Isolation and Identification of Bacteria from Root ...

ISSN 0031-7454

PHILIPP AGRIC SCIENTIST Vol. 100 No. 1, 103–117 March 2017

Isolation and Identification of Bacteria from Root Nodules of Philippine Legumes Using 16S rRNA Gene Sequencing Vernans V. Bautista1,*, Emerson V. Barcellano2, Rosario G. Monsalud3 and Akira Yokota4 1

Microbiology Division, Institute of Biological Sciences, College of Arts and Sciences, University of the Philippines Los Baños, 4031 College, Laguna, Philippines 2 College of Forestry, Kalinga State University, Bulanao, Tabuk City, Kalinga, Philippines 3 National Institute of Molecular Biology and Biotechnology, University of the Philippines Los Baños, 4031 College, Laguna, Philippines 4 Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-Ku, Tokyo 113-0032, Japan * Author for correspondence; e-mail: [email protected]; Tel: +63-926-669-2482 Part of the doctoral dissertation of the first author conducted at the Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-Ku, Tokyo 113-0032, Japan –

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Key Words: rhizobia, root nodules, leguminous plants, 16S rRNA, novel species Abbreviations: BLAST – Basic local alignment research tool, bp – base pair, NCBI – National Center for Biotechnology Information, rRNA – ribosomal ribonucleic acid

The Philippine Agricultural Scientist Vol. 100 No. 1 (March 2017)

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Isolation and Identification of Bacteria from Root Nodules of Philippine Legumes

INTRODUCTION Legumes have been gathered, cultivated, eaten and used in a multitude of other ways by humans for millennia (Lewis et al. 2005). They are also immensely important in forestry and agriculture because many species can colonize marginal or barren lands owing to their capacity to fix atmospheric nitrogen through root nodules (Sprent 2001). The symbiotic relationship between the nodulation bacteria (rhizobia) and the members of the plant family Leguminosae, as well as between the actinomycete Frankia and the actinorhizal plants such as Alnus, Myrica, Ceanothus, Elaeagnus and Casuarina, are essential contributors to nitrogen fixation (Sawada et al. 2003). The Philippines is an agricultural country endowed with a rich source of legumes (Aguilar et al. 1994) and microbial diversity. One agriculturally important group of microorganisms is the rhizobia that are symbiotic to leguminous plants. However, many indigenous rhizobial isolates have not been fully characterized and described. The Philippine National Collection of Microorganisms (PNCM) of BIOTECH, which serves as the national repository of microorganisms, has at least 100 rhizobial strains isolated from soil samples from Taal Volcano and several leguminous plants such as Albizia procera (Roxb.) Benth, Arachis hypogaea L., Calopogonium sp., Cajanus cajan (L.) Millsp., Centrosema pubescens Benth., Enterolobium saman (Jacq.) Prain, Leucaena leucocephala (Lam.) de Witt., Vigna radiata (L.) Wilczek, Glycine max Merrill, Samanea saman Merr., and Pterocarpus indicus Willd. which are locally grown or cultivated (Directory of Culture Collections in the Philippines 2012). Although we have this vast natural resource of leguminous plants and microorganisms, to our knowledge, none of the novel species of bacterium within the order Rhizobiales and Burkholderiales has been described, collected or isolated so far from the Republic of the Philippines (LPSN 2016). The great diversity of bacteria and leguminous plants, environmental differences, and the exclusive interaction between plants and rhizobia, provides a window of opportunity out of which new or novel species of bacteria can still be isolated. In this regard, taxonomic studies on these indigenous isolates are deemed important, not only to properly place these microorganisms in appropriate taxa, but also as basis of comparison for new microbes. This 104

Vernans V. Bautista et al.

study aims to give insights, to strengthen the field of bacterial systematics, not only by identifying and describing novel species, but also through the longterm preservation of isolates. Thus, the objectives of this study are (1) to isolate and identify the species of bacteria derived from root nodules of indigenous Philippine leguminous plants based on partial 16S rRNA sequencing, and (2) to determine the taxonomic position of probable novel bacterial species.

