Madhaiyan et al. Biotechnol Biofuels (2015) 8:222
DOI 10.1186/s13068-015-0404-y
Biotechnology for Biofuels
Open Access
RESEARCH
Leaf-residing Methylobacterium species
ix nitrogen and promote biomass and seed
production in Jatropha curcas
Munusamy Madhaiyan1, Tan Hian Hwee Alex1, Si Te Ngoh1, Bharath Prithiviraj2 and Lianghui Ji1*
Abstract
Background: Jatropha curcas L. (Jatropha) is a potential biodiesel crop that can be cultivated on marginal land
because of its strong tolerance to drought and low soil nutrient content. However, seed yield remains low. To enhance
the commercial viability and green index of Jatropha biofuel, a systemic and coordinated approach must be adopted
to improve seed oil and biomass productivity. Here, we present our investigations on the Jatropha-associated
nitrogen-ixing bacteria with an aim to understand and exploit the unique biology of this plant from the perspective
of plant–microbe interactions.
Results: An analysis of 1017 endophytic bacterial isolates derived from diferent parts of Jatropha revealed that
diazotrophs were abundant and diversely distributed into ive classes belonging to α, β, γ-Proteobacteria, Actinobacteria and Firmicutes. Methylobacterium species accounted for 69.1 % of endophytic bacterial isolates in leaves and
surprisingly, 30.2 % which were able to ix nitrogen that inhabit in leaves. Among the Methylobacterium isolates, strain
L2-4 was characterized in detail. Phylogenetically, strain L2-4 is closely related to M. radiotolerans and showed strong
molybdenum-iron dependent acetylene reduction (AR) activity in vitro and in planta. Foliar spray of L2-4 led to successful colonization on both leaf surface and in internal tissues of systemic leaves and signiicantly improved plant
height, leaf number, chlorophyll content and stem volume. Importantly, seed production was improved by 222.2 and
96.3 % in plants potted in sterilized and non-sterilized soil, respectively. Seed yield increase was associated with an
increase in female–male lower ratio.
Conclusion: The ability of Methylobacterium to ix nitrogen and colonize leaf tissues serves as an important trait for
Jatropha. This bacteria–plant interaction may signiicantly contribute to Jatropha’s tolerance to low soil nutrient content. Strain L2-4 opens a new possibility to improve plant’s nitrogen supply from the leaves and may be exploited to
signiicantly improve the productivity and Green Index of Jatropha biofuel.
Keywords: Culturable endophyte, Nitrogen ixation, Methylobacterium, Jatropha curcas L., Biofuel
Background
Jatropha curcas L. (Jatropha) is a woody perennial,
drought-tolerant shrub belonging to Euphorbiaceae and
is widely distributed in tropical and subtropical regions.
Jatropha seeds contain high level of triacylglyceride with
a fatty composition well suited for biodiesel production
*Correspondence: jilh@tll.org.sg
1
Biomaterials and Biocatalysts Group, Temasek Life Sciences Laboratory,
1 Research Link, National University of Singapore, Singapore 117604,
Singapore
Full list of author information is available at the end of the article
[1]. Jatropha is resistant to drought, able to thrive on
marginal land under climate and soil conditions that are
unsuitable for food crop plantation [2–5]. In addition
to sequestrating CO2 and reducing the world’s reliance
on fossil fuel, Jatropha helps control soil erosion [6] and
detoxify polluted soil [7–9]. As a wild plant, however,
Jatropha seed and oil productivity remains low, particularly when chemical fertilizer input is limited. Apart from
breeding programs for high-yielding Jatropha varieties
[10–12], agronomical practices, such as the application
of inorganic fertilizer [13] and plant growth regulators,
© 2015 Madhaiyan et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License
(http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium,
provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license,
and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/
publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Madhaiyan et al. Biotechnol Biofuels (2015) 8:222
have also been reported to improve seed yield [14, 15].
As Jatropha is targeted to plant on marginal soil with low
nutrient levels, fertilizer requirement would be higher
than other crops. his would signiicantly afect the commercial viability of Jatropha and ofset the Green Index of
Jatropha biofuel.
It has been increasingly realized that plants form close
association with a large population of diverse bacteria,
which are either loosely associated with roots (rhizosphere bacteria), actively colonizing internal plants tissues
(endophyte) and leaf surfaces (epiphyte) [16–21]. Plants
often beneit from such interactions because of nitrogen
ixation; production of plant growth hormones, such as
auxin, cytokinin and gibberellin; delayed senescence
through suppression of ethylene biosynthesis by secreting 1-aminocyclopropane-1-carboxylate (ACC) deaminase; alteration of sugar sensing mechanisms [22–24]
and inhibiting pathogen attacks through production
of hydrolytic enzymes [25], competition for space and
nutrients [26, 27], and induction of systemic defence
mechanisms [28–30]. Bacterial inoculations improved
growth and development of switchgrass seedlings, signiicantly stimulated plant growth, and tiller number on
the low fertility soil, and enhanced biomass accumulation
on both poor and rich soils, with more efective stimulation of plant growth in low fertility soil than in high
fertility soil [31]. Our previous study also showed that
Kosakonia species suitable for limited N-content soil and
signiicantly promoted growth and seed yield of Jatropha
[32]. Here, we present an investigation on the diversity of
culturable endophytic bacteria of Jatropha and a detailed
study on the role of a novel leaf-colonizing diazotroph,
Methylobacterium sp. strain L2-4, on Jatropha biomass
and seed production.
Results and discussion
Culturable endophytic bacterial density in Jatropha tissues
We sampled Jatropha root, stem and leaf tissues from
three diferent germplasm accessions. Endophytic bacterial colonies were established on six diferent solid
media after thorough surface sterilization of the plant
tissues. As expected, endophytic bacterial densities varied amongst tissue types and culture media employed.
he highest density was seen in roots, followed by stems
while leaves showed the lowest density irrespective of the
isolation media used (Additional ile 1: Figure S1). Similar to previous indings [33, 34], use of complete media,
such as KB media and Medium 869, resulted in higher
endophyte density while synthetic media, such as AMS
with methanol as carbon source, Nfb or BAz media with
malic acid or azelaic acid as the carbon source, yielded
lower densities (Additional ile 1: Figure S1). Canonical
discriminant analysis (CDA) of the combined population
Page 2 of 14
data of 3 germplasm accessions showed that the origin
of plant tissues and media employed for the isolation
formed distinct groups (Additional ile 2: Figure S2).
hese results suggest that the distribution of endophytic
population vary within diferent parts of Jatropha and the
bacterial population will be ill-presented if the investigation relies on a single medium.
