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Nodulation and plant-growth promotion by methylotrophic bacteria isolated from tropical legumes
ARTICLE IN PRESS Microbiological Research 164 (2009) 114—120
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Nodulation and plant-growth promotion by methylotrophic bacteria isolated from tropical legumes M. Madhaiyana,, S. Poonguzhalia, M. Senthilkumarb, SP. Sundaramb, Tongmin Saa a
Department of Agricultural Chemistry, Chungbuk National University, 48 Gaesin-dong, Cheongju, Chungbuk 361-763, Republic of Korea b Department of Agricultural Microbiology, Tamilnadu Agricultural University, Coimbatore 641 003, Tamilnadu, India Received 12 July 2006; received in revised form 26 August 2006; accepted 28 August 2006
KEYWORDS Macroptilum atropurpureum; Nodulation; nodA gene; mxaF gene; Methylobacterium sp.
Summary The nitrogen fixing methylotrophic bacteria were isolated from the nodules of tropical legumes. Two isolates CMCJ317 and CMSA322 isolated from Crotalaria juncea and Sesbania aculeata possessing high nitrogenase activities under pure culture conditions and able to form nodules under inoculated conditions were further characterized. The biochemical characteristics revealed their close relationship with Methylobacterium nodulans type strain ORS2060. The PCR amplification of nodA and mxaF genes showed the expected 584 and 555 bp products, respectively, similar to M. nodulans ORS2060 and digestion with restriction enzymes revealed that the two isolates differed. The strains showed significantly higher nitrogenase activity and also improved nodulation and shoot nitrogen of the plants when inoculated to Macroptilum atropurpureum. CMCJ317 and CMSA322 formed nodules on C. juncea and M. atropurpureum under green house conditions and also significantly increased the nitrogen concentration in shoots. These findings show that the ability to establish symbiosis with legumes is more widespread in Methylobacterium. & 2006 Elsevier GmbH. All rights reserved.
Introduction Corresponding author. Tel.: +82 43 261 2561;
fax: +82 43 271 5921. E-mail address: [email protected] (M. Madhaiyan).
The success of legumes is largely debted to their symbiotic relationship with specific nitrogen fixing bacteria known as rhizobia, a name that portray root and stem nodulating bacteria. The symbiosis is
0944-5013/$ - see front matter & 2006 Elsevier GmbH. All rights reserved. doi:10.1016/j.micres.2006.08.009
ARTICLE IN PRESS Nodulation and plant-growth promotion methylotrophic bacteria manifested by the development of nodules on the roots of legumes acting as factories of nitrogen fixation. Phylogenetically rhizobia are very diverse representing several lineages. Though nitrogen fixation by other organisms (Ex. Herbaspirillum) (Baldani et al., 1996) of b-Proteobacteria has been documented, none of these harbored nodulating genes for establishing symbiotic relations. The taxonomic classifications so far have placed rhizobia within three different lineages in a-subclass of Proteobacteria (van Berkum and Eardly, 1998; Young, 1996). The first large branch comprises of the genera Rhizobium, Sinorhizobium, Mesorhizobium, and Allorhizobium, the second branch with Bradyrhizobium and the third branch includes Azorhizobium. Chen et al. (2001) described the nodulating species from Mimosa pudica as Ralstonia taiwanensis, the first formally described b-Proteobacterium capable of N fixation and root nodule formation. In the same year, Moulin et al. (2001) reported two strains of Burkholderia with an ability of nodulating Aspalathus carnosa and Machaerium lunatum, respectively, which were later designated as Burkholderia tuberum and Burkholderia phymatum (Vandamme et al., 2002). Barrett and Parker (2005) reported the predominance of four genotypes of Burkholderia sp. with an ability to form nodules on mimosoid legumes. The above results have changed the long held dogma that only bacteria of a-subdivision are able to nodulate legumes and the terms a-rhizobia and b-rhizobia could be used to distinguish the phylogenetic lineages of nodule symbiotic bacteria (Moulin et al., 2001) extending the diversity of rhizobia. Sy et al. (2001) described a fourth rhizobial branch within a-subclass of Proteobacteria belonging to Methylobacterium. Methylobacterium spp. are a group of bacteria known as pink pigmented facultative methylotrophs or PPFMs, strict aerobic Gram negative rods, able to grow on C1 compounds such as methanol, formate, formaldehyde, and methylamine as well as on a variety of C2, C3 and C4 compounds (Lidstrom, 2001). Methylobacterium strains are ubiquitous and reported from various plants and they are able to influence plant growth promotion through plant growth regulators (Ivanova et al., 2001; Madhaiyan et al., 2005; Omer et al., 2004) and induced systemic resistance against diseases (Madhaiyan et al., 2004). Though Methylobacterium is reported from various tissues and crops, their symbiotic association with plants was first reported in Methylobacterium nodulans (Jourand et al., 2004). The 16S rRNA gene based phylogenetic analysis of this non-pigmented bacterium from the nodules of three Crotalaria
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species, C. glaucoides, C. perrottetii and C. podocarpa placed it in the genus Methylobacterium (Sy et al., 2001). Subsequently, Jaftha et al. (2002) characterized the nitrogen fixing isolates from the root nodules of a shrubby legume Lotononsis bainesii as pigmented methylotrophic bacteria with its closest phylogenetic neighbor as M. nodulans showing a sequence homology to 98%. In this study, we investigated the survival of Methylobacterium as symbionts of tropical legumes, and we now report the presence of two Methylobacterium strains as nodule symbionts. This non-pigmented methylotrophic isolates were able to form nodules on Macroptilum atropurpureum and Crotalaria juncea, two crops so far not reported with nodule forming Methylobacterium, formed a homogenous group of closely related cluster with M. nodulans, widely separating from other diazotrophs.
