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ESTIMATION OF GENETIC DIVERSITY OF PLANT GROWTH PROMOTING RHIZOBACTERIA (PGPR) STRAINS FOUND IN DIFFERENT AREAS OF PAKISTAN USING RAPD AND 16S rRNA ANALYSIS.

Byline: T. Shaheen, A. A. Khan, Mahmood-ur-Rahman, M. T. U. Qamar and M. Rahman

ABSTRACT

The present study was an approach to provide the information about the diversity of rhizobacteria involved directly or indirectly in the plant growth and development. Among molecular markers, RAPD proved to be the most promising tool to delineate rhizobacterial strains. Seventeen bacterial strains of rhizobacteria were isolated from different parts of Pakistan (Sargodha, Mianwali, Karak and Faisalabad). RAPD analysis of these strains has shown a high degree of polymorphism. Range of genetic similarity was observed 0.543-0.927. Cluster analysis resulted into three clusters (I, II and III). Cluster I comprised of bacterial strains collected from Faisalabad, cluster II comprised of Strains collected from Sargodha while cluster III included strains from Mianwali. Strains isolated from the Karak region were not making any cluster. These results have shown that soil conditions and local environment has a significant impact on genetic make-up of bacterial strains.

Further 16S rRNA analysis of the strain was performed to validate the results. Results of 16S rRNA analysis have revealed that most dominating species were Bacillus sp. and two strains were also showing homology with Brevibacterium halotolerans. The findings of this study were very promising for the application of PGPRs for plant growth improvement.

Key words: Rhizobacteria, RAPD, Genetic diversity, Soil, 16S rRNA, Plant growth, Sequencing.

INTRODUCTION

Soil microorganisms subsidize plant growth by enhancing nutrient availability by recycling (Lynch et al. 1985), enhance root health by providing a defense against pathogens (Weller, 1988) or enhancing nutrient uptake (Kennedy and Smith, 1995). Among the microbes the rhizobacteria are the most important group which perform these jobs basically by increasing the soil productivity, and indirectly producing some chemicals which increase the soil fertility. The utility of rhizobacteria for enhancing productivity has been implemented for the better soil fertility (Aragno, 2005). Especially, the biocontrol plant growth-promoting bacteria (biocontrol-PGPB) and plant growth-promoting bacteria (PGPB) are auspicious for their possible use in maintaining ecological balance for agriculture (Davison, 1988; Park and Kloepper, 2000; Hallmann et al. 1997).

Plant growth promoting rhizobacteria (PGPR) are helpful for plant growth (Bashan et al. 1995), working as biocontrol (Bolwerk et al. 2003; Ashrafuzzaman et al. 2009), producing plant hormone-like chemicals and dropping of plant ethylene level (Glick, 1995) and initiation of resistance against pathogens (Van Loon et al. 1998). PGPR have adopted some defensive mechanisms against a variety of toxic metals (Park and Kloepper, 2000; Burd et al. 2000) and provide protection to plants against pathogens and pests (Park and Kloepper, 2000; Ashrafuzzaman et al. 2009).

The microbes inhabiting rhizosphere are influenced by certain characteristics of plant (Grayston et al. 1998; Marilley and Aragno, 1999; Germida and Siciliano, 2001; Marschner et al. 2004) and soil (Latour et al. 1996; Marschner et al. 2001), agronomic applications (Kennedy and Smith, 1995; Alvey et al. 2003) and also infections caused by mycorrhizae (Marschner et al. 2001). Ecological theories suggest that microbial communities with greater microbial diversity would be less susceptible to invasion by pathogens (Matos et al. 2005). Evaluation of environmental risk factor is necessary before utilization of indigenous or non- indigenous microbes for agricultural improvement (Aragno, 2005; Joseph et al. 2012). A comprehensive evaluation of genetic diversity and the environmental impact can be useful for the introduction of a new microorganism into the environment (Van Veen et al. 1997).

