 5 days as c.f.u. 0.1 mg-1 root. Biofilm formation assay Biofilm formation was studied on a abiotic surface by a microtiter plate assay according to Fujishige et al. (2006) with some modifications. The bacterial cultures were grown upto an optical density at k600 nm (OD600) = 2.0 in YEM broth, pelleted by centrifugation at 8,000 rev. min-1 for 2 min, and washed with sterile distilled water. The cells were resuspended in the same medium and maintained at OD600 = 0.2. An aliquot (150 ll) of bacterial cell suspension was added to individual wells in a 96-well polyvinyl chloride (PVC) plate (Fisher, USA). YEM alone was used as the control. The plates were covered with plastic lids and incubated at 28 °C for 24 h for fast growing and 72 h for slow growing bacteria. After the incubation period, the medium was removed and the wells were washed with sterile water. The plates were allowed to dry and the wells were treated with 150 ll of 0.001 % crystal violet for 15 min. The excess of dye was removed and the wells were washed with sterile water. The retained stain was solubilized with 150 ll of 95 % ethanol and amount of dye was quantified by plate reader at absorbance of 570 nm.

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and Doly 1979). The primers used for amplification of full length 16S rRNA gene were universal primer P1 (forward primer, 50 -CGGGATCCAGAGTTTGATCCTGGTCAGA ACGAACGCT-30 ) and P6 (reverse primer, 50 -CGGGATCCT ACGGCTACCTTGTTACGACTTCACCCC-30 ), which correspond to E. coli positions 8–37 and 1,479–1,506, respectively and amplifies 1,500 bp fragment (Tan et al. 1997). Each 25 ll of PCR reaction mixture contained 2.5 ll 109 PCR buffer, 2 ll MgCl2, 1 ll dNTPs (2.5 mM), 1 U of Taq Polymerase (Promega), 1 ll of each primer (100 ng ll-1) and 1 ll template DNA (12.5 ng ll-1). Reaction mixture (25 ll) prepared for 16S rRNA gene amplification, was initially denatured at 94 °C for 2 min followed by 25 cycles consisting of denaturation at 94 °C for 1 min, primer annealing at 52 °C for 1 min and primer extension at 72 °C for 3 min and finally extension at 72 °C for 20 min in a thermal cycler. The amplified 16S rRNA gene was ligated in TA cloning vector pTZ57R/T (Fermentas), which has 2,886 bp length. 30 ll ligation reaction was prepared in sterile water with 1.5 ll T4 DNA ligase, 3 ll ligation Buffer, 3 ll pTZ57R/T vector (Fermentas), 3 ll of PEG 4000 and 4 ll amplified DNA in 1.2 ml tube. Ligation was performed overnight in water bath at 16 °C. Plasmids were transformed chemically in E. coli TOP10 and extraction of recombinant plasmids was carried out using GeneJET Plasmid Miniprep Kit (Fermentas) and clones were confirmed by restriction analysis using EcoRI and BamHI (Fermentas). During molecular studies, amplified products and clones were resolved on 1 % agarose gel and GeneRulerTM 1 kb ladder #SM0313 was used as DNA size marker. Clones were sequenced on ABI Prism 3100 Genetic Analyzer (Hitachi, Japan) using Big Dye Terminator v 1.1 Cycle Sequencing Kit. The gene sequences were compared with others in sequence databases using NCBI BLAST software (Altschul et al. 1990) at http://www.ncbi.n1m.nih.gov/blast/Blast.cgi.

Results Isolation of endophytic bacteria from root nodules

The data were analyzed statistically using MSTAT software. Analysis of variance was computed and means were compared employing the LSD test (Steel and Torrie 1980).

A total of ten bacterial isolations were obtained on the basis of colony morphology from the nodules sweet pea grown in the fields of NIBGE, Faisalabad, Pakistan. Colony and cell morphological characters were highly variable as presented in Table 1. Bacterial isolates were also tested for Gram reaction. Out of ten isolates, two were gram positive while rests of the bacterial isolates were gram negative (Table 1).

