Ann Microbiol (2015) 65:1017–1026 DOI 10.1007/s13213-014-0946-4

ORIGINAL ARTICLE

Isolation and characterization of an endophytic bacterium, megaterium BMN1, associated with root-nodules of Medicago sativa L. growing in Al-Ahsaa region, Saudi Arabia

Ashraf Y. Z. Khalifa & Mohammed A. Almalki

Received: 4 April 2014 /Accepted: 15 July 2014 /Published online: 13 August 2014 # Springer-Verlag Berlin Heidelberg and the University of Milan 2014

Abstract Medicago sativa L. (Alfalfa) is an important forage plants. In conclusion, BMN1 belongs to the group of plant crop legume in Saudi Arabia due to its high nutritive value and growth promoting rhizobacteria and could have significant yield. Soil exist in root or root-nodules of Medicago agricultural applications. sativa in either symbiotic relationships or in associations. In the current study, the endophytic bacterium Bacillus Keywords Bacillus megaterium . Lens esculentus . megaterium BMN1 was isolated from surface-sterilized root- Phaseolus vulgaris . Pisum sativum nodules of Medicago sativa and characterized phenotypically and genotypically. The results indicated that BMN1 consumed a variety of sugars as sole carbon source, and produced catalase and amylase but not urease. BMN1 exhibited some Introduction plant growth-promoting traits, such as production of indole acetic acid and acetoin, and solublization of inorganic phos- Medicago sativa L. (Alfalfa) is an important perennial legumi- phate. In addition, comparative sequence analysis of the 16S nous plant that is cultivated as livestock fodder because it has a rRNA gene showed that BMN1 exhibited 99 % homology high feeding value and wide adaptability. Furthermore, with Bacillus megaterium. In addition, BMN1 could not Medicago sativa is used in production of biofuel and pest nodulate alfalfa when re-inoculated but the strain enhanced management (Samac et al. 2006; Sanderson and Adler 2008), root growth parameters compared to uninoculated plants. Co- and cultivated for land restoration (Guan et al. 2013). In the inoculation of BMN1with Sinorhizobium meliloti increased Kingdom of Saudia Arabia, it has been estimated that not only the number of nodules formed on roots of alfalfa but Medicago sativa represents 64 % of the total area devoted to also root length and root dry weight under greenhouse condi- fodder cultivated area (Agriculture Statistical Yearbook 2012a). tions. Furthermore, the effects BMN1 inoculation on the Medicago sativa roots establish symbiotic relationships growth of Lens esculentus, Phaseolus vulgaris and Pisum with nitrogen-fixing soil bacteria, rhizobia, e.g., sativum were also assessed. The length of the primary root, Sinorhizobium meliloti (Young 2003), resulting in the forma- number of secondary roots and dry weight of roots of the three tion of structures known as root nodules. Inside these nodules, crop legumes were significantly increased upon inoculation rhizobia provide the host with ammonia via reduction of by Bacillus megaterium compared to uninoculated control nitrogen, and receive photosynthate, i.e., reduced carbon, from the host (Peoples et al. 2009; Ferguson et al. 2010). : Therefore, efficient nodules play a pivotal role in sustaining A. Y. Z. Khalifa (*) M. A. Almalki agricultural production as they enable legumes to grow in low- Biological Sciences Department, King Faisal University, Al-Ahsaa, nitrogen soils (Gallego-Giraldo et al. 2014). Saudia Arabia e-mail: [email protected] In addition to rhizobial endosymbionts, arrays of non- nodulating endophytic bacteria, such as Brevibacillus A. Y. Z. Khalifa chosinensis and Microbacterium trichothecenolyticum e-mail: [email protected] (Stajković et al. 2009), Micromonospora (Trujillo et al. A. Y. Z. Khalifa 2010)andEndobacter medicaginis, (Ramírez-Bahena et al. Botany Department, University of Beni-Suef, Beni-Suef, Egypt 2013), inhabit root nodules of Medicago sativa and share this 1018 Ann Microbiol (2015) 65:1017–1026 ecological niche. These bacterial species exhibit plant growth rhizospheric soils, placed in sterile plastic bags and brought promoting activity. Furthermore, a wide taxonomic diversity to the laboratory. The root systems were cleaned of soil by of bacterial communities associated with different parts of gentle washing using tap water and surface-sterilized by im-

