Global Advanced Research Journal of Agricultural Science (ISSN: 2315-5094) Vol. 6(9) pp. 285-292, September, 2017 Issue. Available online http://garj.org/garjas/home Copyright © 2017 Global Advanced Research Journals

Full Length Research Paper

Molecular Characterization Using 16S rRNA Gene of Isolated Bacterial Genera Associated With Mango Cultivation in Jazan province, South West KSA

Asmaa A. Alharbi

Department of Biology, Faculty of Science, Jazan University, Kingdom of Email: [email protected]

Accepted 24 September, 2017

Three hundred and fifty mango soil and root samples were collected from Jazan province south west KSA in order to examine the presence of different bacterial genera distributed in this area. A total of 13 bacterial isolates were identified by 16S rRNA sequencing and using BLAST. The neighbor-joining method was used to produce a Molecular Phylogenetic analysis and a phylogenetic tree was constructed. As a result of this study the following bacteria Bacillus sp. (1), B. firmus (1), B. licheniformis (1), B. subterraneus (2), B. subtilis (1), B. vireti (1), Fictibacillus barbaricus (1), Lysobacter enzymogenes (1), Paenibacillus sp. (1), Paracoccus sp. (1), Streptomyces sp. (1), S. griseorubens (1) were found. Isolated bacteria are significantly important because they regulate soil ecosystem. To the best of our knowledge this is the first report of identified bacterial isolates associated with mango soil samples according to 16S rRNA gene in Jazan province, KSA.

Keywords: Bacteria, 16S rRNA, Survey, Mang, soil, Abu Arish, Sabia,

INTRODUCTION

Fruits are widely cultivated in large quantities in different Soil is the most precious part of nature due to contain parts of the world (Veeraragavathatham et al ., 1996). minerals, organic components and microorganisms. The Mango ( Mangifera indica L.) is one of the world's most physical, chemical and biological properties of the soil important tropical fruits, and is widely regarded as the most profoundly affect life on earth (Dominati et al., 2010). Soil is popular fruit in tropical and subtropical regions (Litz, 2009). an important source of nutrients and food for all living Mango is regarded as king of fruits; it's originated from things and it has large variety of different microorganisms. and Southeast Asia; it is one of the most important The role of these microorganisms in soil is to stabilize the fruits cultivated in tropical countries. Mango cultivation has soil structure for the ecosystem services. Soil now extended to several other parts of the world including microorganisms as bacteria contribute to the cycling of all Africa, the Americas and the Caribbean region (Diedhiou et major elements (e.g. C, N, P) in the recycling of wastes, al ., 2007). In Saudi Arabia mango was introduced only 30 and the detoxification of environmental pollutants (Fierer et years ago, but is popular among Saudis. Some mango al., 2007; Dominati et al., 2010). Bacteria in soil ensure a trees in the Jazan region are reportedly over 100 years old. great contribution to organic matter production and soil (Dawah et al., 2013). formation. In addition some bacteria in soil promote plant growth and products synthesized by these bacteria 286. Glo. Adv. Res. J. Agric. Sci.