Phylogenetic analysis of nodule endophytic bacteria

Nodulation assay

Total genomic DNA of potential bacterial strains MSP9 and MSP10 was isolated by the alkaline lysis method (Birnboim

All the nodule endophytic bacteria were initially tested for plant infectivity assay for nodulation. They were able to

Statistical analysis

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Table 1 Morphological characteristics of nodule endophytic bacteria isolated from sweet pea nodule Isolates

Colony morphology

Cell morphology

Gram reaction

MSP1 MSP2

Round medium, smooth, creamy

Small rods

-ve

Round small, smooth, light yellow

Small rods

-ve

MSP3

Small round, smooth, greenish yellow

Small rods

-ve

MSP4

Large round, smooth, creamy

Small rods

?ve

MSP5

Round medium, wavy, dark creamy

Medium rods

-ve

MSP6 MSP7

Small, wavy, creamy Small, smooth, yellow

Small rods Small rods

?ve -ve

MSP8

Round medium, wavy, dark creamy

Small rods

-ve

MSP9

Round medium, smooth, creamy

Very small rod

-ve

MSP10

Round medium, smooth, light brown

Small rod

-ve

Table 2 Comparison of plant growth promoting attributes of pea nodule endophytic bacterial isolate Isolate

IAA production (lg ml-1)

ARA (n mole C2H2 reduced h-1mg-1 protein)

Phosphate solubilization (lg ml-1)

MSP1

1.38 ± 0.27d

–

6.8 ± 0.33c

MSP2

–

58.9 ± 3.4b

11.73 ± 0.26a

MSP3

_

–

9.3 ± 0.43b

MSP4

3.39 ± 0.33c

–

–

MSP5

–

–

5.57 ± 0.36c

MSP6

–

–

9.33 ± 0.36b

MSP7

1.64 ± 0.29d

–

9.2 ± 0.36b

MSP8

4.13 ± 0.3b

–

6.85 ± 0.12c

MSP9

16.16 ± 0.27a

–

11.73 ± 0.27a

MSP10 LSD 0.05

0.86 ± 0.12e 0.425

348 ± 15.6a 26.9

6.83 ± 0.18c 1.37

Means are followed by the standard error Mean sharing same letter in column do not differ significantly

reinfect roots, but did not develop root nodule on their host, sweet pea. Moreover, PS-I and PS-II (Rhizobium leguminosarum bv. viciae), which was used as positive control, developed nodules on pea plants. Characterization for plant growth promoting attributes All the bacterial endophytes were tested for plant growth promoting properties including IAA production, nitrogen fixation and phosphate solubilization (Table 2).

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Five bacterial isolates were able to produce IAA at varying level, ranged from 0.86 to 16.16 lg ml-1. Isolate MSP9 was most efficient in IAA production by producing 16.16 lg ml-1 (Table 2). Bacterial isolates MSP2 and MSP10 were found positive for nitrogenase activity in acetylene reduction assay by reducing 58.9 ± 3.4 and 348 ± 15.6 n mole C2H2 h-1 mg-1 protein, respectively. Phosphate solubilization data indicated that all the bacterial isolates were able to solubilize phosphate except MSP4. Phosphate solubilization efficiency ranged from 5.57 to 11.73 lg ml-1 (Table 2). Further, these bacterial isolates were checked for legume root surface colonization. All the isolates showed very high colonization on root surface of their respective hosts, which was statistically different. MSP9 was found to be most efficient colonizing bacteria (Fig. 1). Similarly, equally efficient biofilm formation ability on the abiotic surface (PVC) was also observed. Bacterial isolates established a significantly different biofilm. Bacterial isolates MSP9 and MSP10 showed high efficiency in biofilm formation on abiotic surface (Fig. 2). Molecular identification of potential bacterial strains Two bacterial strains were selected for phylogenetic identification on the basis of plant growth promoting potential. Phylogenetic identification was performed using 16S rRNA gene sequencing technique. DNA fragments of *1,500 bp were amplified using primers P1 and P6 set (Fig. 3a). The DNA fragments were cloned in vector pTZ57R/T. When the clones were restricted with EcoRI and BamHI, DNA fragment of *2,900, 900 and 600 bp were observed, which revealed that 16S rRNA gene fragments had the site of either EcoRI or BamHI (Fig. 3b). 16S rRNA gene sequences of the potential strains were compared with others in sequence databases using NCBI BLAST software. MSP9 showed 99 % similarity to different species of genus Ochrobactrum, while bacterial strains MSP10 showed 99 % similarity with the Enterobacter. 16S rRNA gene sequences of MSP9 and MSP10 were deposited at NCBI GenBank under the accession numbers JF313266 and JF313267, respectively.

Discussion Plant growth promoting bacteria is a group of soil bacteria which promote plant growth by developing a positive relationship with roots, endophytically or in the rhizosphere. Endophytic bacteria reside inside plant tissue and can be isolated from surface sterilized plant tissues (Garbeva et al. 2001). Root nodules of legume accommodate a large number of non-nodulating plant growth

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723

80000 a

cfu/0.1 mg root .