MATERIALS AND METHODS Legume Sampling and Nodule Collection Indigenous leguminous plants were identified and collected from remote areas in the provinces of Isabela, Kalinga, Benguet, Laguna, Occidental Mindoro, and Mindanao, Philippines from 2004 to 2008. Herbaceous leguminous plants were uprooted with a shovel. Root nodules from the seedlings of woody/tree legumes such as P. indicus and L. leucocephala or from the surface or secondary roots of the main tree were collected. The roots and root nodules were carefully washed in running water and placed onto properly labeled plastic bags, stored in an ice chest and transported to the laboratory for bacterial isolation. Isolation of the bacteria from the root nodules was performed within 48 h. Alternatively, root nodules were stored in improvised microfuge tubes containing anhydrous silica gel as described by Date and Halliday (1987). Since the plants were initially identified based on a broad morphology of the legumes (leaf shape and patterns, presence of pods, etc.), collected plants were measured and photographed. Fresh samples were submitted to an expert in legume identification to ascertain their proper identities. Surface Disinfection of the Root Nodules and Isolation of Rhizobia Desiccated nodules were transferred aseptically onto sterile plates and rehydrated overnight with 15 mL sterile distilled water while the fresh nodules were removed from the roots and deposited onto sterile disposable plates. Approximately 20 mL of a 10% sodium hypochlorite solution was added and allowed to stand for 10–15 min. The nodules were disinfected twice, first using 10% sodium hypochlorite followed by 70% ethyl alcohol with



rinsing in between five to six times using sterile distilled water. The nodules were transferred to a sterile 2 mL microfuge tube and crushed with a pair of flame-sterilized forceps. The resulting aliquot was diluted up to 10-5, from which the last two dilutions were spread plated on a rhizobium medium (Vincent 1970) supplemented with Congo red (15 mg/L) and Kabicidin™ (Nihon Seiyaku). The plates were incubated in an inverted position at room temperature for 3 d. Initial observation and selection of colonies were done after the second day of incubation. Well-isolated colonies exhibiting typical rhizobium cultural characteristics were carefully picked and purified using the same medium. Gram reaction (BD Gram Stain Kit, Becton Dickinson and Co.) and morphological homogeneity of the cells were determined using microscopy (Olympus BX 60). Maintenance of Pure Cultures Axenic cultures obtained from the root nodules were maintained and periodically transferred in rhizobium agar slants every 4 mo and stored in a cold room at 10–15 °C. For long-term preservation, cultures were maintained in microfuge tubes containing rhizobium broth amended with 20% glycerol and stored at 80 °C. PCR Amplification and Direct Sequencing of 16S rRNA Gene The genomic DNA was extracted following the method of Hiraishi (1992), and was used as template in PCR. For the partial 16S rRNA gene amplification, 50 µL reaction mix consisted of 80 µg/mL DNA, 3 µM of each of the universal primers 8F (5’-AGAGTTTGATCCTGGCTCAG-3’) and 1510R (5’-GGCTACCTTGTTACGA-3’) (Brosius et al. 1978), 1.5 mM MgCL2, 200 µM dNTPs, 1X Bioline Buffer, 0.5 U Bioline Taq Polymerase, and Milli-Q water. The PCR was run for 30 cycles with initial denaturation for 2 min at 94 °C, denaturation for 1 min at 94 °C, annealing for 1 min at 55 °C, extension for 1 min at 72 °C, and final extension for 4 min at 72 °C using a Thermo Hybaid PCR Express Thermal Cycler (Hybaid, Ltd., UK). About 1 µL of the PCR product was electrophoresed in 1% agarose gel (Nacalai Tesque) with 1X TAE buffer. The 100 bp DNA ladder (New England BioLabs Japan Inc.) was used as the size marker. The gel was run at 100v for 35 min, stained with ethidium bromide, and viewed under the UV transilluminator equipped with basic UV