Medicago sativa have been reported using culture- mersing in H2O2 (5 %) for 5 min. To remove traces of H2O2, independent methods (Pini et al. 2012). The term microbial the nodules were washed using sterilized distilled water at endophyte is applied to the microorganisms that are isolated least five times. from plant parts after surface sterilization (Hallmann et al. 1997; Gamalero and Glick 2012). This group of microorgan- Isolation of Bacillus megaterium strain BMN1 isms has been studied widely (Mengoni et al. 2004, 2011, from root-nodules 2012). Several reports have shown that endophytic bacteria exhibit overall plant growth enhancement via different mech- Bacillus megaterium strain BMN1 was isolated from root- anisms, e.g., improving nutrient uptake, solubilizing inorganic nodules of Medicago sativa. Between two sterilized glass phosphate, producing phytohormones and/or protecting plants slides, randomly selected nodules were crushed in drops of against pathogens (Rodríguez-Díaz et al. 2008; Ali et al. 2010; sterilized distilled water and aliquots were streaked onto yeast- Gamalero and Glick 2012; Rashid et al. 2012;Jietal.2013). extract mannitol agar (YMA) (Vincent 1970). Aliquots of the Bacillus spp. have been found to act as growth promoting distilled water used in the final wash of the Medicago sativa bacteria and agents for biological control against plant patho- nodules were also streaked onto YMA plates in order to check gens (Ghosh et al. 2003; Vespermann et al. 2007; Gao et al. the efficiency of the surface sterilization process. The plates 2014). It has been reported recently that the abscisic acid were incubated at 30 °C for 2–3 days. BMN1 was purified by content in tomato plants is essential for the growth promoting streaking on fresh agar plates and maintained in agar slants activity of Bacillus megaterium by maintaining low levels of stored at 4 °C for further use. BMN1 were transferred regu- ethylene production (Porcel et al. 2014). The genome se- larly to new agar slants to keep them viable. quence of Bacillus megaterium has been released recently (Wu et al. 2014). This will open new doors for in-depth Morphological investigations molecular and genetical studies of this interesting bacterium. Although Medicago sativa and, to some extent, the bacteria The morphological characteristics; color, diameter, and edge associated with its root have been studied extensively, few of the colony of strain BMN1 was determined. studies have focused on bacterial endophytes associated with root-nodules of Medicago sativa and their potential role in Characterization of strain BMN1 using API 20E kit enhancing the growth of economically important grain le- gumes in the Al-Ahsa region (Eastern province, Kingdom of The ability of the strain BMN1 to utilize 20 different chemical Saudi Arabia). Grain legumes play a pivotal role in improving substrates was tested using an API20E strip (bioMérieux, food security, nutrition and health and in sustainable agricul- Marcy l’Etoile, France) according to the manufacturer’spro- ture. Saudi Arabia imports more than 100,000 tons of Lens tocol. Incubation was carried out at 30 °C and results were esculentus, Phaseolus vulgaris and Pisum sativum annually scored after 24 h. (Agriculture Statistical Yearbook 2012b); this raises the im- portance of improving locally produced grain legumes. One Catalase test approach is to explore the plant growth promoting bacteria isolated from herb legumes in order enhance the growth of To determine if BMN1 produces catalase, an aliquot of an grain legumes. Therefore, the aim of the current study was to overnight BMN1 culture was smeared on a clean glass slide isolate and characterize the endophytic bacteria inhabiting the and drops of hydrogen peroxide (5 %) were added. Formation root-nodules of Medicago sativa growing in Al-Ahsa, King- of gas bubbles was observed within few seconds. dom of Saudi Arabia. Furthermore, the effects on growth of three economically important crop legumes upon inoculation Production of indole acetic acid with the endophytic bacteria were also investigated. BMN1 was tested for the ability to produce indole acetic acid (IAA) using a standard colorimetric assay (Gordon and Weber Materials and methods 1951). BMN1 was cultured in flasks containing 20 mL Luria Bertani (LB) broth supplemented with 0.0, 0.1, and Collection of Medicago sativa plants 0.2 mg mL−1 tryptophan, for 3 days at 30 °C. After incubation, cells were precipitated by centrifugation at 10,000 rpm for Medicago sativa plants were collected from street sides in Al- 10 min, then 1 mL of the supernatant was mixed vigorously

Ahsaa city. The plants were uprooted along with the with 2 mL Salkowski’ s reagent (150 mL concentrated H2SO4, Ann Microbiol (2015) 65:1017–1026 1019

250 mL distilled H2O, 7.5 mL 0.5 M FeCl3·6H2O). The tubes 7644), Staphylococcus aureus (ATCC 25923), and Staphylo- were incubated in the dark at room temperature for 25 min. coccus saprophyticus (ATCC 15305). Each test bacterial