strengthen the structure of the soil (Aislabie and Deslippe, glycerol nutrient agar slants for later use (Bailey and Scott, 2013). 1966). Although up to now many bacteria have been cultured from soil researches but this subject still continues. Reason Identification of microorganisms for this is these bacteria are used in many industrial fields, especially in agriculture, plastic industry, chemical industry Morphological and cultural identifications (color, form, as enzyme, antibiotic, polysaccharide productions and elevation, margin, size, texture, luster and colony opacity) bacterial plastics (Hayat et al., 2010; Abdulkadir and were made according to the Bergey’s Manual of Waliyu, 2012; Mauti et al., 2013 ; Alves et al., 2014; Systematic Bacteriology (Krieg and Holt,1984). Biradar et al., 2015; Nehra et al., 2015). Nowadays it has The PCR programme consisted of an initial denaturation left this place to molecular methods while using the at 94°C for 3 min followed by 35 cycles, each with 30s at classical for bacterial isolation from soil (Mauti et 94°C for denaturation, 1 min at 50°C for annealing and 1 al., 2013). The diversity of bacteria in the soil is better min at 72°C for elongation. Reaction mixtures were then understood by the use of molecular methods (Janssen, incubated at 72°C for 10 min for final extension. PCR 2006). products were separated on a 1.5% agarose gel in TBE The sequence of the 16S rRNA gene has been widely buffer (Maniatis et al ., 1982), stained with ethidium bromide used as a molecular clock to estimate relationships among and photographed under UV light. bacteria (phylogeny), but more recently, it has also become Moreover, full length of 16S rRNA gene (1550 bp) was important as a mean to identify an unknown bacterium at amplified for all bacterial isolates using two universal the genus or species level (Sacchi et al ., 2002). The primers P0 (5`-GAAGAGTTTGATCCTGGCTCAG-3`) and variable regions of the 16S rRNA gene provide unique P6 (5`-CTACGGCTACCTTGTTACGA-3`). The PCR signature that can be analyzed to provide an identification amplification was carried out in a total volume 25 µL of bacterial species. containing 2 µL of template DNA, 10 mM Tris-HCl (pH 8.8), The aim of this study was to isolate and identify bacterial 50 mM KCl, 1.5 mM MgCl2, 0.2 mM of each dNTP, 10 isolates associated with mango fields cultivated in Abu pmol of each primer and 0.5 unit Dynazyme TM II DNA Arish and Sabia governorates, Jazan province, south west Polymerase. PCR amplification was performed as one of Saudi Arabia, using 16S rRNA sequencing methods, cycle at 95°C for 5 min followed by 34 cycles each with 45 different. s at 95°C for denaturation, 1 min at 50°C for annealing and 2 min at 72°C for elongation. Reactions were then incubated at 72°C for 10 min for final extension. The PCR MATERIALS AND METHODS products were analyzed as above.

Collection of soil samples Phylogenetic analysis of isolates

In our study three hundred and fifty soil samples were Phylogenetic tree was constructed by using the neighbor- taken during mango growing season 2016 from different joining method which generated using MEGA version 5.1 mango fields located in Abu Arish and Sabia governorates, (Tamura and Nei, 1993; Kumar et al., 2016) from Jazan province, south west KSA (211 from Abu Arish and CLUSTAL W alignments. Comparisons with sequences in 139 from Sabia). Samples were collected from different the GenBank database were achieved in BLASTN seven mango cultivars (Hindi, Jolie, Palmer, Sechin, searches at the National Center for Biotechnology Sudany, Tommy and Zell) which cultivated in surveyed Information site (http://www.ncbi.nlm.nih.gov). The 16S mango fields to determine the occurrence of different rDNA sequences have been deposited in the GenBank bacterial genera associated with soil samples. The ground database under the accession numbers which shown in was dug as deep as 10-15 cm for the collection of soil Table (3). samples. These soil samples were kept in sterile polythene bags and brought to the laboratory for bacterial isolation and identification. RESULTS

Bacterial isolation: Data in Table (1) showed that survey in different mango fields which cultivated with seven mango cultivars; Hindi, Isolation trials were carried out from collected mango soil Jolie, Palmer, Sechin, Sudany, Tommy and Zell resulted in samples. A loop full of the resulting suspension was 62 bacterial isolates. streaked on 5% Plain Agar (PA) plates. Single colonies Identification method showed the presence of 13 observed after 24-48 h incubation at 27-37°C were isolated bacterial isolates in all soil samples of the surveyed mango and consequently purified. All isolates were kept on 2% fields. Bacillus subtilis , B. vireti , Lysobacter enzymogenes

Alharbi 287

Table 1. Seven mango cultivars and number of bacterial isolates from each cultivar

