symbiotic specificity and nodulation in the southern ... · i wish to thank dr catherine...
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Symbiotic specificity and nodulation in the
southern African legume clade Lotononis s. l. and
description of novel rhizobial species within the
Alphaproteobacterial genus Microvirga
by
Julie Kaye Ardley
This thesis is presented for the degree of
Doctor of Philosophy
of
Murdoch University
2011
ii
DECLARATION
I declare that this thesis is my own account of my research and contains as its
main content work which has not previously been submitted for a degree at any
tertiary education institution.
Julie Kaye Ardley
iii
TABLE OF CONTENTS
Publications arising from this thesis………………………………………………….ix
Acknowledgements……………………………………………………………………………x
Abstract…………………………………………………………………………………………..xii
Chapter 1: Introduction and literature review……………………………………1
1.1 The legume-rhizobia symbiosis….……………….…….……………………………………..………..3
1.2 The legume-rhizobia symbiosis in Australian agriculture.………………………….……….4
1.2.1 Background…………………………………………………………………………………….…….…..4
1.3 Legumes.……….……………………………………………………………………………………..……….….6
1.3.1 Evolution of legumes…………………………………………….……….…………….…….…….6
1.3.2 Legume taxonomy and phylogeny………………………………………………….…………7
1.3.2.1 Phylogeny of the Papilionoideae…………………………….………….…………..….8
1.4 Rhizobia…..…………………………………………………………………….………………………………..11
1.4.1 History of rhizobial classification……………………………………………………...……..11
1.5 Bacterial systematics….……………………….…………….………………………………….….……..13
1.5.1 Genotypic classification methods………………………………………………..…….……15
1.5.2 Phylogenetic classification methods…………………..…………………..………….…..15
1.5.3 Phenotypic classification methods………………………………………..…..…………...18
1.5.4 The effect of lateral gene transfer on bacterial phylogeny…………………...…19
1.6 General recommendations for classifying and describing bacteria……….………….21
1.7 Rhizobial symbiotic genes………………………………………………………..………………………23
1.7.1 The role of nodulation genes and Nod factors…………………………………………24
1.8 Rhizobial infection and nodule formation in legumes………………………………………25
1.8.1 Infection pathways in legumes……………………………..…………………………….…..26
iv
1.8.2 Nodule types………………………………………………………..……………………….………..29
1.8.2.1 Formation and structure of nodules infected via infection
threads………………………………………………………………………………………….…….……….30
1.8.2.2 Formation and structure of intercellularly infected nodules …………….31
1.9 Specificity and effectiveness in the legume-rhizobia symbiosis………………………..32
1.10 The Lotononis s. l. clade………………………………………………………………………………...34
1.10.1 The genus Listia…………………………………………………………………………………....36
1.10.2 The genus Leobordea…………………………………………………………………….………37
1.10.3 The genus Lotononis s. str……………………………………………………………………..38
1.11. The Lotononis s. l. -rhizobia symbiosis…………………………………………………………..39
1.12 Summary of current knowledge of the symbiotic relationships between
Lotononis s. l. and associated rhizobia……………………………………………………………………44
1.13 Aims of this thesis…………………………………………………………………………………….……46
Chapter 2: Determining symbiotic specificity within Lotononis s.l. ….47
2.1 Introduction…………………………………………………………………………………………………….49
2.1.1 Background……………………………………………………………………………………………..49
2.1.2 Experimental approach……………………………………………………………………………50
2.2 Materials and methods…………………………………………………………………………………...53
2.2.1 Rhizobial strains………………………………………………………………………………………53
2.2.2 Host plants………………………………………………………………………………………………53
2.2.3 Glasshouse experimental design……………………………………………………………..55
2.2.4 General glasshouse procedures……………………………………………………………….58
2.2.4.1 Preparation of plant material……………………………………………………………59
2.2.4.2 Preparation of inoculum……………………………………………………………………60
2.2.4.3 Harvesting…………………………………………………………………………………………61
2.2.5 Statistics………………………………………………………………………………………………….61
2.2.6 Molecular fingerprinting………………………………………………………………………….61
2.2.7 Amplification and sequencing of 16S rRNA genes……………………………………63
2.2.8 Amplification and sequencing of nodA genes………………………………………….66
2.3 Results……………………………………………………………………………………………………………..68
v
2.3.1 Selection of inoculant strains………………………………………………………………..…68
2.3.1.1 Selection of a representative Listia angolensis strain………………...……..68
2.3.1.2 Authentication of non-pigmented Lotononis s. l. strains……………………69
2.3.2 Symbiotic interactions of Lotononis s. l. species with phylogenetically
diverse Lotononis s. l. rhizobia………………………………………………………………………….71
2.3.3 Symbiotic specificity within Listia species………………………………………………..75
2.3.4 Nodule structure in Lotononis s. l. species……………………………………………….75
2.3.5 Amplification and sequencing of 16S rRNA genes……………………………………77
2.3.6 Amplification and sequencing of nodA…………………………………………………….84
2.3.6.1 nodA sequences of WSM2598 and WSM2667…………………………………..84
2.3.6.2 nodA sequences of the remaining Lotononis s. l. rhizobia…………………85
2.4 Discussion………………………………………………………………………………………………………..90
2.4.1 Nodule structure in Lotononis s. l. species……………………………………………….90
2.4.2. Diversity of Lotononis s. l. rhizobia………………………………………………………….90
2.4.2 1 The Methylobacterium lineage………………………………………………………….91
2.4.2.2 The Microvirga lineage……………………………………………………………………..93
2.4.2.3 The Bradyrhizobium, Ensifer and Mesorhizobium lineages…………….….94
2.4.3 Symbiotic relationships within Lotononis s. l. hosts………………………………...96
2.4.4 Phylogeny of nodA genes…………………………………………………………………………98
2.4.5 Environmental factors that may be effectors of diversification in rhizobia
associated with Lotononis s. l. species……………………………………………………………103
2.4.6 Putative evolution of symbiotic patterns within Lotononis s. l. …………….107
Chapter 3 Nodule structure in Lotononis s. l. species and infection and
nodule formation in Listia angolensis and Listia bainesii…………….….111
3.1 Introduction…………………………………………………………………………………………………..113
3.1.1 Modes of infection and nodulation in legumes……………………………………..113
3.2 Materials and methods………………………………………………………………………………….115
3.2.1 Legume hosts, bacterial inoculant strains and growth conditions………….115
3.2.2 Harvesting……………………………………………………………………………………………..117
3.2.3 Nodule initial staining and sectioning, nodule sectioning and light
microscopy…………………………………………………………………………………………………....118
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3.3 Results……………………………………………………………………………………………………………119
3.3.1. Localisation of infection site…………………………………………………………………119
3.3.2 Infection and nodule organogenesis in Listia angolensis and Listia
bainesii…………………………………………………………………………………………………………..121
3.3.3 Localisation of the infection site in Listia angolensis and Listia bainesii
plants inoculated at 85 days old…………………………………………………………………….124
3.3.4 Internal structure of Lotononis s. l. nodules…………………………………………..125
3.4 Discussion………………………………………………………………………………………………………128
Chapter 4 Characterisation and description of novel Listia angolensis
and Lupinus texensis rhizobia……………………………………..…………………133
4.1 Introduction…………………………………………………………………………………………………..135
4.2 Materials and methods………………………………………………………………………………….137
4.2.1 Bacterial strains and culture conditions…………………………………………………137
4.2.2 DNA amplification and sequencing………………………………………………………..137
4.2.2.1 Amplification and sequencing of the 16S rRNA gene………………………137
4.2.2.2 Amplification and sequencing of the nodA gene…………………………….139
4.2.3 Phenotypic characterisation………………………………………………………………….139
4.2.3.1 Morphology…………………………………………………………………………………….139
4.2.3.2 Extraction of pigments……………………………………………………………………140
4.2.3.3 Temperature range…………………………………………………………………………141
4.2.3.4 Optimal growth temperature………………………………………………………….141
4.2.3.5 Growth curves and determination of mean generation time………….141
4.2.3.6 Determination of sodium chloride and pH tolerance………………………141
4.2.3.7 Anaerobic growth……………………………………………………………………………143
4.2.3.8 Antibiotic sensitivity……………………………………………………………………….143
4.2.3.9 Substrate utilisation………………………………………………………………………..144
4.2.3.9.1 Utilisation of sole carbon sources in Biolog GN2 plates…………….144
4.2.3.9.2 Utilisation of substrates in Biolog Phenotype Microarray
plates……………………………………………………………………………………………………..144
4.2.3.9.3 Growth on sole carbon substrates……………………………………………145
4.2.3.10 Biochemical characterisation………………………………………………………..147
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4.2.4 Host range…………………………………………………………………………………………….148
4.3 Results……………………………………………………………………………………………………………149
4.3.1 Amplification and sequencing of the 16S rRNA gene……………………………..149
4.3.2 Amplification and sequencing of nodA…………………………………………………..151
4.3.3 Phenotypic characterisation………………………………………………………………….153
4.3.3.1 Morphology…………………………………………………………………………………….153
4.3.3.2 Characterisation of pigments………………………………………………………….155
4.3.3.3 Growth characteristics……………………………………………………………………156
4.3.3.4 Antibiotic sensitivity……………………………………………………………………….156
4.3.3.5 Substrate utilisation………………………………………………………………………..158
4.3.3.5.1 Utilisation of sole carbon sources in Biolog GN2 plates…………….158
4.3.3.5.2 Utilisation of substrates in Biolog Phenotype Microarray
plates………………………………………………………………………………………………………162
4.3.3.5.3 Growth in sole carbon substrate broths……………………………………168
4.3.3.6 Biochemical characterisation………………………………………………………….169
4.3.4 Host range…………………………………………………………………………………………….171
4.4 Discussion………………………………………………………………………………………………………173
4.4.1 Genotypic characterisation of novel Microvirga species………………………..173
4.4.2 G+C content, DNA:DNA hybridisation and cellular fatty acid analysis…….176
4.4.3 Phenotypic characterisation of novel Microvirga species………………………176
4.4.3.1 Substrate utilisation………………………………………………………………………..181
4.4.3.1.1 Critique of the use of Biolog plates in determining substrate
utilisation………………………………………………………………………………………………..181
4.4.3.1.2 Substrate utilisation in Listia angolensis and Lupinus texensis
strains
4.4.4 Biochemical characteristics of Listia angolensis and Lupinus texensis
strains…………………………………………………………………………………………………………….187
4.4.5 Host range………………………………………………………………………………………….…187
4.5 Summary, emended description of Microvirga and description of Microvirga
lotononidis sp. nov., Microvirga zambiensis sp. nov. and Microvirga lupini sp. nov.
……………………………………………………………………………………………………………………………188
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4.5.1 Emended description of Microvirga (Kanso & Patel, 2003 emend. Zhang et
al, 2009, emend. Weon et al, 2010)……………………………………………………………….189
4.5.2 Description of Microvirga lotononidis sp. nov. ………………………………………189
4.5.3 Description of Microvirga zambiensis sp. nov. ………………………………………190
4.5.4 Description of Microvirga lupini sp. nov. ………………………………………………191
Chapter 5 General discussion………………………………………………………...193
5.1 Symbiotic relationships within Lotononis s. l. ………………………………………………..195
5.1.1 Habitat as a driver of rhizobial diversity………………………………………………..199
5.1.2 Lotononis s. l. rhizobia have diverse nodA sequences……………………………201
5.1.3 Specificity and effectiveness within Lotononis s. l. ……………………………….203
5.1.4 Models of legume-rhizobia symbiosis……………………………………………………204
5.2 Infection and nodule organogenesis in Listia spp. …………………………………………207
5.3 Characterisation of novel rhizobial species of Microvirga………………………………209
Bibliography…………………………………………………………………………………..213
Appendix……………………………………………………………………………………….249
ix
PUBLICATIONS ARISING FROM THIS THESIS
Ardley, J. K., Garau, G., Yates, R. J., Parker, M. A., O’Hara, G. W., Reeve, W.
G., De Meyer, S., Walker R., Dilworth, M. J., Ratnayake, S., Willems, A.,
Watkin, E., and Howieson, J. G. 2011. Variations on a theme: novel rhizobial
strains of Burkholderia, Methylobacterium and Microvirga demonstrate the diversity
of root nodule bacteria/legume symbioses. Advances in Legume Systematics (in
press).
Ardley, J. K., Parker, M. A., De Meyer, S., Trengove, R. D., O'Hara, G. W.,
Reeve, W. G., Yates, R. J., Dilworth, M. J., Willems, A. & Howieson, J. G.
Microvirga lupini sp. nov., Microvirga lotononidis sp. nov., and Microvirga
zambiensis sp. nov. are Alphaproteobacterial root nodule bacteria that specifically
nodulate and fix nitrogen with geographically and taxonomically separate legume
hosts. International Journal of Systematic and Evolutionary Microbiology. Published
online ahead of print December 23, 2011, doi: 10.1099/ijs.0.035097-0.
x
ACKNOWLEDGEMENTS
I would like to take this opportunity to thank all the many people who have
helped or supported me during this PhD.
Firstly, I would like to express my sincerest appreciation and gratitude to my
four supervisors Professor John Howieson, Dr Graham O’Hara, Dr Wayne Reeve
and Dr Ron Yates. You have all given generously of your time and knowledge in
your various areas of expertise and have provided invaluable guidance and support. It
has been a privilege and a pleasure to work with you.
I would also like to thank Professor Mike Dilworth for contributing so much
to this project, whether in the form of scientific expertise on all things rhizobial, or in
the provision of insightful editorial comments during the preparation of this thesis.
Thanks also to Dr Ravi Tiwari and Dr Lambert Bräu for always being prepared to
lend an ear and to Dr Vanessa Melino for being a kind and enthusiastic colleague,
and generator of great discussions. To Regina Carr, many thanks for being a great
friend and for providing valuable advice and assistance during the many glasshouse
trials. Many thanks also to Gordon Thomson for his expert and beautiful histological
preparations and generous advice on microscopy and to Dominie Wright for kindly
giving advice on the preparation of Biolog plates.
I wish to thank Dr Catherine Masson-Boivin, Dr Xavier Perret and Dr Bharat
Patel for kindly providing the strains ORS 2060, NGR234 and FaiI4, respectively. I
also wish to thank Dr Matt Parker, Dr Anne Willems and Dr Sofie De Meyer for the
xi
time, hard work and expertise they have supplied to the collaborative research into
novel rhizobial strains of Microvirga.
I have been lucky enough, during the course of these studies, to meet many
wonderful researchers in the field of nitrogen fixation, none more so than Professor
Janet Sprent, who has been a most generous source of encouragement, ideas and
inspiration.
I also count myself very fortunate to have been able to work with all the past
and present members of the Centre for Rhizobium Studies, who have made the CRS
such an enthusiastic and supportive research environment. Especial thanks to Sharon,
Rebecca, Macarena, Felipe, Liza, Rob, Jason, Will, Yvette and Tina, who have
shared the journey and provided friendship, support, advice, good humour, good
conversation and, on occasion, great times together.
Finally, to my family, many thanks for all your love and patience.
xii
ABSTRACT
Lotononis s. l. is a legume clade within the Crotalarieae tribe, with a centre of
origin in South Africa. After taxonomic revision, the three genera Listia, Leobordea
and Lotononis s. str. are now recognised. The N2-fixing symbiosis between Listia
bainesii and pigmented Methylobacterium rhizobia is known to be highly specific,
while a recent study has shown that Listia angolensis is effectively nodulated by a
novel lineage of root nodule bacteria. The symbiotic relationships of Lotononis s. l.
species outside the Listia genus have not yet been examined. The work presented in
this thesis sought to determine the identity of rhizobia isolated from Listia,
Leobordea and Lotononis s. str. hosts, to examine the phylogeny of their nodA genes
and to quantify the nodulation and N2 fixation capabilities of Lotononis s. l.-
associated rhizobia on eight taxonomically diverse Lotononis s. l. hosts.
Additionally, this research sought to examine the processes of infection and nodule
initiation in L. angolensis and L. bainesii and to validly name and characterise the
novel L. angolensis rhizobia.
Amplification and sequencing of nearly full length fragments of the 16S
rRNA gene showed that the rhizobia isolated from nodules of Lotononis s. l. species
were phylogenetically diverse. Strains isolated from Leobordea and Lotononis s. str.
hosts were most closely related to Bradyrhizobium spp., Ensifer meliloti,
Mesorhizobium tianshanense and Methylobacterium nodulans. The Listia angolensis
microsymbionts, together with closely related Lupinus texensis rhizobia, were
identified as novel species of the Alphaproteobacterial genus Microvirga. The
xiii
phylogeny of the nodA genes correlated more with the rhizobial 16S rRNA genes
than with the taxonomy of the host plants.
The nodulation and effectiveness trials confirmed the symbiotic specificity of
the genus Listia. L. bainesii nodulated only with the representative pigmented
Methylobacterium strain WSM2598. As measured by plant shoot dry weight, this
symbiosis was highly effective. L. angolensis was effectively nodulated only by
Microvirga rhizobia, but formed ineffective nodules with the pigmented
Methylobacterium and M. nodulans strains. WSM2598 was effective only on Listia
species (other than L. angolensis). In contrast, the Microvirga strain WSM3557 was
partially effective on some Leobordea and Lotononis s. str. species. No clear pattern
of symbiotic association was seen in the Leobordea and Lotononis s. str. hosts.
Nodulation in these species was usually promiscuous and often ineffective for N2
fixation.
The nodules formed on Listia species were lupinoid, whereas the nodules of
Leobordea and Lotononis s. str. species were indeterminate. Micrographs of all
sectioned nodules, whether lupinoid or indeterminate, showed a mass of central,
uniformly infected tissue, with no uninfected interstitial cells. Rhizobial infection
and nodulation in L. angolensis and L. bainesii did not appear to involve root hair
curling or the development of infection threads. Nodule organogenesis followed a
process similar to that observed in Lupinus species, with nodule primordia
developing in the outer cortex.
xiv
The polyphasic characterisation of the Microvirga rhizobia associated with
Listia angolensis and Lupinus texensis resulted in the description of three new
rhizobial species: Microvirga lotononidis, M. zambiensis and M. lupini. Microvirga
species possess several phenotypic properties that are unusual in rhizobia, including
the ability to grow at relatively elevated temperatures and the presence of
pigmentation in most strains. The rhizobial Microvirga strains WSM3557T and Lut6
T
have been included in the Genomic Encyclopedia for Bacteria and Archaea Root
Nodule Bacteria (GEBA-RNB) sequencing project (Kyrpides & Reeve, collaborative
CSI project (http://genome.jgi-psf.org/programs/bacteria-archaea/GEBA-RNB.jsf)
and should provide new insights into the evolution of and genomic architecture
required for rhizobial symbionts.
Chapter 1
1
CHAPTER 1
Introduction and literature review
Chapter 1
2
Chapter 1
3
1.1 The legume-rhizobia symbiosis
Biological nitrogen fixation (BNF), in which bacteria reduce atmospheric
nitrogen (N2) to the biologically usable form of ammonia, is a highly important
source of nitrogen (N) in both natural and agricultural systems, providing about 65%
of the biosphere’s N (Lodwig et al., 2003). In terms of the quantity of N2 fixed and in
its importance to agricultural systems, the symbiosis between root nodule bacteria
(RNB, collectively known as rhizobia) and legumes is the most ecologically and
economically important form of BNF. It is estimated to contribute approximately 50
- 70 Tg (Tg = 1012
g) of fixed N to agricultural systems per year (Herridge et al.,
2008). The rate of N2 fixation by nodulated legumes varies from 57 to 600 kilograms
per hectare per year compared with 0.1 to 0.5 kg ha-1
year-1
obtained from free-living
Azotobacter and Clostridium (Evans & Barber, 1977). Unlike the N2-fixing
associations of epiphyte species of Gluconacetobacter, Azospirillum,
Herbaspirillum, and Azoarcus with various grasses, most of the N2 fixed in nodules
is transferred to and assimilated by the plant for its growth (Hirsch et al., 2001).
Legumes are important components of agriculture as both pastures and pulses, not
only for their input of fixed N but also in their contribution to the agronomy of crop
production (Graham & Vance, 2003). Grain legumes rank as the second major type
of field crop after cereals and constitute an important component of people’s diet
(Broughton et al., 2003; Peoples et al., 1995).
Chapter 1
4
1.2 The legume-rhizobia symbiosis in Australian agriculture
1.2.1 Background
Southern Australian agricultural systems are characterised by a Mediterranean
climate, with predominantly winter rainfall varying from 250 – 1000 mm annually
(Nichols et al., 2007). Soils are often acid, have a low clay content and low organic
matter, and tend to be inherently infertile (Howieson et al., 2000; Nichols et al.,
2007). Introduced legumes are established components of these agricultural systems
(Howieson et al., 2000). Traditionally, pasture legumes have been Mediterranean
annuals such as medics and subterranean clover (Loi et al., 2005), but recent
challenges to the sustainability of these practices have emerged. Howieson et al.
(2000) have identified several threats to current practice, such as the emergence of
new pests and diseases, less reliable winter rainfall and, in particular, the
development of dryland salinity.
Dryland salinity occurs when an increase in groundwater recharge raises the
water table, bringing highly saline water to the surface (Hatton et al., 2003). In these
agricultural systems, it is attributable to the replacement of native deep-rooted
perennial vegetation with shallow-rooted annual crops and pastures (Dear et al.,
2003). In Western Australia, approximately 3 million hectares are estimated to be
affected and more than 6 million hectares are potentially at risk (George et al., 1997).
For this reason, greater use of perennials in these farming systems has been
advocated, as a means of preventing the development of dryland salinity (Cocks,
2003; Dear et al., 2003). Lucerne (Medicago sativa) is a perennial pasture legume
that has been shown to lower ground water levels (Cocks, 2003), but its use is
constrained by its poor adaptation to low rainfall, acid soils and waterlogging
Chapter 1
5
(Cocks, 2001; Dear et al., 2003). Researchers have therefore been engaged in an
examination of the global flora for climatically and edaphically adapted perennial
legumes that are potential alternatives to lucerne (Howieson et al., 2008; Li et al.,
2008; Nichols et al., 2007). These legumes include several species of Listia
(previously Lotononis (Boatwright et al., 2011)) that have shown potential as
alternatives to lucerne in areas of high acidity, low rainfall or waterlogging (Dear &
Ewing, 2008; Yates et al., 2007). As part of this development of exotic pasture
legumes, an interspecific crossbreeding program has been undertaken by the Centre
for Rhizobium Studies to obtain Listia hybrids with non-shattering pods and larger
seed size (Anon., 2006; Howieson, 2006).
The introduction of exotic legume pasture plants also requires a concomitant
evaluation of their rhizobial partners, as N2 fixation will be compromised if
inoculants are poorly adapted to the target environment, poorly competitive against
resident rhizobial strains or sub-optimal for fixation effectiveness (Howieson &
Ballard, 2004; Sessitsch et al., 2002). The rhizobiology of Listia is interesting, as
species in this genus are nodulated only by strains of pigmented Methylobacterium
or, in the case of Listia angolensis, by light-pink-pigmented strains that are likely to
belong to a new genus of rhizobia (Yates et al., 2007). Before proceeding with a
description of the research into symbiotic relationships within this group of legumes,
however, it is appropriate to review the current state of knowledge of the legume-
rhizobia symbiosis.
Chapter 1
6
1.3 Legumes
1.3.1 Evolution of legumes
Legumes belong in the Rosid 1 clade of the angiosperms. The ten families of
flowering plants that are able to form endosymbiotic associations with nodulating
N2-fixing bacteria are placed within this clade, according to a phylogeny based on
the chloroplast rbcL gene (Soltis et al., 1995). This suggests that, rather than N2-
fixing symbioses evolving in disparate families, the underlying genetic architecture
necessary for nodulation and fixation is confined to one lineage of closely related
angiosperm taxa (Soltis & Soltis, 2000). Parasponia, a member of the Ulmaceae
(and included in the Rosid 1 clade) is the only non-legume known to form a N2-
fixing symbiosis with rhizobia (Sprent, 2007).
The Leguminosae are the third largest flowering plant family, comprising over
700 genera and some 20,000 species (Lewis et al., 2005). They are found throughout
temperate and tropical regions and are particularly diverse in seasonally dry tropical
forests and xeric climate temperate shrublands. Several species are aquatic (Lewis et
al., 2005). Their morphology is equally diverse, ranging from large trees to
perennials, climbing vines and annual herbs (Doyle & Luckow, 2003).
The first legumes are believed to have evolved some 60 million years ago
(mya) in the Paleocene epoch (Sprent, 2007). It has been hypothesised that the
earliest legumes first appeared in the boreotropical forests of North America (Doyle
& Luckow, 2003), but it is now thought more likely that they arose in a succulent
biome in seasonally dry tropical forest to the north of the Tethys seaway (Figure 1.1)
(Schrire et al., 2005) Their nitrogen-demanding metabolism is thought to be an
Chapter 1
7
adaptation to climatically variable arid or semi-arid habitats (Wojciechowski et al.,
2004). Rapid diversification of the Leguminosae then followed, such that by the
middle Eocene (50 mya) most of the major lineages had arisen and are present in the
fossil record (Cronk et al., 2006; Lavin et al., 2005).
Figure 1.1. Map of the globe 60 million years ago, showing the position of the continents
and the Tethys seaway. Adapted from Scotese (2004).
1.3.2 Legume taxonomy and phylogeny
Using the terminology of Lewis et al. (2005), the Leguminosae are divided into
three subfamilies, the Caesalpinioideae, the Mimosoideae and the Papilionoideae.
These three subfamilies are further divided into groups of genera called tribes. The
Papilionoideae are the largest subfamily, containing 28 tribes, 476 genera and around
14,000 species. The Mimosoideae have 4 tribes, 77 genera and 3,000 species and
there are currently 4 tribes, 171 genera and 2,250 species in the Caesalpinioideae.
(Doyle & Luckow, 2003; Lewis et al., 2005). Both the Papilionoideae and the
Tethys seawayTethys seaway
Chapter 1
8
Mimosoideae are monophyletic. Caesalpinioids, in contrast, appear to be a
paraphyletic grouping of diverse lineages (Lewis et al., 2005).
Recent molecular phylogeny based on analyses of plastid gene sequences
shows the caesalpinioids forming a basal clade within which the other subfamilies
are nested (Lavin et al., 2005; Wojciechowski et al., 2004). The Mimosoideae appear
to have diverged relatively recently (40 mya) and are a sister group to a caesalpinioid
lineage. In contrast, the papilionoids constitute an early branching from the major
caesalpinioid clades. This accords with the fossil evidence; the oldest known legume
fossils, dating from around 56 mya, are of leaves, fruits and flowers that are similar
to extant genera in the papilionoid genistoid clade (Lavin et al., 2005;
Wojciechowski et al., 2004).
1.3.2.1 Phylogeny of the Papilionoideae
Molecular analyses have resolved a number of distinct groups in the
Papilionoideae and can be used, together with fossil evidence, to estimate when
particular legume “crown” (diversification) clades first appeared (Lavin et al., 2005;
Wojciechowski et al., 2004). The phylogenetic analysis of Wojciechowski et al.
(2004) supports seven major sub-clades that are informally recognised as the
Cladrastis clade, genistoid sensu lato, dalbergioid sensu lato, mirbelioid, millettioid,
and robinioid clades, and the inverted-repeat-lacking clade (IRLC) (Figure 1.2.)
(Cronk et al., 2006). This phylogeny is in agreement with that of McMahon &
Sanderson (2006), who have additionally recognised the Amorpheae clade (between
the genistoids and the dalbergioids) and the Indigofereae clade (between the
milletioids and the robinioids). The Cladrastis clade is basal to the other sub-clades.
Chapter 1
9
Figure 1.2. The major monophyletic clades of the papilionoid legumes. Figure taken from
Cronk et al. (2006).
Cladrastis species have not been observed to nodulate and at least one species
appears to lack this capacity (Foster et al., 1998). The genistoid clade dates from
56.4 mya. It includes the agriculturally important genus Lupinus (Sprent, 2007) and
the large, mainly African tribe Crotalarieae (Boatwright et al., 2008). The Lotononis
sensu lato clade, comprising the genera Listia, Leobordea and Lotononis sensu
stricto, is included within tribe Crotalarieae (Boatwright et al., 2011). The
dalbergioid clade is also an early diverging lineage, dating from 55.3 mya, and
Chapter 1
10
includes the stem-nodulated Aeschynomene species and economically important
genera such as Dalbergia, Arachis and the forage legume Stylosanthes (Lavin et al.,
2001). The mirbelioid group consists mostly of endemic Australian tribes and
originated 48 mya (Lavin et al., 2005; Sprent, 2007; Wojciechowski et al., 2004).
The milletioid crown dates from 45.2 mya and is divided into two sub-clades, the
Milletieae and the Phaseoleae. The latter includes the agriculturally important species
Phaseolus vulgaris and Glycine max (Sprent, 2007; Wojciechowski et al., 2004).
The two remaining clades are both included in the Hologalegina group, which
comprises the majority of temperate, herbaceous legumes, including the well-known
peas, chickpeas, clovers and lucerne and the model legumes Medicago truncatula
and Lotus japonicus (Lavin et al., 2005; Wojciechowski et al., 2004). The first clade
in this group is the robinioids, consisting of the Sesbanieae, Loteae and Robineae
tribes and dated at 48 mya. The second clade is the comparatively recent (39 mya)
inverted repeat loss clade (IRLC), named after the loss of one copy of the 25-
kilobase chloroplast inverted repeat and containing members of the tribes Cicereae,
Trifolieae, Hedysareae and Fabeae (Sprent, 2007; Wojciechowski et al., 2000).
The existence of rhizobia predates that of legumes by several hundred million
years (Turner & Young, 2000). In contrast to the monophyletic Leguminosae,
rhizobia are a polyphyletic group and genera capable of nodulating hosts are found in
both the Alpha- and Betaproteobacteria (Sawada et al., 2003). The following is a
brief examination of rhizobial systematics.
Chapter 1
11
1.4 Rhizobia
Rhizobia are defined as Gram-negative, saprophytic soil or water micro-
organisms that can form N2-fixing symbioses with legumes or Parasponia by
eliciting nodules on the roots or stems of their hosts (Masson-Boivin et al., 2009;
Sprent, 2007). Within the nodule, the free-living form of the microsymbiont
differentiates into bacteroids that convert atmospheric N2 to ammonia (Jones et al.,
2007).
1.4.1 History of rhizobial classification
Prior to 1982, all root-nodule bacteria were included in the genus Rhizobium
and were classified according to the legumes they were able to nodulate (Jordan,
1982; Sawada et al., 2003). Six species of Rhizobium were recognised: R.
leguminosarum, R. trifolii, R. phaseoli, R. meliloti, R. japonicum (nodulating Glycine
max) and R. lupini (Fred et al., 1932). These early studies understandably focussed
on temperate, agriculturally important legume hosts and thus were not representative
of the diversity of the Leguminosae, or their microsymbionts (Willems, 2006). The
idea of classifying rhizobia according to their host range and grouping legumes
according to their microsymbionts (the “cross-inoculation concept”) proved to be
flawed, however, as some rhizobial strains are capable of nodulating a wide range of
legumes (Pueppke & Broughton, 1999; Young & Haukka, 1996). Similarly, various
legumes (notably those in tribe Phaseoleae) are nodulated by a broad spectrum of
rhizobia (Perret et al., 2000). That there was some correlation between rhizobial host
range and physiological properties resulted in the proposal that slow-growing strains
be placed in a new genus, Bradyrhizobium, to distinguish them from the fast-growing
Rhizobium (Jordan, 1982; Young & Haukka, 1996). At this time, the classification of
Chapter 1
12
rhizobia, and bacteria in general, was still based on phenotypic properties such as cell
morphology and physiological and biochemical markers (Schleifer, 2009). The
advent of molecular techniques, in particular gene sequencing, revolutionised
bacterial taxonomy by permitting the development of phylogenies that reflect
bacterial genomic relatedness (Woese, 1987). This, along with studies of novel
isolates from indigenous legumes in diverse biogeographical areas, has demonstrated
that rhizobia are a larger and more diverse group that originally supposed (Lindström
et al., 2010; Sawada et al., 2003; Willems, 2006).
Phylogenies based on a comparative analysis of the 16S rRNA gene sequence
show that rhizobia are polyphyletic, being distributed in both the Alpha- and
Betaproteobacteria and intermingled with taxa that do not contain legume
microsymbionts (Figure 1.3) (Masson-Boivin et al., 2009; Sawada et al., 2003).
Currently, 12 rhizobial genera and over 70 species have been described (Weir, 2011)
(http://www.rhizobia.co.nz/taxonomy/rhizobia.html). The majority of the currently
described rhizobial species belong to the genera Rhizobium, Bradyrhizobium,
Mesorhizobium and Ensifer (including former Sinorhizobium), but novel
Alphaproteobacterial microsymbiont strains of Devosia (Rivas et al., 2002),
Methylobacterium (Sy et al., 2001), Ochrobactrum (Trujillo et al., 2005) and
Shinella (Lin et al., 2008) have also recently been characterised, along with rhizobial
Betaproteobacteria belonging to Burkholderia and Cupriavidus (Amadou et al.,
2008; Bontemps et al., 2010; Chen et al.,2007, 2008; Garau et al., 2009).
Chapter 1
13
Figure 1.3. Unrooted phylogenetic tree of 16S rRNA gene sequences from selected Alpha-,
Beta- and Gammaproteobacteria. Genera in bold font contain rhizobia. Figure taken from
Masson-Boivin et al. (2009).
Potential new rhizobial taxa are expected to conform to the proposed minimal
standards for the description of new genera and species outlined by Graham et al.
(1991) and to the current recommendations for the characterisation of prokaryotes
(Stackebrandt et al., 2002; Wayne et al., 1987). To understand what this entails, it is
worth examining the elements and methodologies upon which modern bacterial
systematics – the taxonomy and phylogeny of prokaryotes – are now based.
1.5 Bacterial systematics
A classification system for living organisms should group taxa with similar
genotypes and phenotypes together. Classification is traditionally based on the
“species concept” (Cohan, 2002; Rosselló-Móra & Amann, 2001). This is less well
defined for prokaryotes than for eukaryotes, which are delineated far more by
morphology and reproductive isolation. A further complicating factor in assigning
Chapter 1
14
bacteria to discrete, hierarchical taxonomies is the phenomenon of horizontal (also
known as lateral) gene transfer (HGT or LGT), the process by which genetic material
is transmitted between individual organisms. It contrasts with the vertical
transmission of genes by direct descent from one generation to the next. HGT is a
common and widespread occurrence in prokaryotes and has played an integral role in
their evolution, diversification and speciation (Ochman et al., 2000).
Bacterial systematics now relies on a polyphasic approach that integrates
genotypic data (derived from DNA and RNA present in the cell), phenotypic data
(such as chemotaxonomic markers) and phylogenetic information (for example
sequences of highly conserved genes) (Schleifer, 2009; Vandamme et al., 1996). The
species is considered to be the basic unit of taxonomy and is defined as a group of
strains, including the type strain, that share 70% or greater DNA-DNA relatedness
and with 5ºC or less ∆Tm (difference in the DNA-DNA hybrid melting points)
(Stackebrandt & Goebel, 1994; Wayne et al., 1987). Bacterial classification is
expected to reflect the degree of relatedness of different microorganisms. The
standard method of determining this phylogenetic relatedness is by sequencing the
16S or 23S rRNA gene (Woese, 1987). As a practicality, strains with <97% 16S
rRNA gene sequence similarity to any known taxa are considered to belong to a
different species, as species having 70% or greater DNA similarity usually have
more than 97% sequence identity (Gevers et al., 2005; Stackebrandt & Goebel, 1994;
Vandamme et al., 1996). A phylogenetically based taxonomy must also show
phenotypic consistency (Vandamme et al., 1996; Wayne et al., 1987). The following
is a summary of polyphasic taxonomy methods as reviewed by Vandamme et al.
Chapter 1
15
(1996), as well as a critique of the limitations of some of the methods used in this
approach.
1.5.1 Genotypic classification methods
Two genotypic methods are widely used in describing bacterial taxa. The first
of these is the determination of the DNA base ratio, that is, determination of the mole
percent guanosine plus cytosine. Organisms that differ by more than 10 mol% do not
belong in the same genus and species should be less than 5 mol% different
(Schleifer, 2009; Wayne et al., 1987). Similar DNA base ratios, however, do not
necessarily imply phylogenetic relatedness (Rosselló-Móra & Amann, 2001). The
second method is the percent DNA-DNA hybridisation, which is an indirect measure
of the sequence similarity between two entire genomes. DNA-DNA hybridisation is
used to delineate species; as stated above, strains are considered to belong to the
same species if they share 70% or greater DNA-DNA relatedness (Stackebrandt &
Goebel, 1994). One problem with DNA-DNA hybridisation is that it can sometimes
be difficult to compare results, as these are dependent on the stringency of the
hybridisation conditions (Vandamme et al., 1996). In addition, incremental databases
cannot be developed for this method (Schleifer, 2009).
1.5.2 Phylogenetic classification methods
The development of molecular protocols for sequencing the small subunit
ribosomal RNA (SSU rRNA) genes allowed the construction of valid bacterial
phylogenies (Woese, 1987). The rRNA genes are considered the best means of
studying phylogenetic relationships, as they are present in all bacteria, are
functionally constant and are composed of highly conserved as well as more variable
domains (Schleifer, 2009; Vandamme et al., 1996). The extensive publication of 16S
Chapter 1
16
rRNA gene sequences and the availability of the sequence data in databases such as
the National Centre for Biotechnology Information (NCBI) GenBank allow rapid
comparison of new isolates with named species and the subsequent construction of
phylogenetic trees.
There are several problems though with using the rRNA genes as strict
determinants of bacterial phylogenetic placement. Some bacterial taxa can contain
more than one copy of the 16S rRNA gene and these alleles may have a relatively
high sequence divergence (Acinas et al., 2004; Amann et al., 2000; Rainey et al.,
1996). The sequences of different ribosomal RNA operon (rrn) genes may also
provide divergent phylogenetic information. The topology of a phylogenetic tree
generated from diverse rhizobial 16S rRNA gene sequences was found to be
significantly different from that of the corresponding tree assembled with 23S rRNA
gene sequences (van Berkum et al., 2003). In addition, rrn operons are prone to
homologous recombination, resulting in mosaic structures and consequent difficulties
in accurately determining phylogenies. Evidence of mosaicism in 16S rRNA gene
sequences has been found in species of Bradyrhizobium, Ensifer, Mesorhizobium and
Rhizobium (van Berkum et al., 2003; Vinuesa et al., 2005). Finally, comparative 16S
rRNA gene sequence analysis can also lack resolving power at and below the species
level, due to the extent of gene conservation (Schleifer, 2009). Bradyrhizobia, for
example, share a high level of 16S rDNA sequence similarity and it is therefore
difficult to evaluate the interrelationships of strains (Willems, 2006). For all these
reasons, the rRNA genes should therefore not be used as sole determinants of
bacterial taxonomies.
Chapter 1
17
The difficulties with resolving rRNA gene-based phylogenies led to the
recommendation that sequences of protein-encoding “housekeeping” genes (required
for the maintenance of basic cellular function) be included in the phylogenetic
analysis of a taxon (Stackebrandt et al., 2002). These genes have a higher degree of
sequence divergence than the rRNA genes, allowing greater resolution of taxonomies
at the species or sub-species level (Martens et al., 2008; Palys et al., 2000). Ideally,
several sets of genes that are widely distributed among taxa, in single copy and from
diverse chromosomal loci should be chosen (Gevers et al., 2005; Stackebrandt et al.,
2002). Housekeeping genes such as atpD, dnaK, glnA, gyrB, GSI and GSII, recA and
rpoB have been widely used in phylogenetic studies of rhizobia and other bacteria
(Adékambi & Drancourt, 2004; Gaunt et al., 2001; Martens et al., 2008; Stepkowski
et al., 2003; Turner & Young, 2000; Vinuesa et al., 2005). As with the 16S rRNA
gene, the availability of extensive sequence data for housekeeping genes facilitates
the phylogenetic analysis of a given group of isolates.
The intraspecific variation between bacterial strains can be identified by
amplification of highly conserved repetitive intergenic DNA sequences. These are
short (usually <200bp), non-coding DNA sequences found in extragenic locations
and widely distributed in prokaryotic genomes (Lupski & Weinstock, 1992). PCR
amplification of these sequences generates products which, when separated on an
agarose gel, give a characteristic banding pattern that can be used as a genomic
fingerprint to identify bacteria at the species, sub-species or strain level (Versalovic
et al., 1991). There are several families of these sequences, two of which are the
enterobacterial repetitive intergenic consensus (ERIC) sequences and the BOX
sequences (Lupski & Weinstock, 1992; Martin et al., 1992). These have been used to
Chapter 1
18
distinguish rhizobial strains in a number of studies (Alberton et al., 2006; de Bruijn,
1992; Ormeño-Orrillo et al., 2006).