Materials and methods Bacterial strains, isolation and culture conditions Bacteria were isolated from the root nodules of 14 different legumes from diverse regions of Coimbatore and Dharmapuri districts of Tamilnadu, India. Root nodules were surface sterilized with 0.1% mercuric chloride and 70% alcohol, serially washed in sterile distilled water and suspensions of crushed nodules were plated on minimum mineral medium M72 (Belgian Co-ordinated Collection of Micro-organisms/Laboratorium voor Microbiologie 1998, Bacteria Catalogue, Universiteit Gent, Ghent, Belgium) with 0.5% methanol. After incubating the plates at 28 1C for 3–5 days, single colonies were isolated and purified. The isolates, plant species and the location of sampling are listed in Table 1. M. nodulans ORS2060 and Methylobacterium extorquens AM1 used in this study were kindly provided by Catherine Boivin-Masson, (LSTM, France) and M.A. Holland (Salisbury University, USA). The Methylobacterium strains were routinely grown in M72 medium with 0.5% methanol.
Nitrogenase activity of free living cultures The nitrogenase activity of the isolates was determined through acetylene reduction assay (ARA) according to Hardy et al. (1968). In 100 ml vials containing 20 ml liquid cultures, acetylene was injected to give a 10 KPa and culture was inoculated at 2% level (2 108 cfu ml1). The vials were incubated at 30 1C for 1 h and measured for
ARTICLE IN PRESS 116 Table 1.
M. Madhaiyan et al. Methylotrophic strains isolated from nodules of different tropical legumes
Strain(s) (no. of isolates)
Host plant
Geographical origin
CMPV300 (1) CMCT301 and CMCT302 (2) CMAH303 (1) CMVU304 (1) CMVR305 and CMVR306 (2) CMGM307 and CMGM308 (2) CMVM309 (1) CMMA310 (1)
Field bean (Phaseolus vulgaris L.) Blue pea (Clitoria ternatea L.)
Coimbatorea Coimbatore
Ground nut (Arachis hypogaea L.) Cowpea (Vigna unguigulata (L.) Walp.) Mungbean (Vigna radiatus L.)
Coimbatore Dharmapurib Coimbatore
Soybean (Glycine max (L.) Merr.)
Coimbatore
Blackgram (Vigna mungo L.) Sirato (Macroptilium atropurpureum (DC.) Urb.) Chick pea (Cicer arietinum L.)
Coimbatore Green house, TNAU Coimbatore
Hyacinth bean (Dolichos lablab L.) Garden pea (Pisum sativum L.) Sunnhemp (Crotalaria juncea L.)
Dharmapuri Dharmapuri Coimbatore
Sesbania (Sesbania rostrata Brem. and Obrem.) Dhaincha (Sesbania aculeata Pers.)
Coimbatore Coimbatore
CMCA311 and CMCA312 (2) CMDL313 (1) CMPS314 (1) CMCJ315 to CMCJ319 (5) CMSR320 (1) CMSA321 and CMSA322 (2)
Nitrogenase activity (nmol C2H4 h1 mg1 protein) 9.2170.78 7.2571.25 and 6.2370.26 5.2370.35 25.1773.35 2.1670.17 and 35.7175.25 22.3671.25 and 6.2370.26 11.4571.25 15.6272.61 14.2573.25 and 62.5479.11 4.1270.61 6.3271.25 20.1272.36–162.7278.51 45.1975.42 5.2370.25 and 120.4876.52
a
Samples were collected from Tamilnadu Agricultural University (TNAU) experimental farm, Coimbatore, Tamilnadu, India. Samples were collected from farmer’s field, Dharmapuri, Tamilnadu, India. Each value represents mean7SE of three replicates per treatment.
b
ethylene production. The protein concentration was determined by a modified Lowry method (Lowry et al., 1951) with bovine serum albumin (BSA) as standards. The ethylene produced was measured using a Systronics Gas Chromatograph with a Porapak-Q-column and flame ionization detector connected to a chromatograph data computer system.
Methanol utilization tests and plant inoculation assays for selected isolates Screened with ARA, two isolates CMCJ317 and CMSA322 were selected for further experiments. Methanol utilization by the isolates was checked according to Sy et al. (2001) in M72 medium. The bacterial suspensions were diluted in M72 medium (optical density (OD) ¼ 0.05), and added with one of the following compounds; methanol (MeOH) (10, 50, 100, or 500 mM), pyruvate (10 mM), or succinate (10 mM). Growth was monitored by measuring OD at 620 nm. M. atropurpureum, a tropical legume capable of producing nodules with diverse nodule bacteria was selected for initial tests of nodulation and for
measuring the nitrogenase activity of the isolates under inoculated conditions. To perform the nodulation tests, seeds scarified with concentrated sulfuric acid (10 min) and surface disinfected with 3% sodium hypochlorite (NaOCl) for 10 min were germinated in moist germination papers placed in Petri dishes incubated under dark conditions. The germinated seeds were then transferred to sterilized growth pouches (16 18 cm) containing 20 ml of nitrogen free nutrient solution (Vincent, 1970). On day 2, the root surface was inoculated with bacterial suspension (1 ml each, approximately 108 cells). Effectiveness of the inoculation was observed by visual interpretation of nodules formed at day 30 after inoculation. For nitrogenase assays, M. atropurpureum seedlings were planted in 50 ml narrow-neck McCartney bottles (two seedlings/ bottle) containing 4 g sterile vermiculite (Kenyan exfoliated, vermiculite, average particle diameter 44 mm) and nitrogen free solution. The seedlings were inoculated with 1 ml of a bacterial suspension (1 106 cfu ml1) grown for 40 h at 30 1C in N-free medium at 3–4 days of plant growth. The ARA of the plants was measured after 2 weeks as described earlier. For both experiments, uninoculated plants served as control and plants were grown in a light
ARTICLE IN PRESS Nodulation and plant-growth promotion methylotrophic bacteria chamber with a light-dark cycle of 14 h (27 1C)/10 h (22 1C).
