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14 15
Mesorhizobium shonense sp. nov., Mesorhizobium hawassense sp. nov. and
16
Mesorhizobium abyssinicae sp. nov. isolated from root nodules of different
17
agroforestry legume trees growing in southern Ethiopia
18
Tulu Degefu1, Endalkachew Wolde-meskel1, 2, Binbin Liu1, Ilse Cleenwerck3, Anne Willems4 19
and Åsa Frostegård1 20
21
1Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life 22
Sciences, P.O. Box 5003, NO-1432 Ås, Norway 23
2School of Plant and Horticultural Sciences, Hawassa University, P.O. Box 5, Hawassa, Ethiopia 24
3BCCM/LMG Bacteria Collection, Ghent University, K. L. Ledeganckstraat 35, B-9000 Gent, 25
Belgium 26
2
4Laboratory of Microbiology (WE10), Ghent University, K. L. Ledeganckstraat 35, B-9000 27
Gent, Belgium 28
29
Author for correspondence: Tulu Degefu. E-mail: tulu.degefu@umb.no 30
Contents category: New Taxa-Proteobacteria 31
Running title: New Mesorhizobium species from Ethiopia 32
33
34
35
ABSTRACT 36
Eighteen Mesorhizobium strains, obtained from root nodules of woody legumes growing in 37
Ethiopia, were previously shown by multilocus sequence analysis of five housekeeping genes to 38
form three novel genospecies (Degefu et al., 2011). In the present study, the phylogenetic 39
relationship between representative strains of these three genospecies and the type strains of their 40
closest phylogenetic neighbors Mesorhizobium plurifarium, Mesorhizobium amorphae, 41
Mesorhizobium septentrionale and Mesorhizobium huakuii was further evaluated using a 42
polyphasic taxonomic approach. In line with our earlier MLSA of other house-keeping genes, the 43
phylogenetic trees derived from the atpD and glnII genes grouped the test strains into three well- 44
supported, distinct lineages that exclude all defined Mesorhizobium species. The DNA-DNA 45
relatedness between therepresentative strains of genospecies I─III and the type strains of their 46
3
closest phylogenetic neighbors was low (≤ 59 %). They differed from each other and from their 47
closest phylogenetic neighbors by presence/absence of several fatty acids, or by large differences 48
in the relative amount of particular fatty acids. While showing distinctive features with one or 49
more references, they were generally able to utilize a wide range of substrates as sole carbon and 50
nitrogen sources. The strains belonging to genospecies I, II and III therefore represent novel 51
species for which we propose the names Mesorhizobium shonense sp. nov., Mesorhizobium 52
hawassense sp. nov. and Mesorhizobium abyssinicae sp. nov. The isolates AC39aT (=LMG 53
26966T = HAMBI 3295T), AC99bT (=LMG 26968T = HAMBI 3301T) and AC98cT (=LMG 54
26987T = HAMBI 3306T) are proposed as type strains for the respective novel species.
55 56 57
Rhizobia form, together with their corresponding legume hosts, a beneficial symbiotic 58
association in which nitrogen is fixed inside nodules formed on the root, or occasionally on the 59
stem, of host species. Ultimately, this leads to improved soil fertility and stability. In view of this 60
agronomic benefit, information on the biodiversity of the indigenous rhizobial resources is 61
important for conservation and sustainable utilization of these microsymbionts in agriculture and 62
forestry. Earlier studies of rhizobia from legumes growing on the African continent, particularly 63
West Africa, identified large and hitherto unknown rhizobial diversity. Further characterizations 64
of these strains have led to the description of new genera and species (Sawada et al., 2003).
65
These include Azorhizobium caulinodans (Dreyfus et al., 1988), Allorhizobium undicola (de 66
Lajudie et al., 1998a), Ensifer saheli and Ensifer terangae (de Lajudie et al., 1994), 67
Mesorhizobium plurifarium (de Lajudie et al., 1998b) and Methylobacterium nodulans (Sy et al., 68
2001), all of which were isolated from legumes growing in Senegal. Studies conducted in North 69
4
African countries, notably in Tunisia and Morocco, also indicated that legumes growing in these 70
countries are associated with strains related to the genera Mesorhizobium, Ensifer and Rhizobium 71
(Ba et al., 2002; Khbaya et al., 1998; Mhamdi et al., 2002). Recently, two new species named E.
72
numidicus and E. garamanticus, isolated from root nodules of legumes growing in infra-arid 73
regions in Tunisia were described (Merabet et al., 2010). Information on the biodiversity, 74
phylogeny and taxonomic identity of microsymbionts nodulating legumes in East Africa is 75
scarce compared to the more studied West African region. Nevertheless, there are a few 76
examples from Sudan and Kenya (neighboring Ethiopia in the Northwest and South, 77
respectively), demonstrating the presence of a large number of phenotypic and genotypic 78
clusters. For example, phenotypic numerical analyses conducted on a set of Sudanese strains 79
revealed a large number of phenotypic groups (Zhang et al., 1991). Further characterization of 80
these isolates led to the recognition of three main phylogenetic groups belonging to the rhizobial 81
genera Ensifer, Rhizobium and Mesorhizobium (Haukka & Lindström, 1994; Haukka et al., 82
1996; Nick et al., 1999a; Nick et al., 1999b). Subsequent characterization of selected strains 83
from the same collection using a polyphasic taxonomic approach has led to the description of E.
84
arboris, E. kostiense and M. plurifarium (de Lajudie et al., 1998b; Nick et al., 1999a). Other 85
studies of similar nature, on samples from Kenya, revealed the existence of diverse phenotypic 86
and genotypic clusters related to the genera Agrobacterium, Bradyrhizobium, Mesorhizobium, 87
Rhizobium and Ensifer (Anyango et al., 1995; McInroy et al., 1999; Odee et al., 1995; Odee et 88
al., 1997; Odee et al., 2002).