Various studies have been focused on evaluation of genetic diversity amongst soil bacteria (Yap et al., 1996: Head et al., 1998; Upadhyay et al., 2009; Hayat et al. 2013). Study of genetic diversity of soil bacteria has been found promising in enhancing soil fertility (Saharan and Nehra, 2011). Previous studies using 16S rRNA showed the association of PGPR strains found in Pakistan with genus Bacillus (Hayat et al. 2013).

As the prior sequence information is not pre- requisite in RAPD (Yasmin et al. 2008) it is very promising technique in microbes and has been used extensively for the evaluation of genetic diversity in microbes (Bereswill et al. 1995; Birch et al. 1997; Saharan and Nehra, 2011). The 16S rRNA genes being an associated component of DNA of all organisms and highly conserved can be utilized as biological markers to identify organisms at species, genus and family level. Phylogenetic analysis of soil bacteria can be done with 16S rRNA sequence analysis and their classification into new genera is done (Hayat et al. 2013). Pakistani soil houses millions of microbes including certain unique ones, only found in Pakistan. There are very few reports on genetic diversity of rhizobacteria in Pakistan. Therefore, conservation and characterization of such agriculturally important microbes are suggested for its maximum use by the next generations.

The aim of the present study was the estimation of genetic diversity and to study phylogenetic relationships among rhizobacteria strains habituating different areas in Pakistan.

MATERIAL AND METHODS

The experimental work was accomplished in the Department of Bioinformatics and Biotechnology (GCUF) and Plant Genomic and Molecular Breeding Lab (PGMB), Agricultural Biotechnology Division, National Institute for Biotechnology and Genetic Engineering (NIBGE) to study phylogenetic relationships among different rhizobacterial strains. Isolation of bacterial strain from soil samples collected from different areas was done in GCUF while RAPD and 16S rRNA analysis was conducted in NIBGE.

Collection and isolation of Rhizobacteria strains: The soil samples used in the study for the collection and isolation of rhizobacteria strains were obtained from different cotton crop growing fields of Faisalabad, Sargodha, Mianwali and Karak. Cotton plants up rooted along with adequate amount of non rhizosphere soil in polythene bags (2 kg size: 23 to 32 cm) were brought instantly to the laboratory and were air-dried. The samples were gently shaken to remove the non rhizosphere soil, leaving only the rhizosphere soil, which is strongly stuck to the roots. Under aseptic conditions for the collection of the rhizosphere soil from the roots, the samples were dipped and gently shaken in sterilized water. The glucose peptone agar medium (GPAM) was inoculated with soil suspension and by streaking three to four times in the fresh medium pure cultures were obtained (Mahler and Wollum, 1982). Bacterial culture was grown in LB broth. Prolific growth was observed in 17 bacterial isolates (Figure 1).

These isolates were selected and stored for further studies due to their unique morphological appearance on agar medium (Table 1).

RAPD analysis: A total of 21 decamer primers were selected on the basis of their performance in PCR reactions including OPE-01 through OPE-06, OPE-09, OPE-10, OPE-13 through OPE-16, OPC-17, OPK-08, OPX-08, OPN-02, OPN-05, OPN-06, OPW-07, OPW-02 and OPK-09. Extraction of Genomic DNA was done for each of bacterial strains by the method of Doyle (1987), spectrophotometrically quantified and checked on 0.8% agarose gels. Total 50 ml reaction volume was made for PCR containing 10 mm Tris-HCl (pH 8.3), 50 mm KCl, 3 mm MgCl2, 100 mm, each of dATP, dCTP, dGTP, dTTP, 30 mg of primer, 0.001% (w/v) gelatin, 20 ng of genomic DNA and 2 units of Taq polymerase. PCR reagents Taq polymerase, along with buffer, MgCl2 dNTPs, and gelatin was purchased from MBI Fermentas, USA. 1.2% gel (w/v) was used to visualize the amplification of products. Staining of the agarose gels was done for 30 min with ethidium bromide after electrophoresis.