1.5.3 Phenotypic classification methods
Phenotypic description requires the analysis of morphological, physiological
and biochemical features and is a requirement of a valid bacterial species definition
(Rosselló-Móra & Amann, 2001; Wayne et al., 1987). Perhaps as importantly, the
determination of bacterial phenotypes not only allows for the classification of
bacteria, but also provides important information about the roles played by a species
in the natural environment and its adaptation to a particular environmental niche
(Bochner, 2009; Fenchel & Finlay, 2006).
Classical phenotypic tests constitute the basis for the formal description of
bacterial taxa (Vandamme et al., 1996). These include the determination of bacterial
morphology (cellular shape, presence of endospores, flagella, Gram staining,
inclusion bodies) and colonial characteristics. The physiological and biochemical
analyses provide data on growth at different temperatures, pH values, salt
concentrations or aerobic/anaerobic conditions; in the presence of various antibiotics;
on various compounds, including sole carbon substrates; and data on the presence or
activity of various enzymes (Rosselló-Móra & Amann, 2001; Vandamme et al.,
1996).
Commercial, miniaturised, phenotypic fingerprinting systems, such as Biolog
and API, can provide rapid and reproducible results under standardised conditions
(Rosselló-Móra & Amann, 2001; Vandamme et al., 1996). The Biolog system
(http://www.biolog.com/phenoMicro.html) is based on microplates that can assay up
Chapter 1
19
to nearly 2000 culture traits and in so doing are able to provide an analysis of the
physiology of the cell (Bochner, 2003; Bochner, 2009). The technology uses cell
respiration to reduce a tetrazolium dye, forming a strong colour that can be read by a
computerised microplate reader (Bochner, 1989). The bioMérieux
(http://www.biomerieux.com/servlet/srt/bio/portail/home) API strips typically
contain 20 miniature biochemical tests. Both these methods can be used in
conjunction with a database to identify bacteria, particularly in clinical diagnostics.
Chemotaxonomy – the collection and use of data on the chemical composition
of cell constituents in order to classify the bacteria - is a more recent addition to
phenotypic methods. It makes use of differences in the distribution of particular
chemicals, notably amino acids, proteins, lipids and sugars, amongst specific
bacterial taxa (Rosselló-Móra & Amann, 2001). Two standard methods used are
cellular fatty acid composition and whole-cell protein analysis. Cellular fatty acids
are the major constituents of lipids in cell membranes and lipopolysaccharides and
their variability in chain length, double-bond position and substituent groups has
been useful in characterising bacterial taxa (Vandamme et al., 1996). Whole cell
protein analysis compares the protein patterns obtained from sodium dodecyl sulfate-
polyacrylamide gel electrophoresis. It is a reliable method for comparing and
grouping large numbers of closely related strains (Vandamme et al., 1996).
1.5.4 The effect of horizontal gene transfer on bacterial phylogeny
The effect of HGT on bacterial evolution and the ecological and pathogenic
character of bacterial species is considerable. Bacterial species are open to gene
transfer from many other species, even those that are distantly related (Cohan, 2002)
and it is estimated that 5%–15% of the genes in a typical bacterial genome have been
Chapter 1
20
acquired from other species (Ochman et al., 2000). The impact of such transfer is
that molecular phylogenies calculated for different molecules from the same set of
species are only rarely completely congruent (Gogarten et al., 2002). Perhaps more
importantly, HGT of entire operons has been detected (Omelchenko et al., 2003),
thus providing a mechanism for the potential gain of new metabolic capabilities or to
confer bacterial antibiotic resistance, pathogenicity or photosynthetic or symbiotic
ability (Barcellos et al., 2007; Boucher et al., 2003; Ochman et al., 2000; Ochman &
Moran, 2001; Sullivan et al., 1995).
This phenomenon of dynamic prokaryotic genomes has led some researchers to
suggest that a network model, rather than a tree, is a more accurate depiction of
bacterial phylogeny (Kloesges et al., 2011; Koonin & Wolf, 2008; Kunin et al.,
2005). Others have argued, however, that carefully selected gene sequences can still
provide valid phylogenies of bacterial lineages (Daubin et al., 2003). This view is
based on the concept that bacterial genomes consist of three distinct pools of genes:
the “core” genome, the “character or lifestyle” genes and the “accessory” genes
(Schleifer, 2009). The core genome consists of a group of essential genes that are
common to all genomes of a phylogenetically coherent group of bacteria. It
preferentially contains informational or housekeeping genes that are stable and less
prone to HGT and thus are suitable candidates for phylogenetic analysis. The second
gene pool contains genes, such as those that code for specific metabolic properties,
which allow the bacteria to survive in a particular environment. The accessory genes
are non-essential, less conserved and often strain specific (Schleifer, 2009) and help
to determine the specific ecological properties of an organism (Fraser et al., 2009).
They are notably more A+T-rich, more prone to HGT and more usually found on
Chapter 1
21
mobilisable genetic elements such as plasmids and chromosomal islands (Daubin et
al., 2003; Young et al., 2006).
The frequency of HGT is positively correlated not only with accessory genes,
but also with genome size, with physical proximity (i.e. among species sharing the
same habitat) and with the degree of relatedness of the bacterial species (Kloesges et
al., 2011). In the latter case, phylogenetic distance imposes barriers to HGT due to
the restricted host ranges of transmissible agents such as bacteriophages and
conjugative plasmids, and to differences in the apparatuses of transcription and
translation (Lawrence & Hendrickson, 2003).
Within the framework outlined above, how should the available methodologies
be used to validly describe and classify bacterial (and in particular, rhizobial)
species?
1.6 General recommendations for classifying and describing
bacteria
Although there is no official classification for prokaryotes, the classification
system represented by Bergey’s Manual of Systematic Bacteriology
(http://www.bergeys.org/pubinfo.html) is widely accepted and is the standard
reference work on bacterial classification. Bacterial nomenclature, on the other hand,
is governed by the Bacteriological Code (Lapage et al., 1992). A list of current
prokaryotic names with standing is maintained by J. P. Euzéby at
http://www.bacterio.cict.fr/. Guidance on the use of current methodologies in the
taxonomic characterisation of prokaryote strains is provided in a series of reports and
Chapter 1
22
notes published in the International Journal of Systematic and Evolutionary
Microbiology (Stackebrandt et al., 2002; Tindall et al., 2010; Wayne et al., 1987).
The general recommendations for the description of new genera and species of
rhizobia can be found at http://edzna.ccg.unam.mx/rhizobial-taxonomy/node/12 and
in Graham et al. (1991). The recommendations are summarised as follows:
• New taxon descriptions should be based on a minimum of at least three
distinct strains, as revealed by molecular markers and, where possible, several
populations from different ecological settings should be sampled.
• A range of different molecular markers suitable to uncover and analyse
genetic diversity at different phylogenetic/taxonomic depths should be used.
Generating full-length 16S rDNA sequences for a few carefully selected
strains, along with the partial sequencing of two protein-coding core loci (e.g.
recA and rpoB) and at least one symbiotic locus (e.g. nifH, nodA or nodC) is
recommended.
• Phenotypic tests should include host range as well as pH and temperature
growth-range, salt tolerance, growth on different carbon and nitrogen sources,
antibiotic resistance profiling and fatty acid methyl-ester analysis. Phenotypes
and chemotaxonomic markers that are relevant as ecologically adaptive traits
should also be included.
• DNA-DNA hybridization should be determined for three distinct strains of a
new potential taxon in order to get an estimate of the standard deviations of
homology values (genome heterogeneity) within the new taxon.
Chapter 1
23
1.7 Rhizobial symbiotic genes
As stated above, rhizobia are polyphyletic and intermingled with non-
symbiotic prokaryotes. Symbiotic ability appears to be conferred by a group of
approximately 400 genes that form part of the accessory gene pool and enable
nodulation and N2 fixation with a particular legume (Broughton & Perret, 1999;
Young et al., 2006). These symbiotic genes are clustered together in potentially
transferable genomic elements such as plasmids or megaplasmids in Rhizobium or
Sinorhizobium species, or genomic islands in Azorhizobium, Bradyrhizobium and
some Mesorhizobium species (Freiberg et al., 1997; González et al., 2003; Kaneko et
al., 2002; Lee et al., 2008; Sullivan & Ronson, 1998; Young et al., 2006).
Regions encoding the rhizobial symbiotic genes are marked by the presence of
insertion sequence elements, transposases, phage-related integrases and related genes
that putatively provide mechanisms for HGT (MacLean et al., 2007). HGT of
symbiotic loci between both closely and distantly related rhizobial taxa has been
demonstrated in several studies (Andam et al., 2007; Barcellos et al., 2007;
Cummings et al., 2009; Johnston et al., 1978; Nandasena et al., 2007a; Sullivan &
Ronson, 1998). What is the extent of HGT of symbiotic loci between distantly
related rhizobia? As well as direct evidence that this occurs (Barcellos et al., 2007),
HGT across different genera can be inferred where phylogenetically diverse rhizobia
nodulating the same legume host possess the same or similar nodulation genes, or
where the phylogeny of the nodulation genes reflects the host plant taxonomy rather
than the rhizobial chromosomal lineage (Chen et al., 2003; Haukka et al., 1998;
Laguerre et al., 2001; Lu et al., 2009; Suominen et al., 2001). On the other hand,
there is evidence that HGT is more prevalent in closely related rhizobia and occurs
Chapter 1
24
within, but not between, different genera (Wernegreen & Riley, 1999). Phylogenetic
studies of the symbiotic loci of diverse rhizobia support the idea that the bacterial
chromosomal background is an important determinant in nodulation gene transfer
between rhizobial strains (Haukka et al., 1998; Moulin et al., 2004).
1.7.1 The role of nodulation genes and Nod factors
The rhizobial nodulation (nod, nol and noe) genes are structural and regulatory
genes that confer the ability to infect and nodulate the legume host. They form part
of a molecular dialogue between the host and the rhizobia, in which plant-derived
flavonoids induce expression of the nod genes and the subsequent synthesis of lipo-
chito-oligosaccharide (LCO) Nod factors (NFs) (Perret et al., 2000). The NFs in turn
induce plant genes that control and coordinate the separate processes of bacterial
infection and nodule organogenesis (D’Haeze & Holsters, 2002).
The LCO backbone is composed of usually four or five β1-4-linked N-acetyl
glucosamine residues that carry an N-linked C16 or C18 acyl chain on the terminal
non-reducing sugar (Gough & Cullimore, 2011). It is encoded by the canonical
nodABC genes that have to date been found in all rhizobia (excepting some
bradyrhizobial strains that form stem nodules on Aeschynomene species (Miché et
al., 2010)), whereas other nodulation loci encode substituent groups that “decorate”
the LCO core (Perret et al., 2000) (Figure 1.4). The variations in LCO structure
(number of glucosamine residues, length and degree of saturation of acyl tail and
addition of substituent groups) are characteristic of a rhizobial strain or species
(D’Haeze & Holsters, 2002; Dénarié et al., 1996) and although NF structure alone
cannot be used to predict host range, there is a correlation between the type of NF
produced and the rhizobial host range (Dénarié et al., 1996; Perret et al., 2000).
Chapter 1
25
Rhizobia synthesise a suite of NFs, which can consist of from two to 60 individual
NF structures, according to the rhizobial strain or environmental factors (D’Haeze &
Holsters, 2002; Morón et al., 2005). The quantity and variety of the NF structures is
also a determinant of rhizobial host range: the exceptionally broad host range strain
NGR234 secretes high concentrations of a large family of NF structures, in which a
wide palette of substituent groups variously decorate the LCO core (Schmeisser et
al., 2009).
Figure 1.4. General structure of Nod factors produced by rhizobia. The substituent groups
that decorate the lipo-chito-oligosaccharide core and the genes that code for the
oligosaccharide moiety, the acyl chain and the substituent groups are indicated. Figure taken
from Perret et al. (2000).
Studies of the model legumes Lotus japonicus and Medicago truncatula show
that it is the specific recognition of NFs by LysM receptor-like kinases in the
epidermis of the root hair that triggers the complex signalling pathway controlling
bacterial infection and nodule morphogenesis (Kouchi et al., 2010; Madsen et al.,
2010; Oldroyd & Downie, 2008).
1.8 Rhizobial infection and nodule formation in legumes
A key aspect of the legume-rhizobia symbiosis is the organogenesis of a
specialised structure, the nodule, which develops on the roots or stems of the host
plant. Within the cells of the nodule the differentiated bacteria (bacteroids) are
housed in plant membrane-bound compartments (symbiosomes), where they reduce
A
Chapter 1
26
N2 to ammonia (Oldroyd & Downie, 2008). Nodule shape and structure are
determined by the host plant and have taxonomic value (Corby, 1981; Lavin et al.,
2001; Sprent & James, 2007). The legume host also controls the infection pathway.
Sprent (2007; 2008b) has identified several types of nodule and two modes of
infection that are characteristic of the different legume groups in which they are
found.
1.8.1 Infection pathways in legumes
Within the Papilionoideae, rhizobial infection may proceed intracellularly via
root hair curling, with the bacteria confined to an infection thread (IT) before their
release into nodule primordium cells, or may be directly through the epidermis
without IT involvement in nodulation (Sprent & James, 2007). The majority of
legume taxa, including species in the tribe Phaseoleae and in the hologalegoid clade,
are usually infected via a root hair pathway (Sprent, 2009) and this well-studied
mode of bacterial entry has been the subject of several reviews. Briefly, infection
begins with the root hair curling around and enclosing attached rhizobia. Within the
curl, the plant cell wall is locally hydrolysed, the plasma membrane invaginates and
new cell wall material is deposited to form the IT, which then grows through the
epidermal cell and into the root cortex, while the bacteria within the thread divide
and proliferate. Cytoplasmic bridges (also known as pre-infection threads) form in
the cortical cells to guide the IT towards the developing nodule primordium. Once it
reaches the nodule primordium, the IT ramifies, followed by the release of bacteria
into the primordial nodule cells. The rhizobia, now surrounded by a peribacteroid
membrane (symbiosomes), differentiate into their symbiotic forms (bacteroids) and
begin to fix N2 (Brewin, 2004; Gage, 2004; Maunoury et al., 2008).
Chapter 1
27
The alternative intercellular mode of infection, where rhizobia enter the host
plant directly through the epidermis and are not confined to infection threads, is
found in at least 25% of extant legumes and is characteristic of the more basal
genistoid and dalbergioid clades (Sprent, 2008a). This type of infection (commonly
referred to as crack entry) has been observed in Aeschynomene fluminensis (Loureiro
et al., 1995), Arachis hypogaea (Boogerd & van Rossum, 1997), Cytisus (previously
Chamaecytisus) proliferus (Vega-Hernández et al., 2001) Genista tinctora (Kalita et
al., 2006) Lupinus species (González-Sama et al., 2004; Tang et al., 1992) and
Stylosanthes species (Chandler et al., 1982). Intercellular infection occurs by
penetration of the middle lamella, often at the junction between a root hair base and
an adjoining epidermal cell (González-Sama et al., 2004; Uheda et al., 2001), and in
dalbergioid legumes is associated with the emergence of lateral or adventitious roots
(Lavin et al., 2001). Bacteria then spread through the cortex in an intercellular
matrix. In some legumes, this invasion appears to induce local cell death, creating an
intercellular space filled with rhizobia (Boogerd & van Rossum, 1997; Vega-
Hernández et al., 2001). The rhizobia eventually penetrate the altered cell wall of
nodule primordium cells. Mitotic division of the newly infected cells and
symmetrical distribution of the symbiosomes (similar to host cell organelles), gives
rise to nodules with characteristic uniformly infected central tissue (Boogerd & van
Rossum, 1997; Fedorova et al., 2007; Sprent & James, 2007).
Studies of both IT (Lotus japonicus and Medicago truncatula) and non-IT
(Lupinus albus) legumes show that the infected nodule cells undergo cycles of
endoreduplication, leading to enlarged polyploid cells (González-Sama et al., 2006;
Maunoury et al., 2008). In some legumes, differentiation of the bacteroids also
Chapter 1
28
involves endoreduplication and an increase in cell size. This has been studied most
notably in the Inverted Repeat Lacking Clade legumes, where bacteroid
differentiation is terminal and bacteroids are unable to resume growth after release
from a nodule; conversely bacteroids within phaseoloid nodules are not swollen or
polyploid and remain viable (Maunoury et al., 2008; Mergaert et al., 2006).
Although the type of bacteroid differentiation is dependent upon the host plant,
swollen bacteroids have been found in legume species belonging to five out of the
six major papilionoid subclades and this host-imposed control is considered by Oono
et al. (2010) to have evolved independently within multiple legume clades.
It has been postulated that intercellular infection is likely to be the ancestral
mode of rhizobial entry and it remains a default pathway, as crack entry can occur in
host plants that are usually infected by root hair curling (Sprent & James, 2007;
Sprent, 2008a). An example is the semi-aquatic robinioid legume Sesbania rostrata.
Rhizobial infection in this plant is via root hair curling, but in flooded conditions
(where waterlogged roots are mainly hairless) bacteria enter via epidermal fissures at
the sites of adventitious root emergence. Subsequent IT formation then guides the
rhizobia towards the nodule primordium (Capoen et al., 2010). Similar crack entry
followed by the development of ITs has been observed in aquatic mimosoid
Neptunia species and in flooded Lotus uliginosus plants (James et al., 1992; James &
Sprent, 1999; Subba-Rao et al., 1995). Lonchocarpus muehlbergianus (tribe
Milletieae), which does not produce root hairs, also appears to be nodulated via an
epidermal infection followed by the formation of ITs (Cordeiro et al., 1996).
Strikingly, the Lotus japonicus ROOT HAIRLESS mutant can be effectively
nodulated via crack entry through the cortical surface of the nodule primordium and
Chapter 1
29
subsequent development of ITs (Karas et al., 2005; Madsen et al., 2010). The
symbiosis between rhizobia and the non-legume Parasponia, where crack entry is
followed by the retention of rhizobia in thin-walled ITs called fixation threads
(Becking, 1992), further supports the theory of ancestral intercellular infection.
1.8.2 Nodule types
Nodules are formed when rhizobia induce the dedifferentiation of the root
cortical cells, activating their cell cycle and establishing a cluster of meristematic
cells that give rise to the nodule primordium (Maunoury et al., 2008). Legume
nodules have traditionally been divided into two types: determinate and
indeterminate, according to whether the nodule possesses a persistent meristem
(Gibson et al., 2008). Sprent (2007), however, has identified several major types of
nodule (Figure 1.5), based on nodule structure, meristem persistence and the
presence or absence of ITs and this terminology will be used in this review. Notably,
in legumes infected via ITs, the active N2-fixing nodule tissue contains a mix of
infected and uninfected cells, whereas in intercellularly infected legumes the central
tissue of the N2-fixing nodule is uniformly infected (Sprent & James, 2007; Sprent,
2009).
Chapter 1
30
Figure 1.5. Main features of legume nodules. (a-b) Nodules in which infection threads carry
bacteria to individual cells derived from the meristem and the central tissue contains infected
and uninfected cells. (a) Indeterminate form as found in the Caesalpinioideae, Mimosoideae
and Papilionoideae subfamilies, where infection threads carry bacteria to individual cells
derived from the meristem. There is a gradient of developmental zones and the nodule may
be branched or unbranched. (b–e) Nodules are found only in the Papilionoideae. (b)
Determinate (desmodioid) nodule, rounded in shape, with no age gradient in the infected
tissue. (c-e) Nodules in which infection threads are not associated with nodulation and the
central uniformly infected tissue is formed by division of a few initially infected cells. (c)
Determinate dalbergioid nodule, associated with a lateral root. (d) Indeterminate nodule with
an age gradient, found in many genistoid legumes. It may be unbranched or branched. (e)
The lupinoid nodule, which is a variant of (d) where the meristems grow around the
subtending root. Found in some genistoid legumes such as Lupinus and Listia. IC = infected
cells; UC = uninfected cells; M = meristem; C = cortex; R = subtending root; LR = lateral
root; arrow indicates a gradation in age of infected cells, youngest towards arrowhead.
Figure taken from Sprent (2007).
1.8.2.1 Formation and structure of nodules infected via infection threads
Both determinate and indeterminate nodules are found in legumes that are
infected via ITs and (usually) root hair curling. The model legumes Lotus japonicus
and Medicago truncatula, respectively, provide well-studied examples of each
nodule type (Maunoury et al., 2008). Indeterminate nodules occur in the majority of
Chapter 1
31
legumes. They originate from cell division in the inner cortex and retain a meristem,
giving them a characteristic cylindrical or lobed shape (Gualtieri & Bisseling, 2000).
The nodule cells are divided into a gradient of developmental zones, with a persistent
apical meristem (zone I), an infection zone (zone II), a fixation zone (zone III) and,
in mature nodules, a senescent zone (zone IV) that is established proximal to zone III
(Guinel, 2009; Maunoury et al., 2008). Determinate (or desmodioid) nodules are
found in the phaseoloid clade and some members of tribe Loteae (Sprent, 2009).
They develop from cells in the hypodermal region or outer cortex and the meristem
ceases to divide at an early stage (Guinel, 2009; Szczyglowski et al., 1998). Because
of this, the nodules are spherical in shape and the cells are all at a similar
developmental stage (Gualtieri & Bisseling, 2000).
1.8.2.2 Formation and structure of intercellularly infected nodules
Determinate and indeterminate nodules are also found in the genistoid and
dalbergioid legumes, in which infection threads are absent. The distinctive
aeschynomenoid nodule, with determinate growth, a central mass of uniformly
infected tissue and associated with a lateral or adventitious root, is synapomorphic
for the dalbergioid clade (Lavin et al., 2001). In studied dalbergioid legumes, the
meristematic zone arises in the cortex some distance from the infection site. The
rhizobia penetrate to the cortex via an intercellular matrix (Arachis) or by the
progressive collapse of invaded cells (Aeschynomene and Stylosanthes); infected
meristematic cells then divide repeatedly to form the nodule (Alazard & Duhoux,
1990; Boogerd & van Rossum, 1997; Chandler et al., 1982).
In contrast, genistoid nodules have a persistent meristem and are thus
indeterminate. The lupinoid nodule found in species of Lupinus and Listia is a
Chapter 1
32
variation on this, with lateral meristems forming a “collar” nodule that encircles the
subtending root (Guinel, 2009; Sprent, 2009). Nodule primordia of the genistoid
legume Cytisus proliferus develop in the inner cortex, and rhizobial dissemination
through the cortex is accompanied by the collapse of host cells (Vega-Hernández et
al., 2001). In contrast, the nodule primordium of Lupinus species originates from a
single infected hypodermal or outer cortical host cell (González-Sama et al., 2004;
Tang et al., 1992).
It has been speculated that legume infection via a crack entry pathway is less
specific than that of root hair infection (Boogerd & van Rossum, 1997; Sprent,
2007). Specificity is not always associated with a root hair curl infection, however,
as demonstrated by promiscuous plants such as Phaseolus vulgaris (Laeremans &
Vanderleyden, 1998; Martínez-Romero, 2003). Legume specificity and a narrow
rhizobial host range do seem to be features of the symbiosis between temperate
legumes in the hologalegoid tribes Vicieae and Trifolieae and their cognate rhizobia
(Sprent, 2007). As specificity and effectiveness appear to be linked (Sprent, 2007)
and are critical aspects in the assessment and development of legumes and inoculants
in agricultural systems (Howieson et al., 2008; Sessitsch et al., 2002), it is important
that these terms are defined and explained.
1.9 Specificity and effectiveness in the legume-rhizobia
symbiosis
Both legume hosts and rhizobial microsymbionts vary in their symbiotic
ability. Legumes in tribe Phaseoleae are known to be promiscuous; that is, able to
nodulate with a broad spectrum of rhizobia (Perret et al., 2000). Conversely,
Chapter 1
33
Biserrula pelecinus is known only to nodulate with strains of Mesorhizobium
(Nandasena et al., 2007b; Nandasena et al., 2009). Some legume cultivars (of clover
and soybean, for example) are more promiscuous than others, with important
implications for agriculture (Drew & Ballard, 2010; Graham, 2008). Similarly,
rhizobia such as the strains NGR234 and USDA257 have exceptionally broad host
ranges (Pueppke & Broughton, 1999), while the narrow host range Azorhizobium
caulinodans is compatible only with Sesbania species (Perret et al., 2000).
A fundamental aspect of the legume-rhizobia relationship is the effectiveness
of the symbiosis, i.e. the amount of N2 fixed by the rhizobia and made available to
the plant. A wide variation in the effectiveness of symbiotic interactions can exist
(Sprent, 2007; Thrall et al., 2000). Based on this variation, Howieson et al. (2005)
have defined four categories of symbiotic interaction:
1. no symbiotic interaction, i.e. plants do not nodulate
2. an ineffective or parasitic interaction, where nodules form but there is
no N2 fixation
3. a partially effective symbiosis, where fixation produces 20–75% of
the biomass achieved by a nitrogen-fed control
4. an effective symbiosis, where nodulated plants produce > 75% of the
biomass achieved by a nitrogen-fed control
A greater understanding of the factors that govern specificity and effectiveness
in legume-rhizobia symbioses will be required if agricultural systems are to provide
the sustainable increases in productivity needed to cope with an increasing world
Chapter 1
34
population, higher nitrogen fertilizer prices and other pressures (Howieson et al.,
2008). This is illustrated by the approach used to develop exotic legumes for
southern Australian agricultural systems, in response to environmental and economic
factors (Howieson et al., 2000; Howieson et al., 2008). Several species in the
Lotononis s. l. clade were included in the list of exotic legumes targeted for
evaluation in this approach, in particular several species in the genus Listia.
1.10 The Lotononis s. l. clade
The Lotononis s. l. clade is grouped within tribe Crotalarieae (and thus is in the
genistoid clade), has a centre of origin in South Africa and consists of some 150
species, divided into 15 sections (Figure 1.6) (van Wyk, 1991). The taxonomy has
recently been revised and the three distinct clades within Lotononis s. l. are now
recognised at the generic level as Listia, Leobordea and Lotononis s. str. (Boatwright
et al., 2011). A brief description of genera, sections and species relevant to this thesis
is provided below (Figure 1.7), and is based on the synopsis of van Wyk (1991) and
the revisions to the taxonomy given in Boatwright et al. (2011).
Chapter 1
35
Figure 1.6. Cladogram for Lotononis s. l., showing the relationships of the different sections.
The diagram is based on the cladogram featured in van Wyk (1991). The three numbered
clades are now recognised at the generic level as (1) Listia, (2) Leobordea and (3) Lotononis
s. str., according to the taxonomy of Boatwright et al. (2011). The monospecific Euchlora
section is now strongly supported as being more closely related to Crotalaria and Bolusia
than to Lotononis s. l. (Boatwright et al. (2008; 2011)). Sections relevant to this thesis are in
blue font.
Listia
Digitata
Leptis
Lipozygis
Monocarpa
Cleistogama
Euchlora
Polylobium
Lotononis
Aulacinthus
Krebsia
Buchenroedera
Leobordea
Synclistus
Oxydium
1
2
3
Listia
Digitata
Leptis
Lipozygis
Monocarpa
Cleistogama
Euchlora
Polylobium
Lotononis
Aulacinthus
Krebsia
Buchenroedera
Leobordea
Synclistus
Oxydium
1
2
3
Chapter 1
36
Figure 1.7. Species of Lotononis s. l.: (a) Listia angolensis, (b) Listia bainesii (c) Leobordea
longiflora, (d) Leobordea platycarpa, (e) Leobordea stipulosa, (f) Leobordea bolusii, (g)
Leobordea polycephala, (h) Lotononis crumanina, (i) Lotononis delicata, (j) Lotononis
falcata, (k) Lotononis laxa, (l) Lotononis pungens
1.10.1 The genus Listia
Listia consists of seven species of herbaceous perennials: L. angolensis, L.
bainesii, L. heterophylla (previously Lotononis listii), L. marlothii, L. minima, L.
Chapter 1
37
solitudinis and L. subulata. Species in this section are of particular interest as pasture
plants as they are perennial, lack the poisonous metabolites found in some other
Lotononis s. l. sections (van Wyk & Verdoorn, 1990) and are able to produce
adventitious, almost stoloniferous roots on the lower branches. These adventitious
roots are a property unique to Listia, and are thought to be associated with the
seasonally wet habitats, such as ditches, riverbanks and dambos (shallow grassy
wetlands) where these species are found. Nodulation has been studied in all species
except the very rare L. minima, and the nodules found to be of the lupinoid form
(Yates et al., 2007; R. Yates unpublished data). L. angolensis has a tropical
distribution, occurring in the uplands encircling the Zaire basin. The L. heterophylla
distribution extends from South Africa into southern Central Africa. L. bainesii is
native to Botswana, Mozambique (south), Namibia and South Africa, while the
remaining species are endemic to South Africa.
1.10.2 The genus Leobordea
Leobordea comprises the sections Digitata, Lipozygis, Leptis, Leobordea and
Synclistus. The six species in the Digitata section all have woody perennial bases and
are mostly found in a narrow distribution in the dry mountainous region of the north-
western Cape Province. Leobordea longiflora (syn. Lotononis speciosa) has the
longest flowers in the genus. Lipozygis section species are perennial suffrutescent
pyrophytic herbs, restricted to summer rainfall grassland areas of the eastern parts of
southern Africa. Leobordea foliosa occurs at high altitudes. Species in the Leptis
section are perennial suffrutescent herbs, or shrublets, or annuals; with a disjunct
distribution in central and southern Africa and in the Mediterranean region.
Leobordea calycina is endemic to southern Africa and widely distributed in the
eastern and central interior. Leobordea mollis is found only in the western Cape.
Chapter 1
38
Species in the Leobordea section are all annuals that occur in the dry parts of
southern Africa. The distribution of Leobordea platycarpa extends through eastern
tropical Africa and Mauritania to Pakistan and the Cape Verde Islands, which makes
it the most widespread species of the genus. Leobordea stipulosa is found in the
Transvaal, Zimbabwe and the southern border of Zambia. The group of species in the
Synclistus section differs from all other Lotononis s. l. sections in having heads of
sessile flowers. They are described as annuals, but in the case of Leobordea bolusii
and Leobordea polycephala are more likely to be short-lived herbaceous perennials,
as the plants can be grown from cuttings (see Chapter 2, Materials and Methods).
The section is endemic to the Cape Province in South Africa.
1.10.3 The genus Lotononis s. str.
Lotononis s. str. includes the sections Oxydium, Monocarpa, Cleistogama,
Polylobium, Lotononis, Aulacinthus, Krebsia and Buchenroedera. The genus is
chemically distinct from Leobordea and Listia in that its members are cyanogenic
(except for L. sect. Cleistogama) and accumulate macrocyclic pyrrolizidine
alkaloids. The monotypic section Euchlora was formerly placed within this group,
but recent studies have found Euchlora to be more closely related to Crotalaria and
Bolusia rather than Lotononis s. l., and to merit generic status (Boatwright et al.,
2008; Boatwright et al., 2011). Euchlora hirsuta (formerly Lotononis hirsuta), the
sole species, is a geophytic herb with a north-western distribution in the Cape
Province. Although the GRIN database (http://www.ars-grin.gov/~sbmljw/cgi-
bin/taxnodul.pl) lists E. hirsuta as nodulated, field observations of this species have
so far found no evidence of nodulation (J. Howieson & R. Yates, pers. comm.).
Species in the Oxydium section are herbaceous annuals or perennials. Lotononis
crumanina is a perennial that occurs on limestone or lime-rich soils in the central
Chapter 1
39
parts of southern Africa. Lotononis delicata is an annual; Lotononis falcata is a
common, widespread annual in southern Africa and Lotononis laxa is the most
common and widely distributed perennial species in Lotononis s. l., with a range
extending along the east coast of southern Africa to Ethiopia. The two species in the
Cleistogama section are very common, short-lived perennials that can be found in
most habitats. Their distribution extends into the north-eastern and eastern Cape,
with the range of Lotononis pungens also extending into the Orange Free state.
1.11. The Lotononis s. l. -rhizobia symbiosis
Renewed interest in the potential of Lotononis s. l. species as perennial pasture
plants in southern Australian agricultural systems has prompted recent research into
the rhizobia that nodulate these legumes (Yates et al., 2007). Although the molecular
studies aimed at identifying and characterising these rhizobia and their host
specificity genes have just begun, previous research on Lotononis s. l. rhizobia was
conducted in Australia and Africa some fifty years ago, as part of the development of
Listia bainesii for tropical and sub-tropical pastures (Bryan, 1961; Sandmann, 1970).
Data from this era, while unable to give a precise identification of Lotononis s. l.
rhizobia, provide records of rhizobial phenotypes and cross-inoculation experiments.
Sandmann’s work in Zimbabwe showed that Lotononis s. l. species were
nodulated by phenotypically diverse rhizobia and that the pigmented rhizobia
isolated from Listia bainesii and Listia heterophylla formed a separate cross-
inoculation group to isolates from Listia angolensis. Similar studies were conducted
by the CSIRO in Queensland, Australia in the 1950s and 60s on various Lotononis s.
l. species and cultivars (Eagles & Date, 1999) (Table 1.1).
Chapter 1
40
Table 1.1. The CB Collection accession list of Lotononis s. l. hosts, rhizobia isolated from
these hosts and effectiveness of the rhizobia on Lotononis s. l. species (Eagles & Date,
1999). Data on ineffectiveness are not given in the cited reference.
Original Host Strain Effective on
Listia angolensis CB1297 L. angolensis
Listia angolensis CB1298 L. angolensis
Listia angolensis CB1299 L. angolensis
Listia angolensis CB1321 L. angolensis
Listia angolensis CB1322 L. angolensis
Listia angolensis CB1323† L. angolensis, Leobordea mucronata
Listia angolensis CB2406 L. angolensis
Listia angolensis CB2645 L. angolensis
Listia bainesii CB360 L. bainesii, Lisita heterophylla
Listia bainesii CB376‡ L. bainesii, Lotononis laxa, Lotononis leptoloba
Listia bainesii CB730 L. bainesii, L. heterophylla
Listia bainesii CB1775 L. bainesii, L. heterophylla
Listia bainesii CB1776 L. bainesii
Listia bainesii CB1777 L. bainesii
Listia bainesii CB1778 L. bainesii
Listia bainesii CB1779 L. bainesii
Listia bainesii CB2343 L. bainesii, L. heterophylla
Listia bainesii CB2344 L. bainesii
Listia heterophylla CB768 L. bainesii, L. heterophylla
Leobordea calycina* CB2676 L. calycina
Leobordea mucronata CB2695 L. mucronata
Leobordea mucronata CB2705 L. mucronata
Leobordea platycarpa CB2648 Lotononis laxa
Leobordea stipulosa CB1394 L. stipulosa
*Referred to as Lotononis orthorrhiza in the cited reference.
†Published performance information lists this strain as effective on some accessions of Listia
angolensis and most Leobordea mucronata and Lotononis laxa (Eagles & Date, 1999).
‡ Published performance information lists this strain as effective on Listia bainesii,
Leobordea mucronata, Lotononis laxa and Lotononis leptoloba (Eagles & Date, 1999).
The CB Collection data on the host range and effectiveness of rhizobia isolated
from and/or inoculated onto species of Listia (L. angolensis, L. bainesii and L.
heterophylla), Leobordea (L. calycina, L. mucronata, L. platycarpa and L. stipulosa)
and Lotononis s. l. (L. laxa and L. leptoloba) indicate that:
Chapter 1
41
1) the rhizobia were phenotypically diverse;
2) L. angolensis, L. bainesii and L. heterophylla were not nodulated by
isolates from other Lotononis s. l. species;
3) L. angolensis isolates and the isolates from L. bainesii and L.
heterophylla formed separate cross-inoculation groups.
Most of the literature on Lotononis s. l. rhizobia is based on studies conducted
on Listia bainesii, which is recognised as a model of acute symbiotic specificity
(Pueppke & Broughton, 1999). Its microsymbionts were first formally described by
Norris (1958), who showed that L. bainesii seedlings inoculated with a wide range of
rhizobia would only nodulate with their specific red-pigmented isolate, designated
strain CB360. This rhizobial strain was also highly specific. In glasshouse trials of 31
legume species from 21 genera, it was able to nodulate only species in the tribes
Aeschynomeneae and Crotalarieae and was effective only on L. bainesii, although
other Lotononis s. l. species were not tested (Norris, 1958). Strain CB376, also
isolated from L. bainesii, has similarly been described as having an extremely narrow
host range (Broughton et al., 1986). In Australia, CB360 was used as the commercial
inoculant strain for L. bainesii from 1958 – 1963, when it was superseded by CB376
(Bullard et al., 2005). The colony colour, serological and symbiotic properties of
both these strains remained stable over a 5 – 12 year period following their
introduction in Queensland (Diatloff, 1977). These and other field trials have
indicated that pigmented L. bainesii strains are able to persist in acidic, sandy,
infertile soils (Diatloff, 1977; Yates et al., 2007).
Chapter 1
42
The pigmented L. bainesii rhizobial strains have since been identified as a
species of Methylobacterium (Jaftha et al., 2002; Yates et al., 2007). The only other
rhizobial Methylobacterium species described to date is the non-pigmented M.
nodulans, which specifically nodulates species of Senegalese Crotalaria (Sy et al.,
2001). Methylobacteria are typically pink-pigmented facultative methylotrophs that
are characterised by their ability to utilise methanol and other C1 compounds, as well
as a variety of multicarbon substrates (Green, 1992; Lidstrom, 2006). The pigments
of strain CB360 have been identified as carotenoids (Godfrey, 1972). Similarly,
pigments in strain CB376 were found to be carotenoids (diapolycopenedioic acid
glucosyl ester and diapolycopenedioic acid diglucosyl ester), with structures identical
to those from Methylobacterium rhodinum (formerly Pseudomonas rhodos) (Britton
et al., 2004; Kleinig & Broughton, 1982). Interestingly, bacterial pellets of CB360
extracted from nodules were not pigmented, indicating that carotenoid synthesis is
suppressed in this environment (Godfrey, 1972). Carotenoid pigments are believed to
play a role in protecting bacteria from ultraviolet (UV) radiation (Jacobs et al.,
2005). Law (1979) has noted, however, that while pigmented L. bainesii rhizobia are
highly resistant to UV irradiation, non-pigmented mutants are also resistant to such
treatment. In addition to carotenoid pigments, bacteriochlorophyll a has been found
in the L. bainesii rhizobial strains 4-46 (incorrectly referred to as a synonym of
CB376; D. Fleischman, pers. comm.), 4-144 and xct14 (Fleischman & Kramer, 1998;
Giraud & Fleischman, 2004; Jaftha et al., 2002), which is consistent with the weakly
phototrophic nature of other species of Methylobacterium (Garrity et al., 2005;
Hiraishi & Shimada, 2001). The sequenced genome of strain 4-46, along with other
Methylobacterium strains, is available at the USA Joint Genome Institute
(http://genome.jgi-psf.org/programs/bacteria-archaea/index.jsf).
Chapter 1
43
Strains of pigmented methylobacteria have now been isolated from, and are
effective on, all studied Listia species, with the exception of L. angolensis, which
forms ineffective nodules with these rhizobia (Yates et al., 2007; R. Yates,
unpublished data). Surprisingly, a study of the methylobacterial isolates from L.
bainesii, L. heterophylla and L. solitudinis showed that they are, uniquely for
Methylobacterium spp., unable to grow on methanol as a sole carbon source (Ardley
et al., 2009). It has since been suggested that long term maintenance of C1
metabolism in methylobacteria requires relatively frequent use of C1 compounds to
prevent the rapid loss of this trait (Lee et al., 2009).
Another unexpected finding was that the Listia angolensis microsymbionts,
based on their 16S rRNA gene sequences, belong to a novel genus of rhizobia (Yates
et al., 2007). Strains WSM3674 and WSM3686, isolated from L. angolensis, were
subsequently shown to be closely related to rhizobia that specifically nodulate
Lupinus texensis plants endemic to Texas, USA (Andam & Parker, 2007). The 16S
rRNA-based phylogenetic tree constructed by Andam & Parker (2007) indicated that
the Listia angolensis and Lupinus texensis strains were most closely related to
Microvirga flocculans (previously Balneimonas flocculans) (Weon et al., 2010), a
species described from a strain isolated from a Japanese hot spring (Takeda et al.,
2004). Currently, four other Microvirga species have been named and characterised:
M. subterranea (Kanso & Patel, 2003), M. guangxiensis (Zhang et al., 2009), M.
aerophila and M. aerilata (Weon et al., 2010), isolated from Australian geothermal
waters, Chinese rice field soil and Korean atmospheric samples (two strains),
respectively.