89 90
As for the other parts of Eastern Africa, investigations of rhizobial diversity in Ethiopia are 91
scarce. In an earlier phenotypic and genotypic analysis of a set of 240 rhizobial strains, isolated 92
5
from various herbaceous and woody legume hosts growing in different agro-ecological zones of 93
southern Ethiopia (Wolde-meskel et al., 2004; Wolde-meskel et al., 2005), a large diversity of 94
microsymbionts representing the main phylogenetic branches of the rhizobial genera was 95
demonstrated. The majority of these isolates grouped separately from the defined species using 96
MLSA, revealing three new genospecies of Mesorhizobium (Degefu et al., 2011) and seven 97
genospecies of Ensifer (Degefu et al., 2012) in this collection.
98 99
The genus Mesorhizobium comprises a group of species with distinctive phenotypic properties.
100
Based on the 16S rRNA phylogeny this genus forms a well-defined clade different from the 101
Rhizobium-Ensifer-Agrobacterium clusters (Jarvis et al., 1997). Currently there are 24 validly 102
described species within this genus, including the two recently described species Mesorhizobium 103
silamurunense and Mesorhizobium muleiense currently in press in IJSEM (Zhao et al., 2011;
104
Zhang et al., 2011). Here, we present a consensus result, generated from a polyphasic taxonomic 105
approach (sequence analyses of atpD and glnII genes, DNA-DNA hybridizations, cellular fatty 106
acid profiling and other phenotypic tests), showing that the three unique genospecies reported 107
earlier (Degefu et al., 2011) belong to new species within the genus Mesorhizobium, and propose 108
the new names M. shonense sp. nov., M. hawassense sp. nov. and M. abyssinicae sp. nov. for 109
genospecies I, II and III, respectively.
110 111
In the course of studies of a large number of rhizobial isolates nodulating agroforestry legume 112
species growing in southern Ethiopia, 18 strains nodulating Sesbania sesban and three Acacia 113
species, were found to belong to the Mesorhizobium branch, with distinctive AFLP and 16S 114
rRNA-RFLP patterns (Wolde-meskel et al., 2004; Wolde-meskel et al., 2005). The taxonomic 115
6
diversity of these isolates was further revealed by MLSA of selected housekeeping genes and 116
they were shown to have distinct individual core gene types, thus occupying three distinct 117
positions in the phylogenetic tree (Degefu et al., 2011). In the present study we further extended 118
the characterization of the strains representing the three unique genomic species, following a 119
polyphasic taxonomic approach (analyses of DNA-DNA relatedness, major fatty acids profiles 120
and morphological/phenotypic features), as this is currently suggested for descriptions of new 121
species (Stackebrandt et al., 2002).
122 123
Analyses of housekeeping genes is regarded as a powerful taxonomic tool for prokaryotic 124
systematics and new species description (Martens et al., 2008). This approach provides higher 125
sequence variation than the 16S rRNA gene, thus allowing better discrimination between closely 126
related strains (Hanage et al., 2005; Martens et al., 2007; Martens et al., 2008). It is also believed 127
to dilute the distorting effects that result from horizontal gene transfer and subsequent 128
recombination events. Therefore, in addition to the MLSA of a set of housekeeping and 129
symbiosis-related genes conducted earlier on these strains (Degefu et al., 2011), we sequenced 130
and analyzed the atpD and glnII genes, as the phylogeny generated from these genes sequences 131
has been reported to fully support both the integrity of the Mesorhizobium clade and its 132
phylogenetic placement based on the 16S rRNA gene (Gaunt et al., 2001; Turner & Young, 133
2000). Primers and PCR conditions were as specified by Gaunt et al. (2001) and Vinuesa et al.
134
(2005). The expected PCR products of each gene were excised from gel and purified using 135
E.Z.N.A.TM Gel Extraction Kit based on the manufacturer’s recommendations, and the resulting 136
purified PCR products were sequenced. The corresponding gene sequences for the reference 137
species were retrieved from the NCBI public database. Phylogenetic analyses were conducted 138
7
using MEGA version 5 (Tamura et al., 2011). The maximum likelihood phylogenetic trees were 139
constructed with 100 replications.
140 141
The phylogenetic trees constructed for each gene (Fig. 1), showed that the strains formed three 142
well-supported clusters corresponding to the three unique genospecies previously defined 143
(Degefu et al., 2011). The novel isolates within each of the designated genospecies shared 144
sequence similarity ranging between 99% and 100% with each other for the sequences of the 145
atpD and glnII genes. However, the highest similarities between the three new species proposed 146
and the described Mesorhizobium species, for both housekeeping genes, did not exceed 96.8 %.
147
Based on the sequence analyses of the two housekeeping genes, the closest phylogenetic 148
neighbors were found to be M. plurifarium and M. silamurunense for the atpD gene, while M.
149
septentrionale and M. amorphae were the closest neighbors based on the glnII gene (Table S3).
150
Comparison of the two genes (Figs. 1 a and b) showed different closest phylogenetic neighbors 151
for the proposed new species, depending on which gene was analyzed, but their distinctive 152
position remained unique in both gene trees. Such variations in the relative phylogenetic 153
placement are common and consistent with other, similar studies, for example while describing 154
B. iriomotense (Islam et al., 2008), Enterococcus species (Naser et al., 2005), M. loti (Jarvis et 155
al., 1982) and M. ciceri (Nour et al., 1994). Differences in evolutionary histories of the genes 156
and the inter-species recombination events might explain this phenomenon. While this 157
manuscript was in preparation, at its final stage, two new Mesorhizobium species, namely M.
158
silamurunense (Zhao et al., in press) and M. muleiense (Zhang et al., in press) were described.
159
Therefore, phylogenetic analyses of 16S rRNA, recA and the concatenation of 16S rRNA, recA, 160
glnII and atpD genes were conducted to check the relatedness among these species and the three 161
8
new species proposed in the present study. The results (Fig. S1) were in agreement with the 162
single-gene phylogenies generated in the present study, and also with those in the previous study 163
(Degefu et al., 2011). The phylogenetic analysis of concatenated gene sequences grouped the test 164
strains into three well-supported (100 % BT) monophyletic clades, with M. plurifarium and M.