Characterization of the rhizobial isolates Assimilation of different carbon sources were tested on AMS medium (Patt et al., 1976) containing plates supplemented separately with the various filter sterilized carbon sources, (0.1% w/v) and in case of volatile organic compounds, ethanol 0.5% (v/v) and methanol 0.5% (v/v) were used. Methane was injected in the headspace (25% v/v of methane gas) of liquid media containing vials (Green and Bousifield, 1982). Bacterial suspension prepared from a 48 h old culture was used as the inoculum. Inoculated plates and vials were incubated at 30 1C and examined after 3 days. For methane utilization, bacterial growth was determined by measuring the OD at 660 nm with a spectrophotometer after incubation. Polymerised chain reaction (PCR) amplifications were performed to detect the presence of nodulation genes (nodA) and methanol dehydrogenase (MDH) genes (mxaF) using the primer pairs nodAfbrad (50 -GTYGAGTGGAGSSTKCGCTGGG-30 ), nodArbrad (50 -TCACARCTCKGGCCCGTTCGG-30 ) and f1003 (50 -GCGGCACCAACTGGGGCTGGT-30 ), r1561 (50 GGGCAGCATGAAGGGCTCCC-30 ), respectively, according to Sy et al. (2001). PCR amplifications were performed with an Eppendorf Mastercycler gradient (PCR Thermocycler, Brinkmann Instruments Inc., Westbury, NY) in a total volume 25-ml reaction mixture containing 50 ng of genomic DNA, each deoxynucleotide triphosphate (200 mM), 0.8 mM each primers MgCl2 (1.5 mM), 1.25 U of Taq DNA polymerase (Bangalore Genei, Bangalore, India), and the buffer supplied with the enzyme. The PCR products were analyzed by horizontal agarose gel electrophoresis using 1.5% agarose (Bangalore Genei, India).
Nodulation and plant growth promotion in green house The symbiotic and plant growth promoting properties of the two methylotrophic rhizobial isolates were studied in M. atropurpureum and C. juncea under greenhouse conditions. Seeds were scarified, surface sterilized and pre-germinated in moist germination paper as mentioned earlier. Germinated seedlings inoculated with bacterial suspensions (108 cells ml1) were grown in pots filled with sterilized soil mix and watered with sterile distilled water as required. The pots were arranged in a completely randomized design with
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three replications for each treatment. At harvest, number of nodules, nodule dry weight, shoot dry weight, and shoot nitrogen content were recorded.
Statistical analysis With the design being completely randomized, the mean, standard error and analysis of variance were calculated, and means were separated by LSD using the SAS package, Version 9.1, SAS Institute Inc., and Cary, NC, USA.
Results and discussion Isolation of Methylobacterium nodule symbionts Twenty-three isolates of methylotrophic rhizobia were recovered from the nodulating legumes of tropic regions on M-72 media with 0.5% methanol. The isolates possessed nitrogenase activity measured through ARA ranging from 2.1670.17 to 162.7276.52 nmol C2H4 h1 mg1 protein. The methylotrophic strains CMCJ317 and CMSA322 from the root nodules of C. juncea and Sesbania aculeate, respectively recorded higher nitrogenase activities of 162.7278.51 and 120.4876.52 nmol C2H4 h1 mg1 protein (Table 1) and their nodulation and methylotrophic characteristics were studied. The strains were grown on methanol, purified as a single colony and inoculated onto M. atropurpureum for testing nodulation. The bacteria formed nitrogen-fixing nodules and single colonies reisolated from the nodules were able to grow on methanol. The results clearly confirmed that the strains exhibited nodulation and methylotrophic properties. Methylotrophic rhizobial strains CMCJ317 and CMSA322 when inoculated on to M. atropurpureum, recorded significantly higher nitrogenase activity of 2.8670.27 and 2.7970.12 mmol C2H4 h1 plant1 compared to control (1.0270.04 mmol C2H4 h1 plant1).
Characteristics of methylotrophic rhizobia The isolates CMCJ317 and CMSA322 were Gram negative cocci occurring singly or in rosettes, motile and often contained poly-b-hydroxy butyrate inclusions. Colonies are non-pigmented, smooth, shiny, raised with regular margins and 0.3–0.8 mm in diameter after 96 h at 30 1C. Both the isolates were catalase and oxidase positive, and negative for indole methyl red and Voges Proskauer tests. They were negative for hydrogen sulfide
ARTICLE IN PRESS 118
M. Madhaiyan et al.
production and could not hydrolyze starch, casein and gelatin. The isolates were compared with that of M. nodulans ORS2060 and M. extorquens AM1. The characteristics of the isolates were more related to M. nodulans ORS2060. M. nodulans ORS2060 and CMCJ317, CMSA322 utilized the carbon sources L-arabinose, L-aspartate, citrate, betaine, acetate, and tartarate. CMCJ317 and CMSA322 also utilized dichloromethane and grow on nutrient agar but not utilized methane that differed from M. nodulans ORS2060. CMCJ317 and CMSA322 were further examined for the presence of mxaF and nodA genes using M. nodulans ORS2060, as a reference. Gram negative Methylobacterium spp. oxidizes methanol to formaldehyde via a key periplasmic enzyme MDH and the use of MDH structural gene mxaF as a functional probe for methylotrophs is well explained (McDonald and Murrell, 1997). Therefore, to evaluate the presence of methanol oxidation genes PCR amplification was performed with non-degenerate primers f1003 and r1561, the primers designed corresponding to highly conserved regions of mxaF gene sequences (McDonald and Murrell, 1997). In our study, the PCR conducted for all the twenty three strains produced an amplification product of the expected size 555 bp, except for four strains viz., CMAH303, CMVR306, CMCA312 and CMDL313 indicating that the selected strains CMCJ317 and CMSA322 contain the structural gene mxaF, required for methanol oxidation. No amplification of these primers could be obtained in rhizobial strains from our laboratory collection (results not shown) and the homologs while using other degenerate primers probably do not correspond to the bonafide mxaF ortholog (Sy et al., 2001). Also CMCJ317 and CMSA322 showed positive amplification of 584 bp PCR products similar to M. nodulans ORS2060 for the nodA gene using the selected primers nodAfbrad and nodArbrad defined from conserved parts of nodA sequences (Sy et al., 2001). Bacterial nodulation genes have been shown to play a central role in the molecular dialogue between the plant and the bacterium leading to plant recognition, Table 2.