Given the large endophytic bacterial population found
in various types of tissues, we focused our studies on
selected representatives of culturable species, which
were selected randomly according to colony morphology, color and size. Amongst the 1017 isolates selected
for analyses, 49.4 % was derived from the roots, 29.8 %
from stems and 20.8 % from leaves. 16S rRNA gene analysis assigned 34.4 % of them to α-Proteobacteria, 31.1 %
γ-Proteobacteria and 24.5 % Actinobacteria (Additional
ile 3: Table S1; Additional ile 4: Table S2).
In leaves, α-Proteobacteria, particularly Methylobacterium genus clearly stood out in the population, while
Sphingomonas, Pantoea, Kocuria, Microbacterium and
Curtobacterium genera were also frequently isolated
(Fig. 1; Additional ile 4: Table S2). he stem population
resembled that of leaves, with Curtobacterium, Methylobacterium and Sphingomonas being the top 3 genera
(Additional ile 5: Figure S3). In contrast, Pseudomonadaceae, Enterobacteriaceae and Rhizobiaceae dominated
in roots (Additional ile 6: Figure S4). he growth promoting role of Enterobacter in Jatropha has been demonstrated previously [32]. Surprisingly, 31.2 % of the isolates
potentially represent new taxa (Additional ile 7: Figure
S5).
Cell wall degrading endoglucanase activity was believed
to be critical for endophytes to successfully colonize
plants [35]. We found that 253 strains (24.9 %) exhibited clear zones on CMC plates stained with Congo red,
indicating production of endoglucanase in those strains
(Table 1, Additional ile 3: Table S1). Isolates from genera Cellulosimicrobium, Curtobacterium, Kosakonia and
Pseudomonas were most frequently observed to produce
endoglucanase. Unexpectedly, 55.9 % of the endophytic
isolates did not show obvious endoglucanase activity. As
it has been suggested previously, many of endophytes
may passively entered the system from the root and
spread to aerial parts in a systematic manner [36, 37].
Endophytic nitrogen‑ixing bacteria
Among the 1017 isolates, 111 strains (11 %) were able to
grow in nitrogen-free media. Members of the genus Pseudomonas (35.1 %), Curtobacterium (10.8 %), Methylobacterium (9.0 %), Sphingomonas (8.1 %) and Rhizobium
(8.1 %) were the major taxa overall (Additional ile 4:
Table S2, Additional ile 6: Table S3). To further conirm
the diazotrophic nature of the strains, we analyzed the
Madhaiyan et al. Biotechnol Biofuels (2015) 8:222
Page 3 of 14
Methylobacterium aminovorans JCM 8240T (AB175629) (1 strain)
Methylobacterium aquaticum GR16T (AJ635303) (4 strains)
Methylobacterium gregans 002-074T (AB252200) (3 strains)
Methylobacterium hispanicum GP34T (AJ635304) (2 strains)
Methylobacterium komagatae 002-079 (AB252201) (10 strains)
Methylobacterium platani PMB02T (EF426729) (2 strains)
Methylobacterium populi BJ001T (CP001029) (29 strains)
Methylobacterium radiotolerans JCM 2831 (CP001001) (31 strains)
Methylobacterium suomiense NCIMB 13778T (AB175645) (7 strains)
Methylobacterium thiocyanatum DSM 11490T (AB175646) (2 strains) α-Proteobacteria
Methylobacterium variabile GR3T (AJ851087) (2 strains)
Methylobacterium zatmanii DSM 5688T (AB175647) (1 strain)
L4-311
99
71
96
99
L8-458
99
Rhizobium alkalisoli CCBAU01393T (EU074168)
Aurantimonas ureilytica 5715S-12T (DQ883810) (11 strains);
Aureimonas jatrophae L7-484T; Aureimonas phyllosphaerae L9-753T
97
99
Sphingomonas abaci C42T AJ575817 (27 strains)
99
L3-20
99
Xanthomonas gardneri ATCC19865T (AEQX0100044)
99
Pseudomonas aeruginosa LMG1242T (Z76651) (7 strains)
99
99 L9-782
91
Moraxella osloensis (EU499677)
γ-Proteobacteria
Enterobacter arachidis Ah-143T (EU672801)
99
69
L1-1
99
Pantoea allii (AY530795) (15 strains);
Tatumella morbirosei (EU344769) (8 strains)
74
99
99
Staphylococcus epidermidis (L37605) (4 strains)
Bhargavaea cecembensis DSE10T (AM286423)
Firmicutes
99 L3-146
81 L2-4-1
99 L7-483
99
Williamsia serinedens IMMIBSR-4 (AM283464)
99
L8-494
99
Mycobacterium cosmeticum (AY449728)
99 Corynebacterium singulare (Y10999)
99
L4-295
99 Kocuria koreensis P31T (FJ607312) (15 strains)
93
L1-101
60
L9-805
99
89
94
Actinobacteria
69
L7-617
L4-613
Micrococcus yunnanensis YIM65004T (FJ214355)
98
Microbacterium aquimaris (AM778449) (16 strains);
Curtobacterium luteum (X77437) (10 strains)
0.02
Fig. 1 Phylogenetic positions and diversity of leaf-endophytic species. The tree was constructed based on 16S rDNA sequences using the
Neighbor-Joining method. Bootstrap values (using 1000 replicates) are indicated at the branching points. Scale bar represents % estimated substitutions. Candidates for nitrogen-ixers (shown in blue) indicate the presence of nifH gene as evidenced by PCR ampliications. The number of strains is
shown in parenthesis in red. The arrowhead sizes indicate the relative abundance of the genus
Madhaiyan et al. Biotechnol Biofuels (2015) 8:222
Page 4 of 14
Table 1 Distribution of nitrogen-ixing and endoglucanase-positive strains originating from diferent media
Media
Medium
Total number of isolatesa
Positive isolates (%)
nifHb
Rich media
ARAc
Endoglucanased
M869
257
132 (51.4)
81 (29.5)
98 (38.1)
KB
354
206 (58.2)
143 (40.4)
155 (43.8)
Heterotrophic media
R2A
189
112 (59.3)
58 (30.7)
75 (39.7)
Minimal media
AMS
106
80 (75.5)
49 (46.2)
33 (31.1)
N-free media
Nfb
101
68 (67.3)
45 (45.6)
78 (77.2)
Baz
10
5 (50.0)
5 (50.0)
9 (90.0)
a
Total number of isolates selected for 16S rRNA gene sequencing and phylogeny
b
PCR ampliication of nifH gene fragments was performed using speciic degenerate primers
c
Acetylene reduction activity in pure culture was measured by GC
d
Plate assays for endoglucanase activity using KM solid medium with 0.2 % CMC were spot inoculated with endophytes and incubated at 30 °C for 3 days
nifH gene encoding the dinitrogenase reductase subunit. nifH sequences were detected by PCR in 64.8 % of
the strains that were able to grow in N-free medium [38].