The OD530 was then measured and the quantity of IAA strain was streaked thoroughly on nutrient agar (NA) plates produced estimated from a standard curve. and a loopful of overnight BMN1 culture was placed on the center of each NA plate. The plates were incubated at 30 °C Testing BMN1 for phosphate solubilization for 3 days. After the incubation period, the plates were checked for formation of an inhibition zone surrounding Phosphate solubilization ability of BMN1 was assessed by the BMN1 growth. appearance of clear halo zones surrounding bacterial colonies grown on Pikovskaya agar containing calcium triphosphate as Production of antifungal agents the source of mineral phosphate, after 72 h incubation at 30 °C (Pikovskaya 1948). BMN1 was tested for its antifungal activity by placing an agar disc of the test fungus mycelium of Fusarium oxysporum, Growth of BMN1 under different concentrations of NaCl Aspergillus sp. and Penicillium sp. with agar in the center of a potato dextrose agar (PDA) (Oxoid, Basingstoke, UK) plate BMN1 was tested for its ability to grow under different and streaking BMN1 strain near the edge. After a 3-day concentrations of NaCl by inoculating nutrient agar plates incubation at 30 °C, plates were checked for formation of an supplemented with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 % NaCl. inhibition zone around the bacterial growth (Lambert et al. The plated were incubated at 30 °C for 3 days. After the 1987). incubation period the plates were checked visually for bacte- rial growth. The experiment was performed in triplicate. Preparation of template DNA

Intrinsic antibiotic resistance Genomic DNA of the bacterial strain BMN1 was obtained by boiling the cells in presence of InstaGene Matrix (Bio-Rad, The intrinsic antibiotic resistance test was carried out by Hercules, CA) according to the manufacturer’s instructions. streaking a 24-h-old culture of BMN1 on YMA plates. Anti- Briefly, a small part of a single colony was picked up with a biotic discs were applied to the plates under aseptic condi- sterilized toothpick, suspended in 0.5 mL sterilized saline tions. The following antibiotics were tested, chloramphenicol (0.85 % NaCl) and centrifuged at 10,000 rpm for 10 min. (C, 30 μg), cephradine (CE, 30 μg) ampicillin sulbactam The pellet was resuspended in 0.5 mL InstaGene Matrix, (SAM, 10 μg), erthromycin (E, 15 μg) and tetracycline (TE, incubated at 56 °C for 30 min and then heated 100 °C for 30 μg). The plates were incubated at 30 °C for 24 h and the 10 min. A 1-μL aliquot of the supernatant was used as experiment was carried out in triplicate. The appearance of a template DNA for PCR amplification of the 16S rRNA gene. halo around the antibiotic disc was taken as a sign of sensi- tivity to the antibiotic at the given concentration. Calculating PCR amplification of the 16S rRNA gene the diameter of the halo was estimated by subtracting the diameter of the disc from the total diameter. The 16S rRNA gene was amplified in a total 20 μLPCR reaction using the universal primers; 27F 5′-AGA GTT TGA Starch hydrolysis TCM TGG CTC AG-3′ and 1492R 5′-TACGGYTACCTTGT TACGACTT-3′ (Weisburg et al. 1991). The PCR conditions To test BNMA1 for its ability to utilize starch—a complex were as follows: denaturation at 94 °C for 45 s, annealing at carbohydrate made from glucose—as a source of carbon and 55 °C for 60 s, and extension 72 °C for 60 s in 35 amplifica- energy for growth, an inoculum of BNMA1 was streaked on a tion cycles, followed by a final extension step at 72 °C for starch agar plate and incubated at 30 °C for 24 h. After 10 min. A positive control (E. coli genomic DNA) and a incubation, iodine reagent was added to flood the bacterial negative control (no DNA template) were also used in parallel growth and the presence of clear halos around the growing reactions. PCR products were purified using the Clean-up kit colonies was observed. (Millipore, Fisher Scientific, Loughborough, UK) according to the instructions of the manufacturer. Antibacterial activity of BMN1 16S rRNA sequencing BMN1 was tested for its antibacterial activity against six reference bacterial strains: Escherichia coli (ATCC 25922), Sequencing of the 16S rRNA gene was carried out using a Big Pseudomonas aeruginosa (ATCC 27853), Salmonella Dye terminator cycle sequencing kit (Applied BioSystems, enterica (ATCC 13076), Listeria monocytogenes (ATCC Foster City, CA). Sequencing products were resolved on an 1020 Ann Microbiol (2015) 65:1017–1026