Chapter 1
44
Published data on the nodulation record or symbiotic specificity of other
species of Lotononis s. l. remain scant. Studies on isolates obtained in 2002 from
nodules of South African Lotononis s. l. hosts established that the rhizobia isolated
from species outside the genus Listia were non-pigmented, genotypically and
phenotypically diverse and unable to nodulate either Listia bainesii or Listia
heterophylla (Ardley, 2005; R. Yates, unpublished data).
1.12 Summary of current knowledge of the symbiotic
relationships between Lotononis s. l. and associated rhizobia
A brief summary of previous studies of the Lotononis s. l. -rhizobia symbiosis
indicates the following:
• Lotononis s. l. species are nodulated by diverse rhizobia, although this has not
been formally reported in the literature.
• The symbiosis between Listia bainesii and strains of pigmented
methylobacteria is both effective and highly specific. This effectiveness and
specificity extends to other studied Listia species, with the exception of L.
angolensis.
• L. angolensis can be ineffectively nodulated by pigmented methylobacteria,
but forms effective nodules only with bacteria that belong to a group of novel
and undescribed rhizobia.
There has as yet been no detailed examination of the level of specificity in
the L. angolensis symbiosis or in Lotononis s. l. species outside the genus Listia.
A first step would be to quantify the nodulation and N2 fixation capabilities of
Chapter 1
45
Lotononis s. l. isolates on host species from a range of Lotononis s. l. sections. To
that purpose, the availability of seeds collected from a number of South African
Lotononis s. l. species in 2007, together with a collection of phylogenetically
diverse Lotononis s. l. isolates, allowed the design of glasshouse trials to compare
the symbiotic interactions of different taxonomic groups of Lotononis s. l. hosts
and their cognate rhizobia.
Secondly, given the important role Nod factors play in determining specificity
(Section 1.7), does the phylogeny of the nod genes of Lotononis s. l. rhizobia provide
a clue to the basis of specificity in this genus? Comparison of the phylogeny of the
rhizobial chromosomal background with that of the nod genes can also help elucidate
the evolution and biogeography of the symbiosis between native populations of
Lotononis s. l. host plants and their associated rhizobia.
Another aspect of symbiosis in Listia bainesii that warrants study is the
mechanism of infection and nodule formation. This is of interest, given that:
1. Studies of other legume hosts in the genistoid clade show a crack or
epidermal, rather than a root hair curl, intercellular infection process
with no, or transient, development of infection threads (Section 1.8)
2. If infection in L. bainesii occurs by epidermal entry without IT
formation, it presents an example of unusual symbiotic specificity in a
legume host with a non-root-hair/IT-mediated infection process.
Finally, according to their 16S rRNA gene sequences, the Listia angolensis
isolates belong to a group of novel and as yet uncharacterised rhizobia (Yates et al.,
Chapter 1
46
2007). This presented an opportunity to make a contribution to the literature on
rhizobia by naming and describing these new symbiotic bacteria. Importantly, the
availability of L. angolensis isolates from the WSM Culture Collection, together with
a group of phylogenetically related Lupinus texensis isolates (Andam & Parker,
2007), allowed for a polyphasic description of this novel group of root nodule
bacteria, based on a number of strains from different hosts and widely separate
geographical areas. Professor Matt Parker, whose laboratory at the State University
of New York (SUNY) was responsible for the work on the L. texensis isolates, was
approached to be a part of this collaboration. Professor Anne Willems and her
student Sofie De Meyer at the University of Ghent were included in the collaboration
for their expertise in DNA: DNA hybridization, determination of G + C content and
cellular fatty acid analysis, with the aim of publishing a valid name and description
of the genus and species.
1.13 Aims of this thesis
Accordingly, the aims of this thesis are to:
1. Assess and compare the symbiotic specificity of Lotononis s. l. species and
their diverse rhizobial isolates in glasshouse trials, and determine the
phylogeny of the isolates’ chromosomal and symbiotic backgrounds.
2. Investigate the process of infection and nodule initiation and the nodule
morphology of Listia angolensis and Listia bainesii.
3. Perform a range of genotypic and phenotypic studies as part of the
requirements for validly naming and describing the novel Listia angolensis
and Lupinus texensis isolates.
Chapter 2
47
CHAPTER 2
Determining symbiotic specificity within Lotononis s. l.
Chapter 2
48
Chapter 2
49
2.1 Introduction
2.1.1 Background
Between 2002 and 2007, the Centre for Rhizobium Studies undertook
collections of nodule isolates and seeds from a range of Lotononis s. l. species
growing in diverse sites in South Africa. This collection of legume germplasm and
rhizobia, together with Listia angolensis strains sourced from the CB Strain
Collection, allowed an examination of the nodulation and nitrogen fixation
capabilities of rhizobia associated with Lotononis s. l. across a range of
taxonomically different Lotononis s. l. hosts. Together with a molecular analysis of
the rhizobial core and symbiosis genes, this study sought to answer the following
questions:
1. What is the extent of nodulation and the nodule morphology found
within Lotononis s. l. species?
2. Is the symbiotic specificity of the genus Listia maintained?
3. How does the symbiotic specificity of L. angolensis compare with other
Listia species?
4. Are there patterns of rhizobial specificity in the other Lotononis s. l.
sections?
5. Given that this is a study of native legumes and microsymbionts
obtained from their centre of diversity, does this study shed light on the
mechanisms by which the evolution and diversification of legume
plants affects their relationship with their preferred microsymbionts?
Chapter 2
50
6. Do the diverse Lotononis s. l. rhizobia also possess diverse symbiotic
genes and is there a relationship between host and symbiotic gene
phylogeny?
2.1.2 Experimental approach
The work was divided into four phases:
1. Selection of appropriate inoculant strains and Lotononis s. l. hosts.
2. Design of a glasshouse trial that would determine the nodulation and
nitrogen-fixing capabilities of Lotononis s. l. species inoculated with
diverse Lotononis s. l. rhizobial isolates.
3. Additional glasshouse trials to examine further the degree of symbiotic
specificity within the genus Listia.
4. Sequencing of rhizobial genes.
The Lotononis s. l. isolates available for this study consisted of three groups of
strains:
1. Sixty seven pink-pigmented methylobacterial isolates obtained from
nodules of South African Listia bainesii, Listia heterophylla and Listia
solitudinis and authenticated as effective nodulators of L. bainesii
(Ardley, (2005); R. Yates, unpublished data).
2. Seven non-pigmented genetically and phenotypically diverse isolates
from South African Leobordea and Lotononis s. str. species, i.e.
obtained from Lotononis s. l. species outside the genus Listia. Partial
16S rRNA sequences identified the isolates as species of
Bradyrhizobium, Ensifer (syn. Sinorhizobium), Mesorhizobium or
Methylobacterium (Ardley, 2005). They were unable to nodulate Listia
Chapter 2
51
bainesii or Listia heterophylla, but were authenticated as RNB after
nodulation tests on promiscuous host plants (Ardley, 2005).
3. Seven Listia angolensis strains (six that formed pale pink colonies and
one non-pigmented strain). These were derivatives of the original CB
strains, obtained by inoculating glasshouse grown L. angolensis plants
(accession No. 8639, ARC—LBD Animal Production Institute,
Pretoria, South Africa) with each strain and reisolating from the
nodules according to the methods of Vincent (1970) (Yates et al.,
2007). Two of these strains, WSM3674 and WSM3686, have been
identified as belonging to a novel genus of rhizobia (Yates et al., 2007).
Inoculant strains were selected from these three groups on the basis of primary
host species, ability to nodulate at least one species of Lotononis s. l. and phenotypic
and genotypic diversity. Additionally, where there were a number of strains from the
same host (as in the Listia angolensis strains) or the same cross-inoculation group (as
in the pigmented methylobacteria) a representative strain was chosen on the basis of
its effectiveness on its original host plant.
Previous studies on the pink-pigmented methylobacteria indicated that strain
WSM2598 was highly effective on both Listia bainesii and Listia heterophylla and
was able to ineffectively nodulate Listia angolensis (Yates et al., 2007). As data on
the effectiveness of the L. angolensis strains was available for only two strains
(WSM3674 and WSM3686 (Yates et al., 2007)), a glasshouse trial was set up to
determine the relative effectiveness of all L. angolensis strains on this host plant. The
seven non-pigmented strains isolated from other Lotononis s. l. species had been
Chapter 2
52
authenticated as rhizobia, but in most cases it was not possible to authenticate them
on their original hosts, due to a lack of available seed or to an inability to identify the
host plant to species level. It was therefore important to confirm that these strains
were able to nodulate Lotononis s. l. hosts. This was undertaken in a subsequent
glasshouse trial that evaluated the ability of the strains to nodulate a taxonomically
diverse range of Lotononis s. l. species.
The Lotononis s. l. hosts were selected similarly, according to their taxonomic
group and the availability of germplasm. Where sufficient quantities of germplasm
existed, Lotononis s. l. species were included in the large-scale nodulation and
nitrogen fixation glasshouse experiment. Other Lotononis s. l. species were used to
confirm that the non-pigmented Leobordea and Lotononis s. str. isolates were able to
nodulate Lotononis s. l. hosts. Symbiotic specificity within the genus Listia was
further examined by assessing the ability of a range of rhizobia, including type
strains, to nodulate L. angolensis, L. bainesii and L. heterophylla.
The rhizobial strains inoculated onto the Lotononis s. l. hosts were identified by
amplification and sequencing of a fragment of the 16S rRNA gene. The phylogeny of
the symbiotic genes was assessed by amplifying and sequencing a fragment of the
nodA gene. This gene was chosen as it is one of the genes that is required for
synthesis of the Nod factor and is an important determinant of host range, due to the
role of the Nod factor acyl tail in host specificity (Dénarié et al., 1996; Haukka et al.,
1998; Roche et al., 1996) (Section 1.6.1).
Chapter 2
53
2.2 Materials and Methods
2.2.1 Rhizobial strains
The Lotononis s. l. isolates used in this study were obtained from the Western
Australian Soil Microbiology (WSM) collection, housed at the Centre for Rhizobium
Studies, Murdoch University. The strains and the details of their collection sites are
listed in Table 2.1. A map of southern Africa, showing site locations, is given in
Figure 2.1. Other rhizobial strains used in this chapter are listed in Table 2.2. All
strains were stored at -80ºC as 12% (v/v) glycerol/media stocks. Strains were
routinely subcultured on modified ½ lupin agar (½ LA) (Yates et al., 2007), or on ½
LA with succinate replacing glucose and mannitol as a carbon source, or on TY agar
(Beringer, 1974). Broth cultures were grown in TY or ½ LA. Plate and broth cultures
were grown at 28ºC and shaking cultures were incubated on a gyratory shaker at 200
rpm.
2.2.2 Host plants
The Lotononis s. l. species used in the glasshouse experiments are shown in
Table 2.3, along with details of their taxonomic grouping. Seeds of L. angolensis, L.
bainesii and L. heterophylla were obtained from the Department of Agriculture
Western Australia and cultivar names or line numbers for these species are also given
in Table 2.3. Seed for all other Lotononis s. l. species was collected between 2002
and 2007 from wild plants growing at various sites in South Africa.
Chap
ter
2
54
Tab
le 2
.1. L
ist
of
Loto
nonis
s. l.
iso
late
s use
d i
n t
his
chap
ter.
Str
ain
A
ffil
iati
on
*
(Syn
on
ym
)
Ho
st
Lo
ton
on
is
s.
l. s
ec
tio
n
Ge
og
ra
ph
ica
l
so
urc
e (
Co
lle
cto
r)
Sit
e
No
.
Sit
e
pH
La
titu
de
Lo
ng
itu
de
GH
Ex
pe
rim
en
t R
efe
re
nc
e
1
2
3
4
Pin
k-p
igm
en
ted
me
thylo
ba
cte
ria
WS
M2598
Meth
ylo
bacte
rium
Lis
tia
bain
esii
Lis
tia
South
Afr
ica
(Yate
s,
Real &
Law
)
35
6.0
S
29 0
1 5
40
E29 3
6 4
91
* *
Yate
s e
t al. (
2007)
No
n-p
igm
en
ted
Lo
ton
on
is s
. l.
str
ain
s
WS
M2596
Bra
dyrh
izobiu
m
Leobord
ea
folio
sa
Lip
ozygis
S
outh
Afr
ica
(Yate
s,
Real &
Law
)
34
5.5
S
29 0
4 4
14
E29 3
6 4
91
*
*
Ard
ley (
2005)
WS
M2624
Mesorh
izobiu
m
Loto
nonis
s. l. s
p.
ND
S
outh
Afr
ica
(Yate
s,
Real &
Law
)
20
7.0
S
32 1
4 0
82
E22 5
5 0
81
*
*
Ard
ley (
2005)
WS
M2632
Bra
dyrh
izobiu
m
Loto
nonis
s. l. s
p.
ND
S
outh
Afr
ica
(Yate
s,
Real &
Law
)
41
5.5
S
28 3
8 9
02
E31 5
5 7
93
*
*
Ard
ley (
2005)
WS
M2653
Ensifer
Loto
nonis
s. l. s
p.
ND
S
outh
Afr
ica
(Yate
s,
Real &
Law
)
22
8.0
S
32 2
1 1
09
E22 3
6 1
27
*
*
Ard
ley (
2005)
WS
M2667
Meth
ylo
bacte
rium
Leobord
ea
caly
cin
a
Leptis
South
Afr
ica
(Yate
s,
Real &
Law
)
53
6.0
S
25 3
5 4
32
E28 2
2 6
68
*
*
Ard
ley (
2005)
WS
M2783
Bra
dyrh
izobiu
m
Loto
nonis
s. l. s
p.
ND
S
outh
Afr
ica
(Yate
s,
Real &
Law
)
49
6.5
S
27 1
2 9
79
E31 1
0 6
35
*
*
Ard
ley (
2005)
WS
M3040
Ensifer
Loto
nonis
s. str
. la
xa
Oxydiu
m
South
Afr
ica
(Yate
s,
Real &
Law
)
14
6.5
S
32 1
1 0
75
E23 5
5 6
31
*
*
Ard
ley (
2005)
Lis
tia
an
go
len
sis
str
ain
s
WS
M3557
Mic
rovirga†
(CB
1322)
Lis
tia
angole
nsis
Lis
tia
Chip
ata
‡, Z
am
bia
(Verb
oom
)
N
D
S 1
3.6
5
E 3
2.6
3
*
* *
Eagle
s &
Date
(1999)
WS
M3673
(CB
1321)
Lis
tia
angole
nsis
Lis
tia
Chip
ata
, Z
am
bia
(Verb
oom
)
N
D
S 1
3.6
5
E 3
2.6
3
*
Eagle
s &
Date
(1999)
WS
M3674
Mic
rovirga†
(CB
1323)
Lis
tia
angole
nsis
Lis
tia
Chip
ata
, Z
am
bia
(Verb
oom
)
N
D
S 1
3.6
5
E 3
2.6
3
*
Eagle
s &
Date
(1999)
Yate
s e
t al. (
2007)
WS
M3675
(CB
2406)
Lis
tia
angole
nsis
Lis
tia
Mbale
, Z
am
bia
(Verb
oom
)
N
D
S 0
8.5
1
E 3
1.2
0
*
Eagle
s &
Date
(1999)
WS
M3686
Mic
rovirga†
(CB
1297)
Lis
tia
angole
nsis
Lis
tia
Chip
ata
, Z
am
bia
(Verb
oom
)
N
D
S 1
3.6
5
E 3
2.6
3
*
Eagle
s &
Date
(1999)
Yate
s e
t al. (
2007)
WS
M3692
(CB
1299)
Lis
tia
angole
nsis
Lis
tia
Chip
ata
, Z
am
bia
(Verb
oom
)
N
D
S 1
3.6
5
E 3
2.6
3
*
Eagle
s &
Date
(1999)
WS
M3693
Mic
rovirga†
(CB
1298)
Lis
tia
angole
nsis
Lis
tia
Chip
ata
, Z
am
bia
(Verb
oom
)
N
D
S 1
3.6
5
E 3
2.6
3
*
Eagle
s &
Date
(1999)
* A
s det
erm
ined
by A
rdle
y (
2005).
† D
eter
min
ed f
rom
this
stu
dy (
see
also
Chap
ter
4).
‡ F
orm
erly
know
n a
s F
ort
Jam
eson. N
D =
not
det
erm
ined
.
Chapter 2
55
Figure 2.1. Geographical locations of southern African sites from which Lotononis s. l.
strains were collected. Map image taken from:
http://www.eecrg.uib.no/projects/AGS_BotanyExp/Drakensberg/Pictures/Biomes.jpg
2.2.3 Glasshouse experimental design
Four glasshouse experiments were conducted in this study:
Experiment 1 was an evaluation of the symbiotic abilities of the seven Listia
angolensis strains on their L. angolensis host, with the aim of selecting the most
effective strain for inclusion in Experiment 3. Each treatment consisted of three
replicate pots, initially sown with six plants and thinned to four plants when the
seedlings were three weeks old. Uninoculated nitrogen-free and supplied nitrogen
(N+) controls were included.
RNB strains
WSM2783
WSM2632
WSM2596
WSM2598 WSM3040
WSM2624
WSM2653
WSM2667
L. angolensis strains
RNB strains
WSM2783
WSM2632
WSM2596
WSM2598 WSM3040
WSM2624
WSM2653
WSM2667
L. angolensis strains
Chapter 2
56
Table 2.2. List of rhizobial strains used to inoculate Listia angolensis, Listia bainesii and
Listia heterophylla (Experiment 4 in this chapter).
Strain Identification Original
host
Geographical
origin
Reference/source
ORS 571T Azorhizobium
caulinodans Sesbania rostrata
Senegal Dreyfus et al. (1988)
USDA 76T Bradyrhizobium
elkanii Glycine max USA Kuykendall et al.
(1992)
USDA 6T Bradyrhizobium
japonicum Glycine max USA Jordan (1982)
2281T Bradyrhizobium
liaongense Glycine max China Xu et al. (1995)
WSM3937 Burkholderia sp. Rhynchosia ferulifolia
South Africa Garau et al. (2009)
WSM4187 Burkholderia sp. Lebeckia ambigua
South Africa J. Howieson
(Unpublished data)
TTR 38T
Ensifer arboris Prosopis chilensis
Sudan Nick et al. (1999)
NGR234 Ensifer fredii Lablab purpureus
New Guinea Trinick (1980)
WSM419 Ensifer medicae Medicago murex
Sardinia Howieson & Ewing
(1986)
Sm1021 Ensifer meliloti Medicago sativa
Australia* Meade et al. (1982)
ORS 609T Ensifer saheli Sesbania
cannabina Senegal De Lajudie et al.
(1994)
ORS 1009T Ensifer terangae Acacia laeta Senegal De Lajudie et al.
(1994)
ORS 2060T Methylobacterium
nodulans Crotalaria podocarpa
Senegal Sy et al. (2001)
Lut6 Microvirga lupini †
Lupinus texensis
USA Andam & Parker (2007)
ORS 992T Rhizobium
(Allorhizobium) undicola
Neptunia natans
Senegal De Lajudie et al. (1998b)
Control strains
WSM2598 Methylobacterium sp.
Listia bainesii
South Africa Yates et al. (2007)
WSM3557 Microvirga lotononidis †
Listia angolensis
Zambia This study
T = Type strain
* StreptomycinR derivative of the introduced wild-type; see Terpolilli (2009)
† Determined from this study (see also Chapter 4)
Chapter 2
57
Table 2.3. List of host plants in this chapter and experiments in which they are used.
Accession numbers or cultivars, where relevant, are given in brackets.
Genus
(Lotononis s. l. section)†
Species
(Accession No. /cultivar)
Glasshouse Experiment
1 2 3 4
Listia Listia angolensis (8363) * * *
Listia bainesii (Miles) * *
Listia heterophylla
(2004 CRSL69) *
Leobordea
(Digitata) Leobordea longiflora *
Leobordea
(Leptis) Leobordea mollis *
Leobordea
(Leobordea)
Leobordea platycarpa *
Leobordea stipulosa *
Leobordea
(Synclistus)
Leobordea bolusii *
Leobordea polycephala *
Lotononis s. str.
(Oxydium)
Lotononis crumanina *
Lotononis delicata *
Lotononis falcata *
Lotononis laxa *
Lotononis s. str.
(Cleistogama) Lotononis pungens *
Euchlora Euchlora hirsuta *
†Based on the taxonomy of van Wyk (1991) and Boatwright et al. (2011).
Experiment 2 authenticated the seven strains isolated from Leobordea and
Lotononis s. str. species as able to nodulate Lotononis s. l. species hosts, as a
prerequisite for their inclusion in Experiment 3. Because of the scarcity of
germplasm, only one to four plants of each species were inoculated separately with
each strain. Plants were assessed for nodulation only, which was deemed to be
effective if the plants were green and the nodules pink in colour; an uninoculated
nitrogen-free control was included.
Experiment 3 investigated the symbiotic interactions of strains of diverse
Lotononis s. l. rhizobia inoculated separately onto eight Lotononis s. l. species from a
range of taxonomic groups. As in Experiment 1, nodulation and nitrogen-fixing
Chapter 2
58
capabilities were determined using three replicate pots per treatment. Each pot was
sown with up to six germinated seeds (depending on seed availability) and thinned to
four seedlings three weeks after planting, or was planted with four cuttings.
Uninoculated nitrogen-free and supplied nitrogen (N+) controls were included in the
treatments. The exception to this experimental set-up was Leobordea stipulosa,
where insufficient seedlings germinated to allow statistically valid replicates.
Experiment 4 was used to evaluate symbiotic specificity within the genus
Listia, especially in regard to the specificity of Listia angolensis as compared with
other species in this section. Seedlings of L. angolensis, Listia bainesii and Listia
heterophylla were grown in closed screw-topped polycarbonate vials (500 ml) and
inoculated separately with the rhizobia shown in Table 2.2. Treatments were
duplicated and an uninoculated control was used. Treatments were repeated, using a
pot system rather than vials, for strains that formed nodules or nodule-like structures
on the host.
2.2.4 General glasshouse procedures
All plants, in both pots and vials, were grown in the axenic sand culture system
described in Howieson et al. (1995) and in Yates et al. (2004). Briefly, free-draining
pots were lined with absorbent paper, filled with a 3:2 mix of yellow sand and
washed river sand, moistened, and then sterilised by steam treatment or autoclaving.
Each pot was flushed twice with hot, sterile, deionised (DI) water to remove
inorganic nitrogen. Sterile polyvinyl chloride tubes (25 mm diameter) with lids were
inserted into the sand mix for supply of water and nutrients. Post-inoculation, the soil
surface was covered with sterile alkathene beads (Figure 2.2a). The closed vial
Chapter 2
59
system consisted of mixed sand medium (400g) and DI H2O (50 ml) and was
sterilised by autoclaving (Figure 2.2b).
Figure 2.2. Axenic sand culture system used in glasshouse experiments: a) pots and b)
screw-topped vials. Scale bar = 4 cm.
All experiments were conducted in a naturally lit, controlled temperature
(maximum 24°C) glasshouse. The pots were watered as required with sterile DI
water. Sterile nitrogen-free nutrient solution (20 ml) (Howieson et al., 1995) was
supplied weekly to each pot. All vials had a single dose of nutrient solution (20 ml)
added at the time of planting. Nitrogen-fed (N+) controls received 5 ml of 0.1 M
KNO3 per pot weekly.
2.2.4.1 Preparation of plant material
Seeds were lightly scarified, then surface sterilised by immersion in ethanol
(70% (v/v); 60 s) transferred to hypochlorite (4% (w/v); two min), then rinsed in six
changes of sterile DI water. The seeds were germinated in the dark at room
temperature on water agar (1.0%) plates and aseptically sown into the pots or vials
when the radicles were 1 – 3 mm in length. For two Leobordea species, L. bolusii
Chapter 2
60
and L. polycephala, plant germplasm was obtained by taking tip cuttings from pot-
grown plants maintained in the glasshouse. The cuttings (approximately 6 cm long)
were stripped of their lower leaves and placed in DI water, then transferred to
hypochlorite (1% (w/v); one min) and rinsed twice in sterile DI water. The cut end
was then dipped in striking powder (active ingredient 4-indol-3-yl butyric acid
(0.3%)) and aseptically planted. Pots with cuttings were supplied as required with
sterile DI water for the first three weeks, and then given a single dose (20 ml) of
nutrient solution containing 0.25 g l-1
KNO3. The rooted cuttings were subsequently
supplied with sterile DI water and weekly doses of sterile nitrogen-free nutrient
solution (20 ml). To maintain sterility, all pots were covered with plastic wrap until
inoculation. Vials were sown with four germinated seeds per vial.
2.2.4.2 Preparation of inoculum
For Experiments 1, 2 and 3, inoculant strains were aseptically washed off agar
plates with approximately 60 ml sterile sucrose solution (1% (w/v)) and resuspended
in a sterile 100 ml screw-topped container. Plants were inoculated with 1 ml of this
solution delivered by a sterile syringe to each seedling or cutting. Inocula for
Experiment 4 were prepared by resuspending five loopfuls of plate culture in 10 ml
of sucrose solution. Inocula contained approximately 3.0 x 107 – 1.0 x 10
9 live cells
per ml, determined from colony counts of media plates spread with a series of
inocula dilutions. Pots and vials with germinated seeds were inoculated after the
seedlings had emerged. This was from seven to ten days after sowing, due to the
variable growth rates of the different Lotononis s. l. species. Cuttings were inoculated
six weeks after planting. To avoid cross-contamination during inoculation, all plants
except for Euchlora hirsuta (Experiment 2) were grouped by rhizobial strain. As
only one E. hirsuta seed germinated, this seedling was inoculated with 1 ml of a mix
Chapter 2
61
of all rhizobial strains, prepared by resuspending two loopfuls of each plate culture
into sucrose solution (10 ml).
2.2.4.3 Harvesting
Plants were harvested eight weeks post-inoculation for Experiment 1 and ten
weeks post-inoculation for Experiments 2 and 3. Plants in Experiment 4 were
harvested six weeks post-inoculation. For quantification of nitrogen fixation, the
aboveground biomass was excised and dried at 60°C, then weighed. Nodules were
assessed for colour, morphology, number and distribution on the root system. Where
nodulation was scored, a scale of 1-10 was applied, using the system given in Fettell
et al. (1997). Selected nodules were excised and fixed overnight at 4°C in 3% (v/v)
glutaraldehyde in 25 mM phosphate buffer (pH 7.0) in preparation for nodule
sectioning. Fixed material was washed in phosphate buffer, then dehydrated in a
series of acetone solutions and infiltrated with Spurr’s resin for sectioning. Sections
were stained with 1% (w/v) methylene blue and 1% (w/v) azur II and examined
under an Olympus BX51 photomicroscope.
2.2.5 Statistics
General analyses of variance using a 5% least significant difference (LSD)
were calculated on the data sets using GenStat 12® (Release 12.1, Lawes
Agricultural Trust, Rothamsted Experimental Station).
2.2.6 Molecular fingerprinting
Rhizobia were re-isolated from nodules and identified, where required, by PCR
fingerprinting with ERIC primers (Versalovic et al., 1991) (Table 2.4).
Chapter 2
62
Table 2.4. Primers used in this study.
Primer Sequence Reference
ERIC
ERIC 1R 5'- ATGTAAGCTCCTGGGGATTCAC -3' Versalovic et al. (1991)
ERIC 2F 5'- AAGTAAGTGACTGGGGTGAGCG -3' Versalovic et al. (1991)
16S rRNA
FGPS6 5'- GGAGAGTTAGATCTTGGCTCAGa -3' Normand et al. (1992)
FGPS1509 5'- AAGGAGGGGATCCAGCCGCA -3' Normand et al. (1992)
420F 5’- GATGAAGGCCTTAGGGTTGT -3’ Yanagi & Yamasato (1993)
800F 5'- GTAGTCCACGCCGTAAACGA -3' Yanagi & Yamasato (1993)
1100F 5'- AAGTCCCGCAACGAGCGCAA -3' Yanagi & Yamasato (1993)
520R 5'- GCGGCTGCTGGCACGAAGTT -3' Yanagi & Yamasato (1993)
920R 5'- CCCCGTCAATTCCTTTGAGT -3' Yanagi & Yamasato (1993)
1190R 5'- GACGTCATCCCCACCTTCCT -3' Yanagi & Yamasato (1993)
16S-1924r 5′- GGCACGAAGTTAGCCGGGGC -3′ Sy et al. (2001)
16S-1080r 5′- GGGACTTAACCCAACATCT -3′ Sy et al. (2001)
nodA
M4-46 nodD 89-109fb
5'- ATTGCGGGCTGGCTTAGGTTG -3' This study
M4-46 nodB 29-48r2b
5'- CGCGCCAATGACACAGAACG -3' This study
nodA-1 5’- TGCRGTGGAARNTRNNCTGGGAAA -3’ Haukka et al. (1998)
nodA-2 5'- GGNCCGTCRTCRAAWGTCARGTA -3' Haukka et al. (1998) a Symbols: A, C, G, T – standard nucleotides; R – A, G; W – A, T; N – all.
b The position of the primer in the corresponding sequence of the Methylobacterium sp. 4-46
target gene.
The ERIC primers were also used in PCR fingerprinting to confirm that the
Listia angolensis isolates were separate strains, rather than clones. DNA template
was prepared from whole cells, using fresh plate culture resuspended in 0.89% (w/v)
NaCl to an OD600 of 6.0. Each PCR reaction was set up according to Table 2.5 and
each set of reactions included a negative control, in which DNA template was
replaced with an equal volume of sterile PCR-grade water. The thermal cycling
conditions are shown in Table 2.6. The PCR was performed on an iCycler (Biorad).
Amplification products were visualized by agarose gel electrophoresis, using a 1.5%
(w/v) agarose in TAE gel, which contained 0.01% (v/v) SYBR® Safe DNA gel stain
Chapter 2
63
10,000X (Invitrogen), submerged in TAE running buffer (40 mM Tris acetate, 1 mM
EDTA, pH 8.0). A 5.0 µL aliquot of 6X loading dye (Promega) was added to each
sample prior to electrophoresis and a 1kb DNA ladder (Promega) was used as a
marker. The gels were run at 80V for 3.5 h, then visualised under UV light using the
BIORAD Gel Doc 2000 system.
Table 2.5. Reaction components for ERIC PCR amplification.
Component Volume (µl)
5 X Gitschier buffer 5.0
Bovine serum albumin (BSA) (20 mg/ml) 0.4
Dimethyl sulfoxide (DMSO) 2.5
Sterile PCR-grade H2O (Fisher Biotech) 12.35
dNTPs (25 mM) 1.25
ERIC 1R (50 µM) 1.0
ERIC 2F (50 µM) 1.0
Taq DNA polymerase (Invitrogen) 0.2
DNA template (whole cells, OD600nm = 6.0) 1.0
25 0
Table 2.6. Thermal cycling conditions for ERIC PCR amplification.
Temperature (ºC) Time No. of cycles
94 7 min 1
94 30 s
50 1 min 35
65 8 min
65 16 min 1
14 ∞ 1
2.2.7 Amplification and sequencing of 16S rRNA genes
Nearly full length PCR amplification of the 16S rRNA gene was performed
using the universal eubacterial primers FGPS6 and FGPS1509 (Nesme et al., 1995;
Normand et al., 1992) (Table 2.4). DNA template was prepared as for ERIC PCRs,
Chapter 2
64
but with an OD600nm of 2.0. The optimised PCR reaction and thermal cycling
conditions are shown in Table 2.7 and Table 2.8 respectively. The PCR amplicons
were purified directly, using a QIAquickTM
PCR Purification Kit (Qiagen), or with a
QIAquickTM
Gel Extraction Kit (Qiagen) following electrophoresis in a 1% (w/v)
agarose gel and excision of the amplified gene products.
Table 2.7. Reaction components for PCR amplification of the 16S rRNA gene.
Component Volume (µl)
5x PCR Polymerisation Buffer (Fisher Biotech) 5.0
MgCl2 (15 mM) 2.5
Sterile PCR-grade H2O (Fisher Biotech) 15.3
Forward primer FGPS6 (50 µM) 0.5
Reverse primer FGPS1509 (50 µM) 0.5
Taq DNA polymerase (Invitrogen) 0.2
DNA template (whole cells, OD600nm = 2.0) 1.0
25 0
Table 2.8. Thermal cycling conditions for 16S rDNA PCR amplification.
Temperature (ºC) Time No. of cycles
94 5 min 1
94 30 s
55 30 s 35
70 1 min
72 7 min 1
14 ∞ 1
Sequencing PCRs were performed on purified 16S rDNA in a 96-well
microplate, using FGPS6 and FGPS1509 and internal primers designed by Yanagi &
Yamasato (1993) and Sy et al. (2001) (Table 2.4) and the BigDye Terminator 3.1
mix (Applied Biosystems). The sequencing PCR reaction and the thermal cycling
parameters are shown in Table 2.9 and Table 2.10, respectively. Sequencing
Chapter 2
65
reactions were purified by an ethanol/EDTA and sodium acetate precipitation, as
recommended by Applied Biosystems. Sequence reads were obtained from the ABI
model 377A automated sequencer (Applied Biosystems). Sequences were manually
edited and aligned using Genetool Lite (version 1.0; Double Twist Inc., Oakland,
CA, USA). Searches for sequences with high sequence similarity to the sample 16S
rDNA were conducted using BLASTN (Altschul et al., 1990) against sequences
deposited in the National Centre for Biotechnology Information GenBank database.
A phylogenetic tree was constructed with MEGA version 4.0 (Tamura et al., 2007),
using the neighbour-joining method (Saitou & Nei, 1987) and the Maximum
Composite Likelihood model, and bootstrapped with 1000 replicates. For
comparison, trees were also constructed using the minimum evolution (ME) and
maximum parsimony (MP) methods.
Table 2.9. Reaction components for PCR sequencing of 16S rDNA.
Component Volume (µl)
Purified DNA template (10-40 ng) 4
BigDye Terminator 3.1 mix 4
Sterile PCR-grade H2O (Fisher Biotech) 1
Primer (10 µM) 1
10
Table 2.10. Thermal cycling conditions for 16S rDNA sequencing PCR.
Temperature (ºC) Time No. of cycles
96 2 min 1
96 10 s
50 5 s 25
60 4 min
14 ∞ 1
Chapter 2
66
2.2.8 Amplification and sequencing of nodA genes
Amplification of the nodA gene of Lotononis s. l. rhizobia was initially
attempted by designing primers based on conserved regions of the nodD and nodB
genes in the Listia bainesii microsymbiont Methylobacterium sp. 4-46. The
sequenced genome of this strain is available on the USA Joint Genome Institute’s
Microbial Genomics Program database (http://genome.jgi-psf.org/programs/bacteria-
archaea/index.jsf). The Methylobacterium sp. 4-46 nodD and nodB gene sequences
were aligned and compared with those of other rhizobia that displayed high sequence
identity to the target genes, based on searches conducted using BLASTN (Altschul et
al., 1990) against sequences deposited in GenBank (Appendix I).
Four primers were designed from these alignments. Combinations of primers
were tested: the primer set, PCR reaction components and cycling conditions that
gave optimum results are shown in Table 2.4, Table 2.11 and Table 2.12
respectively. Forward primer M4-46 nodD 89-109f starts from the 5’ end of nodD
(codon 4271507 in the Methylobacterium sp. 4-46 genome sequence) and reverse
primer M4-46 nodB 29-48r2 ends in the 5’ end of nodB (codon 4270473). They
amplify a product of approximately 1000 bp. As these primers yielded appropriate
products with only two of the rhizobial strains, the nodA primers developed by
Haukka et al. (1998) were also trialled (Table 2.4). The optimised cycling conditions
for these primers were modified slightly from the original conditions cited by
Haukka et al. (1998) and are shown in Table 2.13. Purification and sequencing of the
nodA PCR amplicons was as described for the 16S rRNA gene, but with the use of
the requisite nodA primers.
Chapter 2
67
Table 2.11. Reaction components for PCR amplification of nodA.
Component Volume (µl)
5x PCR Polymerisation Buffer (Fisher Biotech) 5
Mg Cl2 (10 mM) 2.5
Sterile PCR-grade H2O (Fisher Biotech) 15.3
Forward primer (50 µM) 0.5
Reverse primer (50 µM) 0.5
Taq DNA polymerase (Invitrogen) 0.2
DNA template (whole cells, OD600nm = 2.0) 1.0
25 0
Table 2.12. Thermal cycling conditions for nodA PCR amplification, using primers based on
the Methylobacterium sp. 4-46 sequenced genome.
Temperature (ºC) Time No. of cycles
94 4 min 1
94 30 s
52 30 s 35
70 30 s
72 5 min 1
14 ∞ 1
Table 2.13. Thermal cycling conditions for nodA PCR amplification, using primers
developed by Haukka et al. (1998).
Temperature (ºC) Time No. of cycles
94 4 min 1
94 45 s
55 45 s 35
68 2 min
70 5 min 1
14 ∞ 1
BLASTN and BLASTX (Altschul et al., 1990) were used to conduct searches
for sequences with high sequence similarity to the sample nodA sequences against
sequences deposited in the National Centre for Biotechnology Information GenBank
database. A protein-coding phylogenetic tree was constructed with MEGA version
Chapter 2
68
4.0 (Tamura et al., 2007), using the neighbour-joining method (Saitou & Nei, 1987)
and the Maximum Composite Likelihood model, and bootstrapped with 1000
replicates. Trees were also constructed using the minimum evolution and maximum
parsimony methods.
2.3 Results
2.3.1 Selection of inoculant strains
Glasshouse trials were used to select the most symbiotically effective Listia
angolensis strain and to confirm that the strains isolated from Leobordea and
Lotononis s. str. species were able to nodulate Lotononis s. l. species.
2.3.1.1 Selection of a representative Listia angolensis strain
The symbiotic effectiveness of the seven L. angolensis strains on their original
host was assessed through visual observation of plant appearance and nodulation,
and by measurement of nodule numbers and the dry weight of plant shoots (Figure
2.3). Plants inoculated with all strains except WSM3692 were green and had
increased biomass compared with the uninoculated N-free control. Plants treated
with WSM3692 were small and pale, with biomass similar to the uninoculated
control. All plants, other than the controls, were nodulated. WSM3692 induced a
large number of nodules, but plants received a low nodulation score, as nodules were
very small and white, with most occurring on the lateral roots. Nodules induced by
the other strains were pink, larger in size and distributed more on the hypocotyl and
taproot. On the basis of these results, WSM3692 was considered to be ineffective for
nitrogen fixation on this accession of L. angolensis. Effectiveness, as measured by
the dry weight of shoots, varied in the other strains, with some being only partially
effective. WSM3557 was chosen as the most symbiotically competent strain, as it
Chapter 2
69
produced both the greatest biomass and the highest nodulation score on the L.
angolensis host. A fingerprinting PCR, using ERIC primers, confirmed that the N2-
fixing L. angolensis rhizobia were separate strains, rather than clones (Appendix II).
2.3.1.2 Authentication of non-pigmented Lotononis s. l. strains
The Leobordea and Lotononis s. str. rhizobia were assessed for their ability to
nodulate a range of Lotononis s. l. hosts (Table 2.14). All strains were authenticated
as able to nodulate at least one species of Lotononis s. l. and were therefore included
in the study of the symbiotic interactions of diverse strains of Lotononis s. l. rhizobia
with Lotononis s. l. species from a range of taxonomic groups (Experiment 3).
Interestingly, the Lotononis laxa isolate WSM3040 was unable to nodulate this host.
All host species in this experiment, with the exception of E. hirsuta, formed nodules
with at least one inoculant strain.
0
1
2
3
4
5
6
7
8
9
10
WSM3557 WSM3673 WSM3674 WSM3675 WSM3686 WSM3692 WSM3693 Uninoc N+
Inoculation treatments
Mea
n n
od
ule
s/n
od
ule
sc
ore p
er p
lan
t
0
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
Sh
oo
t d
ry w
eig
ht
(g/p
lan
t)
a
cc
b
b
b
cc
c
0
1
2
3
4
5
6
7
8
9
10
WSM3557 WSM3673 WSM3674 WSM3675 WSM3686 WSM3692 WSM3693 Uninoc N+
Inoculation treatments
Mea
n n
od
ule
s/n
od
ule
sc
ore p
er p
lan
t
0
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
Sh
oo
t d
ry w
eig
ht
(g/p
lan
t)
a
cc
b
b
b
cc
c
Figure 2.3. Symbiotic ability of Listia angolensis strains on L. angolensis,
assessed by nodule number ( ), nodulation score ( ) and dry weight of
shoots ( ) of plants harvested after eight weeks growth. For shoot dry
weight, treatments which share a letter are not significantly different
according to Fisher’s LSD test (P<0.05).