Notably, only 37.4 % nifH-positive strains displayed nitrogenase activity in vitro (Table 2; Additional ile 3: Table
S1). he discrepancy may be attributed to the presence
of non-functional nifH gene. Alternatively, nitrogenases
were not functional under the in vitro assay conditions.
herefore, the ability to grow on N-free medium and
the presence of a nifH gene does not warrant nitrogenase activity in vitro. his is in accordance with several
previous studies on nitrogen-ixing bacteria [39–41].
he majority of the nifH-positive isolates that failed to
show in vitro nitrogenase activity belonged to the order
Rhizobiales: e.g., Rhizobium, Ensifer, Sinorhizobium,
Bradyrhizobium, and Mesorhizobium genera, which are
known to ix nitrogen efectively only in root nodules
[42, 43]. In contrast, isolates belonging to the genus Cellulomonas, Curtobacterium, Microbacterium, Mycobacterium, Chryseobacterium or Achromobacter showed
AR activity. However, no nifH DNA sequences could be
ampliied under the conditions used, suggesting the nifH
genes in those isolates was more divergent.
Our results demonstrated that Jatropha tissues are
associated with an abundant and diverse population of
diazotrophs. he diferential pattern of diazotrophic population in diferent parts of Jatropha shared high similarity to those of soybean and potato [42, 44], but it was
signiicantly diferent from that of switchgrass and wild
rice [45].
Analyses of nifH genes
We sequenced the nifH PCR products from 42 strains
that appeared to be unique species based on 16S rDNA
sequences. All isolates showed AR activity in vitro culture except Rhizobium and Sinorhizobium groups. Alignment of the predicted NifH amino acid sequences formed
6 major clusters (A–F) (Fig. 2). It is noticeable that the
NifH sequence homology does not consistently correlate
with phylogenetic relationship. his suggests that multiple independent horizontal gene transfer events occurred
in the evolution of nitrogen-ixing bacteria. Notably, cluster F include sequences from leaf isolates only, all belonging to the genus Methylobacterium. hese sequences
were highly divergent from NifH consensus sequence
[46]. In fact, they were more related to the Pfam NifH/
frxC-family protein, i.e., chlorophyllide reductase iron
protein subunit X involved in photosynthesis [47].
Nitrogenase activity of Methylobacterium species in vitro
and in planta
Among 125 Methylobacterium isolates (Additional
ile 4: Table S2) characterized from Jatropha plant tissues, obvious AR activity was observed in 34 strains
(20–634 nmol C2H4/bottle). 52 strains showed weak AR
activity (<20 nmol C2H4/bottle) while 39 strains had no
detectable activity although all strains had the nifH-like
sequences (Additional ile 3: Table S1, Additional ile 4:
Table S2). Phylogenetically, AR-positive strains were
closely related to M. radiotolerans, M. populi, M. komagatae and M. aquaticum (Additional ile 3: Table S1).
Strain L2-4 can be classiied as M. radiotolerans based
on its rDNA sequence and was among the fastest grower
in N-limiting conditions and showed distinct AR activity in vitro (Additional ile 9: Figure S6). Assays of its AR
activity in the presence or absence of iron (Fe2+), molybdenum (MoO42−) and vanadium (V) suggest that the
L2-4 strain nitrogenase used Fe2+ and MoO42−) as cofactors. he highest AR activity was recorded in the presence of FeSO4 (10 mg/l) and Na2MoO4 (5 mg/l) (Fig. 3a).
Vanadium showed weak inhibitory efect. As expected,
ammonium ion strongly inhibited AR activity (Fig. 3b).
he ability of L2-4 strain to ix nitrogen in planta was
conirmed by inoculating the strain to Jatropha by foliar
Madhaiyan et al. Biotechnol Biofuels (2015) 8:222
Page 5 of 14
Table 2 Distribution of nifH-positive representative bacterial taxa in the leaf, stem and root of Jatropha germplasm
accessions
Phylogenetic group
Genera (21)
Number of isolates
AR‑activity positivea
Actinobacteria
Leaf
Stem
10
35
1
1
2
1
1
Cellulomonas
Curtobacterium
AR‑activity negativeb
Root
Leaf
Stem
Root
16
21
4
1
5
1
Microbacterium
Micromonospora
Mycobacterium
Bacteroidetes
Chryseobacterium
Firmicutes
Paenibacillus
α-Proteobacteria
Bradyrhizobium
3
1
Ensifer
1
Herbaspirillum
1
13
Mesorhizobium
Methylobacterium
3
63
21
Pleomorphomonas
β-Proteobacteria
γ-Proteobacteria
30
7
1
Rhizobium
Sphingomonas
2
2
15
16
2
14
52
12
16
4
Achromobacter
11
Burkholderia
4
Klebsiella
1
Kosakonia
1
Enterobacter
8
8
33
11
2
104
1
8
4
12
Pseudomonas
6
16
a
Number of positive isolates showing ethylene peak was measured by GC
b
Number of isolates failed to produce ethylene peak or undetectable quantity
spraying and maintaining the plants under sterile condition. Seedlings treated with strain L2-4 showed strong
AR activity (204.6 nmol C2H4 g−1 dry tissues day−1). Furthermore, L2-4 strain also displayed strong AR activity in
planta in sorghum, rice, cotton, and caster plant (Fig. 3c).
he association between Methylobacterium species and
host plants varies from strong or symbiotic to weak or
epiphytic and to intermediate or endophytic [48, 49].
Methylobacterium nodulans and M. radiotolerans have
been reported to be involved in nitrogen ixation and
nodule formation [50, 51], while other Methylobacterium
species has been reported multiple plant growth promoting traits [52, 53].
1
1
Pantoea
Stenotrophomonas
1
4
39
5
Epiphytic and endophytic colonization
by Methylobacterium
Methylobacterium species have been found in association
with several species of plants, actively colonizing leaves,
stem, branches and roots [54–60]. Methylobacterium cells
were observed in intracellular space of the meristematic
cells of Scots pine and tomato [61, 62]. Endophytic occurrence of Methylobacterium was conirmed in Medicago
truncatula leaves [63]. We found that strain L2-4 colonized on leaf surfaces (epiphytic) as well as inside leaf tissue of Jatropha grown under sterile conditions. On day
45 after leaf spraying, surface-sterilized leaf tissues had
endophytic bacteria counts of 5.2 × 106 cfu/g leaf tissues.