documentation system. The remaining PCR product was purified by using PEG 6000 DNA purification technique, followed by ethanol precipitation. The DNA was then dissolved in 25 µL sterile distilled water to serve as DNA template for Big Dye® PCR. Big Dye® Terminator v3.1 Cycle Sequencing Kit The Big Dye® reaction mixture was prepared following the Big Dye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and the cycle sequencing was performed using the 16S rRNA gene primer set indicated by Kurahashi and Yokota (2002). The Big Dye® PCR was run for 25 cycles with initial denaturation for 2 min at 96 °C, denaturation for 10 s at 96 °C, annealing for 5 s at 50 °C, and extension for 4 min at 60 °C, using Thermo Hybaid PCR Express Thermal Cycler (Hybaid, Ltd., UK). The Big Dye® reaction products were cleaned using Agencourt® CleanSeq® Dye-Terminator Removal (Agencourt Bioscience Corp.) following the manufacturer’s protocol. A 35 µL ‘cleaned’ Big Dye product dissolved in elution buffer (0.01 mM EDTA) was used in sequencing reactions using the ABI PRISM™ 310 Genetic Analyzer (Applied Biosystems). Phenotypic Characterization The capability of the probable novel species (P5b, PAb, M30a, 56b, ELS-4, M9cR1, and T25a) and the reference strains (Rhizobium phaseoli NBRC 14785T; Rhizobium leguminosarum IAM 12609T) of rhizobia to utilize various substrates was assessed using Biolog GN2 Microplate (Biolog) following the manufacturer’s instructions but with some modifications. The cells of the rhizobial species previously grown on modified Beringer’s medium (MBM) (Beringer 1974; Jarvis and Tighe 1994) were washed twice aseptically with sterile 0.85% saline solution and incubated in the microplates for 72 h at 28 °C. The temperature range for growth was determined at incubation temperatures of 4, 10, 17, 25, 28, 30, 37 and 45 °C. The pH range for growth was 4.0 to 10.5 with 0.5 pH unit increments using filter-sterilized (0.45 µm, Advantec) MBM broth. Phylogenetic Tree Construction The 16S rRNA gene sequences were edited and aligned in BioEdit V7.0.5.3 (Hall 1999) and compared with the sequences obtained from GenBank (National Center for Biotechnology


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Information 2016) and DNA Data Bank of Japan (DDBJ 2016). Multiple alignments of the sequences were carried out in Clustal X V1.83 (Thompson et al. 1997). Alignment gaps and ambiguous bases were not taken into consideration when the bases of 16S rRNA gene nucleotides were compared. The phylogenetic and molecular evolutionary analyses were determined using the Mega software version 4 (Tamura et al. 2007). Phylogenetic trees were constructed using the neighbor-joining (NJ) method (Saitou and Nei 1987) utilizing the Kimura 2parameter (Kimura 1983). Evolutionary distances were computed using the Kimura 2-parameter with bootstrap values based on 1,000 replications selected in the program.

RESULTS AND DISCUSSION A total of 39 different species of leguminous and nodule-forming plants were collected from different places in the Philippines. The collected plants belonging to the family Leguminosae were represented by the subfamilies Mimosoideae (5 species) and Papilionoideae (34 species). From these leguminous plants, 364 bacterial isolates (data not shown) were isolated from their root nodules. The axenic bacterial isolates were partially sequenced using the universal primers (8F and 1510R) for the 16S rRNA gene, and the sequences ranging from 620 to 710 bp were compared with the NCBI GeneBank. BLAST similarities that scored the highest Max Score, 100% query coverage, E value of 0.0 and 99–100% identity were selected as the probable identities of the isolates. Isolates showing equal to and/or less than 98% BLAST similarities were further studied and subjected to phenotypic characterization. BLAST analyses showed that not all of the 364 bacterial isolates are members of the rhizobia group. The persistence of bacteria belonging to class Gammaproteobacteria – Pantoea, Stenotrophomonas, Enterobacter, and Pseudomonas that were inadvertently isolated and sequenced were known to have endophytic associations with some plant species providing benefits on plant growth (Lambert et al. 1990; Vega et al. 2005; Idris et al. 2009; Delétoile et al. 2009; Ramos et al. 2011). For example, the presence of Enterobacter cloacae could be beneficial in the rhizosphere and nodulation of legumes by rhizobia. Xu et al. (1994) reported that 106