The electrophoresis documentation and analysis system 120 (Kodak Digital Science, Japan) was used to take photographs (Figure 2).

Statistical analysis: Scoring of all visible and unambiguous fragments amplified by the primers was made by visual observation. Amplifications (band in each position) were scored as present or absent (Alvey et al. 2003). Euclidean distances were estimated with the help of this data. These distances were further utilized to make a dendrogram using the unweighted pair group method of arithmetic means (UPGMA).

16S rRNA analysis: For amplification of the 16S rRNA genes by PCR, forward primer f (5'- AACACATGCAAGTCGAAC-3') (Lane, 1991) and reverse primer 1387r (5'-ACGGGCGGTGTGTACAAG-3') (Marchesi et al. 1998) were used which are designed to amplify the conserved region of bacterial 16S rRNA genes and produce an approximately 1300bp fragment. The total volume of 50 L of PCR mixture comprised of 25 L Taq polymerase buffer, 0.5 L Taq polymerase, 5 L DNA template, 8 L dNTPs, 1 L each primer (10 pmol) and 9.5 L d3H2O. The first step was the initial denaturation at 95 C for 2 min, it was followed by 35 cycles of denaturation at 94 C for 30 Sec, annealing at 55 C for 30 Sec, and elongation at 72 C for 1 min, last step was the final extension for 5 min at 72 C. The presence of specific PCR products was checked on 1% agarose (w/v) gel electrophoresed for 45 min at 70V in TAE 1X buffer. Amplicons were further purified by Gel/PCR DNA fragments extraction kit (Fermentas, USA).

Sequence Analysis of 16S rRNA Gene: DNA fragments extraction kit (Fermentas, USA) was used for the purification of the PCR products of 16S rDNA. Five of the strains were selected for further sequencing and Bioinformatics analysis. The characterization of each sequence compared to the GenBank database was done by The BLASTN program (www.ncbi.nlm.nih.gov). The sequences were aligned with the ClustalW program (www.ebi.ac.uk). ClustalX2 (Larkin et al. 2007) was used to construct the Neighbor-joining tree and bootstrap analysis of samples.

RESULTS AND DISCUSSION

Plant growth promoting Rhizobacteria (PGPRs) colonize plant roots and employ advantageous effects on the development and growth of plants by a variety of processes. The actual procedure behind this is still unknown: however, several theories like phytohormones production, fight against deleterious organisms, initiation of phosphate solubilization (Verma et al. 2007), and enhanced mineral nutrient uptake is most likely involved (Glick, 1995). PGPR have been proven to contribute to the enhancement of the growth, emergence of seeds and augmented crop yield, and also protect plants against certain pathogens and pests (Kokalis-Burelle et al. 2006; Ashrafuzzaman et al. 2009). Randomly amplified polymorphic DNA (RAPD) profiles have proven to be a promising tool for investigation of genetic diversity (Yasmin et al. 2008).

This method was used by different researchers (Gajbhiye et al. 2007) for genetic analysis of Bacillus isolates, (Paffetti et al. 1996) for the study of genetic diversity of an Italian Rhizobium meliloto population (Bevivino et al. 1998) and for characterization of Burkholderia cepacia population. 16S rRNA sequence analysis has also been used to classify and reclassify soil bacteria phylogenetically into new genera (Hayat et al. 2013).

In this study, the genetic diversity of the plants growth promoting rhizobacteria was determined in order to select the better strains. We screened seventeen samples with the RAPDs primers and 16S rRNA, these samples were collected from cotton growing fields of different regions of Pakistan (Faisalabad, Sargodha, Mianwali, Karak). We selected cotton crop because cotton has a significant economic importance (Shaheen et al., 2014). Faisalabad and Sargodha are cotton growing area while in Mianwali and Karak region cotton is grown occasionally. We selected these diverse areas to study genetic diversity of PGPRs. The analysis of genetic diversity of the rhizobacteria isolates was done to validate the diversity amongst the rhizospheric soil. In the present study a total of 21 RAPD primers were utilized to find out the genetic diversity among 17 rhizobacteria strains. Total number of amplicons produced by these 21 decamer primers is 557.