Applied Biosystems model 3730XL automated DNA se- phenotypic traits. Furthermore, the length of the primary root quencing system. and number of secondary roots of three crop legumes in- creased significantly upon inoculation with B. megaterium Construction of phylogenetic tree under greenhouse conditions. Additionally, although BMN1 was unable to nodulate alfalfa roots when re-inoculated, the The 16S rRNA sequence of strain BMN1 was checked visu- strain enhanced root growth parameters compared to uninoc- ally using Chromas version 2.4.2, then identified by BLAST ulated plants. Co-inoculation of BMN1with S. meliloti in- searching against the GenBank nr/nt database (BLASTN) on creased not only the number of nodules formed on roots of the National Center for Biotechnology Information (NCBI) alfalfa but also root length and root dry weight under green- website (http://www.ncbi.nlm.nih.gov/). The 16S rRNA gene house conditions. sequence of BMN1 was deposited with the NCBI database Colonies of BMN1 are white in color, round and smooth in under the accession number KJ461522. 16S rRNA sequences shape and entire in edge. The cells of Bacillus megaterium for recognized bacterial strains that showed high percentage BMN1 are Gram-positive rod-shaped and forming identity to strain BMN1 were retrieved from NCBI. (data not shown). It is worth mentioning that other rhizobial- Evolutionary history was inferred using the Maximum like strains were also isolated from alfalfa nodules and have Likelihood method based on the Tamura-Nei model (Tamura been kept to be investigated in a separate study. and Nei 1993). Evolutionary analyses were conducted in MEGA5.02 (Tamura et al. 2011). Characterization of strain BMN1 using API 20E tests

Effect of BMN1 on growth of alfalfa and some legumes BMN1 utilized 50 % (10 out of 20) of the different chemical substrates contained in the API20E strip. In general, BMN1 The aim of the current study was to assess the effects of the had the ability to ferment/oxidize all the carbohydrate tested endophytic bacterium, Bacillus megaterium strain BMN1 on except rhamnose and the polyol carbohydrate inositol the growth of three economically important crop legumes; (Table 1). BMN1 fermented/oxidized glucose, mannitol, sor- Lens esculentus, Phaseolus vulgaris and Pisum sativum as bitol, sucrose, melibiose, arabinose and amygdalin. Further- well as the original host, alfalfa. The seeds were surface- more, BMN1 consumed ortho-nitrophenyl-β-galactoside sterilized as described previously (Vincent 1970) and planted (ONPG), Na pyruvate and charcoal gelatin. However, none in sterilized agricultural soils on pot trails. Bacterial suspen- of the amino acids tested was utilized by strain BMN1 sions of the strain BMN1 of an optical density of 1.0 at (Table 1). In addition, BMN1 was unable to hydrolyze urea, 660 nm were applied to seeds of legume at a ratio of 5 mL indicating a lack of urease. These findings in are agreement per five seeds for each pot. with those recorded in Bacillus spp. (Guinebretiere et al. 2013; For co-inoculation, a mixture of BMN1 with Chang et al. 2014), and indicate that BNMI has diverse Sinorhizobium meliloti (1:1) was used for inoculation of al- enzymatic systems that enable it to consume many substrates falfa; 1 mL of each bacterial culture was used for treatment. but that it lacks some enzyme systems. Seeds of alfalfa were also inoculated with S. meliloti strain Interestingly, strain BMN1 has the ability to produce 1021 alone. Untreated control plants had 5 mL sterile distilled acetoin (the 3-hydroxy-2-butanone) (Table 1), which plays water applied and the experiment was carried out in triplicate. an important role in induced systemic resistance. Induced The conditions of the growth chamber were adjusted to a 14 h systemic resistance results from the role of rhizobacteria in light/10 h dark cycle and temperature of 25 °C. After 3 weeks, increasing plant resistance to pathogens caused by triggering growth parameters such as height of the primary shoot and the plant defense systems (Kloepper et al. 2004; VanLoon and root length were measured and the number of the root Glick 2004). Acetoin produced by has been branches and nodules counted. The roots, shoots and nodules shown to act as an elicitor in and result- were dried at 80 °C for 3 days and then weighed. Data were ed in reduction of disease severity against Pseudomonas analyzed statistically using Student’s t-test to determine sig- syringae pv. tomato (Rudrappa et al. 2010). Therefore, it can nificance between treatments. be concluded that strain BMN1 might play a pivotal role in activating the resistance of plants against certain pathogens via acetoin production.