strains of Enterobacter cloacae, which were isolated from the surface of root nodules, stimulated the nodulation of Astragalus sinicus cv. Japan (rengesou) induced by Rhizobium huakuii bv. renge. Unwanted microorganisms such as molds and yeast were suppressed by supplementing the medium with an antifungal antibiotic KabicidinTM. The variable antibiotic resistance of rhizobia (Xavier et al. 1998) is the main reason why its isolation medium was not amended with bactericidal agents. Despite this limitation, majority (92%) of the bacterial isolates and the six probable strains were identified as within the order Rhizobiales of the class Alphaproteobacteria. Few of the isolates belong to either class Betaproteobacteria or Gammaproteobacteria. As of March 2016, the nitrogen-fixing and nodulating, considered as “true” rhizobia, consisted of 99 bacterial species found in 14 genera, namely Azorhizobium, Bradyrhizobium, Burkholderia, Cupriavidus, Devosia, Ensifer (formerly Sinorhizobium), Mesorhizobium, Methylobacterium, Microvirga, Ochrobactrum, Phyllobacterium, Rhizobium, Shinella (Weir 2016) and Aminobacter (Maynaud et al. 2012). In this study, partial 16S rRNA gene sequencing revealed that there are nine notable bacterial genera namely, Rhizobium, Bradyrhizobium, Ensifer, Mesorhizobium, Burkholderia, Herbaspirillum, Pleomorphomonas, Crabtreella, and Labrys, isolated from the root nodules of leguminous plants (Table 1). The genus Rhizobium is the most heterogeneous group of the family Rhizobiaceae, and comprises most of the species which form symbiotic relationships with leguminous plants, and also includes plant pathogenic bacteria (Weir 2016). Likewise, the genus Rhizobium is found to have a wide host range. It was isolated in 85% (33/39) of the legumes used in this study namely, L. leucocephala (Lam.) de Witt., M. diplotricha Wright in Sauvalle, M. pudica L. Mimosa sp., Abrus pecatorius L., A. indica L., A. sensitiva P. Beauv., Alysicarpus nummularifolios DC., A. vaginalis (L.) A. DC., A. hypogaea L., C. cajan (L.) Millsp., C. carabaeoides (L.) Thouars, Clitoria ternatea L., Crotalaria incana L., Derris elliptica Benth., Desmodium scorpiurus (Sw.) Desv., Desmodium sp., D. styracifolium, D. triflorum (L.) DC., Glycine tomentosa (Benth) Benth., Macroptilium lathyroides Urb., Phaseolus vulgaris L., Pisum sativum L., Psophocarpus tetragonolobus DC., P. indicus Willd., Pueraria phaseoloides Benth., Pueraria sp., Samanea saman Merr., Senna sp., Sesbania sesban