70000

b

60000

c cd

50000

de

cde

de

MSP6

MSP7

cd

e

40000 f

30000 20000 10000 0 MSP1

MSP2

MSP3

MSP4

MSP5

MSP8

MSP9

MSP10

Isolates Fig. 1 Colonization efficiency of nodule endophytic bacteria on pea roots. Each value is plotted as the mean ± SE (n = 3). Mean sharing same letter in column do not differ significantly. LSD 0.05 = 7,699 0.8 a

Absorbance at 570nm

0.7 0.6

b

0.5 0.4 0.3 c

0.2

cd

d

0.1

cd

cd e

e

MSP6

MSP7

e

0 MSP1

MSP2

MSP3

MSP4

MSP5

MSP8

MSP9

MSP10

Isolates Fig. 2 Biofilm formation efficiency of nodule endophytic bacteria on abiotic surface (PVC). Each value is plotted as the mean ± SE (n = 16). Mean sharing same letter in column do not differ significantly. LSD 0.05 = 0.0354 Fig. 3 a Amplified fragments of 16S rRNA gene. b Confirmation of 16S rRNA gene cloning into vector pTZ57R/T by restriction analysis

Ladder Control MSP9 MSP10 Ladder Control MSP9 MSP10

a promoting bacteria along with rhizobia (Mishra et al. 2009). Occurrence of bacteria other than rhizobia in root nodule was first reported by Beijerinck and Delden (1902)

GeneRuler 1kb

b

and identified as A. radiobacter in the clover plant. Agrobacterium spp. has also been reported to found in the nodules of chick pea (Hameed et al. 2004). In this study,

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we isolated ten fast growing bacteria from the nodules of pea. These fast growing nodule endophytic bacteria were further tested for nodulation assay and it is found they were able to reinfect roots, but were unable to nodulate pea plant. These results strengthen the literature on the occurrence of non-rhizobial bacteria in the root nodules (Taurian et al. 2012). Such a type of non-rhizobial bacteria within nodule establish a synergistic interaction with the rhizobia and promote plant growth (Bansal 2009). Generally, endophytic bacteria have better plant growth promotion abilities to a certain extent than bacteria restricted to the rhizoplane and rhizosphere (Dong et al. 2003). Therefore, non-rhizobial endophytic bacteria were isolated from nodules, which could have plant growth promoting potential. IAA production, nitrogen fixation, phosphate solubilization, biofilm formation and root colonization are some of the major characters to determine the growth promoting potential of plant beneficial bacteria (Bhattacharyya and Jha 2012; Phetcharat and Duangpaeng 2012). IAA produced by the plant associated bacteria improves root development in legumes and non-legumes. Additionally, in legumes IAA is also involved in the nodule development (Fedorova et al. 2005; Patten and Glick 2002). Similarly, nitrogen fixed by bacteria benefits plant by increasing their protein content (Fabre and Planchon 2000). Phosphate solubilizing ability of bacteria converts insoluble form of phosphorus into the useable form, which is an important trait in sustainable farming for increased plant yield (Ekin 2010). Moreover, biofilm formation is a key character of plant beneficial bacteria, which remained unnoticed in the past, enables the plant to withstand various biotic and abiotic stress conditions (Hirsch 2010a, b; Seneviratne et al. 2011). All the isolated bacteria showed potential of different plant growth promoting characters at varying level. Bacterial isolates MSP9 and MSP10 were found highly efficient in almost all the characters, especially in biofilm formation. These finding suggest the use of these bacterial strains as biofertilizers after field testing. Further, 16S rRNA gene sequence analysis of the potential strains revealed that pea nodules were colonized by the non-nodulating bacteria belong to genera Ochrobactrum and Enterobacter. These genera are well known for having endophytic bacteria, which colonize the root nodule of legume plants and improve nodulation when coinoculated with rhizobia (Ibanez et al. 2009; Tripathi et al. 2006; Xu et al. 1994). These results strengthen the literature on the endophytic nature of Ochrobactrum and Enterobacter spp. in pea plant. Generally, literature is fragmentary on the mode of infection of endophytic bacteria to plant. It is hitherto known that endophytic bacteria enter the plant roots in a two steps process. Initially, bacteria colonize at the points of emergence of lateral roots called lateral root cracks, and then bacteria move into

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intercellular space between the cortex and endoderm of roots (Gough et al. 1997). In case of legumes, it is speculated that non-nodulating endophytic bacteria might get entrapped into the pocket of root hair during the process of curling and finally enter the nodule primordial where it may multiply in number, but this area further need to be studied. Considering all the findings, this is the first report on the occurrence of Ochrobactrum and Enterobacter spp. in the nodules of pea plant, which showed enormous potential for plant growth promotion. Furthermore, it is suggested that these bacterial strains can be used as biofertilizers alone or in combination with other plant growth promoting bacteria for legumes and non-legume crops after field evaluation.

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