Results and discussion Catalase test

Bacillus megaterium BMN1 was isolated from surface- The catalase test result is presented in Table 2.Uponaddition sterilized root-nodules of Medicago sativa and characterized of hydrogen peroxide to BNM1 on the slides copious oxygen using PCR-amplified 16S rRNA gene sequences as well as bubbles were evolved, indicating the presence of the enzyme, Ann Microbiol (2015) 65:1017–1026 1021

Table 1 Results of API20, starch hydrolysis and catalase tests for Bacil- Production of indole acetic acid lus megaterium strain BMN1. ONPG Ortho-nitrophenyl-β-galactoside

Test Test Substrate Enzyme involved/ Resulta BMN1 could produce IAA when the growth medium was number code reaction tested supplemented with tryptophan. As can be seen (Table 2), BMN1 produced IAA (51 μgmL−1) when tryptophan 1 ONPG ONPG * Beta-galactosidase + − (0.1 μgmL 1) was added to the growth medium. The quantity − 2 ADH Arginine Arginine dihydrolase of IAA produced increased to 95 μgmL−1 when the added − 3 LDC Lysine Lysine decarboxylase tryptophan was increased to 0.2 mg mL−1. No IAA was − 4 ODC Ornithine Ornithine decarboxylase detected when BMN1 was grown on no-added tryptophan − 5 CIT Citrate Citrate utilization growth medium. IAA is a phytohormone produced from − 6H2S Na thiosulfate H2Sproduction tryptophan that regulates cell division and root elongation in − 7 URE Urea Urea hydrolysis plants (Khan et al. 2009). BMN1 thus possessed a the com- − 8TDATryptophanDeaminase mon plant growth promoting trait, i.e., production of IAA, and 9 IND Tryptophan Indole production + therefore complied with previous studies of Ahemad and 10 VP Na pyruvate Acetoin production + Mohammad (2010) and Bumunang and Babalola (2014). 11 GEL Charcoal gelatin Gelatinase + 12 GLU Glucose Fermentation/oxidation + 13 MAN Mannitol Fermentation/oxidation + Testing BMN1 for phosphate solubilization 14 INO Inositol Fermentation/oxidation − 15 SOR Sorbitol Fermentation/oxidation + BMN1 formed a halo zone when grown on Pikovskaya agar 16 RHA Rhamnose Fermentation/oxidation − containing calcium triphosphate (Fig. 1a), indicating that this 17 SAC Sucrose Fermentation/oxidation + strain has phosphate solubilization ability (Table 2). 18 MEL Melibiose Fermentation/oxidation + Rhizobacteria with this trait secrete organic acids and phos- 19 AMY Amygdalin Fermentation/oxidation + phatases that solubilize insoluble phosphate. Therefore, phos- — — 20 ARA Arabinose Fermentation/oxidation + phorous an essential nutrient for plant growth become 21 STA Starch Amylases + available to be absorbed by plant roots. The ability of Bacillus 22 HYP Hydrogen Catalase + strains to solubilize inorganic phosphate was also reported by peroxide Stajkova et al. (2009). Furthermore, phosphate-solublizing 23 ability was not observed for some rhizobacteria (Rathaur et al. 2012). Along the same lines, Li et al. (2008) noticed a + positive result, BMN1 was able to utilize the corresponding substrate; − negative result, BMN1 was unable to utilize the corresponding substrate that endophytic bacteria of soybean exhibited growth promot- ing activities such as production of IAA and solubilization of mineral phosphate. These characteristics were also observed which plays a protective role by splitting the toxic hydrogen in BMN1 strain isolated from Medicago sativa. peroxide into water and oxygen. Hence, BMN1 with catalase activity could survive in the rhizospheric soil and enhance plant growth indirectly. Production of catalase by BMN1 is in Growth of BMN1 under different concentrations of NaCl agreement with previous studies on endophytic bacteria (Bumunang and Babalola 2014; Chaiharn et al. 2008). The results of the effects of NaCl salinity levels are presented in Table 2. BMN1 could grow under elevated levels of NaCl up to 8 %, after which no growth was observed. These results are in agreement with those obtained with other Bacillus Table 2 Results of biochemical tests of BNM1 strains, which could tolerated elevated levels of NaCl Test Result (Dastager et al. 2014). Tolerance to salinity confers a selective advantage for rhizobacterial species populating the same soil Catalase + localities suffering from elevated levels of salts. Indole-3-acetic acid (IAA) at no-added tryptophan Not detected Indole-3-acetic acid (IAA)in at 0.1 mg mL-1 added 50.94±2.1 μgmL-1 tryptophan Starch hydrolysis Indole-3-acetic acid (IAA) at 0.2 mg mL-1 added 95.84±4.3 μgmL-1 tryptophan Phosphate solubilization + The results of the starch hydrolysis test are presented in NaCl tolerance (9 %) − Table 2. A halo zone was formed around colonies of BMN1 Starch hydrolysis + upon addition of iodine reagent, indicating its ability to utilize starch as a carbon and energy source via α-amylase. 1022 Ann Microbiol (2015) 65:1017–1026