infection and nodulation (Denarie et al., 1996; Schultze and Kondorosi, 1998). The PCR amplification of mxaF and nodA genes in CMCJ317 and CMSA322 resulted in products similar to the reference strain M. nodulans ORS2060. The analysis of the fragments of the 16S rDNA digested with the restriction enzyme BclI by restriction fragment length polymorphic (RFLP) analysis resulted in 4 distinct bands. The bands obtained with CMSA322 showed close similarity to the reference strain M. nodulans ORS2060 where as the products of the strain CMCJ317 were distinct. This reveals that the two strains differ within and also from the reference strain (results not shown).
Nodulation and plant growth promotion by methylobacterial isolates M. nodulans ORS2060 is a well-characterized strain with respect to its methylotrophic and nodulating characteristics and hence in this study we tested the effects of inoculating CMCJ317 and CMSA322 on plant growth under pot culture conditions with the legumes M. atropurpureum and C. juncea. Both CMCJ317 and CMSA322 nodulated and improved the plant growth of C. juncea. Strain CMSA322 formed pink color indeterminate nodules and the number of nodules formed, shoot nitrogen percentage was higher compared to CMCJ317 (Table 2). The efficiency of nodulation of the two strains was greater in M. atropurpureum than in C. juncea. However, the strain CMSA322 formed more number of nodules and increased shoot nitrogen and dry weight higher than CMCJ317 similar to C. juncea (Fig. 1). The rhizobial inoculations significantly increased the shoot nitrogen concentration in the plants when compared to control. These results clearly suggest that CMCJ317 and CMSA322 exhibit methylotrophy and form symbiotic association with legumes improving the plant growth characteristics. Recent discoveries proved that bacteria from outside Rhizobiaceae family also can nodulate legumes and fix nitrogen symbiotically
Growth, nodulation and nitrogen fixation of Crotalaria juncea infected by Methylobacterium isolates
Treatments
Shoot length (cm)
Root length (cm)
Control CMCJ317 CMSA322 LSD (Pp0.05)
125.3275.96b 21.1271.80a 139.4178.32a 22.6373.83a 140.85710.31a 24.0371.75a 8.55 4.65
Shoot dry weight (g)
Shoot N (% dry weight)
Nodules per plant (#)
Nodules dry weight (mg)
25.1271.80a 26.8273.36a 26.971.67a 3.67
0.8270.05c 2.4670.06b 3.2570.14a 0.21
— 56.3275.38b 61.8274.52a 3.73
— 11.2670.85b 12.3671.08a 0.99
Each value represents mean7SE of three replicates per treatment. In the same column, significant differences at Pp0.05 levels are indicated by different letters. Data followed by same letter in the same column are not significantly different from each other.
ARTICLE IN PRESS Nodulation and plant-growth promotion methylotrophic bacteria
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Figure 1. Growth, nodulation and nitrogen fixation of Macroptilum atropurpureum infected by methylotrophic rhizobia. (a) Shoot dry weight, (b) nodule number per plant, (c) shoot nitrogen as percentage of dry weight, and (d) nodule dry weight per plant. Each value represents mean7SD of three replicates.
and one such is Methylobacterium that formed a fourth branch within the a-Proteobacteria. The results clearly reveal that the two root nodule isolates from tropical legumes represent two distinct strains of Methylobacterium, with M. nodulans as their closest neighbour. However, conclusive evidences on their identification could not be reached since molecular characterization such as 16S rDNA sequencing was not performed. This study confirms and extends the capacity of Methylobacterium to effectively nodulate legume plants thus proving the polyphyletic origin of rhizobia within a-Proteobacteria.
Acknowledgements We thank Catherine Boivin-Masson, LSTM, UMR 113 IRD/INRA/AGRO-M/CIRAD, 34398 Montpellier Cedex 5, France for the valuable suggestions and provision of strain. M.M and S.P are indebted to the Korea Research Foundation, Republic of Korea for a
position as invited foreign scientist and Ph.D. research grants, respectively.