No. Mango cultivar Symbol No. of isolates/cultivar 1 Hindi HL 6 2 Jolie JeL 13 3 Palmer PL 11 4 Sechin ChL 17 5 Sudany SL 2 6 Tommy TL 9 7 Zell ZL 4 Total No. of isolates = (62)

Table 2. Number of positive samples and frequency of occurrence % (FO) of isolated bacteria from mango cultivation in Abu-Arish and Sabia governorates

Go vernorate Bacterial genera a Sabia Abu Arish (211 ) (139) Bacillus sp. - 21 b , 15.1 c B. firmus 10 , 4.7 - B. licheniformis 7 , 3.3 - B. subterraneus - 10 , 7.2 B. subterraneus 18 , 8.5 - B. subtilis - 46 , 33.1 B. vireti 49 , 23.2 - Fictibacillus barbaricus - 28 , 20.1 Lysobacter enzymogenes 52 , 24.6 - Paenibacillus sp. 18 , 8.5 - Paracoccus sp. 21 , 10.0 - Streptomyces sp. 36 , 17.2 - S. griseorubens - 34 , 24.5 a= number of total collected mango soil and root samples. b = number of positive samples containing bacterial isolate. c = FO % = (number of positive samples/total number of the collected samples) × 100.

and Streptomyces griseorubens were the most prevalent BLASTN software. Species we found are as following bacteria in all collected soil samples with frequency of Bacillus sp. (1), B. firmus (1), B. licheniformis (1), B. occurrence (FO) 23.2-33.1% followed by Bacillus sp., subterraneus (2), B. subtilis (1), B. vireti (1), Fictibacillus Streptomyces sp. and Fictibacillus barbaricus with 15.1- barbaricus (1), Lysobacter enzymogenes (1), Paenibacillus 20.1% FO. B. subterraneus , Paenibacillus sp. and sp. (1), Paracoccus sp. (1), Streptomyces sp. (1), S. Paracoccus sp. were found in mango soil and root samples griseorubens (1). The analysis of the soil samples of Jazan with FO 7.2-10.0 %. The genera B. firmus and B. provinces showed that there were thirteen bacterial licheniformis were less common with FO 3.3-4.7% Table isolates. All the sequences were submitted to Genbank (2). with accession numbers as illustrated in Table 4. Morphological characters of these bacterial isolates were Alignment of the 16S rRNA sequences of 13 bacterial shown in Table 3. Also, Images of each bacterial genera isolates with the 16S rRNA sequences was carried out which isolated from Abu Arish and Sabia governors were utilizing CLUSTAL W (1.82) at which MEGA version 5.1 placed in figures. 1 & 2. was used to generate the Bootstrap neighbor-joining tree. A total of 350 soil samples from mango cultivation were The cluster analysis of all bacterial isolates identified processed in this study. About 62 isolated bacterial formed two major clusters. In 1st cluster, nine of the 13 colonies were obtained. PCR characterization of 13 isolates were Bacillus sp., B. firmus, B. licheniformis, B. colonies were performed. These colonies resulted in good subtilis, two isolates of B. subterraneus, B. vireti , amplified products with approximately 1,6 kb in length on Fictibacillus barbaricus and Paenibacillus . In the 2 nd cluster agarose gel (Figure. 3). ther were four bacterial isolates include, Lysobacter PCR results of these samples were send to GATC enzymogenes, Paracoccus sp., Streptomyces sp . and S. BioTech, Germany for sequencing. Molecular identification griseorubens (Figure. 4). made by comparing sequence results with Genebank using 288. Glo. Adv. Res. J. Agric. Sci.