Fig. 1 Results of a phosphate solubilization and b antibiotic resistance tests

Intrinsic antibiotic resistance sequence similarities < 97 % relate to different species, while those with sequence similarities > 97 % most likely, but not As seen in Table 3, BNM1 exhibited sensitivity to all the necessarily, belong to the same species (Stackebrandt and antibiotics tested as indicated by the appearance of an inhibi- Goebel 1994). tion zone. The zone of inhibition of growth varied from 1.9 to Comparative sequence analysis of the 16S rRNA gene of 3.2 cm indicating different degrees of sensitivities toward the BMN1showed that this strain was closely related to the genus antibiotics. Compared to other antibiotics tested, chloram- Bacillus. BMN1 exhibited 99 % homology with phenicol (30 μg) and cephradine (30 μg) were very effective B. megaterium strain 29B (accession number KC329822.1), against strain BMN1 as indicated by the diameter of the zone B. megaterium strain Cl-30 (accession number KC178604.1) of inhibition (3.2 cm). Ampicillin sulbactam (10 μg) and and B. megaterium strain TAUC4 (accession number erthromycin (15 μg) formed similar zones of inhibition HQ914779.1). In the 16S rRNA sequence analysis, strain (2 cm) (Fig. 1b). BNM1 formed a monophyletic group with species of the B. megaterium group (Fig. 2), while representatives of other Antibacterial and antifungal activity of BMN1 , i.e., Listeria monocytogenes strain Pyde1(KC852899), Lactobacillus crispatus strain BLB2 No zones of inhibition were observed around BMN1 colonies, (AF243141), Clostridium tetani strain HT1 (DQ978212) and indicating that this strain has no antibacterial or antifungal Bacillus firmus strain UST981101-006 (FJ188300), formed activities against the bacterial and fungal species tested. distinct outgroups. Similar results have been obtained by Stajkovicetal.(2009), who found that nodules of M. sativa 16S rRNA gene sequence analysis and phylogenetic tree growing in central Serbia harboured B. megaterium strains as shown by 16srRNA gene sequences. It is worth mentioning Comparative 16S rRNA gene sequence analysis has been used that although analyses of 16S rRNA sequences can be used to for bacterial identification at the genus and species levels as define species (Wu et al. 2006), this is not always the case well as for inferring phylogenetic relationships among pro- because 16S rRNA genes might contain highly conserved karyotic organisms (Woese 1987; Stackebrandt 2002). As a regions that do not allow the discrimination among some general rule, bacterial strains that exhibit 16S rRNA gene species and subspecies within this group (Shaver et al. 2002; Zhang et al. 2014).

Table 3 Results of intrinsic antibiotic resistance of BNM1 Effect of BMN1 inoculation on legume growth Antibiotic Abbreviation Amount Diameter of zone μ a ( g) of inhibition (cm) The results showing the effects of BMN1 on growth promo- Chloramphenicol C 30 3.2 ±0.05 tion of Lens esculentus, Phaseolus vulgaris and Pisum Cephradine CE 30 3.2±0.05 sativum are presented in Table 4. BMN1 inoculation signifi- Ampicillin sulbactam SAM 10 2±0.0 cantly increased the dry weight of roots, length of the primary Erthromycin E 15 1.9±0.08 root, number of secondary roots and shoot height (Table 4). BMN1 promoted growth of the primary root in Lens Tetracycline 30 TE 30 2.6±0.04 esculentus and the number of the lateral roots in Phaseolus a Diameter of inhibition zone around the antibiotic disc vulgaris and Pisum sativum.InLens esculentus, inoculation Ann Microbiol (2015) 65:1017–1026 1023

Fig. 2 Maximum likelihood phylogenetic tree based on 16S rDNA gene addition, representatives from the Firmicutes, Listeria monocytogenes sequences showing relationships between strain Bacillus megaterium strain Pyde1(accession no. KC852899), Lactobacillus crispatus strain BMN1 (accession no. KJ461522) and related species B. megaterium BLB2 (accession no. AF243141), Clostridium tetani strain HT1 (acces- strain 29B (accession no. KC329822.1), B. megaterium strain Cl-30 sion no. DQ978212) and Burkholderia cepacia (accession 566 no. (accession no. KC329822.1), B. megaterium strain TAUC4 (accession U96927.1), were also included no. HQ914779.1), and Bacillus firmus (accession no. FJ188300). In