165
silamurunense more distantly related (Fig. S1). These analyses thus confirmed the distinctness of 166
our strains.
167 168
DNA-DNA hybridization allows genome-wide comparisons between organisms and is a standard 169
technique for description of new species (Graham et al., 1991; Hanage et al., 2006; Wayne et 170
al., 1987). We designed and conducted the hybridization experiments based on the results from 171
our previous MLSA (Degefu et al., 2011). High-molecular mass DNA for DNA-DNA 172
hybridization studies and DNA base composition determination was extracted using the method 173
of Wilson (1987), with minor modifications (Cleenwerck et al., 2002). The DNA-DNA 174
hybridizations were carried out with the type strains of M. shonense sp.nov. AC39aT, M.
175
hawassense sp. nov. AC99bT, M. abyssinicae sp. nov. AC98cT and M. abyssinicae sp. nov 176
AC100e and the type strains of M. plurifarium LMG 11892T, M. amorphae LMG 18977T, M.
177
septentrionale LMG 23930T and M. huakuii LMG 23930T at 48 °C using a modification 178
(Cleenwerck et al., 2002; Goris et al., 1998) of the microplate method described elsewhere 179
(Ezaki et al., 1989). Reciprocal reactions were performed, and their variation was generally 180
within the limits of this method (Goris et al., 1998). The DNA mol% G+C content of strains 181
AC39aT (=M. shonense sp. nov. AC39aT), AC99bT (=M. hawassense sp. nov. AC99bT) and 182
AC98cT (= M. abyssinicae sp. nov. AC98cT) and AC100e (=M. abyssinicae sp. nov. AC100e) 183
was determined by HPLC according to the method of Mesbah et al. (1989). The DNA-DNA 184
9
relatedness (Table S1) obtained between the type strains of the three new species and the type 185
strains of the four closest phylogenetic neighbours of the Mesorhizobium species was below the 186
threshold limit of 70 % set for genomic species identity (Wayne et al., 1987) and did not exceed 187
59 %. The DNA mol% G+C content for the type strains M. shonense sp. nov. AC39aT, M.
188
hawassense sp. nov. AC99bT and M. abyssinicae sp. nov. AC98cT was 62.2 mol%, 62.5 mol%
189
and 63.5 mol%, respectively (Table S1), which is similar to the values previously reported for 190
other Mesorhizobium species (Jarvis et al., 1997).
191 192
Analysis of cellular fatty acid profiles is a useful tool for identifying and characterizing unknown 193
strains of rhizobia and for establishing taxonomic relationship between species (Nandasena et al., 194
2009; Tighe et al., 2000). The whole-cell fatty acid composition was determined for the three 195
new species proposed, M. shonense sp. nov. AC39aT, M. hawassense sp. nov. AC99bT, M.
196
abyssinicae sp. nov. AC98cT and M. abyssinicae sp. nov. AC100e, and for the type strains M.
197
plurifarium LMG 11892T and M. huakuii LMG 14107T as previously described (Wang et al., 198
2007), using an Agilent Technologies 6890N gas chromatograph (Santa Clara, CA, USA). Cells 199
were harvested from cultures grown for 48 h at 28 °C on a previously described modified TY 200
medium (Jarvis et al., 1996). Cultivation of the strains, extraction and analysis of the fatty acid 201
methyl esters were performed according to the recommendations of the Microbial Identification 202
System, Sherlock version 3.10 (MIDI). The peaks of the profiles were identified using the 203
TSBA50 identification library version 5.0.
204 205
The cellular fatty acid profiles of the type strains of the three new species (including a strain 206
AC100e representing a separate sub-group of A. abyssinicae) and their closest phylogenetic 207
10
neighbors in the genus Mesorhizobium are shown in Table 1. The major fatty acids obtained for 208
M. plurifarium and M. huakuii are in accordance with those previously reported (Wang et al., 209
2007). The dominating fatty acids in strains of Mesorhizobium, reported by Tighe et al (2000) 210
and Nandasena et al (2009) are C16:0 (generally comprising 10 % or more of the total fatty acid 211
content), C18:1ω7c (often comprising 25-84 % of the total fatty acids; sometimes clustered with 212
C18:1 ω 9cis/trans and C18:1ω12trans, both of which are found only in trace amounts in some 213
bacteria (Ratledge and Wilkinson, 1988), and C19:0 cyclo ω8c (often designated as cy19:0, 214
generally comprising 20-30 % of the total fatty acid content). All these fatty acids are common to 215
most Gram-negative bacteria (Ratledge and Wilkinson, 1988). Other fatty acids generally found 216
in Mesorhizobium, and comprising up to a few % of the total fatty acids, include C17:0, C18:0,
217
C17:1ω8c and C17:0cyclo, the latter also being common to many Gram-negative bacteria (Ratledge 218
and Wilkinson, 1988). The two methyl-branched fatty acids 11-methyl C18:1ω7c and 10-methyl 219
C19:0, often comprising > 10 % and around 1 %, respectively, of the fatty acids in Mesorhizobium 220
strains (Tighe et al., 2000; Nandasena et al., 2009), are not commonly reported for other groups 221
of bacteria (Ratledge and Wilkinson, 1988). Of these, 11-methyl C18:1ω7c was detected in the 222
two analysed strains of M. abyssinicae sp. nov. (at lower percentage than reported for most other 223
Mesorhizobium strains), but not in M. shonense sp. nov. or M. hawassense sp. nov., while 10- 224
methyl C19:0 was detected in all of the three proposed novel species, at similar levels as for other 225
Mesorhizobium strains. Thus, the three new species can be differentiated from each other and 226
their closest phylogenetic neighbors by presence/absence or by differences in the relative 227
concentration of particular fatty acids such as C12:0 3-OH, C15:1 ω8c, 11-Methyl C18:1 ω7c and 228
C19:0 cyclo ω8c (Table 1).