Figure 2.3. Symbiotic ability of Listia angolensis strains on L. angolensis,
assessed by nodule number ( ), nodulation score ( ) and dry weight of
shoots ( ) of plants harvested after eight weeks growth. For shoot dry
weight, treatments which share a letter are not significantly different
according to Fisher’s LSD test (P<0.05).
Chap
ter
2
70
Tab
le 2
.14.
Abil
ity o
f non-p
igm
ente
d s
trai
ns
isola
ted f
rom
Leobord
ea a
nd L
oto
nonis
s.
str.
spec
ies
to n
odula
te a
ran
ge
of
Loto
nonis
s.
l. a
nd E
uchlo
ra h
ost
s.
Nodula
tion i
s in
bold
type.
Iso
late
O
rig
inal h
ost
N
od
ula
tio
n o
f L
oto
no
nis
s. l. (
secti
on
) h
ost
Leo
bo
rd
ea
mo
llis
(Lep
tis)
Leo
bo
rd
ea
po
lycep
hala
(Syn
cli
stu
s)
Leo
bo
rd
ea
pu
ng
en
s
(Cle
isto
gam
a)
Lo
ton
on
is
delicata
(Oxyd
ium
)
Lo
ton
on
is
laxa
(Oxyd
ium
)
Eu
ch
lora
hir
su
ta†
(Eu
ch
lora)
Brad
yrh
izo
biu
m
WS
M2596
Leob
ord
ea fo
liosa
(Lip
ozyg
is)
N-
N+
F+
N
- N
+F
- N
- N
-
WS
M2632
Loto
non
is s
. l. s
p.
ND
N
+F
+
N+
F-
N+
F-
ND
N
-
WS
M2783
Loto
non
is s
. l. s
p.
N-
N+
F+
N
+F
- N
+F
+
N-
N-
En
sif
er
WS
M2653
Loto
non
is s
. l. s
p.
N+
F-
N-
N+
F+
N
- N
+F
- N
-
WS
M3040
Loto
non
is laxa
(Oxyd
ium
)
N+
F-
N-
N+
F+
N
- N
- N
-
Me
so
rh
izo
biu
m
WS
M2624
Loto
non
is s
. l. s
p.
N+
F-
N-
N-
N-
N-
N-
Me
thylo
bacte
riu
m
WS
M2667
Leob
ord
ea c
aly
cin
a
(Leptis)
N+
F+
N
- N
- N
+F
- N
- N
-
N+
F+
= n
odula
tion a
nd n
itro
gen
fix
atio
n;
N+
F-
= n
odula
tion, but
no n
itro
gen
fix
atio
n;
N-
= n
o n
odula
tion, N
D =
not
det
erm
ined
.
† T
he
E.
hir
suta
see
dli
ng w
as i
nocu
late
d w
ith a
mix
of
all
the
RN
B i
n t
his
Tab
le,
plu
s W
SM
2598 (
isola
ted f
rom
and e
ffec
tive
on L
isti
a b
ain
esi
i) a
nd
WS
M3557 (
isola
ted f
rom
and e
ffec
tive
on L
isti
a a
ngole
nsi
s).
Chapter 2
71
Although the nodules of the Lotononis s. l. species varied in size and shape, all
were indeterminate in structure (Figure 2.4). Molecular fingerprinting, using ERIC
primers, confirmed nodule occupancy by the inoculant strain (Appendix II).
Figure 2.4. Nodulation in Lotononis s. l. spp.. Scale bar = 5 mm.
a) ineffective nodules on Leobordea mollis inoculated with WSM2624;
b) effective nodules on Leobordea polycephala inoculated with WSM2632;
c) effective nodules on Lotononis delicata inoculated with WSM2783;
d) ineffective nodules on Lotononis laxa inoculated with WSM2653;
e) effective nodules on Leobordea pungens inoculated with WSM2653.
2.3.2 Symbiotic interactions of Lotononis s. l. species with
phylogenetically diverse Lotononis s. l. rhizobia
The symbiotic relationships between Lotononis s. l. species and the rhizobia
that nodulate them are summarised in Table 2.15. Figures for the nodulation and N2-
fixation of rhizobial strains on the individual Lotononis s. l. species are given in
Appendix III (Figures 1 - 8). A notable feature of these symbioses was the extreme
symbiotic specificity exhibited by Listia bainesii (Figure 2, Appendix III). This host
species was nodulated only by the pigmented Methylobacterium strain WSM2598.
The symbiosis was highly effective, producing plant biomass equivalent to that
Chapter 2
72
obtained for the N+ control (Figure 2, Appendix III). Listia angolensis was less
specific, as host plants consistently, or occasionally, formed ineffective nodules with
the methylobacterial strains WSM2598 and WSM2667, respectively. Effective
nodulation, however, was observed only with the novel rhizobial strain WSM3557
(Table 2.15).
The nodulation ability and effectiveness of Lotononis s.l.-associated rhizobia
on Leobordea and Lotononis s. str. species is shown in Appendix III (Figures 3 – 8)
and Table 2.15. In some species, the response of plants to the N+ treatment has been
much greater than their response to rhizobial inoculation, and in these cases a
rescaled figure that omits the N+ treatment has also been included, to reveal partial
rhizobial effectiveness. Statistical analysis has not been performed for the Leobordea
stipulosa results, as apart from the WSM2598 and WSM3557 treatments, too few
plants were available to provide valid data. No uninoculated control data are included
for this species, as the single plant that was available for this treatment died.
Many of the Leobordea and Lotononis s. str. species could be considered
promiscuous, as they were nodulated by a range of Lotononis s. l. rhizobia. There did
not appear to be any obvious taxonomically based pattern of symbiotic specificity in
these species. Leobordea platycarpa, for example, was comparatively specific, being
nodulated by only three strains (WSM2653 and WSM3040 (Ensifer spp.) and
WSM3557), yet Leobordea stipulosa, in the same taxonomic section as L. platycarpa
(Leobordea), was nodulated by all inoculants except the two Ensifer strains.
Nodulation was seldom effective.
Chap
ter
2
73
Tab
le 2
.15. S
um
mar
y o
f nodula
tion a
nd f
ixat
ion a
bil
ity o
f L
oto
nonis
s. l.
spec
ies
inocu
late
d w
ith r
hiz
obia
iso
late
d f
rom
Loto
nonis
s. l.
spec
ies.
Lo
ton
on
is s
. l.
Secti
on
*
Lo
ton
on
is s
. l.
sp
.
Str
ain
Brad
yrh
izo
biu
m
En
sif
er
Me
so
rh
izo
biu
m
Me
thylo
bacte
riu
m
Mic
ro
vir
ga
WS
M
2596
WS
M
2632
WS
M
2783
WS
M
2653
WS
M
3040
WS
M
2624
WS
M
2598
WS
M
2667
WS
M
3557
Lis
tia
Lis
tia. a
ngo
lensis
N
- N
- N
- N
- N
- N
- N
+F
- N
+/-
F-
N+
F+
Lis
tia b
ain
esii
N-
N-
N-
N-
N-
N-
N+
F+
N
- N
-
Dig
itata
Leob
ord
ea
lon
giflo
ra
N+
/-F
- N
+/-
F-
N+
F-
N+
/-F
- N
- N
+/-
F-
N+
F-
N+
F-
N+
F-
Leo
bo
rd
ea
Leob
ord
ea
pla
tycarp
a
N-
N-
N-
N+
F+
N
+F
- N
- N
- N
- N
+F
+
Leob
ord
ea
stipu
losa
N
+F
- N
+F
- N
+F
- N
- N
- N
+F
- N
+F
- N
+F
- N
+F
-
Syn
cli
stu
s
Leob
ord
ea b
olu
sii
N+
/-F
+/-
N
+F
+
N+
F+
N
- N
+/-
F-
N+
/-F
- N
+F
- N
+/-
F-
N+
/-F
+/-
Ox
yd
ium
Loto
non
is
cru
manin
a
N+
/-F
+/-
N
+/-
F-
N+
/-F
- N
+F
+
N+
F+
N
+/-
F-
N-
N-
N+
F+
/-
Loto
non
is fa
lcata
N
+F
- N
+F
- N
+/-
F-
N-
N+
/-F
- N
- N
- N
- N
+F
-
* a
ccord
ing t
o v
an W
yk (
1991)
Chapter 2
74
Nodulation of the Leobordea and Lotononis s. str. species was seldom
effective. This was a not unexpected finding, given that none of these species was an
original host of the inoculant strains used in this experiment. Moreover, the
effectiveness was in most cases only partial as plants were green, but biomass was
much smaller than for the N+ control. There was also variation in the response of
individual plants both to inorganic nitrogen and to inoculation, as evidenced by the
large error bars seen for some treatments. This is illustrated in the case of WSM2653
on Leobordea platycarpa, where the plant response ranged from ineffective to
partially effective and effective nodulation (Figure 2.5).
Figure 2.5. Inoculant response of Leobordea platycarpa plants to WSM2653 (10 weeks
post-inoculation): a) effective nodulation;
b) ineffective nodulation;
c) partially effective nodulation
Similarly, WSM2596 on Leobordea bolusii and WSM3557 on L. platycarpa
were effective or partially effective on some plants but failed to nodulate others. All
inoculant strains were able to nodulate at least two Lotononis s. l. species (Table
2.15). The strain with the broadest host range was the Listia angolensis isolate
Chapter 2
75
WSM3557, which was able to nodulate all Lotononis s. l. species except for Listia
bainesii and was partially effective on Leobordea bolusii, Leobordea platycarpa and
Lotononis crumanina. Nodule occupancy was confirmed in most cases, as inoculant
strains were successfully reisolated from harvested nodules. Reisolates were not
obtained from nodules of Leobordea stipulosa inoculated with WSM2596,
WSM2624, WSM2632 and WSM2783 or Lotononis falcata inoculated with
WSM2632 or WSM3557. Additionally, reisolates were not obtained from nodules of
Leobordea longiflora and Lotononis crumanina inoculated with WSM2624.
2.3.3 Symbiotic specificity within Listia species
The symbiotic specificity of L. angolensis, L. bainesii and L. heterophylla was
evaluated by inoculating seedlings of these species with rhizobia of a diversity of
genera and species (Table 2.2). L. bainesii and L. heterophylla were nodulated only
by the pink-pigmented methylobacterial strain WSM2598, and nodulation was
always effective. L. angolensis was slightly less specific, being nodulated effectively
only by WSM3557, but forming ineffective nodules consistently with WSM2598 and
occasionally with Methylobacterium nodulans strain ORS 2060.
2.3.4 Nodule morphology in Lotononis s. l. species
Nodule morphology clearly differentiated Listia species from the other
Lotononis s. l. taxa. Both Listia angolensis and Listia bainesii formed lupinoid
nodules, primarily on the hypocotyl and taproot (Figure 2.6 a and b), whereas
nodules of Leobordea and Lotononis s. str. species were indeterminate and
distributed throughout the root system (Figure 2.6 c – h).
Chapter 2
76
Figure 2. 6. Nodule morphology in Lotononis s. l. spp. (inoculant strain in brackets). Scale
bar is 10 mm, except for 2.15g and inserts.
a) Listia angolensis (WSM3557); b) Listia bainesii (WSM2598); c) Leobordea longiflora
(WSM2598); d) Leobordea bolusii (WSM2783) e) Lotononis crumanina (WSM2596); f)
Lotononis falcata (WSM3557) (scale bar in insert = 2 mm; g) Leobordea platycarpa
(WSM2653) (scale bar = 5 mm; scale bar in insert = 3 mm); h) Leobordea stipulosa
(WSM2632) (scale bar in insert = 2 mm).
Chapter 2
77
2.3.5 Amplification and sequencing of 16S rRNA genes
The 16S rRNA gene of the pink-pigmented Methylobacterium strain
WSM2598 had previously been sequenced (Yates et al., 2007). Nearly full-length
portions of the 16S rRNA gene were amplified and sequenced for the remaining
eight Lotononis s. l. strains.
BLASTN sequence identity searches confirmed that the Lotononis s. l. strains
were phylogenetically diverse. Strains WSM2596, WSM2632 and WSM2783 were
most closely related to Bradyrhizobium, WSM2653 and WSM3040 were identified
as Ensifer strains, WSM2624 was related to Mesorhizobium and WSM2667 was
most closely related to Methylobacterium nodulans. The Listia angolensis strain
WSM3557 was not related to any currently described rhizobial genus, but instead
grouped with the other L. angolensis strains WSM3674 and WSM3686, and with
root nodule bacteria isolated from Lupinus texensis (Andam & Parker, 2007). The
highest sequence identity to a validly named microbial species was to Microvirga
(formerly Balneimonas) flocculans (Takeda et al., 2004; Weon et al., 2010).
Analysis of a 1381 bp segment of the 16S rRNA gene from the bradyrhizobial
strains showed that WSM2632 and WSM2783 had the same sequence and shared
99.1% sequence identity with WSM2596 (Table 2.16). The sequence identity of
WSM2596 with the type strains of Bradyrhizobium elkanii and Bradyrhizobium
japonicum was 99.4% and 98.8%, respectively, while WSM2632 and WSM2783 had
98.7% and 98.0% sequence identity with these type strains (Table 2.16).
Chapter 2
78
Table 2.16. Pairwise percent identity and number of nucleotide mismatches (in brackets) for
a 1381 bp fragment of the 16S rRNA gene of Lotononis s. l. Bradyrhizobium strains and type
strains of Bradyrhizobium elkanii (USDA 76T) and Bradyrhizobium japonicum (USDA 6
T).
WSM2596 WSM2632 WSM2783 USDA 76T USDA 6
T
WSM2596 99.1
(15)
99.1
(15)
99.4
(11)
98.8
(20)
WSM2632 99.1 (15)
100 (0)
98.7 (19)
98.0 (36)
WSM2783 99.1
(15)
100
(0)
98.7
(19)
98.0
(36)
USDA 76T 99.4
(11)
98.7
(19)
98.7
(19)
98.0
(36)
USDA 6T
98.8
(20)
98.0
(36)
98.0
(36)
98.0
(36)
The current phylogeny of Bradyrhizobium divides this genus into two groups.
The first group includes B. japonicum, B. canariense, B. liaoningense and B.
yuanmingense strains and the second comprises strains related to B. elkanii, B.
pachyrhizi and B. jicamae (Menna et al., 2009). The NJ phylogenetic tree grouped
WSM2596, WSM2632 and WSM2783, with high bootstrap support, in this second
clade (Figure 2. 7). Phylogenetic trees constructed using MP or ME methods gave
similar topologies. The closest relatives to WSM2596 on the basis of 16S rRNA
sequence identity were a group of strains (ApE4.8, LcCT6, aeky10 and jws91-2)
isolated from diverse desmodioid and phaseoloid wild legumes from the USA and
Japan (Parker & Kennedy, 2006; Qian et al., 2003). In contrast, WSM2632 and
WSM2783 were grouped, in a well-supported clade, with rhizobia isolated from
diverse tropical or sub-tropical wild hosts (strains Ai1a-2, Ai4.2 and Cp5-3, from
Andira inermis (Dalbergieae) and Centrosema pubescens (Phaseoleae) in Costa Rica
and Barro Colorado Island, Panama (Parker, 2003; 2004; 2008); and strain ARRI 218
(genospecies Y) from Indigofera linifolia, growing in Kakadu National Park, in
northern Australia (Lafay & Burdon, 2007).
Chapter 2
79
B. sp. ApE4.8 (DQ354611) Apios
B. sp. aeky10 (AF417051) Amphicarpaea
B. sp. jws91-2 (AF514700) Amphicarpaea
B. sp. LcCT6 (DQ354610) Lespedeza
WSM2596 Lotononis s. l.
B. sp. onl92.6 (DQ354612) Amphicarpaea
B. sp. SEMIA 621 (FJ390908) Kummerowia
B. sp. ORS204 (AF514796) Pterocarpus
B. sp. R5 (EU364711) Vigna
B. elkanii USDA 76T (U35000) Glycine
B. pachyrhizi PAC48T (AY624135) Pachyrhizus
B. jicamae PAC68T (AY624134) Pachyrhizus
B. sp. RSB6 (FJ514039) Retama
B. sp. ARRI 218 (AJ785291) Indigofera
B. sp. Cp5-3 (AY187548) Centrosema
B. sp. Ai4.2 (EF569650) Andira
WSM2632 Lotononis s. l.
WSM2783 Lotononis s. l.
B. sp. Ai1a-2 (AF514703) Andira
B. sp. RC2d (EU364716) Vigna
B. iriomotense EK05T (AB300992) Entada
B. canariense BTA-1T (AJ558025) Chamaecytisus
B. japonicum USDA 6T (U69638) Glycine
B. sp. WSM2793 (EU481826) Rhynchosia
B. liaoningense 2281T (AF208513) Glycine
B. sp. GC1d (EU364703) Vigna
B. sp. R10 (EU364714) Vigna
B. yuanmingense B071T (AF193818) Lespedeza
A. caulinodans ORS 571T (D11342)
77
90
65
60
99
93
66
96
85
87
96
89
56
53
65
0.01
B. sp. ApE4.8 (DQ354611) Apios
B. sp. aeky10 (AF417051) Amphicarpaea
B. sp. jws91-2 (AF514700) Amphicarpaea
B. sp. LcCT6 (DQ354610) Lespedeza
WSM2596 Lotononis s. l.
B. sp. onl92.6 (DQ354612) Amphicarpaea
B. sp. SEMIA 621 (FJ390908) Kummerowia
B. sp. ORS204 (AF514796) Pterocarpus
B. sp. R5 (EU364711) Vigna
B. elkanii USDA 76T (U35000) Glycine
B. pachyrhizi PAC48T (AY624135) Pachyrhizus
B. jicamae PAC68T (AY624134) Pachyrhizus
B. sp. RSB6 (FJ514039) Retama
B. sp. ARRI 218 (AJ785291) Indigofera
B. sp. Cp5-3 (AY187548) Centrosema
B. sp. Ai4.2 (EF569650) Andira
WSM2632 Lotononis s. l.
WSM2783 Lotononis s. l.
B. sp. Ai1a-2 (AF514703) Andira
B. sp. RC2d (EU364716) Vigna
B. iriomotense EK05T (AB300992) Entada
B. canariense BTA-1T (AJ558025) Chamaecytisus
B. japonicum USDA 6T (U69638) Glycine
B. sp. WSM2793 (EU481826) Rhynchosia
B. liaoningense 2281T (AF208513) Glycine
B. sp. GC1d (EU364703) Vigna
B. sp. R10 (EU364714) Vigna
B. yuanmingense B071T (AF193818) Lespedeza
A. caulinodans ORS 571T (D11342)
77
90
65
60
99
93
66
96
85
87
96
89
56
53
65
0.010.01
Figure 2.7. NJ phylogenetic tree, bootstrapped with 1000 replicates,
showing the relationships of Bradyrhizobium strains associated with
Lotononis s. l. (in bold type) with related strains and type strains of
Bradyrhizobium, based on aligned sequences of the 16S rRNA genes.
GenBank accession numbers are in brackets. Also shown is the original
host. Geographical origin is indicated if the strain is African ( ) or South
African ( ). Scale bar, 1% sequence divergence (one substitution per 100
nucleotides). A., Azorhizobium; B., Bradyrhizobium. T = type strain.
Figure 2.7. NJ phylogenetic tree, bootstrapped with 1000 replicates,
showing the relationships of Bradyrhizobium strains associated with
Lotononis s. l. (in bold type) with related strains and type strains of
Bradyrhizobium, based on aligned sequences of the 16S rRNA genes.
GenBank accession numbers are in brackets. Also shown is the original
host. Geographical origin is indicated if the strain is African ( ) or South
African ( ). Scale bar, 1% sequence divergence (one substitution per 100
nucleotides). A., Azorhizobium; B., Bradyrhizobium. T = type strain.
Chapter 2
80
The fast-growing strains WSM2653 and WSM3040 had identical (1383 bp
fragment) 16S rRNA gene sequences. The NJ phylogenetic tree grouped these strains
with rhizobia closely related to Ensifer meliloti (Figure 2. 8). They shared 100%
sequence identity with Ensifer strains isolated from Lotus species and Phaseolus
vulgaris from the Canary Islands (León-Barrios et al., 2009; Zurdo-Piñeiro et al.,
2009), Prosopis alba in northern Spain (Iglesias et al., 2007) and Canadian
Medicago sativa (Bromfield et al., 2010) (strains Lma-x, Lse-2, GVPV12, RPA13
and T15, respectively (Figure 2.8)). Strains from Tunisian Argyrolobium uniflorum
(STM4038) and Genista saharae (STM4028) (Mahdhi et al., 2007; 2008) were also
closely related. WSM2653 and WSM3040 shared sequence identities of 99.7% (5 bp
mismatches), 99.7% (4 bp mismatches), 99.7% (5 bp mismatches), and 98.5% (21 bp
mismatches) with the type strains of E. meliloti, Ensifer medicae, Ensifer numidicus
and Ensifer garamanticus, respectively. The WSM2653 and WSM3040 16S rRNA
gene sequences contained the specific primer sequence that allows differentiation of
E. meliloti from E. medicae (Garau et al., 2005).
WSM2624 was most closely related to strains of Mesorhizobium tianshanense
(Figure 2.8). A 1452 bp fragment of the 16S rRNA gene had sequence identity of
99.8% (2 bp mismatches) with the M. tianshanense type strain A-1BST, isolated from
diverse native legumes in arid north-western China (Chen et al., 1995; Jarvis et al.,
1997), and 99.6% (6 bp mismatches) with RCAN03, an effective nodulant of
chickpeas (Cicer arietinum) in northern Spain (Rivas et al., 2007). WSM2624 was
also closely related to the strains LBER1 and Ala-3 that effectively nodulate the
endemic Canary Islands legumes Lotus berthelotii (Lorite et al., 2010) and Anagyris
latifolia (Donate-Correa et al., 2007), respectively.
Chapter 2
81
E. sp. STM 4028 (EF100524) Genista
E. meliloti T15 (EU928877) Medicago
WSM3040 Lotononis s. l.
E. meliloti RPA13 (EF201802) Prosopis
E. sp. STM 4O38 (EF152477) Argyrolobium
E. meliloti Lma-x (FM178847) Lotus
E. meliloti Lse-2 (FM178848) Lotus
E. sp. GVPV12 (FJ462794) Phaseolus
WSM2653 Lotononis s. l.
E. meliloti WSM1022 (EU885227) Medicago
E. meliloti WSM1701 (AF533684) Lotus
E. meliloti IAM 12611T (D14509) Medicago
E. medicae A 321T (L39882) Medicago
E. numidicus ORS 1407T (AY500254) Argyrolobium
E. arboris HAMBI 1552T (Z78204) Prosopis
E. garamanticus ORS 1400T (AY500255) Argyrolobium
E. sp. STM 4015 (EF100514) Genista
E. kummerowiae CCBAU 71714T (AY034028) Kummerowia
E. fredii LMG 6217T (X67231)Glycine
E. saheli LMG 7837T (X68390) Sesbania
E. kostiensis LMG 19227T (AM181748) Acacia
E. terangae LMG 7834T (X68388) Acacia
E. adhaerens LMG 20216T (AM181733)
E. adhaerens LMG 9954 (AM181735) Leucaena
M. alhagi CCNWXJ12-2T (EU169578) Alhagi
M. loti ATCC 33669T (D14514) Lotus
M. ciceri UPM-Ca7T (U07934) Cicer
M. albiziae CCBAU 61158T (DQ100066) Alb izia
M. amorphae SEMIA 806 (FJ025125) Lotus
M. amorphae ACCC 19665T (AF041442) Amorpha
M. huakuii IFO 15243T (D13431) Astragalus
M. sp. AC39e1 (GQ847892) Acacia
M. plurifarium LMG 11892T (Y14158) Acacia
M. mediterraneum UPM-Ca36T (AM181745) Cicer
M. sp. LBER1 (FN563430) Lotus
M. sp. Ala-3 (AM491621) Anagyris
M. tianshanense A-1BST (AF041447) Glycyrrhiza
WSM2624 Lotononis s. l.
M. tianshanense RCAN03 (AY195846) Cicer
A. caulinodans ORS 571T (D11342) Sesbania
98
99
69
97
63
98
72
77
58
68
45
82
97
99
100
52
67
100
60
79
62
61
59
0.01
E. sp. STM 4028 (EF100524) Genista
E. meliloti T15 (EU928877) Medicago
WSM3040 Lotononis s. l.
E. meliloti RPA13 (EF201802) Prosopis
E. sp. STM 4O38 (EF152477) Argyrolobium
E. meliloti Lma-x (FM178847) Lotus
E. meliloti Lse-2 (FM178848) Lotus
E. sp. GVPV12 (FJ462794) Phaseolus
WSM2653 Lotononis s. l.
E. meliloti WSM1022 (EU885227) Medicago
E. meliloti WSM1701 (AF533684) Lotus
E. meliloti IAM 12611T (D14509) Medicago
E. medicae A 321T (L39882) Medicago
E. numidicus ORS 1407T (AY500254) Argyrolobium
E. arboris HAMBI 1552T (Z78204) Prosopis
E. garamanticus ORS 1400T (AY500255) Argyrolobium
E. sp. STM 4015 (EF100514) Genista
E. kummerowiae CCBAU 71714T (AY034028) Kummerowia
E. fredii LMG 6217T (X67231)Glycine
E. saheli LMG 7837T (X68390) Sesbania
E. kostiensis LMG 19227T (AM181748) Acacia
E. terangae LMG 7834T (X68388) Acacia
E. adhaerens LMG 20216T (AM181733)
E. adhaerens LMG 9954 (AM181735) Leucaena
M. alhagi CCNWXJ12-2T (EU169578) Alhagi
M. loti ATCC 33669T (D14514) Lotus
M. ciceri UPM-Ca7T (U07934) Cicer
M. albiziae CCBAU 61158T (DQ100066) Alb izia
M. amorphae SEMIA 806 (FJ025125) Lotus
M. amorphae ACCC 19665T (AF041442) Amorpha
M. huakuii IFO 15243T (D13431) Astragalus
M. sp. AC39e1 (GQ847892) Acacia
M. plurifarium LMG 11892T (Y14158) Acacia
M. mediterraneum UPM-Ca36T (AM181745) Cicer
M. sp. LBER1 (FN563430) Lotus
M. sp. Ala-3 (AM491621) Anagyris
M. tianshanense A-1BST (AF041447) Glycyrrhiza
WSM2624 Lotononis s. l.
M. tianshanense RCAN03 (AY195846) Cicer
A. caulinodans ORS 571T (D11342) Sesbania
98
99
69
97
63
98
72
77
58
68
45
82
97
99
100
52
67
100
60
79
62
61
59
0.010.01
Figure 2.8. NJ phylogenetic tree, bootstrapped with 1000 replicates, showing
the relationships of Ensifer and Mesorhizobium strains associated with
Lotononis s. l. (in bold type) with related strains and type strains of Ensifer and
Mesorhizobium, based on aligned sequences of the 16S rRNA genes. GenBank
accession numbers are in brackets. Also shown is the genus of the original host
(E. adhaerens LMG 20216T was isolated from soil). Scale bar, 1% sequence
divergence (one substitution per 100 nucleotides). A., Azorhizobium; E.,
Ensifer; M., Mesorhizobium. T = type strain.
Chapter 2
82
Analysis of the nearly full-length (1416 bp) 16S rRNA gene sequence of
WSM2667 placed this strain in the genus Methylobacterium (Figure 2.9). The closest
relationship was to the Methylobacterium nodulans type strain ORS 2060T (Sy et al.,
2001), with 99.9% (1 bp mismatch) sequence identity. WSM2667 was also closely
related to Methylobacterium isbiliense DSM 17168T (Gallego et al., 2005; Kato et
al., 2008) and to strains of the pigmented methylobacteria isolated from Listia
species, such as Methylobacterium sp. 4-46 (Renier et al., 2008), WSM2598,
WSM2799 and WSM3032 (Yates et al., 2007), with sequence identities of 98.3%,
98.0%, 98.0%, 98.0% and 97.5%, respectively.
Strain WSM3557 was identified, with high bootstrap support, as being in the
genus Microvirga and family Methylobacteriaceae (Figure 2.9). Its closest
characterised phylogenetic relative was Microvirga flocculans TFBT (Takeda et al.,
2004; Weon et al., 2010). As well as grouping with the Listia angolensis strains
WSM3674 and WSM3686 (Yates et al., 2007) and Lupinus texensis strains Lut5 and
Lut6 (Andam & Parker, 2007), WSM3557 had high sequence identity with several
other root nodule isolates. These included the authenticated strains AC72a, isolated
from nodules of Phaseolus vulgaris growing in Ethiopia (Wolde-Meskel et al., 2005)
and ARRI 185 (genospecies AL), from northern Australian Indigofera linifolia
(Lafay & Burdon, 2007). The 16S rRNA gene sequences of several unpublished
nodule isolates also clustered within this clade. Strain SWF66521 was isolated from
a Sesbania species in Yunnan Province, China, while strain TP1 was obtained from
Tephrosia purpurea growing in the Thar Desert, India (Figure 2.9). Further analysis
of the taxonomy and phylogeny of WSM3557 and the other novel rhizobial isolates
from Listia angolensis and Lupinus texensis is covered in Chapter 4.
Chapter 2
83
Mtb. sp. WSM2598 (DQ838527) Li. bainesii
Mtb. sp. WSM2799 (DQ848137) Li. bainesii
Mtb. sp. 4-46 (GI170738367) Li. bainesii
Mtb. sp. WSM3032 (DQ848137) Li. solitudinis
Mtb. isbiliense DSM 17168T (AB302929)
Mtb. nodulans ORS 2060T (GI220920054) C. podocarpa
WSM2667 Le. calycina
Mtb. variabile GR3T (NR_042348)
Mtb. aquaticum GR16T ((NR_025631)
Mtb. organophilum ATCC 27886T (NR_041027)
Mtb. oryzae CBMB20T (NR_043104)
Mtb. populi BJ001T (NR_029082)
Mtb. extorquens IAM 12631T (AB175632)
Mtb. zatmanii DSM 5688T (NR_041031)
Mi. subterranea DSM14364T (FR733708)
Mi. sp. ARRI 185 (AJ785294) I. linifolia
Mi. sp. AC72a (AY776209) P. vulgaris
Mi. sp. SWF66521 (GQ922064) Sesbania sp.
Mi. sp. TP1 (HM042680) T. purpurea
Mi. flocculans TFBT (NR_036762)
Mi. sp. Lut5 (EF191407) Lu. texensis
Mi. sp. Lut6 (EF191407) Lu. texensis
Mi. sp. WSM3557 (HM362432) Li. angolensis
Mi. sp. WSM3686 (DQ838529) Li. angolensis
Mi. sp. WSM3674 (DQ838528) Li. angolensis
B. japonicum USDA 6T (U69638) G. max
100
62
100
58
53
100
98
55
99
80
86
75
95
79
100
100
83
100
71
100
68
0.01
Mtb. sp. WSM2598 (DQ838527) Li. bainesii
Mtb. sp. WSM2799 (DQ848137) Li. bainesii
Mtb. sp. 4-46 (GI170738367) Li. bainesii
Mtb. sp. WSM3032 (DQ848137) Li. solitudinis
Mtb. isbiliense DSM 17168T (AB302929)
Mtb. nodulans ORS 2060T (GI220920054) C. podocarpa
WSM2667 Le. calycina
Mtb. variabile GR3T (NR_042348)
Mtb. aquaticum GR16T ((NR_025631)
Mtb. organophilum ATCC 27886T (NR_041027)
Mtb. oryzae CBMB20T (NR_043104)
Mtb. populi BJ001T (NR_029082)
Mtb. extorquens IAM 12631T (AB175632)
Mtb. zatmanii DSM 5688T (NR_041031)
Mi. subterranea DSM14364T (FR733708)
Mi. sp. ARRI 185 (AJ785294) I. linifolia
Mi. sp. AC72a (AY776209) P. vulgaris
Mi. sp. SWF66521 (GQ922064) Sesbania sp.
Mi. sp. TP1 (HM042680) T. purpurea
Mi. flocculans TFBT (NR_036762)
Mi. sp. Lut5 (EF191407) Lu. texensis
Mi. sp. Lut6 (EF191407) Lu. texensis
Mi. sp. WSM3557 (HM362432) Li. angolensis
Mi. sp. WSM3686 (DQ838529) Li. angolensis
Mi. sp. WSM3674 (DQ838528) Li. angolensis
B. japonicum USDA 6T (U69638) G. max
100
62
100
58
53
100
98
55
99
80
86
75
95
79
100
100
83
100
71
100
68
0.01
Figure 2.9. NJ model phylogenetic tree, bootstrapped with 1000 replicates,
showing the relationships of Methylobacterium and Microvirga strains isolated
from Listia species (in bold type) with related strains and type strains of
Methylobacterium and Microvirga, based on aligned sequences of the 16S
rRNA genes. GenBank accession numbers are in brackets. The host species of
rhizobial strains are given. Bootstrap values are indicated on branches only
when higher than 50%. Scale bar, 1% sequence divergence (one substitution
per 100 nucleotides). B., Bradyrhizobium; Mi., Microvirga; Mtb.,
Methylobacterium. C., Crotalaria; G., Glycine; I., Indigofera; Le., Leobordea;
Li., Listia; Lu., Lupinus; P., Phaseolus; T., Tephrosia. T = type strain.
Chapter 2
84
2.3.6 Amplification and sequencing of nodA
2.3.6.1 nodA sequences of WSM2598 and WSM2667
PCR amplification using the primers M4-46 nodD 89-109f and M4-46 nodB
29-48r2 yielded products of approximately 1000 bp for the two methylobacterial
strains WSM2598 and WSM2667. All other strains failed to yield an amplification
product with these primers. Sequences of 937 bp and 965 bp were obtained for
WSM2598 and WSM2667, respectively, and contained a portion of nodD, the NodD
nod box binding region, the complete nodA gene and a portion of nodB. Both strains
had nodA genes of 645 bp length, with the proteins deduced from the nodA gene
sequences containing 214 amino acids. The sequence identity between WSM2598
and WSM2667 was less than 80%. WSM2598 had highest sequence identity with the
Listia bainesii symbiont strain Methylobacterium sp. 4-46 (99.4%) while the
WSM2667 nodA sequence was most closely related to that of Methylobacterium
nodulans ORS 2060 (99.5%) (Table 2.17). The nod box region of WSM2598 had the
same sequence as that of Methylobacterium sp. 4-46. Similarly, the WSM2667 and
ORS 2060 sequences shared 100% sequence identity in this region (Figure 2.10).
Table 2.17. Pairwise percent identity and number of nucleotide mismatches (in brackets) for
the 645 bp nodA gene of the Lotononis s. l. methylobacterial strains and the reference strains
Methylobacterium sp. 4-46 and Methylobacterium nodulans ORS 2060T.
WSM2598 4-46 WSM2667 ORS 2060T
WSM2598 99.4
(4) 79.4 79.1
Mtb sp. 4-46 99.4
(4) 79.7 79.4
WSM2667 79.4 79.7 99.5 (3)
ORS 2060T
79.1 79.4 99.5
(3)
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Meth 4-46 GATCGAGGCCCTTGAAGCGCATCTACCCTCCTATCCACCACCCAGATGCG
WSM2598 GATCGAGGCCCTTGAAGCGCATCTACCCTCCTATCCACCACCCAGATGCG
ORS 2060 TATCCATCGTTTGGATATGCTGCATCAAAACAATCGATTTTACAAATGTC
WSM2667 TATCCATCGTTTGGATATGCTGCATCAAAACAATCGATTTTACAAATGTC
pRL1JI TATCCATTCCATAGATGATTGCCATCCAAACAATCAATTTTACCAATCTT
Figure 2.10. Comparison of nod box sequences in the promoter regions of nodA genes from
methylobacterial strains WSM2598, WSM2667 and Methylobacterium sp. 4-46 (isolated
from Lotononis s.l. hosts), and from Methylobacterium nodulans ORS 2060 (associated with
Crotalaria species). A-T-C-N9-G-A-T repeats (or related motifs) are in blue type. The
sequence from the symbiotic plasmid pRL1JI of Rhizobium leguminosarum bv. viciae (Feng
et al., 2003) is included for comparison.
2.3.6.2 nodA sequences of the remaining Lotononis s. l. rhizobia
A partial sequence of the nodA and nodB genes was obtained for WSM2632,
WSM2653, WSM2783, WSM3040 and WSM3557 using the primers nodA-1 and
nodA-2. No amplification products could be obtained for WSM2596 or WSM2624.
The 638 bp sequences of the bradyrhizobial strains WSM2632 and WSM2783 were
identical, while a 631 bp fragment of nodA from the E. meliloti strains WSM2653
and WSM3040 had 100% sequence identity. The 636 bp sequence of the Microvirga
strain WSM3557 had sequence identity of 84.9% or less with WSM2632,
WSM2653, WSM2783 and WSM3040. Rhizobial strains identified by BLASTN as
having nodA sequences that were closely related to WSM3557 had sequence
identities of 83% or less. Alignment of a 565 bp nodA fragment showed that the
Bradyrhizobium, Ensifer, Methylobacterium nodulans, pigmented Methylobacterium
and Microvirga strains all possessed distinct nodA sequences, with 84.9% or less
sequence identity between each of these groups (Table 2.18).
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Table 2.18. Pairwise percent identity for a 565 bp fragment of the nodA gene of Lotononis
s.l. strains.
WSM2598 WSM2667 WSM2632 WSM2783 WSM2653 WSM3040 WSM3557
WSM2598 80.4 71.5 71.5 66.2 66.2 70.1
WSM2667 80.4 83.5 83.5 72.6 72.6 81.0
WSM2632 71.5 83.5 100 79.5 79.5 84.9
WSM2783
71.5 83.5 100 79.5 100 84.9
WSM2653 66.2 72.6 79.5 79.5 100 75.6
WSM3040 66.2 72.6 79.5 79.5 100 75.6
WSM3557 70.1 81.0 84.9 84.9 75.6 75.6
The complete or nearly complete nodA sequences of the Lotononis s. l. strains
were analysed, along with those of rhizobial strains identified as having highest
BLASTN or BLASTX sequence similarity, and several reference strains. The
resulting NJ phylogenetic tree is shown in Figure 2.11. Although the clades within
the tree were clearly defined and mostly well supported by high bootstrap values, the
higher branches were not well supported, as evidenced by low bootstrap values. The
phylogenetic relationships between the nodA genes of the Lotononis s. l. strains and
those of other rhizobia therefore could not be determined with confidence. Trees
generated using the ME and MP methods yielded similar results.
The Lotononis s. l. strains in the NJ nodA tree formed several polyphyletic
clusters that intermingled with other taxonomically diverse rhizobial strains. It is
interesting to note that all strains, except for the methylobacteria WSM2598 and
WSM2667, shared a three base pair deletion with reference Ensifer, Mesorhizobium,
Microvirga and Rhizobium strains that was not present in the nodA sequences of
Bradyrhizobium, Burkholderia and Methylobacterium rhizobia (Figure 2.12).
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WSM2653 and WSM3040 grouped with a cluster of African Ensifer strains that
nodulate Acacia and Prosopis (Ba et al., 2002; Nick et al., 1999), with sequence
identities ranging from 76% to 78%. The Lotononis s. l. bradyrhizobia WSM2632
and WSM2783 formed a separate lineage from Bradyrhizobium strains isolated from
Australian native legumes (WSM1735 and WSM1790) and from the clade of
remaining bradyrhizobia, which included a number of strains isolated from native
African legumes. WSM3557 was a sister group to the Lotononis s. l. bradyrhizobia.
The Methylobacterium strains WSM2598 and WSM2667 were included in a well-
supported clade that contained the other methylobacterial rhizobia strains ORS 2060
and 4-46. There was a large divergence, however, between the Methylobacterium
nodulans strains (ORS 2060 and WSM2667) and the strains isolated from Listia
bainesii (4-46 and WSM2598).
Chapter 2
88
E. sp. ORS1085 (AJ302677) A. tortilis
E. sp. ORS1230 (AJ302681) A. tortilis
E. arboris HAMBI 1396 (EF457955) P. chilensis
E. terangae ORS1009T (Z95237) A. laeta
E. meliloti WSM2653 Lotononis s. l .
E. meliloti WSM3040 Lo. laxa
M. plurifarium CFN ESH18 (EU291996) Ac. angustissima
M. plurifarium ORS1255 (AJ302680) A. tortilis
Mi. sp. Lut6 (EF191414) Lu. texensis
R. tropici CFN299 (X98514 ) Ph. vulgaris
E. meliloti ORS1044 (AJ300232) A. tortilis
E. sp. BR827 (Z95232) Leu. leucocephala
Br. sp. WSM2632 Lotononis s. l.