OPE-03 produced a minimum number of amplicons while OPW-02 produced the maximum number of amplicons. All the bacterial strains elucidated a high degree of polymorphism with decamer primers. RAPD profile based genetic similarity matrix for the seventeen bacterial strains was attained using Nei and Lei's coefficient of similarity (Table 2). The genetic similarity of seventeen bacterial strains was high, ranging from 0.543-0.927%. The genotypes BACT -13 and BACT -17 had the greatest genetic similarity (0.927). The second highest genetic similarity was estimated between BACT -8 and BACT - 10 (0.905).

A dendrogram of 17 genotypes of PGPR bacteria was constructed based on the RAPD primer scoring profile (Figure 3) using Unweighted Pair Group Method of Arithmetic means (UPGMA). RAPD analysis has shown a high degree of genetic similarities among seventeen cultivars. UPGMA analysis resulted into dendrogram with three major clusters I, II and III. The grouping of genotypes into three distinct clusters actually indicates the difference in their lineage and the grouping. The genotypes included in the cluster I are BACT-1, BACT-4, BACT-8, BACT-10, BACT-14. These all strains were collected from Faisalabad region. Whereas, genotypes included in cluster 'II' are BACT-2, BACT-9, BACT-13, BACT-17, BACT-12, BACT-16, BACT-11, and BACT-15. These all strains were collected from Sargodha region. The genotypes BACT-3, BACT-7 were collected from Mianwali region and they form a different cluster III. The genotype BACT-5 and BACT-6 collected from Karak are not making any cluster.

The genetic divergence among these strains was high. The cluster analysis and similarity matrix also indicate that bacterial strains collected from the same region are showing a high degree of similarity with each other. Other studies were also showing the similar results, bacterial strains collected from the same field were showing close resemblance then other field samples (Roesti et al. 2006).

The biodiversity studies have been conducted in microbial communities to assess the effects of environmental agitations mostly at the genus and species level. Truly, the study of factors which influence the biodiversity at the intraspecific level is vital for assessing the effect of exogenous microorganisms on closely related local bacteria. Furthermore, plant and cultivar type can also affect the genetic makeup of rhizobacteria (Paffetti et al. 1996). The strains which are in cluster I are showing similarity and showing genetic divergence from cluster II and III.

Table 1. Rhizobacterial strains with their native regions.

Region###Name of Bacterial Strains

Faisalabad###BACT-1, BACT-4, BACT-8, BACT-10 and

###BACT-14

Sargodha###BACT-2, BACT-9, BACT-13, BACT-17,

###BACT-12, BACT-16, BACT-11, BACT-15

Mianwali###BACT-3, BACT-7

Karak###BACT-5, BACT-6

Table 2. Similarity Matrix for Nei and Lei's Coefficient of 17 Plants Growth promoting rhizobacteria strain. Numbers Both in First Column and Row Represent the plants growth promoting rhizobacteria strain of the table.

###1###2###3###4###5###6###7###8###9###10###11###12###13###14###15###16###17

1###1

2###0.754###1

3###0.746###0.750###1

4###0.832###0.707###0.698###1

5###0.651###0.716###0.672###0.644###1

6###0.616###0.644###0.612###0.621###0.543###1

7###0.720###0.793###0.871###0.690###0.672###0.621###1

8###0.806###0.767###0.698###0.836###0.681###0.638###0.733###1

9###0.767###0.841###0.772###0.737###0.694###0.711###0.806###0.780###1

10###0.815###0.759###0.698###0.853###0.655###0.655###0.733###0.905###0.806###1

11###0.754###0.784###0.776###0.698###0.690###0.629###0.776###0.724###0.806###0.724###1