Table 3: Morphological characteristics of isolated bacteria from mango cultivation in Abu Arish and Sabia governorates

Colonies morphology No Isolate Ele vatio Textur Colony Blast identification . code Colour Form Margin Size Luster n e Opacity JeL2 Irregula Unbonat Wrinkle 1 Off-white Curled Large Dull Opaque Bacillus sp. 8B r e d Irregula Umbonat Undulat Wrinkle 2 HL1A White Medium Dull Opaque B. licheniformis r r e e d Wrinkle 3 SL1 Off-white Circular Flat Entire Small Dull Opaque B. subterraneus d d Unbonat 4 BL2B Off-white Circular Curled Small Rough Dull Opaque B. subterraneus e e CHL2 Irregula Unbonat Wrinkle 5 Beige Curled Medium Dull Opaque B. subtilis 2B r e d Umbonat Wrinkle 6 TL1 Off-white Circular Curled Medium Shiny Opaque B. vireti e e d Wrinkle 7 HL1B Off-white Rhizoid Raised Lobate Large Dull Opaque B. firmus d d Irregula Fictibacillus 8 ZL2 3B Off-white Flat Curled Medium Smooth Shiny Opaque r r barbaricus CH3B1 Lysobacter 9 Orange Circular Convex Entire Small Mucoid Shiny Opaque 0 enzymogenes Very 10 TL3B Off-white Circular Convex Entire Mucoid Shiny Opaque Paenibacillus sp. Small Very 11 JeL3 1 Orange Circular Convex Entire Mucoid Shiny Opaque Paracoccus sp. Small White with gray Unbonat 12 PL3 11 Circular Curled Small Dry Dull Opaque Streptomyces sp. center e JeL2 Transpare 13 Orange Circular Convex Entire Small Smooth Shiny S. griseorubens 7B nt

Bacillus firmus (HL1 B) B. licheniformis (HL1 A) B. vireti (TL1)

B. subterraneus (SL1) Lysobacter enzymogenes (CH3 B10) Paenibacillus sp. (TL3 B)

Streptomyces sp.(PL3 11) Paracoccus sp. (JeL3 1)

Figure 1: Morphological shapes of bacterial species isolated from Abu Arish governorate.

Alharbi 289

Bacillus sp. (JeL2 8B) B. subterraneus (BL2 B) B. subtilis (CHL2 2B)

Streptomyces griseorubens (JeL2 7B) Fictibacillus barbaricus (ZL2 3B)

Figure 2: Morphological shapes of bacterial species isolated from Sabia governorate.

Figure 3: Agarose gel (1.5%) electrophoresis illustrating PCR products utilizing specific primers for thirteen bacterial isolates from mango cultivation in Abu Arish and Sabia governorates. M: A-100 bp DNA marker ladder. The position of 1.6 kb is indicated.

290. Glo. Adv. Res. J. Agric. Sci.

Table 4. Isolate code, genbank accession numbers and blast identification of isolated bacteria from mango cultivation in Abu Arish and Sabia governorates

No. Isolate code Genbank accession Blast identification Governorate 1 TL1 LT601532 Bacillus vireti 2 Ch3B10 LT601528 Lysobacter enzymogenes 3 JeL3 1 LT601529 Paracoccus sp. 4 PL3 11 LT601531 Streptomyces sp. Abu Arish 5 TL3 B LT601533 Paenibacillus sp. 6 HL1 B LT601535 B. firmus 7 SL1 LT601536 B. subterraneus 8 HL1A LT601545 B. licheniformis 9 JeL2 8B LT601541 Bacillus sp. 10 BL2B LT601538 B. subterraneus 11 ZL2 3B LT601539 Fictibacillus barbaricus Sabia 12 JeL2 7B LT601540 Streptomyces griseorubens 13 ChL2 2B LT601543 B. subtilis

Figure 4: Phylogenetic tree constructed upon bootstrap neighbor-joining tree method based on 16S rRNA gene partial sequence of thirteen bacterial isolates from mango cultivation in Abu Arish and Sabia governorates. The scale at the bottom indicates linkage distance.