229 230
11
Phenotypic features of the strains representing the novel species were determined and compared 231
with the type strains of some of the closest phylogenetic neighbors in the defined Mesorhizobium 232
species. The following parameters were included for phenotypic characterization: utilization of 233
sole carbon sources, resistance to antibiotics, tolerance to NaCl, and pH and temperature range 234
for growth. The ability of the test strains to utilize amino acids (L-Alanine, L-leucine, L-aspartic 235
acid, L-glutamic acid, L-phenylalanine, L-proline, L-histidine) as sole nitrogen source was also 236
investigated following the methods described elsewhere (Amarger et al., 1997). The ability of 237
the strains to utilize different substrates as sole carbon sources were previously determined 238
(Wolde-meskel et al., 2004). The results presented in Table S2, show the distinctive 239
phenotypic/physiological features of the new species and also demonstrated that the three novel 240
species were able to utilize a wide range of substrates as sole carbon and nitrogen sources. In 241
addition, the new species could be differentiated from each other based on their positive or weak 242
use of the following substrates as sole carbon sources: formic acid, malonic acid, L-serine, 243
sebacic acid, putrescine, propionic acid, D-serine and p-hydroxy phenyl acetic acid.
244 245
In conclusion, based on previous AFLP, 16S rRNA PCR-RFLP (Wolde-meskel et al., 2005) and 246
MLSA data (Degefu et al., 2011) of eighteen bacterial strains belonging to the genus 247
Mesorhizobium, we distinguished three distinct groups (genospecies) within this genus. In the 248
present study, based on sequence analyses of two additional housekeeping genes, DNA-DNA 249
hybridizations, fatty acid profiling and phenotypic tests, the three unnamed genospecies were 250
clearly differentiated from each other and from their closest phylogenetic neighbors, thus 251
forming three novel lineages. Taken together, the results from the genotypic and phenotypic 252
characterizations in this and earlier studies suggest that the three genospecies represent three new 253
12
species within the Mesorhizobium clade. We propose the names M. shonense sp. nov. for GSI (=
254
strain AC39aT = LMG 26966T = HAMBI 3295T), M. hawassense sp. nov. for GSII (= strain 255
AC99bT = LMG 26968T = HAMBI 3301T) and M. abyssinicae sp. nov. for GSIII (= strain 256
AC98cT = LMG 26967T = HAMBI 3306T). Phenotypic differentiation of these species from their 257
closest phylogenetic neighbors is given in Table S2.
258 259
Description of Mesorhizobium shonense sp. nov.
260
Mesorhizobium shonense (sho.nen’se. N. L. neut. adj. shonense of Shone, referring to Shone in 261
Southern Ethiopia, the location where this species was first isolated).
262 263
Cells are Gram-negative, motile, rod-shaped, 0.34 ± 0.05 μ m wide by 2.59 ± 0.48 μ m long.
264
Colonies on YMA are white, opaque, with generation time of about 6 h. When cultured on YMA 265
this species can grow at pH values ranging between 4.5 and 10.0 and at temperatures up to 35 266
°C, but it does not tolerate NaCl concentrations beyond 0.5 % (w/v). It is sensitive to 267
streptomycin (50 µg/ml), lincomycin (100 µg/ml), novobiocin (10 µg/ml), erythromycin (20 268
µg/ml), neomycin (20 µg/ml), spectinomycin (5 µg/ml) and kanamycin (15 µg/ml). Unlike some 269
representatives of the defined Mesorhizobium species (M. plurifarium, M.huakuii, M. ciceri and 270
M. loti), this species can utilize a wide range of substrates as sole carbon source (based on Biolog 271
system) (Wolde-meskel et al., 2004). However features that discriminate this species from the 272
other two proposed new species but also from the defined Mesorhizobium species are given in 273
Table S2. It grows well on formic acid, malonic acid, L-serine, sebacic acid and p-hydroxy 274
phenyl acetic acid. However it showed slight growth on putrescine, propionic acid and D-serine.
275
Fatty acids included a small amount of C15:1 ω8c and Summed Feature 3 (15 iso 2-OH and/or 276
13
C16:1 ω7c) while no 11-Methyl C18:1 ω7c was detected (Table 1). The type strain is AC39aT (=
277
LMG 26966T = HAMBI 3295T) and its DNA G+C content is 62.2 mol%. This strain was isolated 278
from nodules of Acacia abyssinica, a leguminous tree native to Ethiopia. The accession number 279
of the 16S rRNA gene sequence of the type species is GQ847890.
280 281
Description of Mesorhizobium hawassense sp. nov.
282
Mesorhizobium hawassense (ha.wa’sen.se. N. L. neut. adj. hawassense of Hawassa, referring to 283
Hawassa, the regional capital of Southern Ethiopia where the type strain was isolated).
284 285
Cells are Gram-negative, motile, rod-shaped, 0.30 ± 0.02 μm wide by 2.7 ± 0.45 μm long.
286
Colonies on YMA are white, opaque, with generation time of about 7.21 ± 0.09 h. This species 287
can grow at pH values ranging between 4.5 and 10.0 and at temperature of not more than 35 °C.
288
This species cannot grow on YMA in the presence of NaCl beyond 0.5 % (w/v). It is sensitive to 289
streptomycin (50 µg/ml), lincomycin (100 µg/ml), novobiocin (10 µg/ml), erythromycin (20 290
µg/ml), neomycin (20 µg/ml), spectinomycin (5 µg/ml) and kanamycin (15 µg/ml). It grows on a 291
wide range of substrates as sole sources of carbon sources based on biolog profiling (Wolde- 292
meskel et al., 2004). However this species can also be differentiated from the other two proposed 293
new species and defined Mesorhizobium species by some phenotypic features (Table S2). While 294
showing slight growth on formic acid, malonic acid, L-serine and p-hydroxyphenyl acetic acid, it 295
grew well on putrescine, propionic acid and D-serine. However it did not grow on sebacic acid.