(See igure on next page.)
Fig. 2 Phylogenetic tree of partial NifH sequences. Alignment was made for the 192 amino acid residues corresponding to amino acid 34–184
in Azotobacter vinelandii NifH protein. The tree was constructed using the Neighbor-Joining method. The scale bar denotes 0.05 % of sequence
distance. The retrieved sequences, in bold, were grouped into clusters A, B, C, D, E and F. The arrowhead sizes indicate the relative abundance of the
genera. Bootstrap values above 50 % are indicated at the branching nodes. Jatropha isolates shown in light blue color indicate the presence of nifH
gene as evidenced by PCR ampliications. The position of root isolates and leaf isolates is marked with red and green arrowheads, respectively
Madhaiyan et al. Biotechnol Biofuels (2015) 8:222
Page 6 of 14
90 R5-409 (KR075971)
Bradyrhizobium japonicum (HM057562)
60
Bradyrhizobium elkanii (DQ485701)
Beijerinckia indica subsp. indica (AF296354)
Bradyrhizobium japonicum (GQ289565)
Sphingomonas azotifigens (AB217474)
S8-608 (KR075970)
76
Cluster A
α,β,γ-Proteobacteria
Sphingomonas sp. BR12248 (ACR19159)
Acidithiobacillus ferrooxidans (M15238)
Sphingomonas paucimobilis (KF182367)
Burkholderia rhynchosiae (EU219869)
52
Burkholderia vietnamiensis (EF158810)
R2-127 (KR075947)
84
75 Burkholderia phymatum (FN908422)
R5-408 (KR075960)
83
Herbaspirillum seropedicae (U97121)
98
Herbaspirillum sp. B501 (AB196476)
Rhodobacter capsulatus (M15270)
52
Rhodopseudomonas lichen (AB241413)
P. diazotrophica R5-392T (KC195919)
65 Gluconacetobacter diazotrophicus (AF030414)
Methylobacterium nodulans (AY312969)
59
Azospirillum brasilense (M64344)
Rhizobium etli (NC_004041)
Cluster B
α-Proteobacteria
Rhizobium tropici (JX863573)
R2-708 (KR075967)
Sinorhizobium meliloti (WP_018097454)
54
R7-601 (KR075968)
S6-271 (KR075962)
S2-45 (KR075961)
82 Mesorhizobium tianshanense (GQ167282)
R1-73 (KR075966)
58
Azotobacter vinelandii (M20568)
Pseudomonas stutzeri (AF117977)
68
Kosakonia spp. (R5-395, R5-424, R5-362, R4-323, R5-397,
R5-326, R4-412, R4-724, R4-368, R4-369, R4-414, R4-422,
and R4-381), Klebsiella sp. (R5-431), Pantoea sp. (R4-341)†
93
Cluster C
γ-Proteobacteria
R1-312 (KR075964)
99
Paenibacillus odorifer (AJ223992)
Stenotrophomonas maltophilia (EF620510)
R2-208 (KR075969)
Cluster D
γ-Proteobacteria
Firmicutes
R7-596 (KR075963)
99
MicromonosporaspFN395243
Cluster E
Actinobacteria
MicromonosporalupiniFN395238
Methylobacterium spp. (L2-4, L7-438, S8-665, L4-301,
99
0.1
L6-305, L6-306, L6-314, L7-470, L8-475)‡
Cluster F
α-Proteobacteria
(nifH-like)
Madhaiyan et al. Biotechnol Biofuels (2015) 8:222
Page 7 of 14
Epiphytic population based on leaf-imprinting assay was
>100 cfu (cm2)−1 in treated leaves. As expected, pink color
colonies were not detected in non-treated leaves of Jatropha seedlings grown under sterile conditions.
nmol C2H4 mg of protein-1 h-1
a
1800
1600
1400
1200
1000
800
600
400
200
0
Inoculation of Methylobacterium improved production
of biomass and seeds
minus Fe
Fe/Mo/V
Mo
Fe+Mo Fe+V Mo+V Fe+Mo+V V
nmol C2H4 mg of protein-1 h-1
b
1600
1400
1200
1000
800
600
400
200
0
-200
0
0.1
1
5
2
10
NH4Cl (mM)
in planta AR acvity
(nmol C2H4 g-1 sample day-1)
c
250
200
150
100
50
0
Co on
Sorghum
Castor bean
Rice
Jatropha
Fig. 3 Characterization of nitrogenase. a, b Acetylene reduction
activity in vitro. Methylobacterium L2-4 was cultured in N-free medium
containing various combination of co-factors (Fe/Mo/V) or concentrations of NH4Cl. a Efects of co-factors. b Sensitivity to ammonium
ions. c Nitrogenase activity of in planta. Seedlings of various crops
were inoculated with L2-4 strain and AR assays were done 20 days
after inoculation. Each value represents mean ± standard deviation
(SD) of three replicates
To further conirm the growth promoting efect of strain
L2-4, Jatropha seedlings were inoculated with the bacterial suspension through seed soaking and as foliage
spray. Seed treatment by soaking seeds with L2-4 bacterial suspension for 2 h increased germination rate by
24 %, from 49.6 % in mock-inoculated seeds to 61.5 % in
treated seeds. At 45 days after sowing, the average dry
biomass of inoculated plants was 40.1 % higher than the
mock-inoculated plants and this was associated with signiicantly increased leaf chlorophyll content and seedling
vigor (Table 3). Several studies reported that Methylobacterium inoculation through seed imbibition and phyllosphere spray enhanced seed germination rate, storability,
and seed vigor [64–66]. In another word, Methylobacterium has both nurturing and protecting roles for the
plants [67]. To demonstrate that nitrogen-ixing strain
L2-4 is able to improve seed production of Jatropha,
seedlings were inoculated by leaf spraying, planted in
large pots and maintained in the open air. Again, L2-4
treated plants showed signiicant improvements in plant
height, leaf counts, leaf chlorophyll content and stem volume compared with the untreated control plants (Fig. 4).