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L., Vigna minima (Roxb.) Ohwi and Ohashi, Vigna radiata (L.) R. Wilczek, V. sinensis (L.) Savi ex Hassk and V. unguiculata (L.) Walp) obtained from different places in the Philippines. About 76% (25/33) of these legumes are associated with a single species of Rhizobium in which R. etli is the dominant species isolated from 12 leguminous plants. The remaining legumes (~24%) are associated with different species of Rhizobium and/or with different genera such as Bradyrhizobium and Ensifer. R. rhizogenes (formerly Agrobacterium rhizogenes) was also isolated from legumes solely or with other known “true” rhizobia. It is a plant pathogen causing hairy root disease or tumors in plants. However, it can induce nodulation under natural environmental conditions if it has the symbiotic plasmids (pSym) containing the nod (nodulating) and nif (nitrogen-fixing) genes (Velasquez et al. 2005). The genus Bradyrhizobium (62%) was isolated from the root nodules of A. lebbeck (L.) Benth., L. leucocephala (Lam.) de Witt., A. vaginalis (L.) A. DC., A. hypogaea L., C. cajan (L.) Millsp., C. mucunoides Desv., C. ternatea L., C. incana L., C. pallida Aiton, Derris elliptica Benth., D. scorpiurus (Sw.) Desv., Desmodium sp., D. triflorum (L.) DC., Dolichos falcatus Klein ex Willd., G. max Merr., G. tomentosa Benth., M. lathyroides Urb., P. tetragonolobus DC., P. indicus Willd., P. phaseoloides Benth., Pueraria sp., S. saman Merr., V. radiata (L.) R. Wilczek, and V. unguiculata (L.) Walp. B. elkanii is the predominant species of Bradyrhizobium because it is associated with the 15 aforementioned species of leguminous plants. This result suggests that B. elkanii has a wider plant host range compared with other species of Bradyrhizobium such as B. yuanmingense, B. japonicum and B. liaoningense. A number of leguminous plants (18%) are associated with the genus Ensifer. The nodules of Pterocarpus indicus Willd. and L. leucocephala (Lam.) de Witt. obtained from Laguna were found to contain two different species of Ensifer (E. adhaerens and E. arboris). On the other hand, L. leucocephala (Lam.) de Witt., which was obtained from Mindanao, was found to contain E. freedii, E. mexicanus and E. saheli. E. saheli was the only Ensifer species found in L. leucocephala (Lam.) de Witt., which was obtained from Benguet, Philippines. Moreover, E. mexicanus was also isolated from the root nodules of Alysicarpus nummularifolius DC. and


Enterolobium saman Prain ex King, obtained from Occidental Mindoro and Laguna, respectively. Other host plants where Ensifer species were isolated include A. hypogaea L., Pueraria sp., and S. saman Merr. These findings indicate that several species of nodule-forming plants that are located in different regions of the Philippines are hosted by different species of Ensifer. Few species belonging to the genus Mesorhizobium (10%) (family Phyllobacteriaceae) and Burkholderia (3%) (family Burkholderiaceae) were isolated from the leguminous plants. This result could be attributed to the limited host range of the species. Isolated bacterial strains with 99% similarities to Pleomorphomonas oryzae, Crabtreella saccharophila, and Herbaspirillum seropedicae that were obtained from the root nodules of M. lathyroides Urb, E. saman Prain ex King and D. scorpiurus (Sw.) Desv., respectively, are reported as free living nitrogenfixers and do not possess the nodulating gene (nodA) (Baldani et al. 1986; Elbeltagy et al. 2001; Valverde et al. 2003; Xie and Yokota 2005a, 2005b, 2006; Madhaiyan et al. 2013). The bacterial isolates from root nodules of P. indicus Willd. and V. radiata (L.) R. Wilczek, obtained from Isabela and Kalinga, respectively, shared 99% of 16S rRNA partial gene sequence similarities with L. neptuniae. Although originally isolated from the aquatic legume Neptunia oleracea, this legume does not induce nodulation to its own host and Macroptilium atropurpureum (Chou et al. 2007). Among the collected leguminous plants, L. leucocephala and D. scorpiurus were found to host several genera of rhizobia such as Rhizobium, Bradyrhizobium, Mesorhizobium and Ensifer from different regions in the Philippines. Furthermore, three of the six probable novel isolates found in this study were obtained from three different species of herbaceous legume of the genus Desmodium (D. scorpiurus, D. triflorum and D. stryracifolium). On the other hand, leguminous plants such as Sesbania sesban, and different species of Mimosa, hosted only the species of Rhizobium. According to Peix et al. (2015), the success of the symbiosis between the rhizobia and the legume depends on the specificity, infectivity and effectiveness of rhizobia and follows a series of steps that could yield the expression of different molecules by the bacterium, the host plant or both. However, Mimosa spp. in Brazil, South Africa, Southern China, New Caledonia and Taiwan