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ARTICLE IN PRESS 120 Denarie, J., Debelle, F., Prome, J.C., 1996. Rhizobium lipo-chitooligosaccharide nodulation factors: signaling molecules mediating recognition and morphogenesis. Annu. Rev. Biochem. 65, 503–535. Green, P.N., Bousifield, I.J., 1982. A taxonomic study of some Gram-negative facultatively methylotrophic bacteria. J. Gen. Microbiol. 128, 623–638. Hardy, R.W.F., Holsten, R.D., Jackson, E.K., Burns, R.C., 1968. The acetylene ethylene assay for N2 fixation. Laboratory and field evaluation. Plant Physiol. 43, 1185–1207. Ivanova, E.G., Doronina, N.V., Trotsenko, Y.A., 2001. Aerobic methylobacteria are capable of synthesizing auxins. Microbiology 70, 392–397. Jaftha, J.B., Strijdom, B.W., Steyn, P.L., 2002. Characterization of pigmented methylotrophic bacteria which nodulate Lotononis bainesii. Syst. Appl. Microbiol. 25, 440–449. Jourand, P., Giraud, E., Bena, G., Sy, A., Willems, A., Gillis, M., Dreyfus, B., de Lajudie, P., 2004. Methylobacterium nodulans sp. nov., for a group of aerobic, facultatively methylotrophic, legume root-noduleforming and nitrogen-fixing bacteria. Int. J. Syst. Evol. Microbiol. 54, 2269–2273. Lidstrom, M.E., 2001. The aerobic methylotrophic bacteria. In: Dworkin, M. (Ed.), The Prokaryotes. Springer, Berlin, Heidelberg, New York, Tokyo, pp. 223–244. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with folin-phenol reagent. J. Biol. Chem. 193, 265–275. Madhaiyan, M., Poonguzhali, S., Senthilkumar, M., Seshadri, S., Chung, H.K., Yang, J.C., Sundaram, S.P., Sa, T.M., 2004. Growth promotion and induction of systemic resistance in rice cultivar Co-47 (Oryza sativa L.) by Methylobacterium spp. Bot. Bull. Acad. Sin. 45, 315–324. Madhaiyan, M., Poonguzhali, S., Lee, H.S., Hari, K., Sundaram, S.P., Sa, T.M., 2005. Pink-pigmented facultative methylotrophic bacteria accelerate germination, growth and yield of sugarcane clone Co86032
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Nodulation and plant-growth promotion by methylotrophic bacteria isolated from tropical legumes M. Madhaiyana,, S. Poonguzhalia, M. Senthilkumarb, SP. Sundaramb, Tongmin Saa a
Department of Agricultural Chemistry, Chungbuk National University, 48 Gaesin-dong, Cheongju, Chungbuk 361-763, Republic of Korea b Department of Agricultural Microbiology, Tamilnadu Agricultural University, Coimbatore 641 003, Tamilnadu, India Received 12 July 2006; received in revised form 26 August 2006; accepted 28 August 2006
KEYWORDS Macroptilum atropurpureum; Nodulation; nodA gene; mxaF gene; Methylobacterium sp.
Summary The nitrogen fixing methylotrophic bacteria were isolated from the nodules of tropical legumes. Two isolates CMCJ317 and CMSA322 isolated from Crotalaria juncea and Sesbania aculeata possessing high nitrogenase activities under pure culture conditions and able to form nodules under inoculated conditions were further characterized. The biochemical characteristics revealed their close relationship with Methylobacterium nodulans type strain ORS2060. The PCR amplification of nodA and mxaF genes showed the expected 584 and 555 bp products, respectively, similar to M. nodulans ORS2060 and digestion with restriction enzymes revealed that the two isolates differed. The strains showed significantly higher nitrogenase activity and also improved nodulation and shoot nitrogen of the plants when inoculated to Macroptilum atropurpureum. CMCJ317 and CMSA322 formed nodules on C. juncea and M. atropurpureum under green house conditions and also significantly increased the nitrogen concentration in shoots. These findings show that the ability to establish symbiosis with legumes is more widespread in Methylobacterium. & 2006 Elsevier GmbH. All rights reserved.
Introduction Corresponding author. Tel.: +82 43 261 2561;
fax: +82 43 271 5921. E-mail address: [email protected] (M. Madhaiyan).
The success of legumes is largely debted to their symbiotic relationship with specific nitrogen fixing bacteria known as rhizobia, a name that portray root and stem nodulating bacteria. The symbiosis is
0944-5013/$ - see front matter & 2006 Elsevier GmbH. All rights reserved. doi:10.1016/j.micres.2006.08.009
ARTICLE IN PRESS Nodulation and plant-growth promotion methylotrophic bacteria manifested by the development of nodules on the roots of legumes acting as factories of nitrogen fixation. Phylogenetically rhizobia are very diverse representing several lineages. Though nitrogen fixation by other organisms (Ex. Herbaspirillum) (Baldani et al., 1996) of b-Proteobacteria has been documented, none of these harbored nodulating genes for establishing symbiotic relations. The taxonomic classifications so far have placed rhizobia within three different lineages in a-subclass of Proteobacteria (van Berkum and Eardly, 1998; Young, 1996). The first large branch comprises of the genera Rhizobium, Sinorhizobium, Mesorhizobium, and Allorhizobium, the second branch with Bradyrhizobium and the third branch includes Azorhizobium. Chen et al. (2001) described the nodulating species from Mimosa pudica as Ralstonia taiwanensis, the first formally described b-Proteobacterium capable of N fixation and root nodule formation. In the same year, Moulin et al. (2001) reported two strains of Burkholderia with an ability of nodulating Aspalathus carnosa and Machaerium lunatum, respectively, which were later designated as Burkholderia tuberum and Burkholderia phymatum (Vandamme et al., 2002). Barrett and Parker (2005) reported the predominance of four genotypes of Burkholderia sp. with an ability to form nodules on mimosoid legumes. The above results have changed the long held dogma that only bacteria of a-subdivision are able to nodulate legumes and the terms a-rhizobia and b-rhizobia could be used to distinguish the phylogenetic lineages of nodule symbiotic bacteria (Moulin et al., 2001) extending the diversity of rhizobia. Sy et al. (2001) described a fourth rhizobial branch within a-subclass of Proteobacteria belonging to Methylobacterium. Methylobacterium spp. are a group of bacteria known as pink pigmented facultative methylotrophs or PPFMs, strict aerobic Gram negative rods, able to grow on C1 compounds such as methanol, formate, formaldehyde, and methylamine as well as on a variety of C2, C3 and C4 compounds (Lidstrom, 2001). Methylobacterium strains are ubiquitous and reported from various plants and they are able to influence plant growth promotion through plant growth regulators (Ivanova et al., 2001; Madhaiyan et al., 2005; Omer et al., 2004) and induced systemic resistance against diseases (Madhaiyan et al., 2004). Though Methylobacterium is reported from various tissues and crops, their symbiotic association with plants was first reported in Methylobacterium nodulans (Jourand et al., 2004). The 16S rRNA gene based phylogenetic analysis of this non-pigmented bacterium from the nodules of three Crotalaria
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species, C. glaucoides, C. perrottetii and C. podocarpa placed it in the genus Methylobacterium (Sy et al., 2001). Subsequently, Jaftha et al. (2002) characterized the nitrogen fixing isolates from the root nodules of a shrubby legume Lotononsis bainesii as pigmented methylotrophic bacteria with its closest phylogenetic neighbor as M. nodulans showing a sequence homology to 98%. In this study, we investigated the survival of Methylobacterium as symbionts of tropical legumes, and we now report the presence of two Methylobacterium strains as nodule symbionts. This non-pigmented methylotrophic isolates were able to form nodules on Macroptilum atropurpureum and Crotalaria juncea, two crops so far not reported with nodule forming Methylobacterium, formed a homogenous group of closely related cluster with M. nodulans, widely separating from other diazotrophs.