At 120 DAI, treated plants recorded an increase of 11.5,
57.1, 11.4 and 56.2 % over the mock-inoculated controls
in plant height, leaf counts, leaf chlorophyll content and
stem volume, respectively (Fig. 4a–d). In consistence
with the plant growth promotion, leaf-epiphytic and
endophytic populations were found signiicantly higher
in inoculated plants. Total leaf-associated methylotrophic bacterial density ranged from 7 to 7.5 log cfu g−1
of tissues in treated leaves compared to 6–6.7 log cfu g−1
of tissues in mock-treated plants at 60–120 DAP (Fig. 4e,
f ). Leaf-imprinting conirmed that strain L2-4 was an
Table 3 Efects of L2-4 strain inoculation on the early growth parameters of Jatropha
Treatmentsa
SVIb
Relative chlorophyll content
Seedling dry biomass (per seedling)c
Control
1818.4 ± 69.3
34.91 ± 1.79
3.07 ± 0.27
L2-4
3297.7 ± 153.7
38.81 ± 0.51
4.30 ± 0.18
LSD (P ≤ 0.05)
551.02
3.18
0.32
a
After seed soaking, the seeds (50 seeds/replicate, n = 3) were drained and sown in trays containing non-sterilized soil and maintained in a greenhouse and at 28 °C
b
Seedling vigor index (SVI) was calculated using the formula: SVI = % germination × seedling length (shoot length + root length) in cm
c
Each value represents mean of three replicates and expressed in grams. Samples were measured at 45 DAS
Madhaiyan et al. Biotechnol Biofuels (2015) 8:222
Page 8 of 14
Plant height (cm)
60
50
40
30
20
10
0
*
*
*
CTL
L2-4
60
90
Rel. chlorophyll cont.
b
a
50
30
CTL
20
L2-4
10
0
120
60
c
120
d
35
30
25
20
15
10
5
0
500
*
*
*
CTL
L2-4
Stem volume
Number of leaves (#)
90
DAP
DAP
*
400
*
300
CTL
L2-4
200
NS
100
0
60
90
120
60
DAP
90
120
DAP
f
70
60
50
40
30
20
10
0
*
*
*
CTL
L2-4
60
90
120
DAP
LAB (log cfu/g of ssue)
e
PPFMs density (per cm2)
*
*
40
8.5
8
7.5
7
6.5
6
5.5
5
4.5
4
*
*
*
CTL
L2-4
60
90
120
DAP
Fig. 4 Promotion of Jatropha biomass growth. Jatropha seedlings were inoculated by leaf spraying, planted in large pots and maintained in the
open air. Sterilized and non-sterilized garden soil was used in Trail I and II, respectively. Values are mean ± standard deviation (SD). a Plant height; b
relative chlorophyll content; c number of leaves; d stem volume; e pink-pigmented facultative methylotrophic bacteria population; f leaf-associated
bacteria. Asterisk means signiicant diference at 5 % threshold between treated and control using DMRT. NS not signiicant
epiphyte, displaying 50–60 cfu (cm2)−1 in treated leaves
compared to 8–11 cfu (cm2)−1 in non-treated leaves
(Fig. 5). We analyzed the 16S rRNA sequence of 20 randomly picked colonies from the leaf-imprinting (Fig. 5a)
and 15 of them (75 %) were identical to that of L2-4.
hese results indicate that L2-4 strain competed well
with indigenous phyllosphere microlora under nonsterile conditions. As expected, pink-pigmented Methylobacterium were detected in low density in roots or stems
irrespective of L2-4 treatments (data not shown).
In two independent long-term open-air growth experiments using sterilized and non-sterilized soil, the average
seed set per tree was increased by approximately 213 and
84.3 %, respectively (Table 4). Student’s t test showed that
Fig. 5 Leaf imprinting. The systemic leaves of L2-4 inoculated plants
(at 30 DAI) were printed on an ammonium mineral salts plate supplemented with 0.5 % methanol (v/v) and incubated at 30 °C for
4 days. a, b show a leaf from an inoculated plant and control plant,
respectively
Madhaiyan et al. Biotechnol Biofuels (2015) 8:222
Page 9 of 14
Table 4 Efects of L2-4 strain inoculation on lower sex ratio and seed yield parameters of Jatropha
Treatment
Number of
female lowers
per inlorescencea
Number of
male lowers
per inlorescen‑
cea
Ratio
female:male
lower
Number
of fruitsb
Number
of seedsb
Seed weight
(g)b
per seed
weight (mg)*
Control
1.68 ± 0.14
25.2 ± 1.83
1:15
4.75 ± 0.71
13.5 ± 2.13
6.86 ± 0.91
426.5 ± 41.8
L2-4
4.48 ± 0.62
39.2 ± 4.19
1:9
16.6 ± 1.19
42.3 ± 4.27
22.1 ± 2.04
478.7 ± 20.7
LSD
(P ≤ 0.05)
0.75
4.08
1.13
4.12
1.93
Control
3.5 ± 0.07
55.0 ± 2.08
1:16
10.7 ± 1.15
28.6 ± 3.78
13.6 ± 0.51
424.1 ± 25.2
L2-4
9.28 ± 0.19
72.3 ± 1.99
1:8
18.4 ± 1.98
52.7 ± 4.72
26.7 ± 0.84
472.0 ± 13.0
LSD
(P ≤ 0.05)
1.92
8.18
1.50
3.57
2.39
Trail I
Trail II
Seedlings were inoculated by foliar spraying at 21 days after seed germination. A second spraying was made at the lowering stage. Plants were planted in large pots
(n = 8 in Trial I and n = 12 in Trial II) and maintained in the open air
Values are mean ± standard deviation (SD). Sterilized and non-sterilized garden soil was used in Trail I and II, respectively
a
Data were recorded with 25 and 50 inlorescences at diferent time points for Trail I and II, respectively
b
Number of fruits/plant, number of seeds/plant and seed weight/plant were recorded on 480 and 540 DAI from Trail I and II, respectively
* Student’s t test showed that trees treated with L2-4 had signiicantly higher seed sets and seed yield per plant than non-treated controls (P < 0.05)
the treated plants produced signiicantly more seed sets
than mock-treated ones in both experiments (P < 0.05).
he improvement in seed yield was associated with
an increase of female-male lower ratio and fruit sets
(Table 4). he average single seed weight was increased
by 12.2 % in Trial I and 11.3 % in Trial II, both being very
signiicant according to Student’s t test (P < 0.01). Cytokinin has been shown to improve female-to-male lower
ratio [15] and Methylobacterium has been shown to
change auxin and ethylene levels in plants due to secretion of 1-aminocyclopropane-1-carboxylate (ACC)
deaminase [68]. he cross-talk between cytokinin and
ethylene pathways is well established. Methylobacterium
strain L2-4 appears to promote Jatropha growth and seed
setting via multiple mechanisms, including nitrogen ixation, modulating photosynthesis, leaf senescence and
lower sex diferentiation. he genome of strain L2-4 presents several genes involved in metabolic pathways that
may contribute to promotion of plant growth and adaptation to plant surfaces [46]. Methylobacterium on plant
surfaces beneit from methanol produced by plants by
means of methylotrophy [59, 69, 70]. However, methanol
is not the only carbon substrate that these bacteria are
able to consume in the phyllosphere [63].