Br. sp. WSM2783 Lotononis s. l .
Mi. sp. WSM3557 (HQ435534) Li. angolensis
M. sp. CCNWAX24-1 (EU722471) Alh. sparsifolia
M. sp. CCNWGS0011 (GQ871220) T. lanceolata
M. sp. ICMP 14330 (DQ100401) So. microphylla
M. albiziae CCBAU 61158T (GQ167236) Alb. kalkora
Bur. sp. WSM3937 (EU219867) Rh. ferulifolia
Bur. tuberum STM678T (AJ302321) Cyclopia
Bur. tuberum DUS833 (EF566976) Cyclopia
Mtb. sp. WSM2598 Li. bainesii
Mtb. sp. 4-46 (CP000943) Li. bainesii
Mtb. nodulans WSM2667 Le. calycina
Mtb. nodulans ORS 2060T (CP001349) Cr. podocarpa
Br. sp. WSM1735 (AJ890293) Rh. minima
Br. sp. WSM1790 (AJ890286) I. colueta
Br. japonicum ORS1816 (AJ430722) Cr. hyssopifolia
Br. sp. F3b (AM117517) Les. cuneata
Br. elkanii USDA 76T (AM117554) G. max
Br. sp. USDA3259 (AM117558) Ph. lunatus
Br. sp. ORS1812 (AJ430723) Ab. stictosperma
Br. sp. ORS170 (AJ300258) F. albida
Br. sp. CP283 (AJ920038) Parasponia
Rhizobial sp. STM251 (AJ300259) Ch. sanguinea
E. fredii NGR234 (U00090) L. purpureus
R. leguminosarum WSM1325 (NC_012850) Trifolium sp.
E. medicae WSM419 (NC_009636) M. murex
A. caul. ORS 571T (NC_009937) S. rostrata
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Senegal
E. sp. ORS1085 (AJ302677) A. tortilis
E. sp. ORS1230 (AJ302681) A. tortilis
E. arboris HAMBI 1396 (EF457955) P. chilensis
E. terangae ORS1009T (Z95237) A. laeta
E. meliloti WSM2653 Lotononis s. l .
E. meliloti WSM3040 Lo. laxa
M. plurifarium CFN ESH18 (EU291996) Ac. angustissima
M. plurifarium ORS1255 (AJ302680) A. tortilis
Mi. sp. Lut6 (EF191414) Lu. texensis
R. tropici CFN299 (X98514 ) Ph. vulgaris
E. meliloti ORS1044 (AJ300232) A. tortilis
E. sp. BR827 (Z95232) Leu. leucocephala
Br. sp. WSM2632 Lotononis s. l.
Br. sp. WSM2783 Lotononis s. l .
Mi. sp. WSM3557 (HQ435534) Li. angolensis
M. sp. CCNWAX24-1 (EU722471) Alh. sparsifolia
M. sp. CCNWGS0011 (GQ871220) T. lanceolata
M. sp. ICMP 14330 (DQ100401) So. microphylla
M. albiziae CCBAU 61158T (GQ167236) Alb. kalkora
Bur. sp. WSM3937 (EU219867) Rh. ferulifolia
Bur. tuberum STM678T (AJ302321) Cyclopia
Bur. tuberum DUS833 (EF566976) Cyclopia
Mtb. sp. WSM2598 Li. bainesii
Mtb. sp. 4-46 (CP000943) Li. bainesii
Mtb. nodulans WSM2667 Le. calycina
Mtb. nodulans ORS 2060T (CP001349) Cr. podocarpa
Br. sp. WSM1735 (AJ890293) Rh. minima
Br. sp. WSM1790 (AJ890286) I. colueta
Br. japonicum ORS1816 (AJ430722) Cr. hyssopifolia
Br. sp. F3b (AM117517) Les. cuneata
Br. elkanii USDA 76T (AM117554) G. max
Br. sp. USDA3259 (AM117558) Ph. lunatus
Br. sp. ORS1812 (AJ430723) Ab. stictosperma
Br. sp. ORS170 (AJ300258) F. albida
Br. sp. CP283 (AJ920038) Parasponia
Rhizobial sp. STM251 (AJ300259) Ch. sanguinea
E. fredii NGR234 (U00090) L. purpureus
R. leguminosarum WSM1325 (NC_012850) Trifolium sp.
E. medicae WSM419 (NC_009636) M. murex
A. caul. ORS 571T (NC_009937) S. rostrata
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South Africa
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South Africa
South Africa
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South Africa
South Africa
South Africa
Senegal
NW Aust
NW Aust
Senegal
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USA
USA
Senegal
Senegal
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New Guinea
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Sardinia
Senegal
Figure 2.11. NJ nodA phylogenetic tree, bootstrapped with 1000 replicates, showing
the relationships of the Lotononis s.l.-associated rhizobia (in bold type) and other
rhizobia. GenBank accession numbers are in brackets. Also shown is the original
host and the geographical location of the strain. Scale bar, 5% sequence divergence
(five substitutions per 100 nucleotides). A., Azorhizobium; Br., Bradyrhizobium;
Bur., Burkholderia; E., Ensifer; M., Mesorhizobium; Mtb., Methylobacterium; Mi.,
Microvirga; R., Rhizobium. A., Acacia;Ab., Abrus; Ac., Acaciella; Alb., Albizia; Alh.,
Alhagi; Ch., Chidlowia; Cr., Crotalaria; F., Faidherbia; I., Indigofera; L., Lablab;
Le., Leobordea; Les., Lespedeza; Leu., Leucaena; Li., Listia; Lo., Lotononis s. str;
Lu., Lupinus; M., Medicago; P., Prosopis; Ph., Phaseolus; Rh., Rhynchosia; S.,
Sesbania; So., Sophora; T., Thermopsis. T = type strain.
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A. caulinodans ORS 571 TGCGGATCCATTTTCAGCGCTT---CTGTCGCAA
E. arboris HAMBI1396 TGCAAAGACACGTGGAAAGATT---CGGCCGACA
E. fredii NGR234 TGTACAAGCTTGTGGGCAGACT---CTGCCGAAA
E. medicae WSM419 TGCGGAACCACGTTGAGAGATA---TTGCCAGAA
E. meliloti WSM2653 TGCAGAAGCACATTACCAGATT---CGTCCGACA
E. meliloti WSM3040 TGCAGAAGCACATTACCAGATT---CGTCCGACA
Ensifer sp. BR827 TGCGCAAACATATTGAGAGGTT---CGCCCGGTA
Ensifer sp. ORS 1044 TGCGCAAGCATGTAGAGCGGTT---CGCCCGATA
Ensifer sp. ORS 1085 TGCAAAGGCACGTGGAAAGATT---CGGCCGACA
Ensifer sp. ORS 1230 TGCAAAGGCACGTGGAAAGGTT---CGGCCGACA
E. terangae ORS 1009 TGCAGAAGCACGTCGAAAGATT---CGGTCGGCA
Mes. albizieae CCBAU61158 TGCGGAACCATGTTGAGAGGTT---CTGCAGAGA
Mes. plurifarium ORS 1255 TGCATAAACACGTTGCAAGATT---GGGGCGAGG
Mes. plurifarium CFN ESH18 TGCGCAAACACGTTGCAAGATT---CGAGCGACA
Mes. sp. CCNWAX24-1 TGCGGAACCATCTTGAGAGGTT---TGGCAGACA
Mes. sp. CCNWGS0010 TGCGGAACCATGTTGAGAGGTT---CTGCCGAGG
Mes. sp. CCNWSX426 TGCGGAACCATATGGAGAGGTT---CTGCAGAGA
Mes. sp. ICMP 14330 TGCGAAACCATATTGTAAGATT---CTTCAGAGA
Microvirga WSM3557 TGCAGAAGCATGTTGCAAGGCT---GGGCCGGCA
Microvirga Lut6 TGCAGAAACATGTTGAGAGGTT---CGGCCGATA
R. tropici CFN299 TGCAAAAGCATATCGAAAGGTT---CGGCCGTTA
R. leguminosarum WSM1325 TGCGGAACCACGTGGAGAGATT---CTGCCGAGA
Br. sp. WSM2632 TGCAGAAGCATGTTGAAAGGGT---CGCCCGGCA
Br. sp. WSM2783 TGCAGAAGCATGTTGAAAGGGT---CGCCCGGCA
Br. elkanii USDA 76 TTGAGAAGCATCTGACCAGACTGGTCGGAAGGCG
Br. sp. CP 283 TTGAGAGGCATCTCACCAGGCTGGTCGGAAGGCA
Br. sp. f3b TTGAGAAGCATCTTACCAGGCTGGTCGGCCGGCA
Br. sp. ORS 1812 TTGAGAAGCATCTCACCAGGCTGGTCGGAAGGCA
Br. sp. ORS 1816 TCGAGAAACATATGACGCGACTGGTCCAAAGGCA
Br. sp. ORS 170 TTGAGAAGCATCTCACCAGGCTAGTCGGAAGGCA
Br. sp. USDA 3259 TTGAGAAGCATCTCACCAGGCTGGTCGGAAGGCA
Br. sp. WSM1735 TGAAGGGCCATCTTACAAGGTTGCTGACCCGCCG
Br. sp. WSM1790 TGAAGGGCCATCTTACAAGGTTGCTGACCCGCCG
Burk. tuberum STM678 TGCGGCAACATATTGCAAGGCTGCTCGGCCGACC
Burk. tuberum DUS833 TGCGGCAACATATTGCAAGGCTGCTCGGCCGACC
Burk. sp. WSM3937 TGCGGCAACATATTGCAAGGCTGCTCGGCCGACA
Mtb. sp. 4-46 TTCAGAAACACCTCACCAGGCTGCTCGGTAAGGC
Mtb. sp. WSM2598 TTCAGAAACACCTCACCAGGCTGCTCGGTAAGGC
Mtb. nodulans ORS 2060 TGCAGAAACATCTTACAAGGCTGCTCGGTAAGGC
Mtb. nodulans WSM2667 TGCAGAAACATCTTACAAGGCTGCTCGGTAAGGC
Figure 2.12. Alignment of a portion of the nodA sequences of the Lotononis s. l. rhizobia (in
bold type) and reference strains featured in Figure 2.11. The aligned sequences begin from
position 388 of the complete Azorhizobium caulinodans ORS 571 nodA sequence. The
Lotononis s. l. bradyrhizobial strains WSM2632 and WSM2783 (highlighted) share a three
base pair deletion with reference Ensifer, Mesorhizobium, Microvirga and Rhizobium strains
that is not present in the nodA sequences of Burkholderia, Methylobacterium and other
Bradyrhizobium rhizobia. A. = Azorhizobium; Br = Bradyrhizobium; Burk. = Burkholderia;
E. = Ensifer; Mes. = Mesorhizobium; Mtb = Methylobacterium; R = Rhizobium.
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2.4 Discussion
This study reports on the type and extent of nodulation within Lotononis s. l.
and the diversity of root nodule bacteria associated with this genus. Additionally, it
describes the possible interactions between the symbiotic and the biogeographical
relationships between these host plants and their associated rhizobia.
2.4.1 Nodule morphology in Lotononis s. l. species
Previous authors (Corby, 1981; Lavin et al., 2001; Sprent & James, 2007) have
noted the value of nodule morphology and structure as a taxonomic tool, and this
study shows that nodule type can be used as a taxonomic marker to distinguish Listia
species from other genera in the Lotononis s. l. clade. Listia angolensis, Listia
bainesii and Listia heterophylla form lupinoid nodules. Recent studies have
confirmed that all examined species in the genus Listia have this type of nodule (R.
Yates, unpublished data). Lupinoid nodulation appears to be a property unique to
Listia, whereas Leobordea and Lotononis s. str. species all have indeterminate
nodules. All tested host species formed nodules, with the exception of the geophytic
Euchlora hirsuta. The inability of this host to nodulate with any of the rhizobial
strains used in this study supports previous observations that record a lack of
nodulation in field studies of this species (J. Howieson, R. Yates, unpublished data),
although the GRIN Rhizobial Nodulation Data (http://www.ars-
grin.gov/~sbmljw/cgi-bin/taxnodul.pl) lists it as nodulated.
2.4.2. Diversity of Lotononis s. l. rhizobia
This study has revealed that wild Listia, Leobordea and Lotononis s. str.
species are nodulated by phylogenetically diverse rhizobia. The rhizobia that
nodulate Lotononis s. l. species belong to five different genera in the
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Alphaproteobacteria, one of which (Microvirga) has not previously been described as
containing rhizobial species. Other studies have similarly reported on wild legumes,
such as species of Lotus, Dalbergia, Ononis, Pterocarpus and Sophora, that are
nodulated by natural populations of genotypically diverse rhizobia (Lorite et al.,
2010; Rasolomampianina et al., 2005; Rincón et al., 2008; Sylla et al., 2002; Zhao et
al., 2010). What is unusual about the Lotononis s. l. symbiotic associations, however,
is firstly, the very wide taxonomic diversity of the rhizobia and secondly, the varying
levels of specificity exhibited by the different host species, ranging from the highly
specific Listia bainesii to the more promiscuous Leobordea bolusii.
Some degree of microsymbiont diversity is to be expected, given that South
Africa is the centre of diversity for Lotononis s. l. and rhizobial centres of diversity
are thought to coincide with those of their legume hosts (Andronov et al., 2003; Lie
et al., 1987). The extent of this taxonomic diversity prompts an examination of the
biogeography and symbiotic relations of the rhizobial lineages that are associated
with Lotononis s. l. and of the climatic, edaphic and host plant factors that may have
contributed to the development of microsymbiont diversity in this group of legumes.
2.4.2 1 The Methylobacterium lineage
Methylobacterium is a widespread genus that contains many plant-associated
and endophytic species (Madhaiyan et al., 2007, 2009b; Sy et al., 2005; Van Aken et
al., 2004). Two rhizobial lineages of Methylobacterium have so far been identified:
the pigmented Listia strains (Jaftha et al., 2002; Yates et al., 2007) and the non-
pigmented M. nodulans, which specifically nodulates species of Senegalese
Crotalaria (Jourand et al., 2004; Sy et al., 2001). Nodulating Methylobacterium
strains appear to have both a limited geographical distribution and host range, with
Chapter 2
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an affinity to African crotalarioid host legumes, although a recent study has
described M. nodulans strains isolated from Indian Crotalaria juncea and Sesbania
aculeata (Madhaiyan et al., 2009a). This geographical distribution reflects the
predominantly African distribution of the Crotalarieae tribe, although the large genus
Crotalaria is pantropical (van Wyk, 2005).
The isolation of WSM2667 from Leobordea calycina (and its ability to
nodulate other Lotononis s. l. species) extends the host range of M. nodulans from a
narrow group of Crotalaria species to other genera in the Crotalarieae tribe.
Although M. nodulans and the pigmented Listia methylobacteria have distinct and
highly specific host ranges, WSM2598 and WSM2667 were able to nodulate the
same subset of Lotononis s. l. species, with the notable exception of Listia bainesii.
The methylobacterial strains WSM2598, WSM2667 and ORS 2060 were also the
only rhizobia able to nodulate Listia angolensis other than its cognate Microvirga
microsymbionts, although the Methylobacterium-induced nodules were always
ineffective.
The results of this study support the finding that Listia methylobacteria have a
narrow host range. WSM2598 was effective only on Listia species (other than L.
angolensis) and ineffectively nodulated or failed to nodulate other Lotononis s. l.
hosts. Pigmented methylobacteria have been isolated, over a wide geographical
range, from all studied Listia species except L. angolensis (Yates et al., 2007; R.
Yates, unpublished data). The individual microsymbiont strains vary in their level of
effectiveness, but none has been found to be ineffective on these hosts (R. Yates,
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unpublished data) and these methylobacteria would appear to constitute a
symbiotically stable population of effective and highly specific microsymbionts.
2.4.2.2 The Microvirga lineage
This study is the first report of Microvirga strains that are capable of
nodulation and N2 fixation with legumes. Although the small number of isolates
obtained from L. angolensis precludes definitive conclusions, the data presented here
indicate that rhizobial Microvirga species preferentially nodulate this legume host.
The symbiotic relationship between Microvirga strains and L. angolensis is not as
tight as that of the other Listia species and their associated methylobacteria.
Symbiotic effectiveness appears to be more variable: in this study, some Microvirga
strains were only partially effective on L. angolensis, and Eagles & Date (1999) have
noted that WSM3674 (= CB1323; see Chapter 4) is effective on only some
accessions of L. angolensis. Rhizobial Microvirga strains appear to have a greater
host range and geographical distribution than the Methylobacterium rhizobia.
WSM3557 had the broadest host range of all strains in this study, nodulating all
Lotononis s. l. hosts except L. bainesii in Experiment 3, and being partially effective
on three of these species in addition to effectively nodulating L. angolensis.
Although rhizobial species of Microvirga have not yet been formally described,
published reports indicate that they have a broad tropical or sub-tropical distribution
and nodulate taxonomically diverse legume hosts: Lupinus texensis in Texas, USA,
Phaseolus vulgaris in Ethiopia and northern Australian Indigofera linifolia (Andam
& Parker, 2007; Lafay & Burdon, 2007; Wolde-Meskel et al., 2005).
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2.4.2.3 The Bradyrhizobium, Ensifer and Mesorhizobium lineages
It is not possible to gain a complete picture of the symbiotic relationships
between Lotononis s. l. species and their cognate rhizobia, due to the small number
of isolates obtained from South African Leobordea and Lotononis s. str. species. It is
illustrative of the genotypic diversity of these isolates though, that the seven
collected strains belong to four different genera (Bradyrhizobium, Ensifer,
Mesorhizobium and Methylobacterium) and two separate lineages of bradyrhizobia.
Their identification has been based solely on sequences of the 16S rRNA gene,
however, which is not adequate for delineating rhizobial species (van Berkum et al.,
2003). Although sequencing additional loci, such as housekeeping genes, would be
required for a more accurate determination of taxonomic status, the existing data do
suggest interesting phylogenetic and biogeographical relationships in these lineages.
WSM2653 and WSM3040 are strains of Ensifer meliloti, which is classically
considered to be a specific microsymbiont of the genera Medicago, Melilotus and
Trigonella (Fred et al., 1932). Recent studies, however, have identified strains of E.
meliloti that effectively nodulate Cicer arietinum in Tunisia (Ben Romdhane et al.,
2007), Phaseolus vulgaris in Tunisia and the Canary Islands (Mnasri et al., 2007;
Zurdo-Piñeiro et al., 2009) and endemic Lotus species in the Canary Islands (León-
Barrios et al., 2009). E. meliloti strains have also been isolated from diverse wild
legumes growing in arid regions of northern Africa. Host plants include Acacia
tortilis and Prosopis farcta (tribe Mimoseae) (Ba et al., 2002; Fterich et al., 2011);
Argyrolobium uniflorum, Genista saharae and Retama raetam (Genisteae);
Hippocrepis bicontorta and Lotus spp. (Loteae); Hedysarum carnosum (Hedysareae);
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Ononis natrix (Trifolieae) (Mnasri et al., 2009); and Colutea arborescens (Galegeae)
(Ourarhi et al., 2011).
The predominant Mesorhizobium species that has so far been associated with
African legumes is M. plurifarium, which has been described as nodulating species
of the mimosoid genera Acacia, Leucaena and Prosopis and the caesalpinioid genus
Chaemaecrista (Ba et al., 2002; de Lajudie et al., 1998a). Recent studies have also
reported novel genospecies of Mesorhizobium isolated from Ethiopian Acacia
abyssinica, A. tortilis (Mimoseae) and Sesbania sesban (Sesbanieae) (Degefu et al.,
2011); Anagyris latifolia (Thermopsideae) and Lotus spp. (Loteae) in the Canary
Islands (Donate-Correa et al., 2007; Lorite et al., 2010) and endemic South African
Lessertia spp (Galegeae) (Howieson et al., 2008; M. Gerding Gonzalez, unpublished
thesis). Mesorhizobium tianshanense, the species with highest sequence identity to
WSM2624, has not previously been reported as associated with African legumes. M.
tianshanense was thought to have a limited geographical distribution. Han et al.
(2010) considered this species to be endemic to Xinjiang region, China, although
strains of M. tianshanense have been isolated from nodules of Cicer arietinum
(Cicereae) in Spain and Portugal (Laranjo et al., 2004; Rivas et al., 2007). It appears
to be adapted to diverse legume hosts, as the host range of the originally described
species encompasses legumes in tribe Phaseoleae and in the genistoid and galegoid
clades (Chen et al., 1995).
The Lotononis s. l. bradyrhizobia form two distinct lineages that do not
correspond to any of the diverse bradyrhizobial strains that have previously been
isolated from native South African or African legume hosts (Figure 2. 7) (Boulila et
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al., 2009; Garau et al., 2009; Steenkamp et al., 2008; Sylla et al., 2002). Their
biogeography is interesting, as the rhizobial strains most closely related to the
Lotononis s. l. bradyrhizobia were isolated from host plants that are phylogenetically
and geographically distant from Lotononis s. l. species (Parker & Kennedy, 2006;
Parker, 2008; Qian et al., 2003). Bradyrhizobia have been reported to nodulate
various African Crotalaria species (Moulin et al., 2004; Sy et al., 2001), but in
general there is a paucity of data available on the microsymbionts of native African
crotalarioid legumes. The phylogenetic and biogeographical relationships of
bradyrhizobial microsymbionts of this group of legumes may become clearer with
further sampling. The strain most closely related to the Lotononis s. l. bradyrhizobia
that had both geographical and host plant affinity was RSB6, which nodulates
Algerian Retama (tribe Genisteae) species (Boulila et al., 2009).
What factors are possible drivers of the phylogenetic diversity seen in the
Lotononis s. l.-associated rhizobia? As host plants have been shown to play a key
role in shaping rhizobial diversity (Han et al., 2010) and plants are more likely than
rhizobia to exercise partner choice (Simms & Taylor, 2002), an examination of the
patterns of symbiotic association between the different Lotononis s. l. taxonomic
groups and their associated rhizobia may provide an explanation for this
microsymbiont diversity.
2.4.3 Symbiotic relationships within Lotononis s. l. hosts
The Lotononis s. l. species can be divided into two groups, in terms of their
symbiotic relationships. Listia species have high symbiotic specificity and are
associated with unusual rhizobia. For Listia species other than L. angolensis,
specificity is acute, as these legumes nodulate only with their associated pigmented
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methylobacteria. Nitrogen fixation is linked with specificity: the Listia
methylobacteria are effective or highly effective on all Listia species except L.
angolensis (Yates et al., 2007; R. Yates, unpublished data). In comparison, in the L.
angolensis-Microvirga symbiosis, there has been some diminution of specificity in
the host plant, a broadening of the host range of the rhizobial partner and greater
variability of nitrogen fixation effectiveness.
In contrast to the Listia species, there appears to be no relationship between
host plant taxonomy and rhizobial chromosomal background in the symbiotic
associations seen in Leobordea and Lotononis s. str. species. Most of these host
species could be considered promiscuous, as they were able to nodulate with diverse
rhizobial strains. Similarly, the Bradyrhizobium, Ensifer and Methylobacterium
strains isolated from Leobordea and Lotononis s. str. species had broad host ranges
across this group of legumes. The Mesorhizobium strain WSM2624, while being the
least infective, also had no obvious pattern of host nodulation. Similar high
promiscuity has been noted in other studies of native populations of rhizobia and
legumes (Han et al., 2010; Lafay & Burdon, 1998; Weir, 2006; Zhao et al., 2010).
The effectiveness of the symbioses was generally poor and can be explained by
several factors. Firstly, none of the Leobordea and Lotononis s. str. species, with the
exception of Lotononis laxa, is a homologous host of the inoculant strains used in
this study. Rhizobia that are not the usual partners of these host species would
therefore not be expected to be as symbiotically compatible. Secondly, rhizobial
strains isolated from a population of a wild legume host can vary in their symbiotic
effectiveness (Odee et al., 2002; Singleton & Tavares, 1986). Unlike WSM2598 and
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WSM3557, the remaining rhizobia in this study have not been screened for nitrogen
fixation effectiveness and may be suboptimal strains. Thirdly, the host plants were
usually obtained from a collection of wild seeds. Individual plants within a
population would be expected to have considerable genetic variability, which has
been linked with variations in symbiotic compatibility with rhizobial partners (Abi-
Ghanem et al., 2011; Drew & Ballard, 2010; Wilkinson et al., 1996). This could also
explain the differences in the responses of individual plants to inoculation, and the
ineffective nodulation of WSM3040 on its cognate L. laxa host, which has
previously been noted for L. laxa isolates on various L. laxa accessions (The CB
Collection Database, unpublished data). This putative lack of broad symbiotic
compatibility across a population of the host species is in contrast to the broad-scale
effectiveness of methylobacterial strains across the Listia cross-inoculation group.
The rhizobial host range is usually a product of the nodulation genes that are
present in the genome (Perret et al., 2000). The phylogeny of the rhizobial nod
genotype has been found to correlate with that of the host plant, most notably in
studies of temperate legumes (Mergaert et al., 1997; Suominen et al., 2001).
Conversely, studies of other legume-rhizobia associations have found no clear
association between the host plant and the nod gene systematics (Lafay et al., 2006;
Moulin et al., 2004). Does an examination of the nodA phylogeny of Lotononis s. l.
rhizobia provide insights into the evolution of symbioses in this group of legumes?
2.4.4 Phylogeny of nodA genes
The nodA sequences of the Lotononis s. l. rhizobia are polyphyletic, forming
four separate clades in which the nodA lineages are associated with the rhizobial
genomic species, although the low bootstrap values mean that the topology of the
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tree is not well supported. The nodA sequences of the Lotononis s. l. rhizobia are
intermingled with nodA of rhizobia isolated from host plants that are not closely
related to Lotononis s. l. (Figure 2.11). This indicates firstly, that the symbiotic genes
of Lotononis s. l. rhizobia were derived from different sources or have evolved
divergently and secondly, that those Leobordea and Lotononis s. str. hosts which are
promiscuous nodulators are not stringently selecting the rhizobial symbiotic
genotype.
The nodA sequences of the methylobacterial strains WSM2598 and WSM2667
are related to nodA of Methylobacterium sp. 4-46 (isolated from L. bainesii) and M.
nodulans ORS 2060 (which specifically nodulates Crotalaria podocarpa, Crotalaria
glaucoides and Crotalaria perrottetti (Renier et al., 2008; Sy et al., 2001)). Both
WSM2667 and ORS 2060 are unable to nodulate L. bainesii and L. heterophylla,
although they do form occasional ineffective nodules on L. angolensis (this study).
The nodA and nod box sequences of the Methylobacterium rhizobia (4-46,
WSM2598, ORS 2060 and WSM2667) suggest that while there is a close
phylogenetic relationship between the nodulation genes of M. nodulans and the
Listia methylobacteria, the branch depth indicates their divergence occurred some
time ago. It has also been suggested that the nodulation genes of the
Methylobacterium rhizobia have been acquired by horizontal gene transfer (HGT)
from Bradyrhizobium strains (Moulin et al., 2004). Their considerable divergence
again suggests an ancient origin for this putative HGT event. In this study, the nodA
sequences of the Methylobacterium strains appear to be more closely related to the
main Bradyrhizobium branch than to the other Lotononis s. l. rhizobia.
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The ORS 2060 nod genes are organised in a single operon that encodes NodA,
NodB, NodC, NodI, NodJ and NodH proteins and is almost identical to the nod
operon found in the sequenced genome of Methylobacterium sp. 4-46 (Renier et al.,
2008). It can be hypothesised from this that Listia and Crotalaria hosts that are
nodulated by methylobacteria have similar Nod factor requirements. Crotalaria
species are postulated to belong to two inoculation groups: most are nodulated by
bradyrhizobia that may produce fucosylated Nod factors, while C. podocarpa, C.
glaucoides and C. perrottetti are specifically nodulated by M. nodulans, which
produces sulfated Nod factors (Renier et al., 2008) The Methylobacterium nodA
sequences are in a sister clade to those of several Burkholderia tuberum strains that
nodulate South African fynbos legumes (Elliott et al., 2007; Garau et al., 2009).
Interestingly, Burkholderia tuberum STM678 (previously named as Bradyrhizobium
aspalati; see Elliott et al. (2007)) does not produce sulfated Nod factors (Boone et
al., 1999).
The nodA sequences of the Bradyrhizobium, Ensifer and Microvirga strains do
not group with rhizobia that nodulate other crotalarioid legumes. This may, however,
be more a reflection of the current lack of data on rhizobia associated with this group
of host plants. The nodA sequences of the E. meliloti strains WSM2653 and
WSM3040 are clearly grouped with diverse Ensifer and Mesorhizobium strains that
form a nodulation group for Acacia, Prosopis and Leucaena (Ba et al., 2002). The
nodA lineages of the bradyrhizobial isolates WSM2632 and WSM2783 are grouped
apart from the Ensifer strains. Interestingly, they are also well separated from the
clade containing all other Bradyrhizobium sequences, including ORS 1816, isolated
from Crotalaria hyssopifolia in Senegal (Moulin et al., 2004). A previous study
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found that the nodA phylogeny of African bradyrhizobia placed them in the large,
pan-tropical Clade III (Steenkamp et al., 2008). Moulin et al. (2004) considered
bradyrhizobia to form a monophyletic branch within the nodA tree and their deduced
NodA proteins contain 209–211 amino acids (with the exception of the
photosynthetic stem-nodulating Bradyrhizobium strains, which encode 197 amino-
acid proteins). The published Methylobacterium strains 4-46 and ORS 2060, and
WSM2598 and WSM2667 from this study, along with Burkholderia tuberum strain
STM678 also contain deduced NodA proteins of over 200 residues. In contrast,
NodA in other rhizobial genera is usually composed of 195–198 amino acids. The
greater length of the bradyrhizobial NodA is the result of a 12–13 amino acid
segment at the N-terminal end (Moulin et al., 2004). The primers used in this study
did not amplify this section of nodA, but it would be interesting to obtain the
complete nodA sequences for WSM2632 and WSM2783, to determine whether, as
the phylogenetic tree suggests, they are more closely related to Ensifer and
Mesorhizobium nodA, rather than other bradyrhizobial strains.
The position of the Microvirga strain WSM3557 in the nodA phylogenetic tree
is intriguing. It forms a sister clade to the bradyrhizobial isolates WSM2632 and
WSM2783 and is separate from the only other nodulating Microvirga nodA sequence
described to date, that of Lut6, which specifically nodulates Lupinus texensis, a
species endemic to Texas, USA (Andam & Parker, 2007). Notably, WSM3557 is
also separate from the nodA lineages of the Methylobacterium strains that
specifically nodulate the remaining Listia species. Microvirga has not previously
been described as a genus containing rhizobial species. It is presumed that the
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symbiotic genes that confer the ability to nodulate Listia angolensis have been
acquired from another rhizobial lineage via HGT, but due to the low bootstrap values
for the higher branches of the nodA tree, the source of the donor and the phylogeny
of these nod genes are unclear.
Although the nodA gene is a host range determinant (Debellé et al., 1996;
Lortet et al., 1996), the congruence between nodA phylogeny and the taxonomy of
the legume host is stronger in some associations than others. Host plants in the
Galegeae, Trifolieae and Vicieae tribes (the galegoid clade) are nodulated by diverse
rhizobia with similar nodA sequences; which is related to the requirement of these
legumes for Nod factors that are N-acylated with unsaturated fatty acids (Debellé et
al., 2001; Suominen et al., 2001). Several studies of other legume species that are
nodulated by phylogenetically diverse rhizobia have also found the nod genes to be
highly similar, regardless of rhizobial chromosomal background (Ba et al., 2002;
Haukka et al., 1998; Laguerre et al., 2001; Lu et al., 2009). Conversely, Han et al.
(2010) and Zhao et al. (2010), in studies of rhizobia isolated from wild legumes in
China, concluded that the relations between the microsymbiont chromosomal
background, nod genes and host plant were promiscuous and the nodA or nodC
lineages were clearly associated with the rhizobial genomic background. Canary
Island Lotus species also have Ensifer meliloti and Mesorhizobium microsymbionts
in which distinct nodC lineages are related to the different chromosomal genotypes,
although in this case the E. meliloti strains have a restricted host range (Lorite et al.,
2010).
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Symbiotic relationships between the Lotononis s. l. legumes and their
associated rhizobia appear to follow the latter model, where nodulation gene lineages
are related to the microsymbiont chromosomal background. It is not possible, from
the data on nodA presented in this study, to trace the lines of descent of the symbiotic
genes that allow nodulation of Lotononis s. l. hosts, or say whether they came from a
single, or multiple, ancestors. There appears to be no evidence for a “Lotononis s. l.”
or a “Listia” nodulation genotype as such, although the very high nodA sequence
identity seen within, rather than between, the Lotononis s. l.-associated
Bradyrhizobium, Ensifer, M. nodulans and pigmented Methylobacterium strains
argues for selection pressure on nod genes and possible HGT within those
chromosomal backgrounds. The collection and study of more rhizobial isolates from
Leobordia and Lotononis s. l. species and the acquisition of complete nodA
sequences for all Lotononis s. l. rhizobia could provide more robust phylogenies of
symbiotic loci and a greater understanding of the evolution of symbiotic relationships
within Lotononis s. l. species.
If there is no evidence for selection pressure on Lotononis s. l.-associated
rhizobial for a particular nodulation genotype, what other factors could account for
the range of microsymbiont diversity and specificity seen in this group of host
plants?
2.4.5 Environmental factors that may be effectors of diversification in
rhizobia associated with Lotononis s. l. species
Rhizobial diversity in Lotononis s. l. can also be looked at in the context of the
distribution and evolution of this clade of legume hosts within the Crotalarieae tribe.
Crotalarieae are monophyletic and sub-endemic to Africa (Boatwright et al., 2008),
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with initiation of the crown clade of Crotalarieae dated to approximately 46.3 mya
(Edwards & Hawkins, 2007). Lotononis s. l. has been included in the Crotalarieae
Cape floral clade, which is defined as a clade that is considered to originate in the
remarkably species-rich Cape Floristic Region (CFR) (Edwards & Hawkins, 2007).
The species density of Lotononis s. l. in southern Africa is also notably high (van
Wyk, 1991). In looking at the radiations and subsequent speciation of the Cape
clades, Cowling et al. (2009) have advanced the theory that topo-edaphic
heterogeneity of the CFR, along with relative climatic stability, are factors that
contribute to the exceptional diversity seen in this region. Another consequence of
this edaphic heterogeneity may be that legume species are faced with selecting
symbiotic partners of diverse genotypes, due to variable rhizobial saprophytic
competence across different eco-regions.
Rhizobia are not obligate symbionts (Young & Johnston, 1989). Each
generation of legume hosts is required to select its microsymbiont partner from a
pool of free-living soil rhizobia (Martínez-Romero, 2009). Rhizobial saprophytic
competence is thus important in determining which strains are available to be
selected by the plant host. Environmental selection implies that distinctive microbial
assemblages are maintained in different contemporary environments (Martiny et al.,
2006). In the case of the bacterial soil community, its composition is controlled
primarily by edaphic factors, in particular soil pH (Fierer & Jackson, 2006).
Rhizobial populations in the soil are also known to be affected by pH, in addition to
other factors such as soil fertility and clay content, temperature, rainfall and the host
plant distribution (Dilworth et al., 2001; Hirsch, 1996; Howieson & Ballard, 2004).
Populations of Bradyrhizobium have been shown to increase as soil pH declines
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(Slattery et al. 2004) and Rhizobium tropici and Mesorhizobium loti are noted acid
tolerant species; conversely E. meliloti (and other Ensifer species) are sensitive to
acid stress and more tolerant of alkaline conditions (Graham & Parker, 1964;
Graham, 2008; Herridge, 2008; Zahran, 1999). The Listia methylobacteria have been
found to be well adapted to acid, infertile soils (Diatloff, 1977; Yates et al., 2007).
Studies on diverse legumes have found that the microsymbiont genotype correlates
with eco-regions and edaphic factors (Bala & Giller, 2006; Diouf et al., 2007; Garau
et al., 2005; Han et al., 2009; Lu et al., 2009). In the present study, the number of
rhizobial strains is too small to attempt a correlation between soil pH and
microsymbiont genotype, but it is interesting to note that the soil pH of the sites
where the bradyrhizobial isolates were collected was on average lower than that of
the Ensifer collection sites (Table 2.1).
Similarly, the seasonally waterlogged habitat favoured by Listia species may
be a factor in their utilisation of Methylobacterium and Microvirga strains as
microsymbionts. Non-rhizobial species of these genera are known to inhabit aquatic
environments. Pink-pigmented methylobacteria are frequently found in fresh water
habitats (Green, 1992) and M. isbiliense, which is closely related to the rhizobial
methylobacteria (Figure 2.9), was described from strains isolated from Spanish
drinking water (Gallego et al., 2005). L. angolensis has a seasonally waterlogged
habitat but a more tropical distribution than other Listia species; thus it is interesting
to note that three of the five validly described non-rhizobial Microvirga species are
from thermal waters or (presumably) waterlogged rice field soil (Kanso & Patel,
2003; Takeda et al., 2004; Weon et al., 2010; Zhang et al., 2009).
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The nodulation of the hypocotyl that is seen in Listia species is also unusual in
legumes. Other accounts of hypocotyl nodulation appear to be restricted to
subspecies of Arachis hypogaea (Nambiar et al., 1982) and to semi-aquatic species
of Aeschynomene, which also form stem nodules (Boivin et al., 1997; Loureiro et al.,
1995). These legumes form aeschynomenoid nodules, however, with bacterial
infection proceeding via epidermal cracks created by emerging lateral roots, rather
than the lupinoid type seen in Listia species (Boogerd & van Rossum, 1997; Sprent,
2009). Arachis hypogaea and Aeschynomene species are nodulated by strains of
bradyrhizobia, some of which, in the case of the Aeschynomene microsymbionts,
may be pigmented, photosynthetic bacteria and/or lack canonical nodABC genes
(Miché et al., 2010; Van Rossum et al., 1995; Yang et al., 2005). Pigmentation is
rare in RNB: the photosynthetic bradyrhizobia and the Methylobacterium and
Microvirga strains that nodulate Listia species are the only pigmented rhizobia to be
described so far. The pigments found in the photosynthetic bradyrhizobia and in
Methylobacterium and Microvirga species have been identified as carotenoids
(Giraud et al., 2004; Jaftha et al., 2002; Kanso & Patel, 2003). In the photosynthetic
bradyrhizobia, carotenoids protect against oxidative stress and function as part of the
photosynthetic system, which may facilitate bacterial infection and contribute to
symbiotic effectiveness (Giraud et al., 2000; Giraud & Fleischman, 2004).
Carotenoids are also prevalent in organisms inhabiting the phyllosphere and aquatic
ecosystems, due to their photoprotective effect against ultraviolet radiation (Jacobs et
al., 2005; Laurion et al., 2002). The presence of carotenoids in pigmented rhizobia
may confer a selective advantage for bacterial survival on stems and hypocotyls, and
in aquatic habitats, as compared with non-pigmented, soil-dwelling rhizobia. It
should be noted, though, that other aquatic legumes such as Neptunia natans and
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Sesbania rostrata are nodulated by the non-pigmented rhizobia Devosia neptuniae
and Azorhizobium caulinodans, respectively (Dreyfus et al., 1988; Rivas et al.,
2002).
In summary then, the Lotononis s. l. clade is nodulated by a remarkable
diversity of rhizobia, with symbiotic associations that range from promiscuous
through to highly specific. Given the above, is there a hypothesis that can explain the
evolution of the symbiotic patterns seen in this group of legumes and their associated
microsymbionts?
2.4.6 Putative evolution of symbiotic patterns within Lotononis s. l.
Edaphic heterogeneity driven by geomorphic change in the early and late
Miocene (23.8–5.3 mya), and simultaneous climatic deterioration, are hypothesised
to have triggered the radiation of the Cape floral lineages (including the Lotononis s.
l. clade) and their subsequent speciation (Cowling et al., 2009; Edwards & Hawkins,
2007). The edaphic heterogeneity of this landscape would presumably also promote
the genotypic diversity of rhizobia available as potential symbiotic partners of these
legume species, due to the varying saprophytic competencies of different rhizobial
genera and strains (Graham, 2008).
