Ann Microbiol (2015) 65:1017–1026 DOI 10.1007/s13213-014-0946-4 ORIGINAL ARTICLE Isolation and characterization of an endophytic bacterium, Bacillus 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 bacteria 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