Conclusions
We have provided strong evidence that the dominant
leaf-associated Methylobacterium species were able to
promote Jatropha growth and seed yield, at least in part
due to nitrogen ixation. To the best of our knowledge,
this is the irst report of bacterial nitrogen ixation on
leaf surface although strain L2-4 is also a competent
endophyte in Jatropha. he abundance of endophytic
nitrogen-ixing bacteria in Jatropha may contribute to
Jatropha’s strong tolerance to poor soil nutrient. Our
studies also suggest that strain L2-4 is able to promote
growth and perform nitrogen ixation in a much wider
range of crops.
Methods
Sampling and isolation of endophytic bacteria
Jatropha germplasm accessions collected in the form of
seeds from Maluku Island, Indonesia; Yunnan Province,
China; and Madurai, Tamil Nadu, India, and the plants
were maintained at the Agrotechnology Experimental
Station, Singapore. hese natural germplasms accessions
have been selected in the breeding program of JOil Company (http://www.joil.com.sg/) to generate hybrid plants
on the basis of high productivity [71]. Healthy, symptomless leaves, stems and roots were collected from three
individual plants of each germplasm and treated separately. Lateral roots of approximately 15 cm away from
the primary stems with diameters from 0.5 to 1.5 cm
were collected. Fully expanded leaves with no obvious
pathogenic infections were collected. Similarly, uninfected stem segments of about 2.0–2.5 cm in diameter
were sampled. All tissues were washed with 70 % ethanol
at the cut sites and placed in plastic bags on ice during
transportation. Subsequently, samples were subjected to
a two-step surface sterilization procedure by washing for
5 min in 1 % (w/v) sodium hypochlorite supplemented
with 1 drop of Tween 80 per 100 ml solution followed
Madhaiyan et al. Biotechnol Biofuels (2015) 8:222
by three rinses in 70 % ethanol in sterilized distilled for
1 min each. To ensure complete surface sterilization, a
second treatment was performed by washing the tissues
for 15 min in 15 % H2O2, followed by 1 min in 70 % ethanol, and then rinsed in sterilized distilled water. A 100 µl
sample of the water from the third rinse was plated on
rich medium to verify the eiciency of sterilization.
Surface-sterilized tissues were macerated by grinding in
50 ml 10 mM MgSO4 and serially diluted suspensions
were plated on various solid media with 15 g/l agar or
phytagel, including 869 medium [33], R2A medium [72],
King’s B medium [73] and Ammonium Mineral Salt
(AMS) medium [74], Nfb medium [75] and BAz medium
[76]. Nitrogen-ixing bacterial populations were estimated by the Most Probable Number (MPN) technique
using ive tubes per dilution with duplicate tubes per
dilution [77], and incubated at 30 °C for 4–5 days. Bacterial growth as seen by a ine subsurface pellicle in the
tubes were further puriied by transferring to an N-free
semi-solid medium, and single colonies were isolated by
streaking on respective N-free agar plates. For N-free
semi-solid medium, MPN counts were calculated at a
level of 95 % conidence according to the method previously described [78].
16S rRNA gene ampliication, sequencing,
and strain identiication
Phylogenetic positions of bacterial isolates were determined by sequence analysis of the complete 16S rRNA
genes. Genomic DNAs were prepared as described previously [79]. 16S rRNA genes were ampliied by PCR
using universal primers 27F and 1492R [80] (all primer
sequences are shown in Additional ile 10: Table S4) with
the following cycling conditions: initial denaturation for
10 min at 95 °C; 30 cycles of 1.5 min at 95 °C, 1.5 min
at 55 °C and 1.5 min at 72 °C; and a inal extension for
10 min at 72 °C. PCR products were gel-puriied and
sequenced directly or cloned in pGEM-T Easy (Promega,
Madison, USA) before sequencing with the Big-dye
sequencing method (AB Applied Biosystems, Hitachi)
using primers 27F, 1492R, 785F, 518R and 1100R.
Sequences were aligned with the Megalign program of
DNASTAR and analyzed against the EzTaxon-e Database
(http://www.ezbiocloud.net/eztaxon) [81]. Phylogenetic
analyses were performed by the Neighbor-Joining [82],
Maximum-Likelihood [83] and Maximum-Parsimony
[84] methods using the MEGA version 5.05 [85] with the
bootstrap values set at 1000 replications [83].
Screening for cell wall degrading endoglucanase activity
Endoglucanase activity was determined as described
previously [86] with some modiications. Plates containing Kim-Wimpenny solid medium with 0.2 %
Page 10 of 14
carboxymethyl cellulose (CMC) [87], with or without
0.5 % d-glucose, were spotted with 1 µl of grown cultures
(OD600nm = 1.0), air-dried and incubated at 30 °C for
3 days. Cell colonies were lushed of plates with water
and plates were stained with a 0.1 % Congo red solution
for 30 min, followed by several washes with 1 M NaCl.
he appearance of clear yellow halo around the colony
in a red background indicates positive staining for endoglucanase activity.
Nitrogenase activity assay and nifH gene screening
Nitrogen-ixing capability of isolated strains was screened
by testing their growth in 2 ml nitrogen-free liquid
medium as described previously [32]. Nitrogenase activity of selected strains was conirmed by acetylene reduction assay (ARA) in liquid cultures injected with puriied
acetylene gas (15 % v/v) in gas-tight bottles, which were
incubated up to 96 h at 30 °C. Gas samples (0.5 ml)
were extracted at regular intervals with a PTFE-syringe
(Hewlett-Packard, USA) and analyzed in a Gas Chromatograph (GC 6890 N, Agilent Technologies Inc., USA)
with an FID operated under the following conditions:
carrier gas: He-35 ml/min; detector temperature: 200 °C;
column: GS-Alumina (30 m × 0.53 mm I.D.); pressure:
4.0psi. Ethylene produced by the bacteria was quantiied
using standard ethylene (C2H4, Product Number: 00489,
Sigma-Aldrich) curve prepared in duplicates in concentrations ranging from 1 to 1000 nmol. Protein concentration was determined with a modiied Lowry method
using BSA as the standard. For nitrogenase switch-of/
switch-on assay, Methylobacterium cells were grown in
N-free medium containing diferent levels (0–10 mM) of
ammonium chloride and nitrogenase co-factors FeSO4
(10 mg l−1), Na2MoO4 (5 mg l−1) and VCl2 (18.1 mg l−1).