Materials and methods Bacterial strains, isolation and culture conditions Bacteria were isolated from the root nodules of 14 different legumes from diverse regions of Coimbatore and Dharmapuri districts of Tamilnadu, India. Root nodules were surface sterilized with 0.1% mercuric chloride and 70% alcohol, serially washed in sterile distilled water and suspensions of crushed nodules were plated on minimum mineral medium M72 (Belgian Co-ordinated Collection of Micro-organisms/Laboratorium voor Microbiologie 1998, Bacteria Catalogue, Universiteit Gent, Ghent, Belgium) with 0.5% methanol. After incubating the plates at 28 1C for 3–5 days, single colonies were isolated and purified. The isolates, plant species and the location of sampling are listed in Table 1. M. nodulans ORS2060 and Methylobacterium extorquens AM1 used in this study were kindly provided by Catherine Boivin-Masson, (LSTM, France) and M.A. Holland (Salisbury University, USA). The Methylobacterium strains were routinely grown in M72 medium with 0.5% methanol.
Nitrogenase activity of free living cultures The nitrogenase activity of the isolates was determined through acetylene reduction assay (ARA) according to Hardy et al. (1968). In 100 ml vials containing 20 ml liquid cultures, acetylene was injected to give a 10 KPa and culture was inoculated at 2% level (2 108 cfu ml1). The vials were incubated at 30 1C for 1 h and measured for
ARTICLE IN PRESS 116 Table 1.
M. Madhaiyan et al. Methylotrophic strains isolated from nodules of different tropical legumes
Strain(s) (no. of isolates)
Host plant
Geographical origin
CMPV300 (1) CMCT301 and CMCT302 (2) CMAH303 (1) CMVU304 (1) CMVR305 and CMVR306 (2) CMGM307 and CMGM308 (2) CMVM309 (1) CMMA310 (1)
Field bean (Phaseolus vulgaris L.) Blue pea (Clitoria ternatea L.)
Coimbatorea Coimbatore
Ground nut (Arachis hypogaea L.) Cowpea (Vigna unguigulata (L.) Walp.) Mungbean (Vigna radiatus L.)
Coimbatore Dharmapurib Coimbatore
Soybean (Glycine max (L.) Merr.)
Coimbatore
Blackgram (Vigna mungo L.) Sirato (Macroptilium atropurpureum (DC.) Urb.) Chick pea (Cicer arietinum L.)
Coimbatore Green house, TNAU Coimbatore
Hyacinth bean (Dolichos lablab L.) Garden pea (Pisum sativum L.) Sunnhemp (Crotalaria juncea L.)
Dharmapuri Dharmapuri Coimbatore
Sesbania (Sesbania rostrata Brem. and Obrem.) Dhaincha (Sesbania aculeata Pers.)
Coimbatore Coimbatore
CMCA311 and CMCA312 (2) CMDL313 (1) CMPS314 (1) CMCJ315 to CMCJ319 (5) CMSR320 (1) CMSA321 and CMSA322 (2)
Nitrogenase activity (nmol C2H4 h1 mg1 protein) 9.2170.78 7.2571.25 and 6.2370.26 5.2370.35 25.1773.35 2.1670.17 and 35.7175.25 22.3671.25 and 6.2370.26 11.4571.25 15.6272.61 14.2573.25 and 62.5479.11 4.1270.61 6.3271.25 20.1272.36–162.7278.51 45.1975.42 5.2370.25 and 120.4876.52
a
Samples were collected from Tamilnadu Agricultural University (TNAU) experimental farm, Coimbatore, Tamilnadu, India. Samples were collected from farmer’s field, Dharmapuri, Tamilnadu, India. Each value represents mean7SE of three replicates per treatment.
b
ethylene production. The protein concentration was determined by a modified Lowry method (Lowry et al., 1951) with bovine serum albumin (BSA) as standards. The ethylene produced was measured using a Systronics Gas Chromatograph with a Porapak-Q-column and flame ionization detector connected to a chromatograph data computer system.