It is proposed that in response to this, two specificity groupings have arisen in
Lotononis s. l. species. In the first group, Leobordea and Lotononis s. str. species are
more or less promiscuous and able to nodulate, with varying degrees of
effectiveness, with soil rhizobia that have a diversity of both chromosomal
backgrounds and symbiotic genes. In the second group, the adaptation of Listia spp.
to waterlogged habitats has consequently required the selection of microsymbionts
that are more specialised inhabitants of these environments. The comparative rarity
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of rhizobial species of Microvirga and Methylobacterium suggests that their capacity
for nodulation has been acquired through horizontal transfer of symbiotic genes from
other rhizobial genera.
The mechanisms of HGT are favoured in closely related microorganisms
(Ochman et al., 2000). This concept accords with the results of Wernegreen & Riley
(1999), who have proposed that HGT in rhizobia is restricted across major
chromosomal subdivisions, but supported among congeneric strains. A recent review
of rhizobial symbiovars also supports this concept, as the biovars were nearly always
found in species belonging to the same rhizobial genus (Rogel et al., 2011).
Nandasena et al. (2007) and Sullivan & Ronson (1998) have demonstrated that in
mesorhizobia, HGT between closely related strains can result in the rapid evolution
of symbiotic bacteria. In the Lotononis s. l. rhizobia, the 100% sequence identity of
the bradyrhizobial nodA genes and likewise the 100% sequence identity of nodA in
the Ensifer strains lend weight to the theory of HGT occurring among closely related
strains.
It is presumed therefore that HGT between distantly related bacteria is a rarer
event than that between closely related strains. That HGT between unrelated strains
does occur in rhizobia has been documented in a study of Brazilian soybean isolates,
where symbiotic genes from Bradyrhizobium japonicum inoculant strains were
transferred to a presumably more saprophytically competent indigenous Ensifer
fredii strain (Barcellos et al., 2007). Newly acquired genes require integration into
the existing cellular regulatory circuits (Masson-Boivin et al., 2009). Moulin et al.
(2004) have suggested that the low frequency of nodulation gene transfer between
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Bradyrhizobium and distant rhizobial genera implies that symbiotic genes from the
former require the correct chromosomal background to function. Interestingly, a
study of 165 sequenced microbial genomes shows that B. japonicum USDA110 is an
important “hub” for HGT, as it has one of the highest numbers of HGT partners
(Kunin et al., 2005).
Thus, in the proposed model of the evolution of symbiotic patterns in
Lotononis s. l., the radiation and subsequent speciation of ancestral plants has led to
transference of symbiotic genes from the originally associated rhizobia to diverse,
more saprophytically competent strains. A selective advantage is conferred upon
rhizobial strains that successfully integrate the symbiotic genes into their
chromosomal background and are able to nodulate the host plants. The symbiotic
genes may then be rapidly disseminated via HGT to closely related rhizobial
populations. Over time, the symbiotic genes evolve within each background, leading
to allelic variation.
Species within Leobordea and Lotononis s. str. appear to have maintained the
symbiotic promiscuity that Perret et al. (2000) consider to be the ancestral state. In
contrast, the adaptation of Listia species to waterlogged habitats appears to involve
the selection of microsymbionts that are more saprophytically competent in this
environment. The waterlogged habitat may restrict the number of other rhizobial
species that are available for HGT, in addition to the cellular mechanisms that restrict
gene flow in genetically divergent organisms (Papke & Ward, 2004), thus
contributing to symbiotic isolation for both the microsymbiont and the legume host.
In the Methylobacterium-Listia symbiosis, the extreme symbiotic specificity that has
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developed may be due to coevolution of the plant hosts and the rhizobia. Aguilar et
al. (2004) have previously suggested that coevolution occurs in the centres of host
genetic diversity for the symbiosis between Phaseolus vulgaris and Rhizobium etli.
The symbiosis of the more tropically adapted L. angolensis with rhizobial strains of
Microvirga appears to be a case of symbiont replacement. Such replacement has also
been documented in the Phaseoleae tribe, in which species of Rhizobium have
replaced Bradyrhizobium strains as the preferred microsymbionts in two Phaseolus
species (Martínez-Romero, 2009).
Symbiotic specificity, and the mechanisms that govern rhizobial infection and
nodule initiation have been studied extensively in a small number of legume hosts
(Goormachtig et al., 2004; Madsen et al., 2010; Oldroyd & Downie, 2008), but not
in Listia species. A first step might be to investigate the methods by which the
Methylobacterium and Microvirga rhizobia infect and nodulate their hosts. To this
end, the processes of infection and nodule initiation were studied in L. angolensis
and L. bainesii, and are detailed in Chapter 3.
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CHAPTER 3
Nodule structure in Lotononis s. l. species and infection and
nodule formation in Listia angolensis and Listia bainesii
Chapter 3
112
Chapter 3
113
3.1 Introduction
3.1.1 Modes of infection and nodulation in legumes
The route by which rhizobial infection proceeds and the shape and structure
of the resulting nodule are under the control of the host plant and have taxonomic
value (Corby, 1981; Lavin et al., 2001; Sprent & James, 2007). Two modes of
infection and several nodule types, characteristic of the different legume groups in
which they are found, have been described (Guinel, 2009; Sprent, 2007) (see also
Section 1. 7).
Within the Papilionoideae, rhizobial infection may proceed intracellularly via
root hair curling, with the bacteria confined to an infection thread (IT) before their
release into nodule primordium cells, or may be directly through the epidermis with
no ITs involved in nodulation (Sprent & James, 2007). The former is characteristic of
phaseoloid and hologalegoid legumes, while the latter is found in the more basal
genistoid and dalbergioid clades. The nodules of root-hair-infected legumes contain
central tissue that has a mixture of infected and uninfected cells. These nodules may
be either indeterminate, with a persistent meristem and a gradient of developmental
zones; or determinate (desmodioid), with a short-lived meristem and all cells at a
similar developmental stage (Sprent & James, 2007). The genistoid and dalbergioid
nodules, in which infection threads are absent, are characterised by a central mass of
uniformly infected tissue. Dalbergioid nodules are determinate and associated with a
lateral or adventitious root, while genistoid legumes have indeterminate nodules
(Lavin et al., 2001; Sprent & James, 2007). A variant of the indeterminate form is the
lupinoid nodule, in which lateral meristems encircle the subtending root. In the
genistoid tribe Crotalarieae, examples of indeterminate and lupinoid nodules are
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found in Crotalaria species and Listia species, respectively (Renier et al., 2011;
Yates et al., 2007).
Root hair infection is considered to be a feature of the more phylogenetically
advanced legumes, in which recognition of Nod factors (NFs) by LysM receptor-like
kinases in the epidermis of the root hair triggers a signalling pathway that controls
infection and nodule formation (Madsen et al., 2010; Oldroyd & Downie, 2008).
This mode of infection is believed to provide the legume with a tighter and more
selective control of bacterial passage through the epidermis (Madsen et al., 2010). In
contrast, it has been speculated that the promiscuous nodulation seen in Arachis and
Stylosanthes may be attributed to their crack entry mode of infection (Boogerd & van
Rossum, 1997).
The rhizobial infection pathway in Listia species has not been determined,
but the internal structure of the lupinoid nodules found in studied plants is that of a
typical genistoid legume, with a central mass of uniformly infected tissue (Yates et
al., 2007). Rhizobial infection in other genistoid legumes occurs via epidermal entry,
rather than by root hair curling (González-Sama et al., 2004; Tang et al., 1992;
Vega-Hernández et al., 2001). If the mode of infection in Listia species does indeed
occur via epidermal entry, then the Listia -Methylobacterium symbiosis represents an
interesting example of symbiotic specificity in legumes with a non root-hair-
mediated infection process.
Little is known of the molecular mechanisms that govern specificity and
signalling pathways in non-IT legumes. An examination of the molecular dialogue
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and signal pathways that underlie rhizobial infection and nodule morphogenesis in
the Listia –Methylobacterium and Listia -Microvirga symbioses is beyond the scope
of this study. An investigation of the infection route and the development of nodule
primordia would, however, be a useful first step in determining what mechanisms
may govern specificity in these symbioses. It is also worthwhile to study the internal
structure of the indeterminate nodules found in Lotononis s.l. species outside the
genus Listia, as this may provide further evidence as to whether nodule structure is a
useful taxonomic marker for the genistoid clade. For these purposes, light
microscopy was used to observe the processes of infection and nodulation in Listia
angolensis and Listia bainesii and to examine nodule sections from Lotononis s. l.
species featured in Chapter 2.
3.2 Materials and Methods
3.2.1 Legume hosts, bacterial inoculant strains and growth conditions
Internal nodule structure was examined for selected nodules of the Listia,
Leobordea and Lotononis s. str. host species featured in Chapter 2. The species
examined and their inoculant strains are listed in Table 3.1. The preparation of plant
material, inocula and growth conditions is described in Chapter 2.
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Table 3.1. List of host plants and rhizobial strains featured in this chapter.
Lotononis s. l.
genus and section*
Lotononis s. l.
species
Inoculant strain Nodule
phenotype
Listia L. angolensis Methylobacterium sp. WSM2598
Microvirga sp. WSM3557
Fix-
Fix+
L. bainesii Methylobacterium sp. WSM2598 Fix+
Leobordea
(Leptis)
L. mollis Methylobacterium nodulans
WSM2667
Fix+
Leobordea
(Leobordea)
L. platycarpa Ensifer meliloti WSM2653 Fix+
Leobordea
(Synclistus)
L. bolusii Bradyrhizobium sp. WSM2632 Fix+
Lotononis s. str. (Oxydium)
L. crumanina Ensifer meliloti WSM2653 Fix+
Lotononis s. str. (Cleistogama)
L. pungens Ensifer meliloti WSM2653 Fix+
*Based on the taxonomy of van Wyk (1991) and Boatwright et al. (2011).
To study infection and nodulation, L. angolensis (Accession number
2004CRSL69) and L. bainesii (cv. Miles) were inoculated with their respective
microsymbiont strains WSM3557 and WSM2598. Plants were grown in a naturally
lit, controlled temperature (maximum 24°C) glasshouse, in either pots (using an
axenic sand culture system (Howieson et al., 1995; Yates et al., 2007)) or in
gnotobiotic growth pouches (CYG Seed Germination Pouch, Mega International,
West St. Paul, MN, USA). For the sand culture system, the preparation of pots was
as previously detailed in the Materials and Methods for Chapter 2.
The methods for growing seedlings in growth pouches were based on
protocols described in Journet et al. (2001). Growth pouches were prepared by
adding 10 ml of 1x Fahräeus solution (modified from Vincent (1970) and containing
0.5 mM MgSO4, 0.7 mM KH2PO4, 0.8 mM Na2HPO4, 50 µM Fe-EDTA and 0.1 µg
each of MnSO4, CuSO4, ZnSO4, H3BO3 and Na2MoO4) to each pouch. The pouches
were wrapped in alfoil (to keep plant roots in the dark) and sterilised by autoclaving
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(121ºC, 20 min). After autoclaving and just prior to seeds being sown, 100 µl of 100
mM CaCl2 was added to each pouch, to give a final concentration of 1 mM CaCl2.
Seeds were prepared according to the protocols detailed in Chapter 2. For pot-grown
plants, the seeds were germinated in the dark at room temperature on water agar
(1.0% (w/v)) plates and aseptically sown into pots (six seeds per pot) when the
radicles were 1 – 3 mm in length. Inocula were prepared according to the protocols
detailed in Chapter 2 and applied after the seedlings had emerged (from five to ten
days after sowing). Selected pots of L. angolensis and L. bainesii seedlings were not
inoculated until the plants were 85 days old.
For growth pouches, shaking broth cultures (5 ml) were grown in TY medium
(28ºC, 200 rpm) to late log phase, then diluted with TY medium to an OD600nm of
0.5. Seeds were germinated as above. Three-day-old seedlings were then transferred
to sterile Petri dishes and incubated with 5 ml of the diluted broth cultures for one h,
to allow the bacteria to colonise the seedlings. The seedlings were then planted
aseptically in the growth pouches, which were then placed inside surface sterilised
boxes fitted with clear PVA plastic lids. Moistened paper towels were placed in the
boxes to maintain humidity and prevent drying out of the growth pouches, after
which the boxes were transferred to the glasshouse. At seven days after inoculation
(dai), a further 5 ml of sterile deionised water, 5 ml of 1x Fahräeus solution and 100
µl of 100 mM CaCl2 were added aseptically to each pouch.
3.2.2 Harvesting
Plants were harvested at regular intervals. At harvest, plants grown in growth
pouches were examined for nodulation. The roots of pot-grown plants were washed
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and sprayed with water to remove all soil and organic matter and similarly examined
for nodulation.
3.2.3 Nodule initial staining and sectioning, nodule sectioning and light
microscopy
Sampled plants were stained to highlight root and nodule initials, following a
protocol adapted from O’Hara et al. (1988). The plants were initially stored in a
fixative solution of 7% (v/v) acetic acid in absolute ethanol. After storage, whole
plants were cleared in a 10% (w/v) KOH solution for 4.5 h, followed by washing in
running water to remove any discoloration. They were then acidified in a solution of
0.25 M HCl for five min, before being placed in a 0.1% (w/v) Brilliant Green (No.
C086, ProSci Tech, QLD, Australia) solution for 30 min, followed by destaining
overnight in tap water.
Sections of fresh plants that included the hypocotyl and the upper portion of
the main root were excised and fixed overnight at 4°C in 3% (v/v) glutaraldehyde in
25 mM phosphate buffer (pH 7.0) in preparation for sectioning. Fixed material was
washed in three changes of phosphate buffer. The samples were dehydrated in a
rotator using a series of acetone solutions (30%, 50%, 70%, 90% and 100%) at 41ºC,
with two changes of each solution, each of 15 min duration. Dehydrated samples
were infiltrated with Spurr’s resin mixed with acetone, using an increasing
succession of resin concentrations (5%, 10%, 15%, 20%, 30%, 40%, 50%, 70% and
90%). The material was kept in each solution for a minimum of 2 h. Infiltrated
material was transferred into 100% Spurr’s resin, left at room temperature for 1–2 h
and then transferred into fresh 100% Spurr’s resin for 5–8 h at room temperature or
left overnight at 41ºC. Finally, in order to obtain good polymerisation, the material
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119
was embedded in fresh Spurr’s resin at 60ºC for 24 h. For light microscopy, 1 µm
sections were cut using a Reichert-Jung 2050 microtome with a glass knife. Sections
were dried onto glass slides at 60ºC and stained with 1% (w/v) methylene blue and
1% (w/v) azur II in 1% (w/v) sodium tetraborate (Richardson et al., 1960) for 3–5
min at room temperature. Stained sections were rinsed in water then dried. Excised
nodules from selected Lotononis s. l. host species (Chapter 2) were similarly
prepared for sectioning and microscopy. Specimens were examined under an
Olympus BX51 photomicroscope and photographed with an Olympus DP70 camera.
3.3 Results
3.3.1. Localisation of infection site
Pouch-grown seedlings of L. angolensis and L. bainesii were examined daily
under a stereomicroscope for nodule initials. They were also examined under a
photomicroscope for nodule and lateral root initials, after first being cleared and
stained with Brilliant Green. Seedlings grown in pots were examined under a
photomicroscope at seven, ten and 16 days after inoculation (dai) for nodule and
lateral root initials, after clearing and staining. In both species and in both growth
systems, nodule initials could be clearly distinguished at 16-17 dai. On seedlings of
both L. angolensis and L. bainesii, nodule initials occurred most frequently on the
plant hypocotyl. This was usually just above the root-shoot transition zone, which
was marked by large numbers of root hairs, whereas the hypocotyl was mostly
devoid of these (Figure 3.1). Nodule initials were also found further down the
taproot. The root hairs did not appear to be involved in the infection process, as they
were not seen on the epidermal surface of most of the hypocotyl nodule initials,
although two root hairs seem to have arisen from basal cells on the epidermal surface
of the nodule initial in Figure 3.1 e. Curled or deformed root hairs were not observed.
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Figure 3.1. Light microscopy of cleared and stained Listia angolensis and Listia bainesii
plants, showing lateral root and nodule initials. The root zone is marked by large numbers of
root hairs, which can be seen just below the hypocotyl.
a, b) L. angolensis 17 days after inoculation (dai).
c, d) L. bainesii 16 dai.
e) L. bainesii 16 dai, showing lateral root and nodule initial.
f) L. bainesii 16 dai showing lateral roots and nodule initials.
H, hypocotyl; LR, lateral root; LRI, lateral root initial; NI, nodule initial; RH, root hair; S,
stele.
Lateral root initials could also be seen along the hypocotyl and taproot, but
could be distinguished from nodule initials by their position adjacent to the plant
stele (Figure 3.1 e). In contrast, nodule initials arose in the outer cortex. Nodule
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121
initials in L. angolensis and L. bainesii were not associated with lateral roots, as they
did not form in the junctions of these roots and the main root, although some nodules
could be found close to emerging lateral roots (Figure 3.1 f).
3.3.2 Infection and nodule organogenesis in Listia angolensis and Listia
bainesii
Transverse sections of the shoot/root junction of L. angolensis and L. bainesii
seedlings showed root hairs, although these were not curled or deformed and
infection threads were not observed (Figures 3.2 and 3.3). In L. angolensis the nodule
primordium developed in the outer cortical layer immediately beneath an enlarged
epidermal cell (EE) (Figure 3.2 b). At six dai, there was a proliferation of cells in the
outer cortex directly under the enlarged epidermal cell. Both anticlinal and periclinal
cell division was observed, and cells in the infection zone had prominent nuclei and
dense cytoplasm. There was no simultaneous division of pericycle cells; rather, cell
division appeared to spread from the outer to the inner cortex. The orientation of the
plane of division of inner cortex cells was more commonly anticlinal. A similar
pattern of nodule development was observed in L. bainesii. At six dai, a cluster of
newly divided cells with prominent nuclei had formed in the outer cortex (Figure 3.3
a and b). At ten dai, the central tissue of the nodule primordium was uniformly
infected with bacteria that did not appear to have differentiated into bacteroids
(Figure 3.3 c, d and e). Infection pockets, caused by the collapse and death of cortical
cells, were not observed in either L. angolensis or L. bainesii.
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122
Figure 3.2. Light micrographs of Listia angolensis hypocotyl sections (transverse to the
primary root axis) showing nodule primordia development after inoculation with WSM3557.
a) Whole section, six days after inoculation.
b) Higher magnification of the same section.
c) Section from the same plant (resin has not been etched). Cells in the nodule primordium
have divided repeatedly and show cytoplasmic staining and prominent nuclei (arrow). Cell
division can also be seen in the inner cortex (arrowheads).
C, cortex; E, epidermis; EE, enlarged epidermal cell; EN, endodermis; NP, nodule
primordium; RH, root hair; S, stele; X, xylem
200 µm
RH
E
NP
C X
S
C
EN
EN
S
C
C
EE NP NP
RH
b c
a
50 µm 50 µm
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123
Figure 3.3. Light micrographs of Listia bainesii hypocotyl sections (transverse to the
primary root axis) showing nodule primordia development after inoculation with WSM2598.
a) Whole section, six days after inoculation (dai) and b) at higher magnification. Cells in the
nodule primordium have divided repeatedly and show cytoplasmic staining and prominent
nuclei (arrow). Cell division can be seen in the inner cortex (arrowhead).
c) Nodule primordium, ten dai and d) section from the same nodule (resin has not been
etched). Cells in the nodule primordium are uniformly infected with bacteria. Another locus
of infection is visible in the bottom left hand corner.
e) The same section at higher magnification, showing details of rhizobia (arrow) infecting
nodule primordium cells.
C, cortex; E, epidermis; EN, endodermis; NP, nodule primordium; R, rhizobia; S, stele
50 µm
200 µm 50 µm
EN S
E
C
C
NP
NP NP
S
NP
S
d
b a
c
50 µm
50 µm
e
R
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124
3.3.3 Localisation of the infection site in Listia angolensis and Listia
bainesii plants inoculated at 85 days old
Uninoculated 85-day-old L. angolensis and L. bainesii plants were not much
larger than young seedlings, possibly due to the small size of Listia spp. seeds. This
growth pattern is typical of uninoculated L. angolensis and L. bainesii plants, in
which leaves grow, but the internodes of the stem remain very short, resulting in a
growth form similar to that of a rosette plant. The nodulation pattern in the plants
inoculated at 85 days old (Figure 3.4) did not appear to differ from that observed in
seedlings inoculated at seven days old (Figure 2.15 a, b; Chapter 2). The location of
the infection site did not change, as collar nodules developed on the hypocotyl and
main root, and occasionally on lateral roots, just as in the seedlings inoculated at
seven days old.
Figure 3.4. Nodulation pattern in Listia angolensis and Listia bainesii plants inoculated at 85
days old and harvested 33 days after inoculation.
a) L. angolensis
b) L. bainesii.
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125
3.3.4 Internal structure of Lotononis s. l. nodules
Nodules taken from ten-week old inoculated Listia, Leobordea and Lotononis
s. str. host species (Chapter 2) were sectioned and examined by light microscopy to
compare their internal structure. In all cases, effective nodules contained uniformly
infected central tissue with no uninfected interstitial cells, whether from lupinoid
nodules of L. angolensis and L. bainesii (Figure 3.5 a – f) or from the indeterminate
nodules of Lotononis s. l. species outside the genus Listia (Figure 3.6 a – f). The
degree of vacuolation of bacteroid-containing cells appeared to vary between species
(Figure 3.5 a and f; Figure 3.6 a and d). In some nodules (Figures 3.5 b and 3.6 f),
amyloplasts could be seen in the cortical uninfected cells next to the nodule central
tissue. L. angolensis inoculated with WSM2598 produced ineffective nodules in
which the bacteria were not differentiated into nitrogen-fixing bacteroids (Figure 3.5
c – e). Interestingly, bacteroids of WSM2598 in nitrogen-fixing nodules of L.
bainesii (Figure 3.7 a) appeared to be far more swollen than the WSM3557
bacteroids in effective L. angolensis nodules (Figure 3.7 b).
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126
Figure 3.5. Light microscopy of sections of ten-week-old Listia angolensis and Listia
bainesii nodules. In both species, the central tissue (*) of effective nodules contains infected
cells only.
a, b) Nitrogen-fixing nodule of L. angolensis inoculated with WSM3557. Amyloplast
structures (arrow) can be seen in the peripheral cortical uninfected cells.
c, d, e) Non-fixing nodule of L. angolensis inoculated with WSM2598.
f) Nitrogen-fixing nodule of L. bainesii inoculated with WSM2598.
S, stele; VB, vascular bundle
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127
Figure 3.6. Light microscopy of sections of ten-week-old Leobordea spp. and Lotononis s.
str. spp. nodules. In all nodules, the central tissue (*) of effective nodules contains infected
cells only.
a) Leobordea bolusii inoculated with WSM2632.
b) Leobordea mollis inoculated with WSM2667.
c) Leobordea platycarpa inoculated with WSM2653.
d) Lotononis crumanina inoculated with WSM2653
e, f) Lotononis pungens inoculated with WSM2653. Structures resembling amyloplasts
(arrow) can be seen in the peripheral cortical uninfected cells.
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128
Figure 3.7. Light microscopy of sections of ten-week-old nodules, showing bacteroids.
a) Listia bainesii inoculated with WSM2598.
b) Listia angolensis inoculated with WSM3557.
3.4 Discussion
Nodulation in L. angolensis and L. bainesii occurs mainly on the hypocotyl
and taproot. Remarkably, the infection site appears to be independent of the age of
the plant at the time of inoculation, as the same pattern of nodulation was observed
for plants inoculated at 85 days old as those inoculated at seven days old. This is in
contrast to other studies of both root-hair-curl- and epidermally-infected legumes.
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129
Rhizobial invasion in legumes infected via root hair curling is confined to a small
zone of root hairs that have nearly finished growing (root hair zone II) and are
susceptible to deformation and infection (Bhuvaneswari et al., 1980, 1981; Gage,
2004; Heidstra et al., 1994; Maunoury et al., 2008). In vetch, about 80% of zone II
root hairs were deformed three hours after exposure to NodRlv factor (Heidstra et al.,
1994). Similarly, rhizobial invasion in the epidermally infected genus Lupinus
appears to be localised within a transient zone. In Lupinus angustifolius, nodules
appeared most frequently in the region between the smallest emergent root hairs and
the root tip at the time of inoculation. Epidermal root cells aged 13 h or over
appeared not to be infected (Tang et al., 1992). Rhizobia preferentially accumulate in
the root hair portion of the main root of Lupinus albus seedlings, c. 10–15 mm from
the root tip (González-Sama et al., 2004).
Although infection and nodule formation in dalbergioid and genistoid
legumes does not proceed via root hair curling and infection thread development,
root hair deformation and transient infection threads have been observed in some
species belonging to these clades. Bradyrhizobium strain BTA-1 induced root hair
deformation and curling, and the development of infection threads (that subsequently
aborted), in the genistoid legume tagasaste (Cytisus proliferus; formerly
Chamaecytisus proliferus) (Vega-Hernández et al., 2001). Deformation and curling
of root hairs has been observed in Arachis, Stylosanthes and Lupinus species
(Boogerd & van Rossum, 1997; Chandler et al., 1982; González-Sama et al., 2004;
Łotocka et al., 2000). Structures similar to short, wide infection threads have
occasionally been found in L. angustifolius (Tang et al., 1992) and L. albus nodules
(James et al., 1997). Infection threads were not found during the nodulation process
Chapter 3
130
in the Listia species (this work) and similarly were not seen in Lupinus albus
nodulation (González-Sama et al., 2004) or in the invasion zone of Crotalaria
podocarpa nodules (Renier et al., 2011).
Root hair cells seem to be important in the initial infection process in some
dalbergioid and genistoid legumes. Rhizobia invade the middle lamella between
enlarged basal hair cells and adjacent root hair or epidermal cells in Arachis and
Lupinus species (Boogerd & van Rossum, 1997; González-Sama et al., 2004; Tang et
al., 1992). In Arachis, rhizobial infection is associated with tufts of root hairs that
arise in the axils of young lateral roots and non-nodulation is strongly correlated with
an absence of these hairs. Successful infection is restricted to penetration sites where
enlarged root hair basal cells are found (Boogerd & van Rossum, 1997; Uheda et al.,
2001). Conversely, in Aeschynomene afraspera stem-nodulation sites, root hairs are
not observed in the epidermis of the lateral root primordium (Alazard & Duhoux,
1990). Further study is required to determine whether or not root hair cells are
involved in rhizobial infection of the hypocotyl in Listia angolensis and Listia
bainesii. It could be hypothesised that in these Listia species, the ability of the
hypocotyl to remain potent for infection may be correlated with the putative lack of a
role for root hair cells.
Nodule organogenesis in Listia angolensis and Listia bainesii appears to
follow a process similar to that observed in Lupinus albus and Lupinus angustifolius
(González-Sama et al., 2004; Tang et al., 1992). In the Lupinus studies, rhizobia
penetrate intercellularly at the junction between epidermal cells and subsequently
invade a cortical cell immediately beneath the epidermis. The infected cell divides
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131
rapidly, as do the rhizobia within the cell, to produce a nodule with characteristic
uniformly infected cells in the central infected zone (Fernández-Pascual et al., 2007).
The incidence of cell division spreads progressively from the infection focus towards
the inner cortex, with cellular division in the deeper cortical layers occurring several
days after the initial infection (González-Sama et al., 2004; Tang et al., 1992). The
same pattern was seen for nodule morphogenesis in the Listia species. The
proliferation of outer cortical cells formed the nodule primordium and cellular
division subsequently spread to the inner cortex. In this, the lupinoid nodule
primordium is more similar to that of the desmodioid nodule, in that desmodioid
nodule initiation (seen for example in Lotus japonicus) is typically found in the first
outer cortical layer, immediately beneath the primary infection site (Guinel, 2009;
Szczyglowski et al., 1998). Conversely, both aeschynomenoid and indeterminate
hologalegoid nodules arise from divisions in the root inner cortex (Alazard &
Duhoux, 1990; Guinel, 2009; Voroshilova et al., 2009). As well, in the aquatic
robinioid legume Sesbania rostrata (where infection can occur intercellularly at
lateral root bases) and in the aeschynomenoid genera Aeschynomene and
Stylosanthes, the infection process is associated with the collapse and death of
cortical plant cells (Alazard & Duhoux, 1990; Chandler et al., 1982; D'Haeze et al.,
2003). In contrast, cell death does not appear to occur during the nodulation process
in Listia species.
In keeping with the morphology found in typically genistoid nodules (Sprent,
2009) nodule sections of all Lotononis s. l. host species, whether of lupinoid or
indeterminate nodules, had a mass of central, uniformly infected tissue, with no
uninfected interstitial cells. It is interesting to note that nitrogen-fixing bacteroids of
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132
WSM2598 within L. bainesii nodules should appear to be more swollen than those of
WSM3557 within L. angolensis nodules. It has been suggested that swollen
bacteroids may confer advantages to the host, such as enhanced nitrogen fixation
capability (Mergaert et al., 2006; Oono & Denison, 2010). The symbiosis between
WSM2598 and L. bainesii is highly effective (Yates et al., 2007), but further study
would be required to determine if a relationship between bacteroid size and
effectiveness exists in this system.
In summary, the mechanism of morphogenesis in lupinoid nodules, in which
cell division is initiated in the outer cortex beneath the infection site, appears to be
the same for both genistoid Lupinus and crotalarioid Listia species. The salient
characteristics of infection and nodule organogenesis in Listia species – epidermal
infection, the lack of infection threads and a central mass of uniformly infected tissue
- are consistent with the nodulation process observed in the dalbergioid and genistoid
clades. The ability of the root and hypocotyl epidermal cells to remain potent for
infection is an interesting feature of the infection process in Listia, and one that
merits further study.
Chapter 4
133
CHAPTER 4
Characterisation and description of novel Listia angolensis
and Lupinus texensis rhizobia
Chapter 4
134
Chapter 4
135
4.1 Introduction
The light-pink-pigmented rhizobial strains WSM3674 and WSM3686,
isolated from nodules of Zambian Listia angolensis, have previously been identified
as belonging to a novel lineage of root nodule bacteria (Yates et al., 2007). The 16S
rRNA gene sequences of these strains showed them to be closely related to rhizobia
that specifically nodulate Lupinus texensis plants growing in Texas, USA (Andam &
Parker, 2007). According to Andam & Parker’s phylogenetic tree, the closest relative
of the Listia angolensis and Lupinus texensis strains was Microvirga flocculans
TFBT (previously Balneimonas flocculans), a strain isolated from a Japanese hot
spring (Takeda et al., 2004; Weon et al., 2010).
The results of the present study have shown that strain WSM3557, an
effective isolate of Lupinus angolensis, is also closely related to WSM3674,
WSM3686, the Lupinus texensis strains and M. flocculans (Chapter 2). These strains
all grouped, with high bootstrap support, within the genus Microvirga (Chapter 2,
Figure 2.9), a member of the Methylobacteriaceae family and a bacterial lineage in
which no strain has previously been identified as a legume symbiont. Microvirga was
first described by Kanso & Patel (2003) from a single strain (FaiI4T) isolated from a
bore sample of geothermal waters from the Australian Great Artesian Basin and
given the name Microvirga subterranea. FaiI4T
is thus the type strain of the genus.
The remaining described species are Microvirga guangxiensis (strain 25BT), obtained
from rice field soil in Guangxi Province, China (Zhang et al., 2009) and Microvirga
aerophila (5420S-12T) and Microvirga aerilata (5420S-16
T), which were isolated
from atmospheric samples taken in Suwon region, Republic of Korea (Weon et al.,
2010).
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136
The availability of six authenticated N2-fixing Listia angolensis strains in the
WSM Culture Collection (Chapter 2), together with 28 isolates from Lupinus
texensis, presented an opportunity to develop polyphasic descriptions of this novel
group of root nodule bacteria. A collaboration to achieve this aim comprised:
a) Professor Matt Parker at the State University of New York (SUNY),
responsible for most of the sequencing of rRNA, housekeeping, nod and nif genes
and the subsequent construction of phylogenetic trees.
b) Professor Anne Willems and her student Ms Sofie De Meyer at the
University of Ghent, included in the collaboration for their expertise in DNA: DNA
hybridization, determination of G + C content and cellular fatty acid analysis.
c) Phenotypic data and additional DNA sequences, obtained by Ms Julie
Ardley at the Centre for Rhizobium Studies and detailed in this chapter.
Together, the data provide a basis to validly name and describe these novel
rhizobial species of Microvirga.
Phenotypic studies were conducted initially on all Listia angolensis strains.
More detailed phenotyping was subsequently performed on the putative type strains
WSM3557T and WSM3693
T. WSM3557
T was chosen as it was the most effective
strain for nodulation and nitrogen fixation on L. angolensis (Chapter 2), while the
phenotypic and molecular studies performed on WSM3693T identified it as being
sufficiently different to warrant consideration as a separate species. Lut5 and Lut6T
were chosen as representative of the Lupinus texensis isolates because of the body of
phenotypic and molecular data already obtained for these strains (Andam & Parker,
Chapter 4
137
2007), and Lut6T, as a putative type strain, was subsequently used for additional
phenotyping.
4.2 Materials and Methods
4.2.1 Bacterial strains and culture conditions
The strains used in this study are listed in Table 4.1. The proposed type
strains Lut6T (= LMG26460
T; = HAMBI3236
T), WSM3557
T (= LMG26455
T, =
HAMBI3237T) and WSM3693
T (= LMG26454
T, = HAMBI3238
T) have been
deposited in the BCCM/LMG and HAMBI Culture Collections. All strains were
stored at -80ºC as 12% (v/v) glycerol/media stocks. Strains were routinely
subcultured on modified ½ lupin agar (½ LA) (Yates et al., 2007), on ½ LA with
succinate replacing glucose and mannitol as a carbon source, or on TY agar
(Beringer, 1974). Broth cultures were grown in TY or ½ LA. Plate and broth culture
was grown at 28ºC or 37ºC and shaking cultures were incubated on a gyratory shaker
at 200 rpm.
4.2.2 DNA amplification and sequencing
4.2.2.1 Amplification and sequencing of the 16S rRNA gene
Amplification and sequencing of the WSM3557T 16S rRNA gene has been
detailed in Chapter 2. Nearly full length PCR amplification and sequencing of the
16S rRNA gene from WSM3693T was performed using the universal eubacterial
primers FGPS6 and FGPS1509 (Nesme et al., 1995; Normand et al., 1992) and
internal primers designed by Yanagi & Yamasato (1993) and Sy et al. (2001),
according to the protocols given in Chapter 2.
Chapter 4
138
Table 4.1. List of strains used in this chapter.
Strain Synonym Host Geographical source
(Collector)
Reference or
source
WSM3557T
CB1322
LMG26455T
Listia angolensis
Chipata (Fort
Jameson), Zambia (Verboom)
Eagles & Date
(1999)
WSM3673 CB1321 Listia angolensis
Chipata (Fort Jameson), Zambia
(Verboom)
Eagles & Date (1999)
WSM3674 CB1323 Listia angolensis
Chipata (Fort Jameson), Zambia
(Verboom)
Eagles & Date (1999)
Yates et al (2007)
WSM3675 CB2406 Listia angolensis
Mbale,Zambia
(Verboom)
Eagles & Date
(1999)
WSM3686 CB1297 Listia angolensis
Chipata (Fort Jameson), Zambia
(Verboom)
Eagles & Date (1999)
Yates et al (2007)
WSM3693T CB1298
LMG26454T
Listia angolensis
Chipata (Fort
Jameson), Zambia
(Verboom)
Eagles & Date
(1999)
Lut5 Lupinus texensis
Texas, USA
(Parker)
Andam & Parker
(2007)
Lut6T LMG26460
T Lupinus
texensis Texas, USA
(Parker)
Andam & Parker (2007)
Reference strains
Strain Host Comments Reference or
source
Bacillus subtilis Hydrolyses starch
Produces acetoin
Murdoch collection
Escherichia coli DH5α Negative control for
antibiotic tests
Bethesda Research
Laboratories (1986)
Escherichia coli KS272 Facultative anaerobe Guzman et al. (1995)
Mesorhizobium cicerae WSM4114
Biserrula pelecinus
SmR derivative of
WSM1497
Nandasena et al. (2007)
Mesorhizobium sp WSM4116
SpR derivative of CJ4 Sullivan et al. (1998)
Methylobacterium sp
WSM2598
Listia bainesii
Pigmented Yates et al. (2007)
Pseudomonas fluorescens
Obligate aerobe Murdoch collection
Ensifer fredii NGR234 Lablab purpureus
RifR Trinick (1980)
Ensifer medicae WSM419
Medicago murex
AmpR, Cm
R, Nal
R W. Reeve (unpublished data)
Ensifer medicae MUR2110
Medicago murex
Derivative of WSM419
GmR, Km
R, Tc
R
G. Garau
(unpublished data)
Chapter 4
139
Searches for sequences with high sequence identity to the sample 16S rDNA
were conducted using BLASTN (Altschul et al., 1990) against sequences deposited
in the National Centre for Biotechnology Information GenBank database. Genetool
Lite was used to align and calculate the pairwise percent sequence identity of a 1396
bp fragment of the 16S rRNA gene from the Listia angolensis and Lupinus texensis
strains and the Microvirga species type strains. A phylogenetic tree was constructed
with MEGA version 4.0 (Tamura et al., 2007), using the neighbour-joining (NJ)
method (Saitou & Nei, 1987) and the Maximum Composite Likelihood model, and
bootstrapped with 1000 replicates.
4.2.2.2 Amplification and sequencing of the nodA gene
Amplification and sequencing of the WSM3557T nodA gene has been detailed
in Chapter 2. Amplification and sequencing of the nodA gene from WSM3693T was
performed using the nodA primers developed by Haukka et al. (1998) and according
to the protocols given in Chapter 2.
4.2.3 Phenotypic characterisation
4.2.3.1 Morphology
Colony morphology was studied on ½ LA plates. Strains were also assessed
for growth on nutrient agar. Gram staining was performed according to standard
methods (Vincent, 1970). Motility of exponential phase broth cultures was observed
using a light microscope and the hanging drop method. To try to induce motility in
the Lut5 and Lut6T strains, they were also grown, using a method modified from
Bowra & Dilworth (1981), on JMM minimal media plates (O'Hara et al., 1989)
containing 0.1 mM of succinate as a carbon source, 0.05 % (w/v) yeast extract, 0.1
mM EDTA and 0.3% agar. One drop of 0.3 mM MgSO4 solution was applied to the
Chapter 4
140
edge of the resulting two day old culture and the cells resuspended by gentle
pipetting, then examined for motility as previously described. The motile isolate
WSM3557T served as a positive control.
For electron microscopy, resuspended cells were collected from overnight ½
LA slopes to which 100 µl of sterile deionised (DI) water had been added. Scanning
electron microscope (SEM) samples were prepared from aliquots of two day old
plate culture resuspended in sterile reverse osmosis deionised (RODI) water. Strains
were examined for spore formation by light microscopy after staining of stationary
phase broth and plate cultures with malachite green (Beveridge et al., 2007).
Stationary phase cultures were also heated to 70ºC for 10 min, and then reinoculated
onto fresh media and observed for growth.
4.2.3.2 Extraction of pigments
Plate cultures of WSM3557T and Lut6
T were incubated in a 28ºC growth
cabinet fitted with a Sylvania T8 F15W/GRO lamp that emitted radiation in the blue
and red spectrum. The cultures were grown for nine days in total, with light supplied
for 15 h per day for the first six days and the cultures subsequently grown without
light. Plate culture (0.1 ml volume) was resuspended in 0.89% (w/v) saline, and
centrifuged at 10, 000 x g for one minute to collect the pellet. Pigments were
extracted with aliquots (3 x 1 ml) of a 7:2 (v/v) acetone: methanol mix at 0ºC under
dim light. Absorption spectra of the extracts were recorded from 400-850 nm with a
Shimadzu UVmini-1240 scanning spectrophotometer and compared with the
absorption spectrum obtained for the pink-pigmented methylobacterial strain
WSM2598 (Table 1) grown and extracted under the same conditions.
Chapter 4
141
4.2.3.3 Temperature range
The growth of strains was assessed over a range of temperatures (10-50ºC, at
intervals of 5ºC, with 1ºC intervals from 38-46ºC) by resuspending a loopful of fresh
plate culture in ½ LA broth and streaking the resulting culture onto ½ LA plates.
Plates were examined for growth up to 15 days after streaking.
4.2.3.4 Optimal growth temperature
Cultures were grown in TY broths to mid-log phase, and then subcultured
into duplicate 50 ml TY broths in 250 ml conical flasks to give an initial optical
density at OD600nm of approximately 0.05. The flasks were incubated in a shaking
water bath (200 rpm) pre-set to the desired temperature and growth was measured by
reading the OD600nm at regular intervals.