Acetylene reduction activity in planta was performed
as described previously [32]. Briely, samples from each
replication were collected from the glass house and most
of the adhering soil was removed by shaking. Seedlings
were inserted into the 125 ml glass bottles, closed with a
20 mm red stopper sleeve. After removing an equivalent
volume of air, acetylene was injected into these bottles to
give a inal concentration of 15 % and incubated at 30 °C
for 24 h. In planta acetylene reduction activity was measured by GC and value is expressed in nmol C2H4 released
day−1 seedlings−1 after subtracting plant’s background
C2H4 emission.
PCR ampliication of nifH gene fragments was performed using primers nif-Fo and nif-Re under stringent
cycling conditions as described [88], i.e., 95 °C/5 min,
40 cycles of 94 °C/11 s, 92 °C/15 s, 54 °C/8 s, 56 °C/30 s,
74 °C/10 s and 72 °C/10 s, and inal extension for
10 min/72 °C. PCR products were puriied with QIAquick
gel extraction kit (Qiagen, USA) and sequenced.
Madhaiyan et al. Biotechnol Biofuels (2015) 8:222
Leaf colonization by Methylobacterium
Surface sterilization of Jatropha seeds was done by washing coat-less seed kernels in 90 % ethanol (v/v) for 1 min
and 10 % H2O2 (v/v) for 60 min followed by 3-5 rinses
in sterilized distilled water. After soaking overnight at
28 °C in darkness, they were germinated on a hormonefree seed germination medium [32] in Petri dishes and
incubated at 25 °C with 16/8 h light–dark cycles. After
10 days, healthy seedlings (10 seedlings/replica, n = 3)
were transferred to Phytatrays (Sigma, USA) containing sterile sand (autoclaved) with 40 ml of plant nutrient
solution [89]. Jatropha leaves were sprayed with L2-4 suspension (108 cfu/ml) till completely wet. On day 45 and
60, epiphytic population was determined from leaves of
inoculated plants were printed on an AMS agar plate
supplemented with 0.5 % methanol (v/v) and incubated
at 30 °C for 3–5 days. Endophytic colonization was determined from surface-sterilized leaves and homogenize
with sterile pestle and mortar followed by serial dilution methods. Serially diluted samples were plated on an
AMS agar plates, incubate at 30 °C for 3–5 days and pinkpigmented colonies counted from 10−3 to 10−4 dilutions.
Efect of Methylobacterium L2‑4 on Jatropha seedling early
growth
Seedling vigor test was performed with Jatropha to study
efects of foliar spray with ACC deaminase producing Methylobacterium. Strain L2-4 was cultured in 2YT
broth supplemented with 1 % methanol (v/v) until exponential growth phase and harvested by centrifugation.
After washing once with sterile distilled water, inoculants
were made by re-suspending the pellets in water to an
OD600nm of 1.2 (~108 cfu ml−1). To assess the impact on
seed germination and early growth of seedlings, imbibed
seeds (50 seeds/replica, n = 3) were sown in plastic pots
individually and allowed to develop into seedlings. Foliar
application was done after seed germination and growth
parameters were recorded at 45 DAS.
Efect of Methylobacterium on Jatropha growth and seed
yield
Seeds of J. curcas cv. MD44 were used throughout the experiments. Surface sterilization of seeds was done by washing coat-less seed kernels in 75 % ethanol (v/v) for 1 min
and 10 % H2O2 (v/v) for 60 min followed by 3–5 rinses in
sterilized distilled water. After soaking overnight at 28 °C in
darkness, they were germinated on a hormone-free seed germination medium (1/2 MS salt, B5 vitamins, 5 g l−1 sucrose,
0.5 g l−1 MES and 2.2 g l−1 phytagel, pH 5.6) in Phytatrays
(Sigma, USA) in a tissue culture room with a temperature of
25 ± 2 °C and 16/8 h light–dark cycles. To assess the efects
of bacterial inoculation on the growth and yield of Jatropha
under natural conditions, two pot culture experiments were
Page 11 of 14
conducted with garden soil. Plants were planted in pots
(one plant per pot) in sterilized soil (compost/sand mix at
1:1 ratio and in ɸ23 cm, 18 cm height pots; named as Trial
I) or non-sterilized soil (nutrient poor clay soil in ɸ30 cm,
28 cm height pots; named as Trial II). Trial I and Trial II were
maintained in diferent locations and started in diferent seasons. L2-4 cell suspension (1.2 OD600) was applied as foliar
spray till wetting of the leaves at 21 days after seed germination. Commercial NPK Fertiliser was applied once in 15 days
at about half of the recommended dose of approximately
50:30:30 g−1 plant−1 year−1. Biometric observations were
recorded once in 30 days. After lowering, yield parameters
were recorded once in 30 days. Seed set numbers per plant
(n = 9 in Trial I and n = 12 in Trial II) were measured at 480
and 520 DAI in Trail I and Trail II, respectively, and single
seed weight was calculated based on the average of 180 randomly selected seeds per treatment were measured.
Triplicate leaf samples were randomly picked from three
plants on 30 DAI. For methylotrophic bacterial enumerations, homogenates were serially diluted using 1X PBS and
plated on to AMS media with 0.5 % methanol to determine
the methylotrophic population. Pink-pigmented colonies
were counted after incubating the plates for 5 days at 30 °C.
Further conirmation, 20 pink color colonies per replica
were picked from 10−5 dilution and streaked on AMS agar
plates and puriied. Puriied colonies were sequenced by
16S rRNA sequencing and identiied using the EzTaxon
server [81] on the basis of sequence data and sequencing
results compared with pairwise identity of strain L2-4.
Statistical analysis
Statistical analyses were carried out using the Statistical Analysis System (SAS) Version 9.2 (SAS Institute
Inc., Cary, North Carolina, USA). Analysis of variance
(ANOVA) for the endophytic and total bacterial population was carried out using the General Linear Model,
GLM in SAS. he bacterial population data were log transformed before being subjected to further analysis. he
means of the treatment results were subjected to ANOVA
and presented using Fisher’s protected Least Signiicant
Diference (LSD). he model adopted was A [log CFU (g/
FW)] = C (cultivar) Pt (plant tissue) M (medium) C*Pt
C*M Pt*M to check the efect of individual factors and the
interactions between them. A canonical discriminant analysis was carried out to discriminate the variations among
the cultivars or plant tissue with reference to the endophytic and total population. Given two or more groups of
observation with measurements on several quantitative
variables, CDA derives a linear combination of the variables that have the highest possible multiple correlation
with the groups. Endophytic bacterial inoculation data
were subjected to analysis of variance and testing of means
by Duncan’s Multiple Range Test (DMRT) at P ≤ 0.05
Madhaiyan et al. Biotechnol Biofuels (2015) 8:222
using SAS package. Student’s t test was done using the
JavaScript maintained by Professor Hossein Arsham, Johns
Hopkins Carey Business School (http://home.ubalt.edu/
ntsbarsh/Business-stat/otherapplets/MeanTest.htm).