Table 4 Effects on growth of Lens esculentus, Phaseolus vulgaris and Pisum sativum of inoculation with BMN1. Values shown represent the average of the replicates ± standard deviation

Parameter Lens esculentus Phaseolus vulgaris Pisum sativum

Control Inoculated Control Inoculated Control Inoculated

Stem length (cm) 27.80±3.5 27.47±3.8 18.42±3.46 19.20±3.44 11.06±3.04 10.21±1.33 Root length (cm) 6.90±1.26 12.0*±1.36 8.80±3.05 11.90±1.06 7.44±3.14 9.57±2.05 Lateral root number plant−1 9.20±1.30 10.40±1.75 15.67±3.57 29.00*±4.82 11.00±2.50 20.57*±3.99 Dry weight root plant −1 (mg) 47±6 121*±3 365±13 498*±32 437±23 548*±16 Dry weight shoot per plant−1 (mg) 91±5 90±4 576±44 591±30 289±9 219±16

*Statistically significant differences estimated by t test (P<0.05) with BMN1 enhanced significantly (P<0.001) root length These findings are in accordance with those of López- (12 cm) and the dry root weight per plant (121 mg plant−1) Bucio et al. (2007), who found that Bacillus megaterium compared to uninoculated control plants (6.9 cm) and (47 mg isolated from the rhizosphere of maize plants enhanced the plant−1), respectively. Furthermore, a significant (P<0.01) growth and development of Phaseolus vulgaris and increase in the number of lateral roots in both Phaseolus Arabidopsis thaliana. Similarly, Bacillus megaterium en- vulgaris (29 cm) and Pisum sativum (20.57 cm) was observed hanced the growth of Arachis hypogaea (Kishore et al. in BMN1-inoculated plants compared to controls (15. 67 cm 2005)andZea mays growing under conditions of water defi- and 11. cm, respectively). Likewise, the dry weights of roots ciency (Marulanda et al. 2009, 2010) and in nutrients of both plants exhibited a significant increase (P<0.001). It (Marulanda et al. 2008). Single inoculation of alfalfa with can be concluded that the growth activation activity of BMN1 either BMN1 or S. meliloti 1021 significantly enhanced the is more clearly distinguishable in the root system than in the root length (19.4 cm and 16.22 cm), number of lateral roots ( shoot system. 15.9 and 16.67) and root dry weights (21.75 and 22.6 mg root

Table 5 Effects of inoculation with BMN1 and co-inoculation with Sinorhizobium meliloti on nodulation and growth parameters of alfalfa. Values shown represent the average of the replicates ± standard deviation

Strain Stem length Root length Lateral root Nodules Dry weight root Dry weight shoot Dry weight nodules (cm) (cm) number plant−1 number plant−1 plant −1 (mg) plant−1 (mg) plant−1 (mg)

BMN1 24.3±1.2 19.4*±1.17 15.9*±1.67 0 21.75*±2.1 58.44±3.22 0 1021 25.52±2.4 16.22*±1.39 16.67*±1.92 9.66± 22.6*±1.89 56.31±3.81 1.26±0.05 BMN1 and 1021 26.73±1.6 17.43*±1.83 15.31*±1.55 14.35± 22.46*±2.04 57±4.1 1.49*±0.07 Uninoculated control 23.22±2.2 13.81±1.54 11.73±1.23 0 16.27±1.63 54.7± 0