4.2.3.5 Growth curves and determination of mean generation time
Cultures were incubated at the optimum temperature, with growth measured as
per Section 4.2.3.4 and the results plotted on a log/linear graph. Mean generation
time was determined from reading the graph during the exponential growth phase;
i.e. when the logarithm of absorbance against time produced a straight line.
4.2.3.6 Determination of sodium chloride and pH tolerance
Inocula were prepared by resuspending a loopful of fresh plate culture in ½
LA broth and incubating the cell suspension at 28ºC for 24 h to obtain stationary
phase cultures. Aliquots (1 ml) were standardised to an OD600nm of 1.0 by the
addition of ½ LA broth and serial dilutions made. An aliquot of 10 µl of the 10-1
, 10-
3, 10
-5 and 10
-6 (or 10
-7) dilutions was plated onto ½ LA media containing 0.0, 0.01,
0.5, 1.0, 1.5, 2.0, 2.5 or 3.0% (w/v) NaCl as shown in Figure 4.1. Plates were
incubated at 28ºC and examined for growth daily, up to 6 days.
Chapter 4
142
Figure 4.1. Layout of aliquots of culture on NaCl plate.
To determine the ability of the strains to grow over a range of pH values, TY
plates were prepared in the following manner: Universal indicator (Vogel, 1962) (5
ml l-1
) was added to the TY medium and the following buffers, at 20 mM working
concentration, were added for the pH values indicated:
MES (2-[N-morpholino]-ethane-sulfonic acid; pKa = 5.96): pH 5.5, 6.0
HEPES (4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid; pKa = 7.31): pH
7.0, 8.0, 8.5
CHES (N-Cyclohexyl-2-aminoethanesulfonic acid; pKA = 9.07): pH 9.00, 9.5,
10.0
Homopipes (Homopiperazine-n,n'-bis-2- (ethanesulfonic acid); pKa = 4.55):
pH 4.0, 4.5 and 5.0
The pH was adjusted by adding HCl or NaOH to the media. Agar (15 g l-1
(w/v) was added prior to autoclaving. For media at pH 4.0, 4.5 and 5.0, agarose was
used as the setting agent, as agar plates did not set firmly enough at these values.
Media were prepared by combining separately autoclaved solutions of agarose and
10-1
10-3
10-5
10-7
Strain 1 Strain 2
10-1
10-3
10-5
10-7
Strain 1 Strain 2
Chapter 4
143
medium, as autoclaving the medium with agarose was found to affect the final pH.
Aliquots of culture were plated and examined for growth as for NaCl plates.
4.2.3.7 Anaerobic growth
Loopfuls of four day old plate culture were resuspended in 0.89% (w/v)
saline, standardised to an OD600nm of 1.0 and serial dilutions made. Aliquots (100 µl)
of the 10-5
dilutions were spread on plates of modified Hugh & Leifson’s medium
(Hugh & Leifson, 1953) containing either glucose or pyruvate as a carbon source.
One set of plates was placed in an anaerobic jar (BBL GasPac 100 Non-vented
system), then two anaerobic generators (bioMérieux GENbox anaer, Ref 96 125) and
an anaerobic indicator strip (bioMérieux, Ref 96 118) were added and the jar sealed.
The other set of plates was grown aerobically. Plates were incubated at 28ºC and
examined for growth after ten days. Escherichia coli strain KS272 and Pseudomonas
fluorescens (Table 4.1) served as positive and negative controls respectively.
4.2.3.8 Antibiotic sensitivity
Intrinsic antibiotic resistance was assessed for nine antibiotics at the
following concentrations: ampicillin (50 and 100 µg ml-1
), chloramphenicol (10, 20
and 40 µg ml-1
), gentamycin (10, 20 and 40 µg ml-1
), kanamycin (50 and 100 µg ml-
1), nalidixic acid (50 and 100 µg ml
-1), rifampicin (50 and 100 µg ml
-1),
spectinomycin (50 and 100 µg ml-1
), streptomycin (50 and 100 µg ml-1
) and
tetracycline (10 and 20 µg ml-1
). Aliquots of culture were pipetted onto ½ LA plates
containing antibiotics and examined for growth as per Section 4.2.2.4. Growth was
compared with that of cultures inoculated onto TY plates devoid of antibiotics, and
with positive and negative bacterial controls (Table 4.1).
Chapter 4
144
4.2.3.9 Substrate utilisation
4.2.3.9.1 Utilisation of sole carbon sources in Biolog GN2 plates
GN2-MicroPlates (Biolog Inc, CA, USA) were used to assess the ability of
the strains to utilise sole carbon sources. The Biolog GN2 microplate consists of 96
wells containing 95 carbon sources and one blank well and is based on the catabolic
production of NADH, which reduces a tetrazolium dye and results in a colour change
from clear to purple in the microplate well (Bochner, 1989). In general, the
manufacturer’s instructions for preparation and reading of the samples were
followed, according to the guidelines recommended by Biolog for Ensifer meliloti
strains. R2A agar (Reasoner & Geldreich, 1985), was used in place of the standard
Biolog Universal Growth (BUG) agar, as the strains grew poorly on the latter.
Fresh plate culture was streaked onto R2A agar plates, incubated for 24 h at
37ºC then resuspended in 15 ml of Biolog GN/GP Inoculation Fluid (IF) to a
concentration of 85% ± 2% transmittance, as measured on a spectrophotometer.
Aliquots (150 µl) of the suspension were added to each well in duplicate microplates
and incubated in a sealed container at 37ºC. Moistened paper towels were placed in
the container to minimise evaporative loss from the wells. Colour development was
monitored and microplate readings taken at 40, 72, 96 and 137 h using a Biorad 680
microplate reader with 595nm filter. The mean of the duplicate raw absorbance value
for each well was expressed as a positive, negative or partial reaction, based on the
cutoff values given for the Biorad software.
4.2.3.9.2 Utilisation of substrates in Biolog Phenotype Microarray plates
Additional phenotyping tests were performed on the provisional type strains
WSM3557T and Lut6
T. Catabolism of carbon, nitrogen, phosphorus and sulfur
Chapter 4
145
compounds was assessed using Biolog Phenotype Microarray (PM) microplates
(panels PM1- 4 respectively). The PM technology is a high-throughput system that
allows global analysis of cellular phenotypes (Bochner et al., 2001) The tetrazolium
dye-based methodology is similar to that of the GN2 plates, but is a more sensitive
system and uses a much wider range of substrates. Testing was performed by Biolog
PM Services (http://www.biolog.com/PM_Services.html) using the following
protocol: The strains were grown on R2A agar prior to being suspended in
inoculation fluid containing vitamin B12, biotin and dye mix G, with pyruvate as a
carbon source in the phosphorus and sulfur panels. Duplicate cell suspensions at a
concentration of 40% ± 2% transmittance were pipetted at 100 µl per well into the 96
well Biolog PM panels. Incubation at 37ºC and recording of phenotypic data over 56
h was performed by an OmniLog instrument. Utilisation of the substrate was scored
as being either positive or negative, or was given as raw absorbance values.
4.2.3.9.3 Growth on sole carbon substrates
Growth supplement requirements were determined using JMM broths with
succinate (20 mM) as the sole carbon source and NH4Cl (10 mM) replacing
glutamate as a nitrogen source. The media contained either no vitamins; the standard
vitamins added to JMM (biotin, thiamine and pantothenic acid); a vitamin solution
containing all B group vitamins, as described in Egli & Auling (2005) for growth of
Chelatococcus asaccharovorans (Appendix IV); the B group vitamin solution plus
casamino acids (0.01% (w/v)); or yeast extract (0.05% (w/v)). Fresh plate culture
was resuspended in JMM medium devoid of vitamins, and then added to duplicate 5
ml broths to a final OD600nm of 0.05. The broths were incubated at 28ºC with shaking
and observed for growth over six days. The minimal amount of yeast extract required
for growth was similarly determined by adding 0.05%, 0.01%, 0.005% or 0.001%
Chapter 4
146
(w/v) yeast extract to the media. Glassware used to grow cultures (McCartney
bottles) was soaked in a 10% (v/v) hydrochloric acid solution for at least 24 h and
rinsed twice in RODI water prior to use. Lids of McCartney bottles were wrapped
with parafilm prior to incubation to prevent contamination.
Strains were examined for growth on L (+) arabinose, D (+) cellobiose, β-D-
fructose, α-D-glucose, glycerol, D-mannitol, acetate, succinate (all at 20 mM
concentration), benzoate, p-hydroxybenzoate (both 3 mM), glutamate (10 mM),
methanol (0.5%, v/v) and ethanol (20 mM) as sole carbon sources in JMM medium
devoid of galactose and arabinose and with NH4Cl (10 mM) as a nitrogen source.
Glutamate (10 mM) was also added to JMM devoid of NH4Cl to determine the
ability of the strains to utilise glutamate as a nitrogen source. Stock solutions of the
carbon substrates (adjusted to pH 7.0 where necessary) were filter sterilised (0.22 µm
filter) and added to the autoclaved JMM medium prior to inoculation.
Inocula were prepared by growing fresh plate culture in 5 ml broths of JMM
medium containing sodium pyruvate (10 mM) as a carbon source and NH4Cl (10
mM) as a nitrogen source and supplemented with yeast extract (0.1% (w/v)).
Cultures were grown for 50 h to stationary phase, then centrifuged (20 800 x g for 30
s), washed twice with 0.89% (w/v) saline, resuspended in JMM medium devoid of
carbon source and added to duplicate 5 ml broths of JMM containing one of the
carbon substrates to a final OD600nm of 0.05. Inoculated culture media were incubated
for 14 days before a visual assessment was made. Two negative controls were used:
an uninoculated control containing JMM medium and various carbon sources, and a
control devoid of carbon substrate but containing bacterial inoculant. Growth on the
Chapter 4
147
carbon substrate was assessed as being no growth (OD600nm was the same as for the
minus carbon substrate control), poor (0.1 < OD600nm < 0.2), moderate (0.2 < OD600nm
< 0.5) or abundant (OD600nm > 1.0).
4.2.3.10 Biochemical characterisation
API –20E (bioMérieux) test strips were used to determine acid production
from sugars and utilisation of various substrates. Fresh plate culture was resuspended
in sterile RODI water containing either vitamin solution (Egli & Auling, 2005) or
yeast extract (0.005% (w/v)) for the Listia angolensis and Lupinus texensis strains,
respectively, and used to inoculate the strips. Strips were incubated at 28ºC for 40 h
and then read in accordance with the manufacturer’s instructions.
Oxidase activity was detected by applying fresh plate culture to filter paper
impregnated with a solution of 1% (w/v) tetra-methyl p-phenylene diamine HCl and
0.1% (w/v) ascorbic acid. Catalase activity was determined on fresh plate culture
using 3% (v/v) hydrogen peroxide solution. Determination of nitrate reduction was
performed by inoculating stationary phase TY broth culture into shaking TY broths
supplemented with KNO3 (1 g l-1
) and growing for 24 h at 28ºC. Nitrate reduction to
nitrite was revealed by adding 1 ml of 0.8% (w/v) sulphanilic acid in 5N acetic acid
to the culture, followed by the addition of 1 ml of 0.8% (w/v) 8-aminonaphthalene-2-
sulphonic acid (Cleve’s acid). The development of a red colour indicated the
presence of nitrite. Powdered zinc was added to solutions that remained colourless; a
subsequent development of red colour revealed residual nitrate, whereas the further
reduction of nitrite was demonstrated by the solution remaining colourless.
Determination of starch hydrolysis was performed on TY agar supplemented with
0.4% (w/v) soluble starch. Bacillus subtilis and Escherichia coli (Table 4.1) served
Chapter 4
148
as positive and negative controls respectively. Oxidative or fermentative catabolism
was determined using Hugh & Leifson’s basal medium (Hugh & Leifson, 1953),
with L-arabinose, α-D-glucose or pyruvate as a carbon source. Test tubes containing
5 ml of medium were stab-inoculated with fresh plate culture. Anaerobic conditions
were created by overlaying the stab culture with sterile paraffin and cultures were
examined for growth and colour change in the medium after incubation at 37ºC for
48 h. The Voges-Proskauer test for production of acetoin from glucose was
performed using Smith’s medium (Leone & Hedrick, 1954) supplemented with 0.5 g
l-1
yeast extract. Test tubes containing 10 ml of medium were inoculated with 100 µl
of fresh plate culture resuspended in 0.89% saline to an OD600nm of 1.0. The cultures
were grown at 28ºC for 42 h, then aliquots of culture (1 ml) were placed in a test
tube, to which was added 15 drops of 5% (w/v) α-naphthol in 100% ethanol followed
by 5 drops of 40% (w/v) KOH in RODI water. The samples were vortexed after the
addition of each reagent, then allowed to stand and monitored for colour
development. A red colour was scored as positive, pink was weakly positive and
amber was negative. B. subtilis and E. coli (Table 4.1) served as positive and
negative controls respectively.
4.2.4 Host range
The host range of WSM3557T, WSM3693
T, Lut5 and Lut6
T was determined
on taxonomically diverse legume hosts (Table 4.2) in glasshouse trials, using either a
closed vial or open pot system according to the methods given in Chapter 2, but with
the pots being sterilised by autoclaving, rather than steam treatment. Duplicate pots
or vials were used for each treatment, along with uninoculated controls.
Confirmation of nodule occupancy was determined by reisolating the inoculant strain
Chapter 4
149
and verifying its identity with fingerprinting PCR, using ERIC primers (Versalovic et
al., 1991) according to the methods given in Chapter 2.
Table 4.2. Legume species used to determine the host range of novel rhizobial species of
Microvirga.
Tribe Host Geographical origin
Acacieae
(Mimosoideae) Acacia saligna Temperate Australia
Crotalarieae
Crotalaria juncea India and tropical Asia
Listia angolensis Tropical Africa
Listia bainesii South Africa
Listia heterophylla South Africa
Genisteae Lupinus angustifolius
Mediterranean
Indigofereae
Indigofera frutescens
South Africa
Indigofera patens South Africa
Loteae Lotus corniculatus Africa and Asia
Phaseoleae
Macroptilium atropurpureum
Pantropical
Phaseolus vulgaris Tropical and warm temperate Americas
Vigna unguiculata Africa
4.3 Results
4.3.1 Amplification and sequencing of the 16S rRNA gene
A nearly full-length portion of the 16S rRNA gene was amplified and
sequenced for WSM3557T (1457 nt; GenBank Accession No. HM362432) and
WSM3693T (1452 nt; GenBank Accession No. HM362433). BLASTN analysis of
these sequences showed that WSM3557T and WSM3693
T were members of the
Alphaproteobacteria and, along with WSM3674, WSM3686, Lut5 and Lut6T, were
most closely related to species of Microvirga. The pairwise percent sequence identity
of a 1396 bp fragment of the 16S rRNA gene for the Listia angolensis and Lupinus
texensis strains and the Microvirga species type strains showed that all strains in this
clade shared at least 96.1% sequence identity (Table 4.3).
Chap
ter
4
150
Tab
le 4
.3.
Pai
rwis
e per
cent
iden
tity
and n
um
ber
of
bas
e pai
r m
ism
atch
es (
in b
rack
ets)
for
1396 b
p f
ragm
ent
of
16S
rR
NA
gen
e fr
om
Lis
tia a
ngole
nsi
s st
rain
s
(WS
M n
um
ber
s),
Lupin
us
texen
sis
stra
ins
(Lut5
and L
ut6
T),
Mic
rovi
rga f
locc
ula
ns
TF
BT,
Mic
rovir
ga a
eri
lata
5420S
-16
T,
Mic
rovi
rga a
ero
phil
a 5
420S
-12
T,
Mic
rovi
rga g
uangxie
nsi
s 25B
Tan
d M
icro
virg
a s
ubte
rranea F
aiI4
T.
L
ut5
L
ut6
T
WS
M3
55
7T
W
SM
367
4
WS
M3
68
6
WS
M3
69
3T
5
42
0S
-16
T
54
20
S-1
2T
TF
BT
25
BT
Fa
iI4
T
Lu
t5
1
00
98
.3
(24
) 9
8.2
(2
5)
98
.2
(25
) 9
8.0
(2
9)
97
.9
(30
) 9
6.9
(5
1)
98
.0
(29
) 9
7.1
(4
4)
97
.5
(35
)
Lu
t6T
1
00
9
8.3
(24
)
98
.2
(25
)
98
.2
(25
)
98
.0
(29
)
97
.9
(30
)
96
.9
(51
)
98
.0
(29
)
97
.1
(44
)
97
.5
(35
)
WS
M3
55
7T
9
8.3
(2
4)
98
.3
(24
)
99
.9
(1)
99
.9
(1)
96
.9
(44
) 9
7.0
(4
2)
96
.1
(56
) 9
7.6
(3
4)
97
.1
(46
) 9
6.8
(4
5)
WS
M3
67
4
98
.2
(25
) 9
8.2
(2
5)
99
.9
(1)
1
00
96
.9
(45
) 9
6.9
(4
3)
96
.1
(57
) 9
7.6
(3
5)
97
.1
(45
) 9
6.7
(4
6)
WS
M3
68
6
98
.2
(25
) 9
8.2
(2
5)
99
.9
(1)
10
0
9
6.9
(4
5)
96
.9
(43
) 9
6.1
(5
7)
97
.6
(35
) 9
7.1
(4
5)
96
.7
(46
)
WS
M3
69
3T
9
8.0
(2
9)
98
.0
(29
) 9
6.9
(4
4)
96
.9
(45
) 9
6.9
(4
5)
9
8.8
(1
8)
96
.4
(51
) 9
8.1
(2
8)
96
.5
(52
) 9
7.1
(4
2)
54
20
S-1
6T
97
.9
(30
) 9
7.9
(3
0)
97
.0
(42
) 9
6.9
(4
3)
96
.9
(43
) 9
8.8
(1
8)
9
6.7
(5
3)
98
.1
(26
) 9
7.4
(4
0)
97
.4
(37
)
54
20
S-1
2T
96
.7
(51
) 9
6.7
(5
1)
96
.1
(56
) 9
6.1
(5
7)
96
.1
(57
) 9
6.4
(5
1)
96
.7
(53
)
96
.4
(56
) 9
7.2
(
41
) 9
7.9
(3
0)
TF
BT
98
.0
(29
) 9
8.0
(2
9)
97
.6
(34
) 9
7.6
(3
5)
97
.6
(35
) 9
8.1
(2
8)
98
.1
(26
) 9
6.4
(5
6)
9
6.4
(5
5)
97
.0
(42
)
25
BT
97
.1
(44
)
97
.1
(44
)
97
.1
(46
)
97
.1
(45
)
97
.1
(45
)
96
.5
(52
)
97
.4
(40
)
97
.2
(41
)
96
.4
(55
)
97
.6
(33
)
Fa
iI4
T
97
.5
(35
) 9
7.5
(3
5)
96
.8
(45
) 9
6.7
(4
6)
96
.7
(46
) 9
7.1
(4
2)
97
.4
(37
) 9
7.9
(3
0)
97
.0
(42
) 9
7.6
(3
3)
Chapter 4
151
The Listia angolensis and Lupinus texensis strains formed three distinct
groups. In the first group, the sequences of WSM3674 and WSM3686 were identical
and shared 99.9% sequence identity (1 bp difference) with WSM3557T. They shared
98.2-98.3% sequence identity (24-25 bp difference) with the Lut5 and Lut6T strains
that formed the second group. The Lut5 and Lut6T sequences were identical. Both
these groups were most closely related to M. flocculans, having 97.6-98.0% sequence
identity with strain TFBT (29-35 bp difference). In contrast, the L. angolensis strain
WSM3693T was most closely related to M. aerilata strain 5420S-16
T, with 98.8%
sequence identity (18 bp difference) and sharing only 96.9% sequence identity (44-
45 bp difference) with the other L. angolensis strains.
The NJ phylogenetic tree obtained from a 1396 bp fragment of the 16S rDNA
sequences (Figure 4.2) demonstrated that the Listia angolensis and Lupinus texensis
strains, along with the Microvirga species type strains, formed a monophyletic group
that was clearly separated from Methylobacterium, Bosea and Chelatococcus
lineages and supported by high (100%) bootstrap values. Microvirga subterranea
was a basal member of this clade.
4.3.2 Amplification and sequencing of nodA
The 635 bp sequence obtained for WSM3693T, containing most of the nodA
gene and part of the 5’ region of nodB, was identical to the sequence obtained for
WSM3557T (detailed in Chapter 2). A Bayesian phylogenetic tree of the Microvirga
nodA genes is shown in Figure 2, Appendix VIII (Matt Parker unpublished data).
Chap
ter
4
152
Fig
ure
4.2
. N
J phylo
gen
etic
tre
e bas
ed on a
com
par
ativ
e an
alysi
s of
16S
rR
NA
gen
e se
quen
ces,
show
ing th
e re
lati
onsh
ips
bet
wee
n novel
sy
mbio
tic
Mic
rovi
rga s
trai
ns
(indic
ated
in b
old
) an
d c
lose
ly r
elat
ed s
pec
ies.
Num
ber
s at
the
nodes
of
the
tree
indic
ate
boots
trap
val
ues
(ex
pre
ssed
as
per
centa
ges
of
1000 r
epli
cati
ons)
. G
enB
ank a
cces
sion n
um
ber
s ar
e giv
en i
n p
aren
thes
es. B
radyrh
izobiu
m j
aponic
um
US
DA
6T
was
use
d a
s an
outg
roup. S
cale
bar
for
bra
nch
length
s sh
ow
s 0.0
1 s
ubst
ituti
ons
per
sit
e.
WS
M3
67
4 (
DQ
838
528
)
WS
M3
68
6 (
DQ
838
529
)
WS
M3
55
7T
(HM
36
24
32
)
Lu
t5 (
EF
19
14
07
)
Lu
t6T
(EF
19
14
08
)
Mic
rovir
ga
flo
ccu
lan
sT
FB
T(A
B0
98
51
5)
WS
M3
69
3T
(HM
36
24
33
)
Mic
rovir
ga
ae
rila
ta5
420
S-1
6T
(GQ
42
184
9)
Mic
rovir
ga
gu
ang
xie
nsis
25
BT
(EU
72
717
6)
Mic
rovir
ga
ae
roph
ila5
420
S-1
2T
(GQ
42
184
8)
Mic
rovir
ga
sub
terr
ane
aF
ail4
T(F
R7
33
708
)
Me
thylo
ba
cte
rium
nod
ula
ns
OR
S 2
060
T(A
F2
20
763
)
Me
thylo
ba
cte
rium
org
an
op
hilu
mD
SM
76
0T
(AB
17
56
39
)
Me
thylo
ba
cte
rium
exto
rqu
en
sIA
M1
26
31
T(A
B1
75
632
)
Me
thylo
ba
cte
rium
pop
uli
BJ0
01
T(A
Y2
51
81
8)
Ch
ela
toco
ccu
s a
sa
cch
aro
vora
ns
TE
2T
(AJ2
943
49
)
Ch
ela
toco
ccu
s d
ae
gue
nsis
K10
6T
(EF
58
450
7)
Bo
se
a m
ina
titla
nen
sis
DS
M1
30
99
T(A
F2
73
081
)
Bo
se
a th
ioo
xid
an
sD
SM
96
53
T(A
J2
507
96
)
Bo
se
a e
nea
e3
46
14
T(A
F2
88
30
0)
Bra
dyrh
izob
ium
jap
on
icum
US
DA
6T
(U6
96
38
)
100
94
100
99
98
99
90
92
77
100
100
73
83
100
93
87
57
99
0.0
1
WS
M3
67
4 (
DQ
838
528
)
WS
M3
68
6 (
DQ
838
529
)
WS
M3
55
7T
(HM
36
24
32
)
Lu
t5 (
EF
19
14
07
)
Lu
t6T
(EF
19
14
08
)
Mic
rovir
ga
flo
ccu
lan
sT
FB
T(A
B0
98
51
5)
WS
M3
69
3T
(HM
36
24
33
)
Mic
rovir
ga
ae
rila
ta5
420
S-1
6T
(GQ
42
184
9)
Mic
rovir
ga
gu
ang
xie
nsis
25
BT
(EU
72
717
6)
Mic
rovir
ga
ae
roph
ila5
420
S-1
2T
(GQ
42
184
8)
Mic
rovir
ga
sub
terr
ane
aF
ail4
T(F
R7
33
708
)
Me
thylo
ba
cte
rium
nod
ula
ns
OR
S 2
060
T(A
F2
20
763
)
Me
thylo
ba
cte
rium
org
an
op
hilu
mD
SM
76
0T
(AB
17
56
39
)
Me
thylo
ba
cte
rium
exto
rqu
en
sIA
M1
26
31
T(A
B1
75
632
)
Me
thylo
ba
cte
rium
pop
uli
BJ0
01
T(A
Y2
51
81
8)
Ch
ela
toco
ccu
s a
sa
cch
aro
vora
ns
TE
2T
(AJ2
943
49
)
Ch
ela
toco
ccu
s d
ae
gue
nsis
K10
6T
(EF
58
450
7)
Bo
se
a m
ina
titla
nen
sis
DS
M1
30
99
T(A
F2
73
081
)
Bo
se
a th
ioo
xid
an
sD
SM
96
53
T(A
J2
507
96
)
Bo
se
a e
nea
e3
46
14
T(A
F2
88
30
0)
Bra
dyrh
izob
ium
jap
on
icum
US
DA
6T
(U6
96
38
)
100
94
100
99
98
99
90
92
77
100
100
73
83
100
93
87
57
99
0.0
1
Chapter 4
153
4.3.3 Phenotypic characterisation
4.3.3.1 Morphology
Colonies of five of the six Listia angolensis strains grown on ½ LA were light
pink, convex and smooth (Figure 4.3a, b). Pigmentation developed after several days.
The sixth strain, WSM3693T, was cream coloured. Cultures grown with glucose and
mannitol as carbon sources typically produced more exopolysaccharide than those
grown with succinate, and developed characteristic banding patterns (Figure 4.3 c).
Colonies of the Lupinus texensis strains were a pale orange on ½ LA (Figure 4.4 d).
All strains grew to 0.5 – 1.5 mm in three days at 28°C and grew well on nutrient
agar. All cultures stained Gram-negative. No spores were observed, nor did cultures
grow after heat treatment at 70°C. Cells of the Listia angolensis strains, observed by
the hanging drop method, were motile. The Lupinus texensis strains Lut5 and Lut6T
were grown on a range of media, but motility was never observed. Electron
micrographs of the L. angolensis strains and Lut5 and Lut6T showed rod shaped cells
(0.4 - 0.5 µm x 1.0-2.2 µm), surrounded by a prominent capsule (Figures 4.4 a and b
and 4.5 a, b and c). The L. angolensis strains all had at least one polar flagellum.
Flagella were not observed on Lut5 or Lut6T.
Chapter 4
154
Figure 4.3. Colony morphology and pigmentation of Listia angolensis and Lupinus texensis
rhizobial strains: a) Three day old colony of WSM3557
T on ½ LA
b) Seven day old culture of WSM3557T on ½ LA, with succinate replacing glucose and
mannitol as the carbon source
c) Seven day old culture of WSM3557T on ½ LA, showing characteristic banding patterns
d) Aged culture of Lut6T on ½ LA
Figure 4.4. Transmission electron micrograph of a) WSM3693T and b) Lut6
T.
1 mma b
c d
1 mm1 mma b
c d
a500 nmb500 nm
a500 nmb500 nm b500 nm
Chapter 4
155
Figure 4.5. Scanning electron micrograph of a) WSM3557T; b) Lut6
T; c) Lut6
T, showing a
clearly visible capsule, due to shrinkage of the cytoplasm.
4.3.3.2 Characterisation of pigments
The pigment extracted from the Listia angolensis isolate WSM3557T was light
pink in colour. The main absorption peak at 495nm (absorbance, 0.071A) (Figure 4.6
a) appeared similar to that obtained for the pigmented Methylobacterium strain
WSM2598 (absorbance, 0.834A) (Figure 4.6 b). The Lut6T pigment extract was
bright yellow and had an absorption peak at 458 nm (absorbance 0.474A) (Figure 4.6
c). No absorption peaks were seen at wavelengths between 700 – 800 nm for
WSM3557T and Lut6
T, whereas the data for WSM2598 indicated a very slight
absorption peak at around 770 nm.
Figure 4.6. Absorption spectra of 7:2 acetone:methanol extracts from rhizobial strains.
a) WSM3557T; b) WSM2598; c) Lut6
T.
10 µm a
5 µm
b1 µm
c10 µm a10 µm a
5 µm
b
5 µm
b1 µm
c1 µm
c
Chapter 4
156
4.3.3.3 Growth characteristics
The pink-pigmented L. angolensis strains grew at a temperature range of
15°C to 44/45°C, while WSM3693T had a growth range of 15°C to 38°C. For Lut5
and Lut6T, the range was 10°C to 43°C. The optimal growth temperature for Lut6
T
was 39°C and the mean generation time (MGT) at this temperature was 1.8 h. The
optimal temperature and MGT for WSM3557T were 41°C and 1.6 h and for
WSM3693T these were 35°C and 1.7 h (Figure 4.7 a-f).
All strains grew optimally at pH 7.0 – 8.5 and over a range of pH 5.5 – 9.5 (or
pH 6.0 – 9.5 for WSM3693T). They were not salt tolerant; best growth was observed
at 0 – 0.5% (w/v) NaCl and isolates did not grow at > 2.0% (w/v) NaCl (Appendix
V). Colonies growing at 1.0 and 1.5% (w/v) NaCl were noticeably smaller.
WSM3557T, WSM3693
T, Lut5 and Lut6
T were strictly aerobic: no growth was
observed on Hugh & Leifson’s medium incubated for ten days in an anaerobic jar.
4.3.3.4 Antibiotic sensitivity
All the L. angolensis strains showed high resistance to gentamycin.
WSM3557T, WSM3674, WSM3675 and WSM3686 were partially resistant to
kanamycin and spectinomycin. WSM3674, WSM3675 and WSM3686 were
additionally partially resistant to chloramphenicol and WSM3674 and WSM3675 to
ampicillin. Lut5 and Lut6T were partially resistant to ampicillin, chloramphenicol,
gentamycin and streptomycin and were sensitive to kanamycin, nalidixic acid,
rifampicin, spectinomycin and tetracycline.
Chapter 4
157
Figure 4.7. Log/linear growth curves at temperature optima to determine mean generation
time: a) Lut6T at 39°C; b) WSM3557
T at 41°C; c) WSM3693
T at 35°C.
a
b
c
a
b
c
Chapter 4
158
4.3.3.5 Substrate utilisation
4.3.3.5.1 Utilisation of sole carbon sources in Biolog GN2 plates
The utilisation of carbon substrates by Listia angolensis and Lupinus texensis
strains inoculated into Biolog GN2 microplates is shown in Table 4.4. The substrates
have been categorised and divided into 11 groups, according to Garland & Mills
(1991). Readings from the 96-h samples were used, as colour development was poor
until 40 h incubation. Dextrin (well A3) was scored as a negative, as the initial
absorbance readings were slightly above the threshold value but did not increase and
no colour development was apparent.
In general, all strains had similar substrate utilisation patterns. A total of 35 of
the 95 carbon sources gave positive results. The carbon sources utilised spanned
most of the 11 designated categories, with none of the polymer, alcohol,
phosphorylated chemical or amine substrates being used. The range of substrates
utilised within each category was, however, quite narrow. Only 9 of 28
carbohydrates and 7 of 24 carboxylic acids were metabolised. The Lupinus texensis
strains utilised 6 of the possible 20 amino acid sources. The pink-pigmented Listia
angolensis strains were less specific in their utilisation, with 12 amino acids giving a
positive result for at least one strain. WSM3693 utilised five amino acids.
Chapter 4
159
Table 4.4. Ability of Listia angolensis strains (WSM numbers) and Lupinus texensis strains
(Lut5, Lut6T) to metabolise sole carbon sources in Biolog GN2 microplates. Carbon sources
are categorised according to Garland & Mills (1991). Data is from 96 hr duplicate reads.
Scored mean values (+ve = green; Weak = yellow; -ve = red)
Carbon source Strain
WSM
3557T
WSM
3674
WSM
3686
WSM
3693T
Lut5 Lut6T
Carbohydrates
N-Acetyl-D-galactosamine
N-Acetyl-D-glucosamine
Adonitol
L-Arabinose
D-Arabitol
D-Cellobiose
i-Erythritol
D-Fructose
L-Fucose
D-Galactose
Gentiobiose
α-D-Glucose
m-Inositol
α-Lactose
Lactulose
Maltose
D-Mannitol
D-Mannose
D-Melibiose
β-Methylglucoside
Psicose
D-Raffinose
L-Rhamnose
D-Sorbitol
Sucrose
D-Trehalose
Turanose
Xylitol
Esters
Pyruvic acid methyl ester
Succinic acid methyl ester
Polymers
α-Cyclodextrin
Dextrin
Glycogen
Tween 40
Tween 80
Chapter 4
160
Table 4.4. (cont)
Carbon source Strain
WSM
3557T
WSM
3674
WSM
3686
WSM
3693T
Lut5 Lut6T
Carboxylic acids
Acetic acid
cis-Aconitic acid
Citric acid
Formic acid
D-Galactonic acid lactone
D-Galacturonic acid
D-Gluconic acid
D-Glucosaminic acid
D-Glucuronic acid
α-Hydroxybutyric acid
β-Hydroxybutyric acid
γ-Hydroxybutyric acid
p-Hydroxyphenylacetic acid
Itaconic acid
α-Ketobutyric acid
α-Ketoglutaric acid
α-Ketovaleric acid
D,L-Lactic acid
Malonic acid
Propionic acid
Quinic acid
D-Saccharic acid
Sebacic acid
Succinic acid
Alcohols
2,3-Butanediol
Glycerol
Amides
Succinamic acid
Glucuronamide
Alaninamide
Phosphorylated chemicals
D,L-α-Glycerol phosphate
Glucose-1-phosphate
Glucose-6-phosphate
Chapter 4
161
Table 4.4. (cont)
Carbon source
Strain
WSM
3557T
WSM
3674
WSM
3686
WSM
3693T
Lut5 Lut6T
Amino acids
D-Alanine
L-Alanine
L-Alanyl-glycine
L-Asparagine
L-Aspartic acid
L-Glutamic acid
Glycyl-L-aspartic acid
Glycyl-L-glutamic acid
L-Histidine
Hydroxy-L-proline
L-Leucine
L-Ornithine
L-Phenylalanine
L-Proline
L-Pyroglutamic acid
D-Serine
L-Serine
L-Threonine
D,L-Carnitine
γ-Aminobutyric acid
Aromatic chemicals
Urocanic acid
Inosine
Uridine
Thymidine
Brominated chemicals
Bromosuccinic acid
Amines
Phenylethylamine
Putrescine
2-Aminoethanol
Substrates that gave a positive reading for at least one of the pink-pigmented
Listia angolensis strains included adonitol, L-arabinose, D-cellobiose, D-fructose, α-
D-glucose, inositol, D-mannose, D-melibiose, L-rhamnose, pyruvic acid methyl
ester, succinic acid methyl ester, acetic acid, D-galacturonic acid, β-hydroxybutyric
acid, γ-hydroxybutyric acid, α-ketoglutaric acid, D,L-lactic acid, succinic acid,
succinamic acid, urocanic acid and bromosuccinic acid. The Lut strains utilised all
these substrates with the exception of D-mannose, succinic acid methyl ester and
urocanic acid, and were additionally able to metabolise saccharic acid and xylitol.
WSM3693 utilised p-hydroxyphenylacetic acid but was unable to metabolise D-
Chapter 4
162
mannose, succinic acid methyl ester, β-hydroxybutyric acid, succinamic acid methyl
ester, urocanic acid and bromosuccinic acid. L-alanine, L-alanyl-glycine, L-
asparagine, L-aspartic acid, L-glutamic acid, glycyl-L-aspartic acid, glycyl-L-
glutamic acid, L-histidine, hydroxy-L-proline, L-ornithine, L-phenylalanine, L-
proline, L-serine and γ-aminobutyric acid were used as sole carbon substrates by the
pink-pigmented Listia angolensis strains. Lut5 and Lut6T utilised L-alanine, L-
glutamic acid, L-histidine, hydroxy-L-proline, L-ornithine and L-proline. WSM3693
utilised L-aspartic acid, L-glutamic acid, L-histidine, hydroxy-L-proline and L-
ornithine.
4.3.3.5.2 Utilisation of substrates in Biolog Phenotype Microarray plates
Biolog PM plates 1 to 4 were used to test the metabolic abilities of
WSM3557T and Lut6
T on 379 substrates, including 190 carbon sources, 95 nitrogen
sources, 59 phosphorus sources and 35 sulfur sources. The complete list of substrates
is shown in Table 1 (Appendix VI). The absorbance values of substrates giving a
positive phenotype are shown in Table 4.5. It is possible that some of the carbon
source readings are false positives. Dye G reacts with some pentoses and other
carbon sources, including D and L-arabinose, D-glucosamine, dihydroxyacetone, L-
lyxose and D-xylose (Biolog PM Services, pers. comm.). Absorbance readings for
the C5 carbohydrates were generally higher than for other substrates. Conversely,
some of the carbon source readings may be false negatives. Succinic acid and L-
glutamic acid, for example, gave negative results, but were utilised (in the form of
succinate and glutamate) as sole carbon substrates for WSM3557T grown in minimal
media. D-mannitol and acetate supported the growth of all tested strains in minimal
media, but also gave negative results on the PM plates.
Chapter 4
163
Table 4.5. Biolog PM microplate readings for substrates that gave positive scores for
inoculated WSM3557T or Lut6
T. Brackets ( ) around the reading indicate a negative score.
Plate Well Substrate Substrate Type Average Reading
WSM3557T
Lut6T
PM2 B05 D-Arabinose C source; C5 carbohydrate 120.5 110.5
PM1 A02 L-Arabinose C source; C5 carbohydrate 124.0 115.5
PM1 H06 L-Lyxose C source; C5 carbohydrate 145.0 117.5
PM1 C04 D-Ribose C source; C5 carbohydrate 150.0 108.0
PM2 B09 2-Deoxy-D-Ribose C source; C5 carbohydrate 36.0 43.0
PM1 B08 D-Xylose C source; C5 carbohydrate 135.5 107.5
PM1 E09 Adonitol (=Ribitol) C source; C5 carbohydrate (11.5) 36
PM1 C07 D-Fructose C source; C6 carbohydrate (12.0) 37.0
PM1 E04
D-Fructose-6-
Phosphate C source; C6 carbohydrate 20.5 13.5
PM1 C09 α-D-Glucose C source; C6 carbohydrate 24.0 35.5
PM2 E05 D-Glucosamine C source; C6 carbohydrate 150.0 170.0
PM1 F03 m-Inositol
C source; C6 carbohydrate
(polyol of cyclohexane) (13.0) 31.5
PM1 A11 D-Mannose C source; C6 carbohydrate 23.5 39.5
PM1 H05 D-Psicose C source; C6 carbohydrate 34.5 64.0
PM1 C06 L-Rhamnose C source; C6 carbohydrate 38.5 65.0
PM2 D06 D-Tagatose C source; C6 carbohydrate 79.0 65.5
PM1 F11 D-Cellobiose C source; C12 carbohydrate (13.5) 26.0
PM2 C12 Palatinose C source; C12 carbohydrate 42.0 38.5
PM2 E05 Arbutin C source; C12 carbohydrate (Glucosylated hydroquinone) 26.5 38.0
PM1 B09 L-Lactic acid C source, C3 carboxylic acid (16.0) 35.0
PM2 F02 D-Malonic acid C source, C3 dicarboxylic acid (0.0) 44.0
PM1 H08 Pyruvic acid C source, C3 carboxylic acid 67.0 52.5
PM1 F06 Bromosuccinic acid C source, C4 dicarboxylic acid (0.5) 74.5
PM1 F05 Fumaric acid C source, C4 dicarboxylic acid (0.0) 79.5
PM1 C03 D,L-Malic acid C source, C4 dicarboxylic acid (3.0) 87.0
PM1 G11 D-Malic acid C source, C4 dicarboxylic acid (5.0) 58.0
PM1 G12 L-Malic acid C source, C4 dicarboxylic acid (17.0) 78.5
PM1 A05 Succinic acid C source, C4 dicarboxylic acid (19.0) 94.5
PM2 F10 Succinamic acid C source, C4 carboxylic acid (10.5) 30.5
PM1 D06 α-Ketoglutaric acid C source, C5 carboxylic acid (20.5) 28.0
PM1 H10 D-Galacturonic acid C source, C6 carboxylic acid 55.0 39.5
PM2 E12
5-Keto-D-Gluconic
acid C source, C6 carboxylic acid 78.5 90.0
PM2 F05 D-Oxalomalic acid C source, C6 tricarboxylic acid 29.0 38.0
PM1 A04 D-Saccharic acid C source, C6 dicarboxylic acid (0.0) 44.5
PM1 G09
Mono-
Methylsuccinate
C source, methyl ester of
carboxylic acid 78.5 12.5
PM2 H09 Dihydroxyacetone C source, C3 alcohol 123.5 133.5
PM1 H07 Glucuronamide C source, C6 amide 45.0 40.5
PM1 A07 L-Aspartic acid C.source, amino acid (5.0) 33.0
PM1 B12 L-Glutamic acid C.source, amino acid (23.0) 52.5
PM1 G03 L-Serine C.source, amino acid 45.0 0.0
PM1 C05 Tween 20 C source, fatty acid (18.5) 27.0
PM2 A12 Pectin C source, polymer 24.0 37.0
Chapter 4
164
Table 4.5. (cont.)