12###0.720###0.802###0.750###0.690###0.681###0.621###0.793###0.741###0.823###0.741###0.767###1

13###0.793###0.832###0.806###0.780###0.731###0.659###0.841###0.797###0.862###0.815###0.841###0.832###1

14###0.823###0.810###0.741###0.819###0.698###0.655###0.776###0.853###0.858###0.905###0.767###0.793###0.858###1

15###0.759###0.806###0.772###0.711###0.677###0.608###0.789###0.728###0.819###0.780###0.901###0.772###0.871###0.789###1

16###0.746###0.802###0.776###0.707###0.681###0.621###0.784###0.733###0.823###0.759###0.793###0.845###0.823###0.819###0.797###1

17###0.815###0.905###0.836###0.784###0.759###0.698###0.871###0.810###0.918###0.828###0.862###0.888###0.927###0.897###0.875###0.879###1

16S rRNA gene analysis for each isolate resulted in amplification of a single band of approximately 1300 base pairs (Figure 4). Five strains from each cluster resulted from the RAPD analysis were used for sequencing and bioinformatics analysis to classify the rhizobacteria. The Bioinformatics analysis of the sequences using the BLASTN program (Table 3) has shown a high degree of similarity, the highest value for each isolate was more than 85% by E-value 0. The similarities of the isolates were found with genetically diverse species, mostly of Bacillus sp., like Bacillus mojavensis, Bacillus safensis, Bacillus pumilus, Lactobacillus murinus, Bacillus amyloliquefaciens, Bacillus subtilis, Bacillus vallismortis, Brevibacterium halotolerans, Bacillus tequilensis, Lysinibacillus fusiformis, Bacillus subtilis, Bacillus axarguiensis. The evolutionary relationships were elucidated by constructing the phylogenetic tree using 16S rRNA gene partial sequences (Figure 5).

The phylogenetic tree has shown that the 5 isolates were showing a high degree of relatedness with each other. Three of the isolated strains were found to belong to bacillus sp. While two of the strains were showing highest similarity with Brevibacterium halotoleras which was in accordance with the previous studies (Hayat et al. 2013).

Table 3. Selected strains of rhizobacteria for 16S rRNA and their highest similarities.

Bacterial isolate(Region)###Sequence length###Highest Blast Score###Bacterial strain

01 (Faisalabad)###1310bp###99%###Bacillus mojavensis

16 (Sargodha)###1322bp###92%###Bacillus safensis, Bacillus pumilus,

###Lactobacillus murinus

06 (Karak)###1324bp###98%###Bacillus amyloliquefaciens, Bacillus

###subtilis, Bacillus vallismortis

05 (Karak)###1315bp###98%###Brevibacterium halotolerans, Bacillus

###tequilensis, Lysinibacillus fusiformis,

###Bacillus subtilis, Bacillus axarguiensis

07 (Mianwali)###1315bp###98%###Brevibacterium halotolerans, Bacillus

###mojavensis, Bacillus tequilensis,

###Lysinibacillus fusiformis, Bacillus subtilis

Conclusions: A high degree of similarity was observed in the isolates; hence it was indispensable to study the strains at the DNA level for providing a perfect fingerprint for its identification. RAPD being a simple technique was used to delineate the bacterial strains and 16S rRNA was utilized further to authenticate the results.

Most of the strains were found to belong to Bacillus sp. which is found to be dominating PGPR species in Pakistan. This study will prove to be a crucial part of studies being conducted to improve soil health in Pakistan.

Acknowledgments: The authors would like to express sincere gratefulness to Higher Education Commission (HEC) for the provision of funding, National Institute of Biotechnology and Genetic Engineering (NIBGE) and Government College University Faisalabad (GCUF), Pakistan, for providing a research facility throughout the study.

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