Methanol utilization tests and plant inoculation assays for selected isolates Screened with ARA, two isolates CMCJ317 and CMSA322 were selected for further experiments. Methanol utilization by the isolates was checked according to Sy et al. (2001) in M72 medium. The bacterial suspensions were diluted in M72 medium (optical density (OD) ¼ 0.05), and added with one of the following compounds; methanol (MeOH) (10, 50, 100, or 500 mM), pyruvate (10 mM), or succinate (10 mM). Growth was monitored by measuring OD at 620 nm. M. atropurpureum, a tropical legume capable of producing nodules with diverse nodule bacteria was selected for initial tests of nodulation and for
measuring the nitrogenase activity of the isolates under inoculated conditions. To perform the nodulation tests, seeds scarified with concentrated sulfuric acid (10 min) and surface disinfected with 3% sodium hypochlorite (NaOCl) for 10 min were germinated in moist germination papers placed in Petri dishes incubated under dark conditions. The germinated seeds were then transferred to sterilized growth pouches (16 18 cm) containing 20 ml of nitrogen free nutrient solution (Vincent, 1970). On day 2, the root surface was inoculated with bacterial suspension (1 ml each, approximately 108 cells). Effectiveness of the inoculation was observed by visual interpretation of nodules formed at day 30 after inoculation. For nitrogenase assays, M. atropurpureum seedlings were planted in 50 ml narrow-neck McCartney bottles (two seedlings/ bottle) containing 4 g sterile vermiculite (Kenyan exfoliated, vermiculite, average particle diameter 44 mm) and nitrogen free solution. The seedlings were inoculated with 1 ml of a bacterial suspension (1 106 cfu ml1) grown for 40 h at 30 1C in N-free medium at 3–4 days of plant growth. The ARA of the plants was measured after 2 weeks as described earlier. For both experiments, uninoculated plants served as control and plants were grown in a light
ARTICLE IN PRESS Nodulation and plant-growth promotion methylotrophic bacteria chamber with a light-dark cycle of 14 h (27 1C)/10 h (22 1C).
Characterization of the rhizobial isolates Assimilation of different carbon sources were tested on AMS medium (Patt et al., 1976) containing plates supplemented separately with the various filter sterilized carbon sources, (0.1% w/v) and in case of volatile organic compounds, ethanol 0.5% (v/v) and methanol 0.5% (v/v) were used. Methane was injected in the headspace (25% v/v of methane gas) of liquid media containing vials (Green and Bousifield, 1982). Bacterial suspension prepared from a 48 h old culture was used as the inoculum. Inoculated plates and vials were incubated at 30 1C and examined after 3 days. For methane utilization, bacterial growth was determined by measuring the OD at 660 nm with a spectrophotometer after incubation. Polymerised chain reaction (PCR) amplifications were performed to detect the presence of nodulation genes (nodA) and methanol dehydrogenase (MDH) genes (mxaF) using the primer pairs nodAfbrad (50 -GTYGAGTGGAGSSTKCGCTGGG-30 ), nodArbrad (50 -TCACARCTCKGGCCCGTTCGG-30 ) and f1003 (50 -GCGGCACCAACTGGGGCTGGT-30 ), r1561 (50 GGGCAGCATGAAGGGCTCCC-30 ), respectively, according to Sy et al. (2001). PCR amplifications were performed with an Eppendorf Mastercycler gradient (PCR Thermocycler, Brinkmann Instruments Inc., Westbury, NY) in a total volume 25-ml reaction mixture containing 50 ng of genomic DNA, each deoxynucleotide triphosphate (200 mM), 0.8 mM each primers MgCl2 (1.5 mM), 1.25 U of Taq DNA polymerase (Bangalore Genei, Bangalore, India), and the buffer supplied with the enzyme. The PCR products were analyzed by horizontal agarose gel electrophoresis using 1.5% agarose (Bangalore Genei, India).
Nodulation and plant growth promotion in green house The symbiotic and plant growth promoting properties of the two methylotrophic rhizobial isolates were studied in M. atropurpureum and C. juncea under greenhouse conditions. Seeds were scarified, surface sterilized and pre-germinated in moist germination paper as mentioned earlier. Germinated seedlings inoculated with bacterial suspensions (108 cells ml1) were grown in pots filled with sterilized soil mix and watered with sterile distilled water as required. The pots were arranged in a completely randomized design with
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three replications for each treatment. At harvest, number of nodules, nodule dry weight, shoot dry weight, and shoot nitrogen content were recorded.
Statistical analysis With the design being completely randomized, the mean, standard error and analysis of variance were calculated, and means were separated by LSD using the SAS package, Version 9.1, SAS Institute Inc., and Cary, NC, USA.
Results and discussion Isolation of Methylobacterium nodule symbionts Twenty-three isolates of methylotrophic rhizobia were recovered from the nodulating legumes of tropic regions on M-72 media with 0.5% methanol. The isolates possessed nitrogenase activity measured through ARA ranging from 2.1670.17 to 162.7276.52 nmol C2H4 h1 mg1 protein. The methylotrophic strains CMCJ317 and CMSA322 from the root nodules of C. juncea and Sesbania aculeate, respectively recorded higher nitrogenase activities of 162.7278.51 and 120.4876.52 nmol C2H4 h1 mg1 protein (Table 1) and their nodulation and methylotrophic characteristics were studied. The strains were grown on methanol, purified as a single colony and inoculated onto M. atropurpureum for testing nodulation. The bacteria formed nitrogen-fixing nodules and single colonies reisolated from the nodules were able to grow on methanol. The results clearly confirmed that the strains exhibited nodulation and methylotrophic properties. Methylotrophic rhizobial strains CMCJ317 and CMSA322 when inoculated on to M. atropurpureum, recorded significantly higher nitrogenase activity of 2.8670.27 and 2.7970.12 mmol C2H4 h1 plant1 compared to control (1.0270.04 mmol C2H4 h1 plant1).