296
Fatty acids analysis did not yield any C12:0 3OH, C15:1 ω8c, 11-Methyl C18:1 ω7c or Summed 297
Feature 3 (15 iso 2-OH and/or 16:1 ω7c) (Table 1). The type strain is AC99bT (= LMG 26968T = 298
HAMBI 3301T). It was isolated from root nodules of S. sesban growing at Wondogenet, around 299
14
Hawassa, the regional capital of Southern Ethiopia. Its G+C content is 62.5 mol%. The accession 300
number of the 16S rRNA gene sequence of the type species is GQ847899.
301 302 303
Description of Mesorhizobium abyssinicae sp. nov.
304
Mesorhizobium abyssinicae (a.by.si.ni.cae L. gen. n. of abyssinica, referring to Acacia 305
abyssinica, the host species indigenous to Ethiopia which these bacteria were first isolated from).
306 307
Cells are Gram-negative, motile, rod-shaped, 0.33 ± 0.05 μ m wide by 2.61 ± 0.43 μ m long.
308
Colonies on YMA are white, opaque, with generation time of between 6.89 ± 0.08 h. This 309
species can grow at pH values ranging between 4.5 and 9.0 and at temperature of ≤35°C. This 310
species cannot grow on YMA in the presence of NaCl beyond 0.5 % (w/v). It is sensitive to 311
streptomycin (50 µg/ml), lincomycin (100 µg/ml), novobiocin (10 µg/ml), erythromycin (20 312
µg/ml), neomycin (20 µg/ml), spectinomycin (5 µg/ml), kanamycin (50 µg/ml). The distinctive 313
features that separate this species from the other two proposed new species including the defined 314
Mesorizobium species is presented in Table S2. This species grew well on formic acid, malonic 315
acid, L-serine, sebacic acid, putrescine, propionic acid and D-serine. But it showed weak growth 316
on p-hydroxy phenyl acetic acid. Fatty acids included small amounts of C12:0 3-OH and 11- 317
Methyl C18:1 ω7c (Table 1). The type strain is AC98cT (= LMG 26967T = HAMBI 3306T). It was 318
isolated from root nodules of Acacia abyssinica and A. tortilis growing at Wondogenet and Leku 319
sampling locations in southern Ethiopia. Its DNA G+C content is 63.5 mol%. The accession 320
number of the 16S rRNA gene sequence of the type species is GQ847896.
321 322
15 Acknowledgments
323
This work was supported by a grant from the Norwegian Programme for Development, Research 324
and Education (NUFU) and by a PhD stipend (T. Degefu) from the Norwegian State Educational 325
Loan Fund. The BCCM/LMG Bacteria Collection is supported by the Federal Public Planning 326
Service – Science Policy, Belgium. The authors wish to acknowledge Katrien Engelbeen for 327
technical assistance.
328 329
REFERENCES 330
Amarger, N., Macheret, V. & Laguerre, G. (1997). Rhizobium gallicum sp. nov. and 331
Rhizobium giardinii sp. nov., from Phaseolus vulgaris nodules. Int J Syst Bacteriol 47, 996- 332
1006.
333
Anyango, B., Wilson, K. J., Beynon, J. L. & Giller, K. E. (1995). Diversity of rhizobia 334
nodulating Phaseolus vulgaris L. in two Kenyan soils with contrasting PHs. Appl Environ 335
Microbiol 61, 4016–4021.
336
Ba, S., Willems, A., de Lajudie, P., Roche, P., Jeder, H., Quatrini, P., Neyra, M., Ferro, M., 337
Prome, J.-C. & other authors (2002). Symbiotic and taxonomic diversity of rhizobia isolated 338
from Acacia tortilis subsp. raddiana in Africa. Syst App Microbiol 25, 130–145.
339
Cleenwerck, I., Vandemeulebroecke, K., Janssens, D. & Swings, J. (2002). Re-examination 340
of the genus Acetobacter, with description of Acetobacter cerevisiae sp. nov. and Acetobacter 341
malorum sp. nov. Int J Syst Evol Microbiol 52, 1551–1558.
342
Degefu, T., Wolde-meskel, E. & Frostegård, Å. (2011). Multilocus sequence analyses reveal 343
several unnamed Mesorhizobium genospecies nodulating Acacia species and Sesbania sesban 344
trees in Southern regions of Ethiopia. Syst Appl Microbiol 34, 216–226.
345
16
Degefu, T., Wolde-meskel, E. & Frostegård, Å. (2012). Phylogenetic analyses of multilocus 346
sequences identify seven novel Ensifer genospecies isolated from less explored biogeographical 347
region in East Africa. Int J Syst Evol Microbiol In press DOI 10.1099/ijs.0.039230-0.
348
de Lajudie, P., Laurent-Fulele, E., Willems, A., Torck, U., Coopman, R., Collin, M. D., 349
Kersters, K., Dreyfus, B. & Gillis, M. (1998a). Allorhizobium undicola gen. nov., sp. nov., 350
nitrogen-fixing bacteria that efficiently nodulate Neptunia natans in Senegal. Int J Syst Bacteriol 351
48, 1277–1290.
352
de Lajudie, P., Willems, A., Nick, G., Moreira, F., Molouba, F., Hoste, B., Torck, U., Neyra, 353
M., Collins, M. D. & other authors (1998b). Characterization of tropical tree rhizobia and 354
description of Mesorhizobium plurifarium sp. nov. Int J Syst Bacteriol 48, 369–382.
355
de Lajudie, P. d., Willems, A., Pot, B., Dewettinck, D., Maestrojuan, G., Neyra, M., Collins, 356
M. D., Dreyfus, B., Kersters, K. & other authors (1994). Polyphasic taxonomy of rhizohia:
357
emendation of the genus Sinorhizobium and description of Sinorhizobium meliloti comb. nov., 358
Sinorhizobium saheli sp. nov., and Sinorhizobium terangae sp. nov. Int J Syst Bacteriol 44, 715–
359
733.