*Statistically significant differences estimated by t test (P<0.05) 1024 Ann Microbiol (2015) 65:1017–1026 plant −1) compared to control plants. No significant differ- Agriculture Statistical Yearbook (2012b) Ministry of Agriculture. Volume ences in the shoot length and shoot dry weight were observed 25, Saudi Arabia Ahemad M, Mohammad SK (2010) Plant growth promoting activities of in alfalfa treated with BMN1 or S. meliloti 1021 alone. Similar phosphate-solubilizing Enterobacter asburiae as influenced by fun- results were obtained when alfalfa seeds were co-inoculated gicides. EurAsia J BioSci 4:88–95 with both BMN1 and S. meliloti 1021 (Table 5). However, the Ali B, Sabri AN, Hasnain S (2010) Rhizobacterial potential to alter auxin content and growth of Vigna radiate (L.). World J Microbiol number of nodules formed on roots of alfalfa (14.35 nodules – −1 −1 Biotechnol 26:1379 1384 plant ) and the nodule dry weights (1.26 mg plant )were Bashan Y (1998) Inoculants of plant growth-promoting bacteria for use in increased in comparison to alfalfa inoculated with meliloti agriculture. Biotechnol Adv 16:729–770 1021 alone (9.66 nodules plant −1 and 1.26 mg plant −1, Bumunang EW, Babalola OO (2014) Characterization of Rhizobacteria respectively). from field grown genetically modified (GM) and non-GM maizes. Braz Arch Biol Technol 57:1–8 These results are in general agreement with those ob- Chaiharn M, Chunhaleuchanon S, Kozo A, Lumyong S (2008) Screening tained in many other studies such as that of Tilak et al. of rhizobacteria for their plant growth promoting activities. Sci (2006), who reported that co-inoculation of pigeonpea Technol J 1:18–23 (Cajanus cajan)withbothBacillus cereus and Rhizobium Chang YC, Choi D, Takamizawa K, Kikuchi S (2014) Isolation of Bacillus spp. strains capable of decomposing alkali lignin resulted in a significant increase in plant growth and nod- and their application in combination with lactic acid bacteria ulation over Rhizobium-inoculated and uninoculated con- for enhancing cellulase performance. Bioresour Technol 152: trol plants. However, Bacillus sp. enhanced nodule num- 429–436 bers on roots of alfalfa when inoculated with S. meliloti but Dastager SG, Mawlankar R, Tang SK, Srinivasan K, Ramana VV, Shouche YS (2014) Bacillus enclensis sp. nov., isolated from sedi- no significant differences were observed in plant growth ment sample. Antonie Van Leeuwenhoek, 105: 199–206 parameters between single and dual inoculation (Stajković Ferguson BJ, Indrasumunar A, Hayashi S, Lin M, Lin Y, Reid DE, et al. 2009). In our study, strain BMN1 was found to Gresshoff PM, Lin MH, Lin YH (2010) Molecular analysis of increase not only the number of nodules but also plant root legume nodule development and autoregulation. J Integr Plant Biol 52:61–76 growth, indicating that the growth-promoting activities of Gallego-Giraldo L, Bhattarai K, Pislariu C, Nakashima J, Jikamura Y, some bacteria are highly specific to certain plant species, Kamiya Y, Dixon R (2014) Lignin modification leads to increase cultivar and genotype (Bashan 1998). The enhancement of nodule numbers in alfalfa (Medicago sativa L.). Plant Physiol 164 : – root growth in the three plants studied could be attributed 1139 1150. doi:10.1104/pp.113.232421 Gamalero E, Glick BR (2012) Plant growth-promoting bacteria and metal to one or more of the multifunctional plant growth promot- phytoremediation. In: Anjum NA, Pereira ME, Ahmad I, Duarte ing traits exhibited by BMN1, via direct or indirect means. AC, Umar S, Khan NA (eds) Phytotechnologies: remediation of It seems that multiple plant-growth promoting activities are environmental contaminants. Taylor and Francis, Boca Raton, – a common feature among rhizobacteria. pp 359 374 Gao X, Han Q, Chen Y, Qin H, Huang L, Kang Z (2014) Biological Collectively, BNM1 exhibited a phenotypic similarity to control of oilseed rape Sclerotinia stem rot by Bacillus subtilis strain that of Bacillus spp., based on morphological, cultural and Em7. Biocontrol Sci Technol 24:39–52 biochemical characteristics and 16S rDNA gene sequence Ghosh S, Penterman N, Little RD, Chavez R, Glick BR (2003) Three (Bergey’s Manual of Systematic Bacteriology, Logan De Vos newly isolated plant growth-promoting facilitate the growth of canola seedlings. Plant Physiol Biochem 41:277–281 2009). The results indicate that this bacterial strain belongs to Gordon SA, Weber RP (1951) Colorimetric estimation of indole-acetic the plant growth promoting rhizobacteria (PGPB) and could acid. PlantPhysiol 26:192–195 have significant agricultural applications to increase plant Guan XK, Zhang XH, Turner NC, Xu BC, Li FM (2013) Two perennial productivity and reduce the use of synthetic fertilizers and legumes (Astragalus adsurgens Pall. and Lespedeza davurica S.) adapted to semiarid environments are not as productive as lucerne pesticides. (Medicago sativa L.), but use less water. Grass Forage Sci 68:469–478 Guinebretiere MH, Auger S, Galleron N, Contzen M, De Sarrau B, De Buyser M, Lamberet G, Sorokin A (2013) Bacillus cytotoxicus sp. nov. is a novel thermotolerant species of the group Acknowledgments The authors are indebted to the Biological Sciences occasionally associated with food poisoning. 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