DISCUSSION 2006). Most common soil bacteria were isolated from mango soil samples. Six bacterial genus were observed, The distribution of bacteria in root rhizosphere of plants are Bacillus, Fictibacillus, Lysobacter, Streptomyces, influenced by many factors, including the host plant, the Paracoccus and Paenibacillus . Only Bacillus, Lysobacter stage of plant growth and cropping practices (Bosco et al. , and Streptomyces species occurred in most of soil Alharbi 291

samples having the highest percentage occurrence. Some CONCLUSION of the bacteria isolated were also reported by Wemedo and Onolleka (2012) from mango soil rhizosphere. This research showed that morphological methods are not In the present work, all the bacterial isolates from mango always adequate and confidential for identification of soil samples PCR product was approximately 1,6 kb in species. Thus, both morphological and molecular methods length as shown in Figure. 1. Data is in agreement with for identification of bacteria were used. It can be seen that, other reports (Khan et al ., 2014; Asif et al ., 2016). in recent years, molecular identification gained more Bacillus was an endophytic bacteria most commonly importance. found in mango soil and root samples. Bacillus plays role as a biocontrol agent in plant and stimulates plant growth. Bacillus serve as antipathogen because of the ability to REFERENCE inhibit the growth of specific pathogens. Bacillus was also Abdulkadir M, Waliyu S (2012). Screening and isolation of the soil able to induce the growth of plant by producing auxin and bacteria for ability to produce antibiotics. European J. Appl. Sci., 4 gibberellin, and able to adapt to drought (Forchetti et al. , (5):211-215. 2007). Pereira et al. (2012) reported that some Bacillus Aislabie J, Deslippe JR (2013). Soil microbes and their contribution to soil species were able to produce IAA, siderophore, and services. In Dymond JR ed. Ecosystem services in New Zealand – conditions and trends. Manaaki Whenua Press, Lincoln, New Zealand. proven to improve plant growth. Besides being able to Al-Askar AA, Abdulkhair MA, Rashad YM, Hafez EE, Ghoneem KM, Baka induce the growth of plants, IAA was also known to inhibit ZA (2014). Streptomyces griseorubens E44G: A potent antagonist the growth of pathogens. isolated from soil in Saudi Arabia. J. Pure & Applied Microbiology, 8 The analysis of mango soil samples using 16S rRNA (2):221-230. Alves PDD, Siqueira FF, Facchin S, Horta CCR, Victória JMN, sequencing showed the presence of 13 bacterial isolates. Kalapothakis E (2014). Survey of microbial enzymes in soil, water, and Many researchers have isolated and used molecular plant microenvironments. The Open Microbiol. J., 8:25-31. methods such as 16s rRNA-PCR, ERIC-PCR, REP-PCR in Asif H, Studholme DJ, Khan A, Aurongzeb M, Khan IA, Azim MK (2016). identifying bacteria in different soil samples. Hayat et al. Comparative genomics of an endophytic Pseudomonas putida isolated from mango orchard. Genetics and Molecular Biology, 39, 3, 465-473. (2010) isolated gram positive Bacillus strains from legumes Bailey WR, Scott EG (1966). Diagnostic Microbiology, Second Edition. rhizosphereic soil and identified using 16S rRNA gene Toppan Company Ltd., Japan, 342 pp. sequencing for plant growth promoting activities in legume. Bello