Nucleotide sequence accession numbers
All 16S rRNA gene sequences determined in this study
have been submitted to NCBI under the accession numbers JQ659304 to JQ660320 and the numbers are also
listed in Additional ile 3: Table S1. nifH gene sequences
have been submitted to NCBI under the accession numbers KR075947-KR075982, KC195919 and CP005991.
Additional iles
Additional ile 1: Figure S1. Culturable endophytic bacteria densities in
various Jatropha tissues. Surface-sterilized tissues (roots, stems and leaves)
were grinded into ine powder; diluted in water in series and plated on to
diferent media. Values shown are the average of three individual plants
originating from the same germplasm collection. (a) Germplasm from
Indonesia, (b) Germplasm from China and (c) Germplasm from India. Each
value represents the mean ± SD, n = 3.
Additional ile 2: Figure S2. Discriminant function analysis. Ordination plots of variables resulting from the irst (CAN1) and second (CAN2)
canonical functions for diferent plant tissue types (a) and media (b). The
variables were generated based on the total populations from diferent
plant tissues (leaf, stem and root) and media (HTM, NFM and MM).
Additional ile 3: Table S1. Complete table of taxonomic units (strains)
derived from surface sterilized Jatropha tissues. Table includes, strains,
cultivars, plant tissue, growth media, close relatives, pairwise similarity,
class, GenBank ID number, nifH positive, endoglucanase and N-ixing
activity. *EzBioCloud software to identify closely related type strains (Kim
et al., 2012). The media 869, R2A and King’S B medium used for isolation of
heterotrophic bacteria; AMS medium used for isolation of methylotrophic
bacteria; N-free semisolid used for isolation of diazotrophic bacteria.
Additional ile 4: Table S2. Distribution of representative bacterial taxa
in the leaf, stem and root of Jatropha biodiesel plants. Relative abundance
of diferent taxon at genus level based on 16S rRNA gene sequences
under diferent classes or phyla.
Additional ile 5: Figure S3. Phylogenetic positions of stem endophytes. The tree was constructed based on the 16S rDNA sequences using
the Neighbor-Joining method. Bootstrap values (using 1000 replicates)
are indicated at the branching points. Scale bar indicates % estimated
substitutions. Candidate for nitrogen-ixers (shown in blue) indicate the
presence of nifH gene as evidenced by PCR ampliications. The number of
strains is shown in parenthesis in red. The green arrowhead sizes indicate
the relative abundance of the genus.
Additional ile 6: Figure S4. Phylogenetic positions of root endophytes.
The tree was constructed based on the 16S rDNA sequences using the
Neighbor-Joining method. Bootstrap values (using 1000 replicates) are
indicated at the branching points. Scale bar represents % estimated
substitutions. Candidate for nitrogen-ixers (shown in blue) indicate the
presence of nifH gene as evidenced by PCR ampliications. The number of
strains is shown in parenthesis in red. The yellow arrowhead sizes indicate
the relative abundance of the genus.
Additional ile 7: Figure S5. Cultivable endophytic bacterial diversity of
Jatropha. Distribution of the phylotypes (%) over the diferent phyla and
classes. The bar of each phylogenetic group is subdivided according to
the diferent identiication levels of the phylotypes. The group “identiied
at species level” contains those phylotypes that belong to existing species
with a 16S rDNA homology threshold of ≥ 99.0 %. The group “identiied
Page 12 of 14
at genus level” contains phylotypes that, based on phylogeny, belong to
a particular genus and may represent a new species within that particular
genus or an existing species within this genus showing < 99 % 16S rRNA
gene sequence pairwise similarity with the type strain of this species. The
group “potential gen. nov.” contains those phylotypes that could not be
assigned to a particular genus (< 97 % pairwise similarity) based on the
phylogeny of the 16S rRNA gene.
Additional ile 8: Table S3. Distribution of representative bacterial
taxa in the diferent germplasm of Jatropha biodiesel plants. Relative
abundance of diferent taxon at genus level based on 16S rRNA gene
sequences under diferent classes or phyla.
Additional ile 9: Figure S6. Gas chromatography chromatogram
showing ethylene and acetylene peaks. (a) 0.5 ml of 4.85 μmol ethylene
(C2H4, Product Number: 00489, Sigma-Aldrich) standard was injected
in GC. (b) Strain L2-4 inoculated in N-free medium (40 ml) and ARA was
performed by injecting puriied acetylene into the bottles sealed with
gas-tight serum stoppers to yield 15 % acetylene (v/v); this was followed
by incubation for up to 48 h at 30 °C. (c) ARA was performed without
strain L2-4 (blank).
Additional ile 10: Table S4. List of primers used in this study.
Abbreviations
ACC: 1-aminocyclopropane-1-carboxylate; AMS: ammonium mineral salt
medium; ANOVA: analysis of variance; ARA: acetylene reduction activity; CDA:
canonical discriminant analysis; CFU: colony forming units; CMC: carboxymethyl cellulose; DAI: days after inoculation; DMRT: Duncan’s multiple range
test; LSD: least signiicant diference; MPN: most probable number; PGP: plant
growth promotion; RG: rate of germination; SAS: statistical analysis system; SVI:
seedling vigor index.
Authors’ contributions
MM and LJ conceived experiments and drafted the manuscript. MM performed strain isolation, characterization and bioassays for bacteria and plants.
NST and THHA participated in plant inoculation experiments, data analysis
and helped to revise the manuscript. BP participated in the bioinformatic,
statistical analysis and helped to revise the manuscript. All authors read and
approved the inal manuscript.
Author details
1
Biomaterials and Biocatalysts Group, Temasek Life Sciences Laboratory, 1
Research Link, National University of Singapore, Singapore 117604, Singapore.
2
Plant Biology Division, The Samuel Roberts Noble Foundation, Ardmore, OK
73401, USA.
Acknowledgements
This work was supported by the Temasek Foundation and the Singapore
Economy Development Board (EDB).
Competing interests
The authors declare that they have no competing interests. Temasek Life Sciences Laboratory has an interest in using selected nitrogen-ixing strains for
applications in agriculture.
Received: 9 June 2015 Accepted: 30 November 2015
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Bharath Prithiviraj