360
Dreyfus, B., Garcia, J. L. & Gillis, M. (1988). Characterization of Azorhizobium caulinodans 361
gen.nov.sp.nov., a stem-nodulating nitrogen-fixing bacterium isolated from Sesbania rostrata.
362
Int J Syst Bacteriol 38, 89–98.
363
Ezaki, T., Hashimoto, Y. & Yabuuchi, E. (1989). Fluorometric deoxyribonucleic acid- 364
deoxyribonucleic acid hybridization in microdilution wells as an alternative to membrane filter 365
hybridization in which radioisotopes are used to determine genetic relatedness among bacterial 366
strains. Int J Syst Bacteriol 39, 224–229.
367
17
Gaunt, M. W., Turner, S. L., Rigottier-Gois, L., Lloyd-Macgilp, S. A. & Young, J. P. W.
368
(2001). Phylogenies of atpD and recA support the small subunit rRNA-based classification of 369
rhizobia. Int J Syst Evol Microbiol 51, 2037–2048.
370
Goris, J., Suzuki, K., De Vos, P., Nakase, T. & Kersters, K. (1998). Evaluation of a 371
microplate DNA-DNA hybridization method compared with the initial renaturation method. Can 372
J Microbiol 44, 1148–1153.
373
Graham, P. H., Sadowsky, M. J., Keyser, H. H., Barnet, Y. M., Bradley, R. S., Cooper, J.
374
E., Deley, D. J., Jarvis, B. D. W., Roslycky, E. B. & other authors (1991). Proposed minimal 375
standards for the description of new genera and species of root and stem-nodulating bacteria. Int 376
J Syst Bacteriol 41, 582–587.
377
Hanage, W. P., Fraser, C. & Spratt, B. G. (2005). Fuzzy species among recombinogenic 378
bacteria. BMC Biol 3, doi:10.1186/1741-7007-3–6.
379
Hanage, W. P., Fraser, C. & Spratt, B. G. (2006). Sequences, sequence clusters and bacterial 380
species. Phil trans R Soc B 361, 1917–1927.
381
Haukka, K. & Lindström, K. (1994). Pulsed-field gel electrophoresis for genotypic comparison 382
of Rhizobium bacteria that nodulate leguminous trees. FEMS Microbiol Lett 119, 215–220.
383
Haukka, K., Lindström, K. & Young, J. P. W. (1996). Diversity of partial 16S rRNA 384
sequences among and within strains of African rhizobia isolated from Acacia and Prosopis. Syst 385
Appl Microbiol 19, 352–359.
386
Islam, M. S., Kawasaki, H., Muramatsu, Y., Nakagawa, Y. & Seki, T. (2008).
387
Bradyrhizobium iriomotense sp. nov., isolated from a tumor-like root of the legume Entada 388
koshunensis from Iriomote island in Japan. Biosci Biotechnol Biochem 72, 1416–1429.
389
18
Jarvis, B. D. W., Pankhurst, C. E. & Patel, J. J. (1982). Rhizobium loti, a new species of 390
legume root nodule bacteria. Int J Syst Bacteriol 32, 378–380.
391
Jarvis, B. D. W., Sivakumaran, S., Tighe, S. W. & Gillis, M. (1996). Identification of 392
Agrobacterium and Rhizobium species based on cellular fatty acid composition. Plant Soil 184, 393
143–158.
394
Jarvis, B. D. W., van Berkum, P., Chen, W. X., Nour, S. M., Fernandez, M. P., Cleyet- 395
Marel, J. C. & Gillis, M. (1997). Transfer of Rhizobium loti, Rhizobium huakuii, Rhizobium 396
ciceri, Rhizobium mediterraneum, and Rhizobium tianshanense to Mesorhizobium gen. nov. Int J 397
Syst Bacteriol 47, 895-898.
398
Khbaya, B., Neyra, M., Normand, P., Zerhari, K. & Filali-Maltouf, A. (1998). Genetic 399
diversity and phylogeny of rhizobia that nodulate Acacia spp. in Morocco assessed by analysis of 400
rRNA genes. Appl Environ Microbiol 64, 4912–4917.
401
Martens, M., Delaere, M., Coopman, R., De Vos, P., Gillis, M. & Willems, A. (2007).
402
Multilocus sequence analysis of Ensifer and related taxa. Int J Syst Evol Microbiol 57, 489–503.
403
Martens, M., Dawyndt, P., Coopman, R., Gillis, M., De Vos, P. & Willems, A. (2008).
404
Advantages of multilocus sequence analysis for taxonomic studies: a case study using 10 405
housekeeping genes in the genus Ensifer (including former Sinorhizobium). Int J Syst Evol 406
Microbiol 58, 200–214.
407
McInroy, S. G., Campbell, C. D., Haukka, K. E., Odee, D. W., Sprent, J. I., Wen-Jun, W., 408
Young, J. P. W. & Joan, M. S. (1999). Characterisation of rhizobia from African acacias and 409
other tropical woody legumes using Biolog and partial 16S rRNA sequencing. FEMS Microbiol 410
Lett 170, 111–117.
411
19
Merabet, C., Martens, M., Mahdhi, M., Zakhia, F., Sy, A., Le Roux, C., Domergue, O., 412
Coopman, R., Bekki, A. & other authors (2010). Multilocus sequence analysis of root nodule 413
isolates from Lotus arabicus (Senegal), Lotus creticus, Argyrolobium uniflorum and Medicago 414
sativa (Tunisia) and description of Ensifer numidicus sp. nov. and Ensifer garamanticus sp. nov.
415
Int J Syst Evol Microbiol 60, 664–674.
416
Mesbah, M., Premachandran, U. & Whitman, W. B. (1989). Precise measurement of the G+C 417
content of deoxyribonucleic acid by high-performance liquid chromatography. Int J Syst 418
Bacteriol 39, 159–167.