OS, Utang GA (2011). An Ecological Survey of Microorganisms Also, Kasa et al. (2015) researched isolation, screening, associated with Plantain Roots Rhizoshere. American Journal of Agricultural and Biological Sciences 6 (4): 567–570. and molecular characterization of plant growth promoting Biradar GG, Shivasharana CT, Kaliwal BB (2015). Isolation and rhizobacteria isolates. Someya et al. (2013) and Kumar et characterization of polyhydroxybutyrate (PHB) producing Bacillus al. (2015) isolated and identified Paenibacillus sp. using species from agricultural soil. Europ. J. Exp. Biol., 5(3):58- 65. 16S rDNA gene sequences of isolated strain from forest Bosco M, Baruffa E, Picard C (2006). Organic breeding should select for plant genotypes able to efficiently exploit indigenous probiotic soil. rhizobacteria. Proceeding of European Joint Organization Congress, Lysobacter is known as a common soil bacterium Vienna, Austria, pp 376-377. (Christensen and Cook 1978). The strong lipase and Christensen P, Cook FD (1978). Lysobacter , a new genus of non fruiting, protease activity may be involved in the lytic activity that gliding bacteria with a high base ratio [Soil and water organisms]. Int. J. Syst. Bacteriol., 28: 367–393. most of the Lysobacter spp. exhibit against a variety of Dawah HA, Alkahtani SA, Hobani AH, Sahloli SN (2013). The First Gram-positive, Gram-negative and blue-green bacteria, Occurrence of aedificator (Fabricius) (Coleoptera: fungi, yeasts, algae and nematodes (Christensen and Cerambycidae) a pest of cultivated mango In South-Western Saudi Cook 1978). Protease activity of Lysobacter sp. may be Arabia, Journal of Jazan University- Applied Sciences Branch, 2 (2). Diedhiou PM, Mbaye N, Drame A, Samb PI (2007). Alteration of important for biocontrol of different root rot fungi (Dunne et postharvest diseases of mango Mangiferaindica, through production al. 2000). Also, several antibiotics have been found in practices and climatic factors. Afr. J. Biotechnol., 6(9):1087-1094. Lysobacter sp., such as lysobactin, a dibasic peptide Dominati E, Patterson M, MacKay A (2010). A framework for classifying (Egner and Bradley 1997; O’Sullivan et al. 1988). and quantifying natural capital and ecosystem services of soils. Ecol. Economics, 69:1858-1868. Streptomyces spp. have the ability to produce enzymes Dunne, C., Moenne Loccoz, Y., De Bruijn F. J. and O’Gara, F. 2000. included cellulases, laccase, lignin peroxidase and man- Overproduction of an inducible extracellular serine protease improves ganese peroxidase (Wang and Wang, 2003). biological control of Pythium ultimum by Stenotrophomonas maltophilia Streptomyces found in soil samples collected from different strain W81. Microbiology (Read) 146: 2069–2078. Egner B, Bradley M (1997). Monitoring the solid phase synthesis of places (Feng et al ., 2013; Prasad et al ., 2013; Al-Askar et analogues of Lysobactin and the Katanosins using in situ MALDI-TOF al ., 2014). MS. Tetrahedron 14021–14030. Al-Askar et al . (2014) reported the presence of S. Feng HW, Zhi YE, Shi WW, Mao L, Zhou P (2013). Isolation, griseorubens in soil samples collected from Jazan area identification and characterization of a straw degrading Streptomyces griseorubens JSD-1. African J. Microbiology Research, 7(22): 2730- and another places in K. S. A. 2735.