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are nodulated mostly by Betaproteobacteria in the genera of Burkholderia and Cupriavidus (Chen et al. 2001 and 2005; Liu et al. 2012; Gyaneshwar et al. 2011; Klonowska et al. 2012). Gyaneshwar et al. (2011) suggested that genetic and physiological factors determine the apparent preference for nodulation by Betaproteobacteria. The physical environment may also play an important role in the distribution of Burkholderia because acidic soils with low levels of inorganic nitrogen will likely favor symbiosis of Burkholderia with Mimosa spp. (Garau et al. 2009). The presence of nitrate (0.5 mM KNO3) has been found to effectively reduce the competitive domination (nodule occupation) of Burkholderia mimosarum PAS44 on Mimosa spp. when co-inoculated with Cupriavidus taiwanensis LMG 19424 (Elliott et al. 2009). Comparative 16S rRNA gene-sequence (1370 bp) analyses revealed that the six probable novel species have 89.5–98.8% sequence similarities with the type species of Rhizobium. Moreover, phylogenetic analysis (Fig. 1) indicated that they belong in the order Rhizobiales more specifically as members of the family Rhizobiaceae. Strains designated as P5b, M30a, T25a, M9cR1, 56b, P-Ab and ELS-4, which were isolated from the root nodules of P. indicus Willd., A. indica L., V. radiata (L.) R. Wilczeck., C. cajan (L.) Millsp., D. scorpiurus (Sw.) Desv., D. triflorum (L.) DC., and D. stryracifolium Merr., respectively, were obtained from different parts of the Philippines (Tables 1 and 2). Hence, the 16S rRNA gene sequence similarity between strains M9CR1 and T25a is 99.9%. This similarity value is high enough to suggest that both strains belong to a probable single novel strain of Rhizobium. This value is also supported in the constructed phylogenetic tree wherein both strains are positioned in the same clade nearest to the branch of Rhizobium undicola LMG 11875T. The phenotypic characteristics of the probable novel species of rhizobia and the reference strains (Rhizobium phaseoli NBRC 14785T and Rhizobium leguminosarum IAM 12609T) are presented in Table 2. The probable novel species have a wider pH range for growth compared with the reference strains of rhizobia. Among the probable novel species, only strain P5b is capable of growing in pH 4.5. However, reference strains have a wider temperature range (10– 35 °C) for growth compared with the probable novel species. Strain P5b has the narrowest temperature range (25–30 °C) for growth. All of the probable novel species of rhizobia and the reference strains have different phenotypic 112


characteristics in terms of their utilization of lipids, amino acids and carbohydrates (Table 2). The exact similarities of the phenotypic characteristics of strains M9cR1 and T25a further suggest that they represent only a single probable novel strain of Rhizobium.

SUMMARY AND CONCLUSION Partial 16S rRNA gene sequencing revealed that there are nine notable bacterial genera namely, Rhizobium, Bradyrhizobium, Ensifer, Mesorhizobium, Burkholderia, Herbaspirillum, Pleomorphomonas, Crabtreella, and Labrys, isolated from the root nodules of 39 different species of leguminous and nodule-forming plants collected from different places in the Philippines. Majority of these plants were associated with the genera of Rhizobium (85%) and Bradyrhizobium (62%) suggesting their wide host range. Among the collected leguminous plants, L. leucocephala and D. scorpiurus were found to host several genera of rhizobia such as Rhizobium, Bradyrhizobium, Mesorhizobium and Ensifer, which were isolated from their root nodules obtained from different regions in the Philippines. On the other hand, leguminous plants such as S. sesban, and different species of Mimosa hosted only the species of Rhizobium. On the basis of the 16S rRNA sequence similarities of 89.5–98.8% with the known type species of Rhizobium, phylogenetic analysis and the distinctive phenotypic characteristics indicate that strains designated as P5b, P-Ab, 56b, M30a, ELS-4, M9cR1 and T25a are probable novel species of Rhizobium belonging to the heterogeneous members of the family Rhizobiaceae. The isolation of these probable novel species is a strong indication that there is still a chance to obtain novel species of rhizobia owing to their heterogeneity in nature.

ACKNOWLEDGMENTS We would like to thank the Ministry of Education, Culture, Sports, Science and Technology of Japan for the scholarship grant to the senior author, Ms. Karen S. Bautista for the valuable suggestions, and the Biodiversity Management Bureau, Department of Environment and Natural Resources, Republic of the Philippines (formerly the Protected Areas and Wildlife Bureau) for the Gratuitous Permit.




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