Characteristics of methylotrophic rhizobia The isolates CMCJ317 and CMSA322 were Gram negative cocci occurring singly or in rosettes, motile and often contained poly-b-hydroxy butyrate inclusions. Colonies are non-pigmented, smooth, shiny, raised with regular margins and 0.3–0.8 mm in diameter after 96 h at 30 1C. Both the isolates were catalase and oxidase positive, and negative for indole methyl red and Voges Proskauer tests. They were negative for hydrogen sulfide
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M. Madhaiyan et al.
production and could not hydrolyze starch, casein and gelatin. The isolates were compared with that of M. nodulans ORS2060 and M. extorquens AM1. The characteristics of the isolates were more related to M. nodulans ORS2060. M. nodulans ORS2060 and CMCJ317, CMSA322 utilized the carbon sources L-arabinose, L-aspartate, citrate, betaine, acetate, and tartarate. CMCJ317 and CMSA322 also utilized dichloromethane and grow on nutrient agar but not utilized methane that differed from M. nodulans ORS2060. CMCJ317 and CMSA322 were further examined for the presence of mxaF and nodA genes using M. nodulans ORS2060, as a reference. Gram negative Methylobacterium spp. oxidizes methanol to formaldehyde via a key periplasmic enzyme MDH and the use of MDH structural gene mxaF as a functional probe for methylotrophs is well explained (McDonald and Murrell, 1997). Therefore, to evaluate the presence of methanol oxidation genes PCR amplification was performed with non-degenerate primers f1003 and r1561, the primers designed corresponding to highly conserved regions of mxaF gene sequences (McDonald and Murrell, 1997). In our study, the PCR conducted for all the twenty three strains produced an amplification product of the expected size 555 bp, except for four strains viz., CMAH303, CMVR306, CMCA312 and CMDL313 indicating that the selected strains CMCJ317 and CMSA322 contain the structural gene mxaF, required for methanol oxidation. No amplification of these primers could be obtained in rhizobial strains from our laboratory collection (results not shown) and the homologs while using other degenerate primers probably do not correspond to the bonafide mxaF ortholog (Sy et al., 2001). Also CMCJ317 and CMSA322 showed positive amplification of 584 bp PCR products similar to M. nodulans ORS2060 for the nodA gene using the selected primers nodAfbrad and nodArbrad defined from conserved parts of nodA sequences (Sy et al., 2001). Bacterial nodulation genes have been shown to play a central role in the molecular dialogue between the plant and the bacterium leading to plant recognition, Table 2.
infection and nodulation (Denarie et al., 1996; Schultze and Kondorosi, 1998). The PCR amplification of mxaF and nodA genes in CMCJ317 and CMSA322 resulted in products similar to the reference strain M. nodulans ORS2060. The analysis of the fragments of the 16S rDNA digested with the restriction enzyme BclI by restriction fragment length polymorphic (RFLP) analysis resulted in 4 distinct bands. The bands obtained with CMSA322 showed close similarity to the reference strain M. nodulans ORS2060 where as the products of the strain CMCJ317 were distinct. This reveals that the two strains differ within and also from the reference strain (results not shown).
Nodulation and plant growth promotion by methylobacterial isolates M. nodulans ORS2060 is a well-characterized strain with respect to its methylotrophic and nodulating characteristics and hence in this study we tested the effects of inoculating CMCJ317 and CMSA322 on plant growth under pot culture conditions with the legumes M. atropurpureum and C. juncea. Both CMCJ317 and CMSA322 nodulated and improved the plant growth of C. juncea. Strain CMSA322 formed pink color indeterminate nodules and the number of nodules formed, shoot nitrogen percentage was higher compared to CMCJ317 (Table 2). The efficiency of nodulation of the two strains was greater in M. atropurpureum than in C. juncea. However, the strain CMSA322 formed more number of nodules and increased shoot nitrogen and dry weight higher than CMCJ317 similar to C. juncea (Fig. 1). The rhizobial inoculations significantly increased the shoot nitrogen concentration in the plants when compared to control. These results clearly suggest that CMCJ317 and CMSA322 exhibit methylotrophy and form symbiotic association with legumes improving the plant growth characteristics. Recent discoveries proved that bacteria from outside Rhizobiaceae family also can nodulate legumes and fix nitrogen symbiotically
Growth, nodulation and nitrogen fixation of Crotalaria juncea infected by Methylobacterium isolates
Treatments
Shoot length (cm)
Root length (cm)
Control CMCJ317 CMSA322 LSD (Pp0.05)
125.3275.96b 21.1271.80a 139.4178.32a 22.6373.83a 140.85710.31a 24.0371.75a 8.55 4.65
Shoot dry weight (g)
Shoot N (% dry weight)
Nodules per plant (#)
Nodules dry weight (mg)
25.1271.80a 26.8273.36a 26.971.67a 3.67
0.8270.05c 2.4670.06b 3.2570.14a 0.21
— 56.3275.38b 61.8274.52a 3.73
— 11.2670.85b 12.3671.08a 0.99
Each value represents mean7SE of three replicates per treatment. In the same column, significant differences at Pp0.05 levels are indicated by different letters. Data followed by same letter in the same column are not significantly different from each other.
ARTICLE IN PRESS Nodulation and plant-growth promotion methylotrophic bacteria
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Figure 1. Growth, nodulation and nitrogen fixation of Macroptilum atropurpureum infected by methylotrophic rhizobia. (a) Shoot dry weight, (b) nodule number per plant, (c) shoot nitrogen as percentage of dry weight, and (d) nodule dry weight per plant. Each value represents mean7SD of three replicates.
and one such is Methylobacterium that formed a fourth branch within the a-Proteobacteria. The results clearly reveal that the two root nodule isolates from tropical legumes represent two distinct strains of Methylobacterium, with M. nodulans as their closest neighbour. However, conclusive evidences on their identification could not be reached since molecular characterization such as 16S rDNA sequencing was not performed. This study confirms and extends the capacity of Methylobacterium to effectively nodulate legume plants thus proving the polyphyletic origin of rhizobia within a-Proteobacteria.
Acknowledgements We thank Catherine Boivin-Masson, LSTM, UMR 113 IRD/INRA/AGRO-M/CIRAD, 34398 Montpellier Cedex 5, France for the valuable suggestions and provision of strain. M.M and S.P are indebted to the Korea Research Foundation, Republic of Korea for a
position as invited foreign scientist and Ph.D. research grants, respectively.
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