292. Glo. Adv. Res. J. Agric. Sci.

Fierer N, Bradford MA, Jackson RB (2007). Towards an ecological Pereira GVM, Karina TM, Emi RL, Thiago PS, Rosane FC (2012). A classification of soil bacteria. Ecology, 88:1354–1364. multiphasic approach for the identification of endophytic bacterial in Forchetti G, Masciarelli O, Alemano S, Alvarez D, Abdala G (2007). strawberry fruit and their potential for plant growth promotion. Microb Endophytic bacteria in sunflower ( Helianthus annuus L.): isolation, Ecol., 63:405-417. characterization, and production of jasmonates and abscisic acid in Prasad P, Singh T, Bedi S (2013). Characterization of the cellulolytic culture medium. Appl Microbiol Biotechnol., 76:1145-1152. enzyme produced by Streptomyces griseorubens (Accession No. Green MR, Sambrook J (2012). Molecular Cloning: A Laboratory Manual, AB184139) isolated from Indian soil. J. King Saud University-Science, 4th edn., Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. 25, 245–250. Hayat R, Ali S, Amara U, Khalid R, Ahmed I (2010). Soil beneficial Sacchi CT, Whitney AM, Mayer LW, Morey R, Steigerwalt A (2002). bacteria and their role in plant growth promotion: A Review. Annals Sequencing of 16S rRNA gene: A rapid tool for identification of Bacillus Microbiol., 60(4): 579–598. anthracis . Emerging Infect. Dis., 8: 1117-1123. Janssen PH (2006). Identifying the dominant soil bacterial taxa in libraries Someya N, Kobayashi YO, Tsuda S, Ikeda S (2013). Molecular of 16S rRNA and 16S rRNA genes. Appl. Environ. Microbiol., 72:1719– characterization of the bacterial community in a potato phytospher. 1728. Microbes Environ., 28(3):295–305. Kasa P, Modugapalem H, Battini K (2015). Isolation, screening, and Tamura K, Nei M (1993). Estimation of the number of nucleotide molecular characterization of plant growth promoting rhizobacteria substitutions in the control region of mitochondrial DNA in humans and isolates of Azotobacter and Trichoderma and their beneficial activities. chimpanzees. Mol. Biol. Evol.,10:512-526. J. Nat. Sci. Biol. Med., 6(2):360–363. Veeraragavathatham D, Jawaharlal M, Jeeva S, Rabindran R (1996). Khan IA, Asif H, Jiskani MM, Muhlbach HP, Azim MK (2014). Isolation and Scientific Fruit culture, Suri Associates, Coimbatore. pp. 227. 16S rDNA sequence analysis of bacteria from dieback affected mango Wang, H. L. and Wang, Z. Y. 2003. Three important enzymes for lignin orchards in southern Pakistan. Pak. J. Bot., 46(4): 1431-1435. degradation. J. Biol., 20 (5): 9–11. Krieg NR, Holt JG (1984). Bergey’s Manual of Systematic Bacteriology. Wemedo SA, Onolleka B (2012). Evaluation of rhizospheree bacteria of Williams and Wilkins, Baltimore, MD. Mangifera Indica (mango) and Terminalia Catappa (almond). J. Kumar S, Stecher G, Tamura K (2016). MEGA7: Molecular evolutionary Emerging Trends in Engineering and Applied Sciences, 3(5): 791-794. genetics analysis version 7.0 for bigger data sets. Mol. Biol. Evol., 33(7):1870-1874. Kumar SN, Jacob J, Reshma UR, Rajesh RO, Kumar BSD (2015). Molecular characterization of forest soil based Paenibacillus elgii and optimization of various culture conditions for its improved antimicrobial activity. Frontiers Microbiol., 6:1-11. Litz RE (2009). The mango botany, production and uses (2 nd edition) CBI International Wallingford. Maniatis T, Fritsch EF, Sambrook J (1982). Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, New York. Mauti EM., Mauti GO, Ouno GA, Mabeya BM, Kiprono S (2013). Molecular identification of soil bacteria by 16srDNA sequence. J. Natural Sci. Res., 3(14):51-58. Nehra K, Jaglan A, Shaheen A, Yadav J, Lathwal P (2015). Production of Poly-β-Hydroxybutyrate (PHB) by bacteria isolated from rhizosphereic soils. Int. J. Microbial Resource Techn., 2(3):38-48. O’Sullivan J, McCullough J, Tymiak A, Kirsch D, Trejo W, Principe P (1988). Lysobactin, a novel antibacterial agent produced by Lysobacter sp. I. Taxonomy, isolation and partial characterization. J. Antibiot. 41: 1740–1744. Patel JB (2001). 16S rRNA gene sequencing for bacterial pathogen identification in the clinical laboratory. Mol. Diagn., 6:313–321.