419
Mhamdi, R., Laguerre, G., Aouani, M. A., Mars, M. & Amarger, N. (2002). Different 420
species and symbiotic genotypes of field rhizobia can nodulate Phaseolus vulgaris in Tunisian 421
soils. FEMS Microbiol Ecol 41 77–84.
422
Nandasena, K. G., O'Hara, G. W., Tiwari, R. P., Willems, A. & Howieson, J. G. (2009).
423
Mesorhizobium australicum sp. nov. and Mesorhizobium opportunistum sp. nov., isolated from 424
Biserrula pelecinus L. in Australia Int J Syst Evol Microbiol 59, 2140–2147 425
Naser, S. M., Thompson, F. L., Hoste, B., Gevers, D., Dawyndt, P., Vancanneyt, M. &
426
Swings, J. (2005). Application of multilocus sequence analysis (MLSA) for rapid 427
identification of Enterococcus species based on rpoA and pheS genes. Microbiology 151, 2141–
428
2150.
429
Nick, G., de Lajudie, P., Eardly, B. D., Suomalainen, S., Paulin, L., Zhang, X., Gillis, M. &
430
Lindström, K. (1999a). Sinorhizobium arboris sp. nov., and Sinorhizobium kostiense sp. nov., 431
isolated from leguminous trees in Sudan and Kenya. Int J Syst Bacteriol 49, 1359–1368.
432
Nick, G., Jussila, M., Hoste, B., Niemi, M., Kaijalainen, S., de Lajudie, P., Gillis, M., de 433
Bruijn, F. J. & Lindström, K. (1999b). Rhizobia isolated from root nodules of tropical 434
20
leguminous trees characterized using DNA-DNA dot-blot hybridisation and rep-PCR genomic 435
fingerprinting. Syst App Microbiol 22, 287–299.
436
Nour, S., Fernandez, M. P., Normand, P. & Cleyet-Marel, J. C. (1994). Rhizobium ciceri sp.
437
nov., consisting of strains that nodulate chickpeas (Cicer arietinum L.). Int J Syst Bacteriol 44, 438
511–522 439
Odee, D. W., Sutherland, J. M., Kimiti, J. M. & Sprent, J. I. (1995). Natural rhizobial 440
populations and nodulation status of woody legumes growing in diverse Kenyan conditions.
441
Plant Soil 173, 211–224.
442
Odee, D. W., Sutherland, J. M., Msksyisni, E. T., McInroy, S. G. & Sprent, J. I. (1997).
443
Phenotypic characteristics and composition of rhizobia associated with woody legumes growing 444
in diverse Kenyan conditions. Plant Soil 188, 65–75.
445
Odee, D. W., Haukka, K., McInroy, S. G., Sprent, J. I., Sutherland, J. M. & Young, J. P.
446
W. (2002). Genetic and symbiotic characterization of rhizobia isolated from tree and herbaceous 447
legumes grown in soils from ecologically diverse sites in Kenya. Soil Biol Biochem 34, 801–811.
448
Ratledge, C., & Wilkinson, S. G. (1988). Microbial lipids. Academic Press, London.
449
Sawada, H., Kuykendall, L. D. & Young, J. M. (2003). Changing concepts in the systematics 450
of bacterial nitrogen-fixing legume symbionts. J Gen Appl Microbiol 49, 155–179.
451
Stackebrandt, E., Frederiksen, W., Garrity, G. M., Grimont, P. A., Kämpfer, P., Maiden, 452
M. C., Nesme, X., Rossello-Mora, R., Swings, J. & other authors (2002). Report of the ad hoc 453
committee for the reevaluation of the species definition in bacteriology. Int J Syst Evol Microbiol 454
52, 1043–1047.
455
21
Sy, A., Giraud, E., Jourand, P., Garcia, N., Willems, A., de Lajudie, P., Prin, Y., Neyra, M., 456
Gillis, M. & other authors (2001). Methylotrophic Methylobacterium bacteria nodulate and fix 457
nitrogen in symbiosis with legumes. J Bacteriol 183, 214–220.
458
Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. & Kumar, S. (2011). MEGA5:
459
Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and 460
maximum parsimony methods. Mol Biol Evol 28 2731–2739.
461
Tighe, S. W., de Lajudie, P., Dipietro, K., Lindström, K., Nick, G. & Jarvis, B. D. W.
462
(2000). Analysis of cellular fatty acids and phenotypic relationships of Agrobacterium, 463
Bradyrhizobium, Mesorhizobium, Rhizobium and Sinorhizobium species using the Sherlock 464
microbial identification system. Int J Syst Evol Microbiol 50, 787–801.
465
Turner, S. L. & Young, J. P. W. (2000). The glutamine synthetases of rhizobia: phylogenetics 466
and evolutionary implications. Mol Biol Evol 17, 309–319.
467
Vinuesa, P., Leon-Barrios, M., Silva, C., Willems, A., Jarabo-Lorenzo, A., Perez-Galdona, 468
R., Werner, D. & Martínez-Romero, E. (2005). Bradyrhizobium canariense sp. nov. an acid- 469
tolerant endosymbiont that nodulates endemic genistoid legumes (Papilionoideae: Genisteae) 470
from the Canary islands, along with Bradyrhizobium japonicum bv. genistearum, 471
Bradyrhizobium genospecies alpha and Bradyrhizobium genospecies beta. Int J Syst Evol 472
Microbiol 55, 569–575.
473
Wang, F. Q., Wang, E. T., Liu, J., Chen, Q., Sui, X. H., Chen, W. F. & Chen, W. X. (2007).
474
Mesorhizobium albiziae sp. nov., a novel bacterium that nodulates Albizia kalkora in a 475
subtropical region of China. Int J Syst Evol Microbiol 57, 1192–1199.
476
Wayne, L. G., Brenner, D. J., Colwell, R. R., Grimont, P. A. D., Kandler, O., Krichevsky, 477
M. I., Moore, L. H., Moore, W. E. C., Murray, R. G. E. & other authors (1987). Report of 478
22
the ad hoc committee on reconciliation of approaches to bacterial systematics. Int J Syst 479
Bacteriol 37, 463–464.
480
Wilson, K. (1987). Preparation of genomic DNA from bacteria. In Current protocols in 481
molecular biology, pp. 241–245. Edited by F. M. Ausubel, R. Brent, R. E. Kingston, D. D.
482
Moore, J. G. Seidman, J. A. Smith & K. Struhl. New York: Green Publishing and Wiley- 483
Interscience.
484
Wolde-meskel, E., Terefework, Z., Lindström, K. & Frostegård., Å. (2004). Metabolic and 485
genomic diversity of rhizobia isolated from field standing native and exotic woody legumes in 486
southern Ethiopia. Syst Appl Microbiol 27, 603–611.
487
Wolde-meskel, E., Terefework, Z., Frostegård, Å. & Lindström, K. (2005). Genetic diversity 488
and phylogeny of rhizobia isolated from agroforestry legume species in southern Ethiopia. Int J 489
Syst Evol Microbiol 55, 1439–1452.
490
Zhang, J. J., Liu, T. Y., Chen, W. F., Wang, E. T., Sui, X. H., Zhang, X. X., Li, Y., Li, Y. &
491
Chen, W. X. (2011). Mesorhizobium muleiense sp. nov., nodulating with Cicer arietinum L. in 492
Xinjiang, China. Int J Syst Evol Microbiol In press DOI 10.1099/ijs.0.038265-0.
493
Zhang, X. P., Harper, R., Karsisto, M. & Lindström, K. (1991). Diversity of Rhizobium 494
bacteria isolated from the root nodules of leguminous trees. Int J Syst Bacteriol 41, 104–113.
495
Zhao, C. T., Wang, E. T., Zhang, Y. M., Chen, W. F., Sui, X. H., Chen, W. X., Liu, H. C. &
496
Zhang, X. X. (2011). Mesorhizobium silamurunense sp. nov., a novel species nodulated with 497
Astragalus species in China. Int J Syst Evol Microbiol In press DOI 10.1099/ijs.0.031229-0.
498 499
Table legends 500
23
Table 1. Fatty acid composition of the novel species and the phylogenetically closest 501
Mesorhizobium species. 1. M. plurifarium LMG 11892T; 2. M. amorphae ACCC 19665T; 3. M.
502
septentrionale SDW014T; 4. M. huakuii LMG 14107T; 5. M. shonense AC39aT sp. nov. (= LMG 503
26966T = HAMBI 3295T); 6. M. abyssinicae AC98cT sp. nov. (= LMG 26967T = HAMBI 504
3306T)(b) and M. abyssinicae AC100e (= HAMBI 3308 (a)); 7. M. hawassense AC99bT sp. nov.
505
(= LMG 26968T = HAMBI 3301T). Values are percentages of total fatty acids. Data in columns 506
1, 4-7 were generated in the frame of this study. Data in columns 2 and 3 were taken from Wang 507
et al. (2007). All listed data were generated under the same conditions. M= Mesorhizobium, ND=
508
not detected 509
510
Figure legend 511
Figure 1. Maximum likelihood phylogenetic trees based on atpD (a) and glnII (b) genes showing 512
the relationships among the three new species (shown in boldface type) and recognized species 513
of the genus Mesorhizobium. Bootstrap values of ≥ 70 % (based on 100 replications) are shown 514
at each node. Scale bar indicates the number of estimated nucleotide substitution per site. M=
515
Mesorhizobium.
516
24 Table 1
Fatty acids 1 2 3 4 5 6 (a) 6 (b) 7
Unknown 9.531 0.4 ND ND ND ND ND ND ND
C12:0 1.9 ND ND ND ND ND ND 0.6
C12:0 3-OH 0.6 ND 0.3 1.0 3.2 2.8 2.0 ND
C13:0 iso 3-OH 2.3 0.5 0.7 3.0 2.7 2.8 2.9 2.0
C14:0 ND ND 0.5 ND ND ND ND ND
C15:0 iso ND 0.4 0.7 ND ND ND ND ND
C15:1 ω8c ND ND ND ND 2.0 0.8 0.7 ND
C16:0 13.7 14.3 12.9 15.3 21.3 17.9 14.8 11.6
C16:0 iso ND ND 0.3 ND ND ND ND ND
C17:0 ND 1.9 2.0 ND 1.8 0.6 0.9 0.7
C17:0 iso 7.0 3.6 3.2 5.7 1.4 1.9 2.8 5.1
C17:0 cyclo ND 0.5 ND ND ND ND ND ND
C17:1 ω8c N 0.5 0.6 ND 2.0 0.3 0.6 ND
25 Table 1 cont…
Fatty acids 1 2 3 4 5 6 (a) 6 (b) 7
C18:0 4.2 6.9 5.5 3.7 2.1 4.1 2.9 4.8
11-Methyl C18:1 ω7c ND 15.9 22.8 3.7 ND 3.6 3.7 ND
C18:1 ω7c (or summed feature 7*
, when marked with ¶) 51.9 34.2¶ 42.3¶ 50.5 53.1 58.2 63.7 71.2
C18:1 ω9c ND 0.7 0.8 ND ND ND ND ND
C19:0 ND ND 0.4 ND ND ND ND ND
C19:0 cyclo ω8c 16.4 18.4 1.7 13.7 5.1 2.2 1.0 3.4
10-Methyl C19:0 1.7 0.5 0.6 3.4 1.9 3.3 0.9 0.6
C20:0 ND 0.4 0.5 ND ND ND ND ND
C20:1 ω7c ND 0.4 0.6 ND ND ND ND ND
C20:1 ω9c ND ND ND ND ND 0.9 ND ND
Summed feature 3* ND 0.9 3.7 ND 3.5 0.7 0.8 ND
*Summed feature 3 contains 15 iso 2-OH and/or 16:1 ω7c.
*Summed feature 7 contains 18:1 ω7c/ω9t/ω12t and/or 18:1 ω7c/ω9c/ω12t.
26