Plate Well Chemical Mode of Action Average Reading
WSM3557T
Lut6T
PM3 C03 D-Alanine N.source, amino acid (0.0) 27.5
PM3 A07 L-Alanine N.source, amino acid (1.0) 37.0
PM3 A08 L-Arginine N.source, amino acid (0.0) 55.5
PM3 A09 L-Asparagine N.source, amino acid (19.5) 96.5
PM3 C05 D-Aspartic acid N.source, amino acid (0.0) 38.0
PM3 A10 L-Aspartic acid N.source, amino acid (23.5) 96.0
PM3 C10 L-Citrulline N.source, amino acid (0.0) 25.0
PM3 A11 L-Cysteine N.source, amino acid 46.0 58.0
PM3 A12 L-Glutamic acid N.source, amino acid 48.5 111.0
PM3 B01 L-Glutamine N.source, amino acid 22.0 103.0
PM3 B03 L-Histidine N.source, amino acid (11.0) 69.0
PM3 B06 L-Lysine N.source, amino acid (0.0) 38.5
PM3 C12 L-Ornithine N.source, amino acid (4.0) 56.5
PM3 B09 L-Proline N.source, amino acid (0.5) 91.0
PM3 B10 L-Serine N.source, amino acid (7.0) 34.5
PM3 B12 L-Tryptophan N.source, amino acid 32.5 51.0
PM3 E11 N-Acetyl-D-Glucosamine N.source, other (1.0) 46.5
PM3 G05 Allantoin N.source, other (22.5) 31.5
PM3 G04 Alloxan N.source, other 46.5 68.5
PM3 G08
γ-Amino-N-Butyric
acid N.source, other (2.5) 72.0
PM3 G10
D,L-α-Amino-
Caprylic acid N.source, other 28.5 24.5
PM3 G11
d-Amino-N-Valeric
acid N.source, other (1.0) 48.0
PM3 E09 D-Galactosamine N.source, other 101.0 124.0
PM3 E08 D-Glucosamine N.source, other 101.0 133.5
PM3 E06 Glucuronamide N.source, other (4.5) 26.0
PM3 E10 D-Mannosamine N.source, other 136.0 157.0
PM3 G01 Xanthine N.source, other 19.5 59.0
PM3 H01 Ala-Asp N.source, peptide (18.5) 53.5
PM3 H02 Ala-Gln N.source, peptide 26.5 71.5
PM3 H03 Ala-Glu N.source, peptide (20.5) 75.5
PM3 H04 Ala-Gly N.source, peptide (2.0) 45.5
PM3 H05 Ala-His N.source, peptide (0.5) 52.0
PM3 H08 Gly-Asn N.source, peptide (8.0) 63.0
PM3 H09 Gly-Gln N.source, peptide (20.0) 78.0
PM3 H10 Gly-Glu N.source, peptide (18.5) 63.0
PM4 B01 Thiophosphate P source, inorganic 25.5 26.5
PM4 D03 Cysteamine-S-Phosphate P source, organic, amine (1.0) 22.0
PM4 D05
O-Phospho-D-
Serine P source, organic, amino acid (0.0) 23.5
PM4 D06
O-Phospho-L-
Serine P source, organic, amino acid (0.0) 32.5
PM4 D07
O-Phospho-L-
Threonine P source, organic, amino acid (0.0) 25.0
PM4 E02
O-Phospho-L-
Tyrosine P source, organic, amino acid (0.0) 22.0
PM4 D04 Phospho-L-Arginine P source, organic, amino acid (0.0) 39.5
PM4 B03
D,L-a-Glycerol
Phosphate
P source, organic, C3
carbohydrate (0.0) 42.5
Chapter 4
165
Table 4.5. (cont.)
Plate Well Chemical Mode of Action Average Reading
WSM3557T
Lut6T
PM4 B06 D-2-Phospho-Glyceric Acid
P source, organic, C3 carbohydrate (0.0) 23.0
PM4 C05
2-Deoxy-D-Glucose
6-Phosphate
P source, organic, C6
carbohydrate (0.0) 24.0
PM4 C06
D-Glucosamine-6-
Phosphate
P source, organic, C6
carbohydrate (6.0) 39.0
PM4 C03
D-Glucose-1-
Phosphate
P source, organic, C6
carbohydrate (0.0) 24.5
PM4 C07
6-Phospho-
Gluconic Acid
P source, organic, C6
carbohydrate (0.0) 25.5
PM4 E11
Inositol
Hexaphosphate P source, organic, C6 polyol 24.0 30.0
PM4 A09 Adenosine 3`-Monophosphate P source, organic, purine (0.0) 20.5
PM4 A10
Adenosine 5`-
Monophosphate P source, organic, purine (0.0) 29.0
PM4 A11
Adenosine 2`,3`-
Cyclic
Monophosphate P source, organic, purine (0.0) 26.0
PM4 B08
Guanosine 2`-
Monophosphate P source, organic, purine (0.0) 27.0
PM4 B09 Guanosine 3`-Monophosphate P source, organic, purine (0.0) 26.0
PM4 B10
Guanosine 5`-
Monophosphate P source, organic, purine (0.0) 27.0
PM4 B11
Guanosine 2`,3`-
Cyclic
Monophosphate P source, organic, purine (0.0) 21.0
PM4 C08
Cytidine 2`-
Monophosphate P source, organic, pyrimidine (0.0) 31.0
PM4 C09
Cytidine 3`-
Monophosphate P source, organic, pyrimidine (0.0) 37.0
PM4 C10 Cytidine 5`-Monophosphate P source, organic, pyrimidine (0.0) 31.0
PM4 C11
Cytidine 2`,3`-
Cyclic
Monophosphate P source, organic, pyrimidine (0.0) 34.5
PM4 E09
Thymidine 3`-
Monophosphate P source, organic, pyrimidine (0.0) 38.0
PM4 E10
Thymidine 5`-
Monophosphate P source, organic, pyrimidine (0.0) 37.0
PM4 E12
Thymidine 3`,5`-Cyclic
Monophosphate P source, organic, pyrimidine (0.0) 25.5
PM4 D08
Uridine 2`-
Monophosphate P source, organic, pyrimidine (0.0) 29.0
PM4 D09
Uridine 3`-
Monophosphate P source, organic, pyrimidine (0.0) 41.5
PM4 D10
Uridine 5`-
Monophosphate P source, organic, pyrimidine (0.0) 36.0
PM4 D11
Uridine 2`,3`-Cyclic
Monophosphate P source, organic, pyrimidine (0.0) 38.5
PM4 F08 D-Cysteine S-source, amino acid (0.0) 32.0
PM4 H04 D,L-Lipoamide S-source, fatty acid (17.5) 25.5
Chapter 4
166
The results for the PM3 and PM4 plates (nitrogen, phosphorus and sulfur
sources) may also not be a true indication of WSM3557T and Lut6
T metabolic
abilities. The lack of a positive reaction for the substrates ammonia (PM3 plate, well
A02) and phosphate (PM4 plate, well A02) for either strain is illustrative, as these
substrates have been used to grow the strains in minimal media, and ammonia is
supplied as the nitrogen source in PM plates 1, 2 and 4 (Biolog PM Services, pers.
comm.). Colour production in plates PM1 and PM2 only requires carbon oxidation,
whereas plates PM3 and PM4 behave differently due to the defined media and may
not work for all organisms because of this (Biolog PM Services, pers. comm.).
WSM3557T utilised 24 of the carbon sources, 10 of the nitrogen sources and
two phosphorus sources. None of the sulfur sources gave a positive reading. Lut6T
utilised 34 of the carbon sources, 35 of the nitrogen sources, 32 phosphorus sources
and two sulfur sources. A variety of substrates were used as carbon sources. For
WSM3557T, these included carbohydrates (D- and L-arabinose, α-D-glucose, L-
lyxose, D-mannose, palatinose, D-psicose, L-rhamnose, D-ribose, D-tagatose, D-
xylose, 2-deoxy-D-ribose, D-fructose-6-phosphate, D-glucosamine and arbutin);
carboxylic acids (D-galacturonic acid, 5-keto-D-gluconic acid, mono-
methylsuccinate, oxalomalic acid and pyruvic acid); alcohols (dihydroxyacetone);
amides (glucuronamide); amino acids (L- serine) and polymers (pectin). Lut6T
metabolised all the above except for D-fructose-6-phosphate, mono-methylsuccinate
and L- serine and was additionally able to utilise D-cellobiose, D-fructose, adonitol
and m-inositol (carbohydrates); bromosuccinic acid, fumaric acid, α-ketoglutaric
acid, L-lactic acid, D- and L-malic acid, D-malonic acid, D-saccharic acid, succinic
Chapter 4
167
acid and succinamic acid (carboxylic acids); L-glutamic acid and L-aspartic acid
(amino acids) and Tween 20 (fatty acid).
WSM3557T was able to metabolise four of 33 amino acids (L-cysteine, L-
glutamic acid, L-glutamine and L-tryptophan), one peptide (Ala-Gln) and five other
substrates (alloxan, D,L-α-amino-caprylic acid, D-galactosamine, D-glucosamine
and D-mannosamine) as nitrogen sources. Thiophosphate and inositol hexaphosphate
(phytic acid), were utilised as phosphorus sources. Lut6T utilised the same substrates
as WSM3557T and additionally twelve other amino acids, seven other peptides, six
other nitrogen sources, and thirty other phosphorus sources. D-cysteine and D,L-
lipoamide were utilised as sulfur sources.
Most of the carbon substrates in the GN2 plates form a subset of those
available in the PM carbon source plates (PM 1 and 2). The results obtained for
WSM3557T and Lut6
T on the PM plates are not directly comparable with GN2 plate
data because of differences in media formulation and redox dyes, and different
inoculation concentrations and incubation times (40% transmittance and 56 h for the
PM plates versus 85% transmittance and 96 h for the GN2 plates). Substrate
utilisation in the PM plates was consistent with some GN2 substrate results, but
differed in several instances, notably with amino acid substrates. A comparison of
results is included in Table 1, Appendix VI. Substrates inoculated with WSM3557T
that tested positive on GN2 and negative on PM plates included D-cellobiose, D-
fructose and m-inositol (carbohydrates); bromosuccinic acid, L-lactic acid, methyl
pyruvate, succinamic acid and succinic acid (carboxylic acids) and L-asparagine, L-
aspartic acid, L-glutamic acid, L-histidine, hydroxy L-proline, ornithine and L-
Chapter 4
168
proline (amino acids). Carbon substrates that tested negative in WSM3557T-
inoculated GN2 plates but positive in PM plates included psicose, glucuronamide
and L-serine. For Lut6T, substrates that were positive on GN2 and negative on PM
plates were methyl pyruvate, L-alanine and L-proline. Substrates that were negative
on GN2 and positive on PM plates included D-mannose, D-psicose, α-ketoglutaric
acid, glucuronamide and L-aspartic acid.
4.3.3.5.3 Growth in sole carbon substrate broths
The Biolog system measures the cellular respiration of an organism, rather than
its ability to actually grow on a given substrate. The strains WSM3557T, WSM3693
T,
Lut5 and Lut6T were therefore grown in minimal media broths to confirm that
growth was possible on selected carbon substrates that gave positive results for the
Biolog GN2 plates. Several other substrates not present in the GN2 plates were also
tested. As Microvirga subterranea had been shown to have an obligate requirement
for yeast extract in minimal media (Kanso & Patel, 2003), all Listia angolensis and
Lupinus texensis strains were first assessed for their supplementary requirements for
growth in minimal medium.
Yeast extract was an absolute requirement for growth of Lut5 and Lut6T in
minimal media. The Listia angolensis strains required either yeast extract or the
vitamin mix described by Egli & Auling (2005) for growth of Chelatococcus
asaccharovorans in minimal media. The minimal amount of yeast extract required
was 0.005% (w/v); this amount supported growth in media with carbon substrates
added, but not growth in media devoid of carbon substrate. In minimal broth media
WSM3557T grew on L-arabinose, D-cellobiose, D-fructose, α-D-glucose, glycerol,
D-mannitol, succinate, glutamate and acetate. WSM3693T grew on all the above and
Chapter 4
169
additionally was able to grow on p-hydroxybenzoate. Neither strain could grow on
benzoate, ethanol or methanol. Lut5 and Lut6T grew on L-arabinose, D-cellobiose,
D-fructose, α-D-glucose, D-mannitol, succinate, glutamate, p-hydroxybenzoate and
ethanol, but were unable to grow on benzoate, glycerol or methanol (Table 4.6).
Table 4.6. Growth of WSM3557T, WSM3693
T, Lut5 and Lut6
T on sole carbon substrates in
minimal media broths, measured 21 days after inoculation.
Carbon Substrate Type of
substrate
Strain
WSM
3557T
WSM
3693T Lut5 Lut6
T
L + Arabinose
(20 mM) C5 carbohydrate xxx xxx xx xx
D + Cellobiose
(20 mM) C12 carbohydrate xxx xxx x x
β D Fructose
(20 mM) C6 carbohydrate xxx xxx x x
D Glucose
(20 mM) C6 carbohydrate xxx xxx xx xx
D Mannitol
(20 mM)
C6 carbohydrate
(polyol) xxx xxx xx xx
Acetate
(20 mM) C2 carboxylate xxx xxx xx xx
Succinate
(20 mM) C4 dicarboxylate xxx xxx xxx x
Benzoate
(3 mM)
C7 aromatic
carboxylate 0 0 0 0
p-hydroxybenzoate
(3 mM)
C7 aromatic
carboxylate 0 xx x x
Glutamate –NH4Cl
(10 mM) Amino acid xxx xxx x xxx
Glutamate +NH4Cl
(10 mM) Amino acid xxx xxx xxx xxx
Methanol
(0.5% (v/v)) C1 alcohol 0 0 0 0
Ethanol
(20 mM) C2 alcohol 0 0 xxx xx
Glycerol
(20 mM) C3 alcohol xxx xxx 0 0
4.3.3.6 Biochemical characterisation
On API 20E strips, all strains were positive for urease and weakly positive for
acetoin production from pyruvate. All except WSM3693T were weakly positive for
tryptophan deaminase. β-Galactosidase, arginine dihydrolase, lysine decarboxylase,
Chapter 4
170
ornithine decarboxylase, indole and hydrogen sulphide were not produced by any
strain. Citrate was not utilised and gelatin was not hydrolysed. Acid was produced
from arabinose but not from glucose, mannitol, inositol, sorbitol, sucrose or
amygdalin. Weak acid production was observed for WSM3673 and WSM3674 on
rhamnose and for WSM3557T on melibiose. All strains were catalase positive and
oxidase negative. Nitrate was reduced to nitrite by WSM3557T, WSM3674,
WSM3686 and WSM3693T, but not by WSM3673, Lut5 or Lut6
T. Starch was not
hydrolysed. Stab cultures of WSM3557T, WSM3693
T, Lut5 and Lut6
T in Hugh &
Leifson’s medium with arabinose, glucose or pyruvate as a carbon source grew in
aerobic conditions and in test tubes overlaid with paraffin. Cultures grown in this
medium acidified arabinose, but not glucose or pyruvate. Weak acetoin production
was observed for WSM3557T, but not for WSM3693
T, Lut5 or Lut6
T, in Smith’s
medium (Leone & Hedrick, 1954) containing glucose as a carbon source (Figure
4.8).
Figure 4.8. Production of acetoin from growth on glucose in Smith’s medium by 1)
WSM3557T (weakly positive); 2) WSM3693
T, 3) Lut5, 4) Lut6
T, 5) Escherichia coli (all
negative); 6) Bacillus subtillus (positive).
1 2 3 4 5 61 2 3 4 5 6
Chapter 4
171
4.3.4 Host range
The symbiotic ability of WSM3557T, WSM3693
T, Lut5 and Lut6
T was
examined on a range of legumes from different phylogenetic clades (Table 4.7).
WSM3557T and WSM3693
T only formed effective nodules on their original Listia
angolensis host, but were able to induce occasional ineffective nodulation on several
other legumes. WSM3693T was the more infective strain, forming nodules on Acacia
saligna, Indigofera frutescens, Phaseolus vulgaris and Vigna unguiculata. Bacteria
were reisolated from nodules of all these hosts, thus confirming nodule occupancy.
WSM3557T induced nodulation only on P. vulgaris and the nodules appeared devoid
of bacteria, as the inoculant could not be reisolated from harvested nodules. Lut5 and
Lut6T did not induce nodulation on L. angolensis, Listia bainesii or Listia
heterophylla. On the other legume hosts, they were able to nodulate only A. saligna
and P. vulgaris, albeit ineffectively. Inoculant was reisolated from nodules only in
the case of Lut5 on A. saligna. PCR amplification using ERIC primers confirmed the
identity of the reisolated strains. Gel images of the fingerprinting patterns obtained
from electrophoresis of the amplification products are shown in Appendix VII.
Chapter 4
172
Table 4.7. Nodulation (average number of nodules per plant out of eight* inoculated plants)
and effectiveness of the Listia angolensis strains WSM3557T and WSM3693
T and Lupinus
texensis strains Lut5 and Lut6T on legume hosts harvested six weeks post inoculation.
Tribe Host Inoculant strain
WSM3557T
WSM3693T
Lut5 Lut6T
Acacieae (Mimosoideae)
Acacia saligna
N- F-
N+/- F-
(0.875) Reisolated
N+/- F-
(0.125) Reisolated
N+/- F-
(0.125) No
reisolates
Crotalarieae
Crotalaria juncea
N- F- N- F- N- F- N- F-
Listia angolensis
N+ F+ (3.0)
Reisolated
N+ F+ (3.12)
Reisolated N- F- N- F-
Listia bainesii N- F- N- F- N- F- N- F-
Listia heterophylla
N- F- N- F- N- F- N- F-
Genisteae Lupinus angustifolius
N- F- N- F- N- F- N- F-
Indigofereae
Indigofera frutescens
N- F-
N+/- F-
(0.8) Reisolated
ND ND
Indigofera patens
N- F- N- F- ND ND
Loteae Lotus corniculatus
N- F- N- F- N- F- N- F-
Phaseoleae
Macroptilium atropurpureum
N- F- N- F- ND ND
Phaseolus vulgaris
N+/- F- (4.5)
No reisolates
N+/- F- (15.0)
Reisolated
N+/- F- (7.5)
No reisolates
N+/- F- (6.75)
No reisolates
Vigna unguiculata
N- F- N+/- F- (3.0)
Reisolated
N- F- N- F-
*I. frutescens: four plants were inoculated with WSM3557T; five plants were inoculated with
WSM3693T. I. patens: four plants were inoculated with WSM3557
T; two plants were
inoculated with WSM3693T.
Chapter 4
173
4.4 Discussion
4.4.1 Genotypic characterisation of novel Microvirga species
The 16S rRNA gene sequences of the Listia angolensis and Lupinus texensis
strains show them to be most closely related to the type strains of Microvirga
species. Based on the 95 % 16S rRNA gene sequence similarity that has been
proposed as a ‘practicable border zone for genus definition’ (Ludwig et al., 1998),
these Listia angolensis and Lupinus texensis rhizobia belong within the genus
Microvirga. It is notable that the lineages of the strains are not based on their
geographical separation or on their symbiotic hosts, as WSM3557T, WSM3674 and
WSM3686 are more closely related to Lut5 and Lut6T than they are to WSM3693
T.
Strains sharing less than 97% 16S rRNA gene sequence similarity are not
considered to be members of the same species (Tindall et al., 2010), although
Stackebrandt & Ebers (2006) have suggested that this be changed to 98.7-99.0%
shared sequence similarity. The pink-pigmented Listia angolensis strains
WSM3557T, WSM3674 and WSM3686 are members of the same species, on the
basis of 99.9% sequence identity of their 16S rRNA gene sequences. Lut5 and Lut6T
share 100% sequence identity and form a second species. WSM3693T is clearly
separated from both the Lupinus texensis and the other Listia angolensis strains. As
its closest relative is M. aerilata strain 5420S-16T, with 98.8% sequence identity, it
merits consideration as a separate species.
As the 16S rRNA gene should not be used as a sole determinant of bacterial
phylogenetic placement (Chapter 1, Section 1.5.2) portions of the housekeeping loci
dnaK, gyrB, recA and rpoB were sequenced in five symbiotic Microvirga strains and
Chapter 4
174
in two non-symbiotic Microvirga species (M. flocculans TFBT and M. subterranea
DSM 14364T (= FaiI4
T)) to further analyse the relationships in this group of bacteria.
A combined analysis of concatenated sequences was performed, using a phylogenetic
tree inferred by MrBayes (Ronquist & Huelsenbeck, 2003), with nucleotide sites
partitioned by codon position and a HKY substitution model. The program was run
for a 250, 000 generation burn-in period and then results were sampled every 250
generations for an additional 250 000 generations. The topology of the phylogenetic
tree obtained from this analysis supports that of the 16S rRNA gene tree and
confirms that the Listia angolensis and Lupinus texensis strains belong in the genus
Microvirga (Matt Parker unpublished data, Figure 1, Appendix VIII).
The phylogeny of the rhizobial Microvirga symbiotic genes was also examined. A
Bayesian phylogenetic analysis of the nodA gene indicated that Microvirga nodA
sequences were derived from two different sources (Matt Parker unpublished data,
Figure 2, Appendix VIII). The WSM3557T and WSM3693
T nodA genes clustered in
a strongly supported clade with reference strains in the genera Bradyrhizobium,
Burkholderia and Methylobacterium. The host plant species of the Bradyrhizobium
strains (ORS 938, USDA 76T, USDA 110 and Lcamp8) are Rhynchosia minima,
Glycine max, Glycine max and Lupinus campestris, respectively (Stepkowski et al.,
2007). The Burkholderia strains (B. tuberum STM678T and Burkholderia sp.
WSM3930) are effective and specific nodulators of Cyclopia spp. (tribe Podalyrieae)
and Rhynchosia ferulifolia (tribe Phaseoleae), respectively, which are legumes native
to the South African fynbos vegetation (Elliott et al., 2007; Garau et al., 2009).
Methylobacterium nodulans ORS 2060T specifically nodulates Senegalese
Chapter 4
175
Crotalaria spp. (Sy et al., 2001), while Methylobacterium sp. 4-46 nodulates the
highly specific Listia bainesii (Fleischman & Kramer, 1998) (see also Chapter 2).
The Lut6T nodA sequence was placed in an equally strongly supported clade with
reference strains in the genera Rhizobium, Mesorhizobium and Ensifer (formerly
Sinorhizobium) isolated from diverse legume hosts (Acacia senegal, Acacia tortilis,
Acaciella angustissima, Phaseolus vulgaris, Prosopis chilensis) growing in Africa or
the Americas (Matt Parker unpublished data, Figure 2, Appendix VIII). These results
suggest that the rhizobial Microvirga strains from Listia angolensis and those from
Lupinus texensis acquired their nodA genes in separate horizontal gene transfer
events and from different donor lineages.
In contrast to the nodA phylogeny, the Bayesian analysis of concatenated
sequences for nifD and nifH clustered both the Listia angolensis and the Lupinus
texensis rhizobia into a single well-supported group with affinities to Rhizobium etli
CFN42T (Matt Parker unpublished data, Figure 3, Appendix VIII). This clade, along
with other Rhizobium, Mesorhizobium and Ensifer strains, was grouped separately
from the nifDH sequences of Azorhizobium and Bradyrhizobium strains. Microvirga
is not a close relative of Rhizobium for housekeeping gene loci (Matt Parker
unpublished data, Figure 1, Appendix VIII). The close affinity of Microvirga nif
genes to those of Rhizobium, Mesorhizobium and Ensifer therefore suggests that
these genes were acquired through horizontal transfer.
Chapter 4
176
4.4.2 G+C content, DNA:DNA hybridisation and cellular fatty acid
analysis
The DNA G+C content of strains Lut5, Lut6T, WSM3557
T and WSM3693
T
ranges from 61.9-63.0%, which is consistent with values obtained for other
Microvirga spp. (Table 1, Appendix IX, Anne Willems and Sofie De Meyer,
unpublished data). A DNA:DNA hybridization (DDH) value equal to or higher than
70% has been recommended as the threshold for the definition of members of a
species (Tindall et al., 2010). DDH values confirmed that WSM3557T, WSM3693
T,
Lut6T and M. flocculans LMG 25472
T, with 19-34.5% hybridization, represented
separate species; while Lut5 and Lut6T, with 97% DDH, belong to the same species
(Anne Willems and Sofie De Meyer, unpublished data, Table 1, Appendix IX).
The major cellular fatty acids were 18:1 w7c (52.58-53%) and 19:0 CYCLO
w8c (17.25-17.65%) for WSM3557T and WSM3693
T and 18:1 w7c (68.94-69.71%)
and SF2 (15.41-16.06%) for Lut5 and Lut6T (Anne Willems and Sofie De Meyer,
unpublished data, Table 2, Appendix IX). Cellular fatty acid composition was similar
for all Microvirga spp. and is a feature that distinguishes this group of bacteria from
the phylogenetically related Chelatococcus asaccharovorans LMG 25503T (Table 2,
Appendix IX and Weon et al. (2010)).
4.4.3 Phenotypic characterisation of novel Microvirga species
The phenotypic features that distinguish Lut6T, WSM3557
T and WSM3693
T
from other Microvirga spp. are given in Table 4.8.
Chap
ter
4
177
Tab
le 4
.8.
Dif
fere
nti
atin
g p
hen
oty
pic
char
acte
rist
ics
of
the
novel
str
ains
Lut6
T,
WS
M3557
T a
nd W
SM
3693
T a
nd t
he
type
stra
ins
of
close
ly r
elat
ed s
pec
ies
of
the
gen
us
Mic
rovi
rga.
Str
ains:
1,
M.
lupin
i sp
. nov.
Lut6
T;
2,
M.
loto
nonid
is s
p.
nov.
WS
M3557
T;
3,
M.
zam
bie
nsi
s sp
. nov.
WS
M3693
T;
4,
M.
floccula
ns
TF
BT (
Tak
eda
et
al.
, 2004);
5, M
. su
bte
rranea F
aiI4
T (
Kan
so &
Pat
el, 2003);
6, M
. guangxi
ensi
s 25B
T (
Zhan
g e
t al.
, 2009);
7, M
. aero
phil
a 5
420S
-12
T (
Weo
n
et
al.
, 2010);
8,
M.
aeri
lata
5420S
-16
T (
Weo
n e
t al.
, 2010).
All
str
ains
are
rod-s
hap
ed,
stri
ctly
aer
obic
and p
osi
tive
for
cata
lase
but
neg
ativ
e fo
r ar
gin
ine
dih
ydro
lase
and i
ndole
pro
duct
ion. (+
= p
osi
tive,
w =
wea
k,
- =
neg
ativ
e, N
D =
not
det
erm
ined
)
Ch
ara
cte
ris
tic
1
2
3
4
5
6
7
8
Isola
tion
sourc
e
Root
nod
ule
R
oot
nod
ule
R
oot
nod
ule
H
ot sprin
g
Therm
al
aqu
ifer
Soil
Air
A
ir
Colo
ny
Pale
ora
nge
Lig
ht p
ink,
mucila
gin
ous
Cre
am
,
mucila
gin
ous
White,
rough
Lig
ht p
ink,
sm
ooth
,
Lig
ht p
ink,
sm
ooth
,
Lig
ht p
ink,
sm
ooth
Lig
ht p
ink,
sm
ooth
Fla
ge
lla
Non-m
otile
P
ola
r flage
lla
Pola
r flage
lla
Pola
r
flagella
N
on-m
otile
N
on-m
otile
N
on-m
otile
N
on-m
otile
Cell
siz
e (µ
m)
0.4
- 0
.5 x
1.0
- 2
.2
0.4
- 0
.5 x
1.0
- 2
.2
0.4
- 0
.5 x
1.0
- 2
.2
0.5
- 0
.7 x
1.5
- 3
.5
1 x
1.5
-
4.0
0.6
– 0
.8 x
1.3
– 2
.1
0.8
– 1
.1 x
1.6
– 4
.2
1.2
– 1
.5 x
1.6
– 3
.3
Optim
um
tem
p (◦C
) 39
41
35
40 -
45
41
37
N
D
ND
Gro
wth
ran
ge (◦C
) 10 -
43
15 -
44
15 –
38
20 –
45*
25 -
45
16 -
42
10 -
35
10 -
35
MG
T
1.8
hrs
1.6
hrs
1.7
hrs
N
D
4.5
hrs
230 m
in
ND
N
D
Optim
um
pH
7.0
– 8
.5
7.0
– 8
.5
7.0
– 8
.5
7.0
7.0
7.0
N
D
ND
PH
gro
wth
ra
nge
5.5
–9.5
5.5
–9.5
6.0
–9.5
N
D
6 –
9*
5.0
– 9
.5
7.0
– 1
0.0
7.0
– 1
0.0
Optim
um
NaC
l %
0.0
– 0
.5
0.0
– 1
.0
0.0
– 0
.5
ND
0
ND
N
D
ND
NaC
l gro
wth
rang
e (
%)
0 –
1.5
0 –
2.0
0 –
2.0
0 –
1.5
* 0 –
1%
0 –
2.0
0 –
2.0
0 –
3.0
Gro
wth
supple
ment
require
d
Yeast
extr
act
Vitam
ins o
r
ye
ast
extr
act
Vitam
ins o
r
ye
ast
extr
act
No
Y
east
extr
act
No
N
D
ND
Antibio
tic s
ensitiv
ity
Gm
R
Gm
R
Gm
R
ND
V
mR
AztR
Ery
R
Km
R
ND
N
D
DN
A G
+C
conte
nt (%
mol)
61.9
62.9
± 0
.1
62.6
64
63.5
± 0
.5
64.3
62.2
61.5
Sym
bio
tic n
itro
ge
n f
ixatio
n
Yes
Yes
Yes
ND
N
D
ND
N
D
ND
Chap
ter
4
178
Tab
le 4
.8. (c
ont.
)
Ch
ara
cte
ris
tic
1
2
3
4
5
6
7
8
Carb
on
so
urces
uti
lised
L-A
rabin
ose
D
-Ce
llobio
se
D
-Fru
cto
se
α
-D-G
lucose
S
uccin
ate
E
than
ol
G
lycero
l
M
ann
ito
l
p-H
ydro
xyb
en
zo
ate
+
+
+
+
+
+ - +
+
+
+
+
+
+ - +
+ -
+
+
+
+
+
- +
+
+
ND
ND
- - - - - - -
ND
- - - - - -
ND
ND
ND
- - +
ND
- - +
ND
- -
ND
-
ND
ND
ND
-
ND
- -
ND
-
ND
ND
ND
-
ND
Hydro
lysis
of
gela
tin
-
- -
+*
+
- -
W
Hydro
lysis
of
sta
rch
- -
- -*
-
- +
+
Acid
pro
duction
fro
m
α-D
-Glu
cose
-
- -
- W
-
ND
N
D
Oxid
ase
-
- -
+
- +
+
+
Ure
ase
+
+
+
-
- +
-
-
Try
pto
pha
n d
eam
inase
W
W
- N
D
- N
D
ND
N
D
Aceto
in p
roduction
W
W
W
-
- -
ND
N
D
Nitra
te r
eduction
-
+
+
- +
+
-
-
Azt
= a
ztre
onam
; E
ry =
ery
thro
myci
n;
Gm
= g
enta
mic
in;
Km
= k
anam
yci
n;
Vm
= v
anco
myci
n
* D
ata
taken
fro
m W
eon e
t al.
(2010)
Chapter 4
179
The morphological and physiological characteristics of the Listia angolensis
and Lupinus texensis strains are consistent with those recorded for Microvirga spp.,
but somewhat atypical when compared with other Alphaproteobacterial rhizobia.
Motility is a variable character in Microvirga spp.; M. flocculans and M. subterranea
are motile, while M. aerilata, M. aerophila and M. guangxiensis are not (Kanso &
Patel, 2003; Takeda et al., 2004; Weon et al., 2010; Zhang et al., 2009). Motility and
chemotaxis are important in the ecology of rhizobia, allowing the bacteria to colonise
the comparatively nutrient rich rhizosphere and conferring advantages in competition
and symbiotic efficiency (Caetano-Anollés et al., 1988; Tambalo et al., 2010). All
the Listia angolensis strains were motile in both plate and broth culture. It was
therefore surprising that under the same growth conditions Lut5 and Lut6T were non-
motile. It is possible that motility and flagellar synthesis are inducible rather than
constitutive properties in Lut5 and Lut6T and culture media do not provide the
appropriate conditions for such induction. Non-motility of rhizobial strains in
laboratory conditions has also previously been noted for type IIA strains of
Rhizobium tropici grown on soft agar (Martínez-Romero et al., 1991).
In keeping with other species of Microvirga, the Listia angolensis and
Lupinus texensis strains have comparatively high temperature optima of 35ºC
(WSM3693T), 39ºC (Lut6
T) and 41ºC (WSM3557
T) and are able to tolerate
temperatures of 38-45ºC. The optimal temperature for most rhizobia is 25 – 30ºC
(Garrity et al., 2005), although this may be a reflection of the preponderance of
species isolated from temperate biomes. Strains of Ensifer saheli, Ensifer terangae
and Rhizobium tropici, isolated from tropical legumes, are able to grow at 40 – 44ºC
(de Lajudie et al., 1994), while Methylobacterium nodulans grows optimally at 30 –
Chapter 4
180
37ºC (Jourand et al., 2004). Photosynthetic bradyrhizobial strains isolated from
nodules of the semi-aquatic legume Aeschynomene indica are also able to grow at
41ºC (van Berkum et al., 2006). The mean generation time of 1.6 – 1.8 h for the
Listia angolensis and Lupinus texensis strains is faster than that recorded for M.
subterranea and M. guangxiensis and faster than many other Alphaproteobacterial
rhizobia. Mean generation time for the fast-growing rhizobial genera Azorhizobium,
Rhizobium and Ensifer is between 1.5 – 6 h (Garrity et al., 2005).
Neither the Listia angolensis nor the Lupinus texensis strains were salt
tolerant, or acid tolerant, and they grew best in neutral to slightly basic conditions.
Their NaCl tolerance and pH growth range are consistent with reports for other
Microvirga species. Strictly aerobic growth is also characteristic of Microvirga, but
growth in stab cultures indicates that WSM3557T, WSM3693
T and Lut6
T (and by
implication the other Listia angolensis and Lupinus texensis strains) are
microaerobic. Intrinsic antibiotic resistance is a distinguishing phenotypic feature:
the Listia angolensis and Lupinus texensis strains are resistant to gentamycin, while
M. subterranea strain FaiI4T was vancomycin-resistant and M. guangxiensis strain
25BT was resistant to aztreonam, erythromycin and streptomycin sulphate (Kanso &
Patel, 2003; Zhang et al., 2009).
Another feature that distinguishes these novel strains is their pigmentation,
which has previously been noted in only two other rhizobial species: the pigmented
methylobacteria associated with species of Listia (previously Lotononis (Boatwright
et al., 2011)) and the photosynthetic bradyrhizobia isolated from Aeschynomene spp.
(Jaftha et al., 2002; Molouba et al., 1999; Norris, 1958; Yates et al., 2007). The
Chapter 4
181
pigments extracted from WSM3557T and Lut6
T are probably carotenoids, based on
the absorption spectra, which show peaks at 450nm (Lut6T) and 495nm
(WSM3557T) (Figure 4.6). This accords with results obtained for M. subterranea
(Kanso & Patel, 2003). Carotenoids typically absorb at between 400–500nm and
individual carotenoids possess unique absorption spectra (Britton et al., 2004). The
absorption spectrum (and thus the type of pigment) in Lut6T is clearly different from
that found in WSM3557T. In addition to carotenoids, the photosynthetic
bradyrhizobia and the pigmented methylobacteria also contain bacteriochlorophyll,
although the quantity of bacteriochlorophyll in the Listia methylobacteria is small,
relative to that found in the bradyrhizobia (Evans et al., 1990; Jaftha et al., 2002).
The absence of absorption peaks at 700–800nm indicates that WSM3557T and Lut6
T
do not contain bacteriochlorophyll, which is consistent with previous findings for M.
subterranea (Kanso & Patel, 2003). The absence of pigmentation in WSM3693T
suggests that this trait is not essential for either saprophytic or symbiotic competence
in this novel group of rhizobia.
4.4.3.1 Substrate utilisation
4.4.3.1.1 Critique of the use of Biolog plates in determining substrate
utilisation
The Biolog systems of phenotype analysis (the GN2 and PM microarrays)
allow rapid metabolic testing of microorganisms and have a wide variety of
applications (Bochner, 1989; Bochner, 2003). Biolog microarrays have been
employed in several previous examinations of rhizobial metabolism. These include
phenotypic characterisation of novel Rhizobium and Burkholderia species (Berge et
al., 2009; Chen et al., 2007), examination of the metabolic diversity of rhizobia
Chapter 4
182
associated with particular legume hosts (Wolde-Meskel et al., 2004) and assessment
of phenotypic variability in Ensifer meliloti populations (Biondi et al., 2009).
In this study, Biolog GN2 and PM plates were used to characterise the
metabolic properties of the novel Listia angolensis and Lupinus texensis strains.
Although the results were generally consistent, some caution needs to be exercised in
the interpretation, as a number of substrates have shown a potential to give false
positive or false negative readings. These potential false readings fall into three
categories:
1) Substrates where the results for the PM plate carbon substrates have
differed from those obtained using the GN2 plates, or from growth
studies in minimal media;
2) The surprisingly large differences in the substrate utilisation of
WSM3557T and Lut6
T on the PM plates, particularly for nitrogen,
phosphorus and sulfur sources;
3) Substrates that have been reported in the literature to give potential
false positives.
The differences in the GN2 plates as compared with the PM plates may be
partially explained by the data from the PM plates being read after 56 h, as compared
with the 96-h read for the GN2 plates, with the extra incubation time allowing more
readings to reach a threshold level in the latter. Substrates that were demonstrably
false negatives for WSM3557T on the PM plates include cellobiose, fructose, acetate,
succinate and glutamate, as these carbon sources supported growth of the strain in
Chapter 4
183
minimal media. Lut6T similarly grew on acetate, but returned a negative result for
acetic acid in the PM plates.
The results for WSM3557T on the PM plates, as compared with those of
Lut6T, may have been affected by the differences in exopolysaccharide production in
these strains. WSM3557T is noticeably more mucilaginous than Lut6
T, and excessive
mucopolysaccharide has been advanced as a possible reason for less than optimal
signal to noise ratios in PM plate reads (Biolog PM Services, pers comm). This may
account for the reduced number of positive results for WSM3557T. The negative
result obtained with WSM3557T and Lut6
T for sulfate as a substrate in the PM plates,
or indeed obtained by WSM3557T for any sulfur source, is probably an artefact,
given that sulfur is essential for bacterial protein synthesis, including nitrogenase
(Krusell et al., 2005).
Potential false positive reactions have previously been reported in PM plates
for the substrates dihydroxyacetone (a triose), and the pentose sugars D-xylose, D-
ribose, L-lyxose, and D- and L-arabinose (Line et al., 2010). The utilisation of L-
arabinose by the Listia angolensis and Lupinus texensis strains has been confirmed
by subsequent growth in minimal media on this substrate (this study).
4.4.3.1.2 Substrate utilisation in Listia angolensis and Lupinus texensis
strains
The putative type strains WSM3557T, WSM3693
T and Lut6
T differ from
other species of Microvirga on the basis of carbon substrate assimilation, in
particular their growth on D-cellobiose and D-fructose. Their ability to grow on a
variety of sole carbon sources markedly distinguishes them from M. subterranea
Chapter 4
184
FaiI4T and M. flocculans TFB
T, which both utilise a comparatively narrow range of
carbon substrates. This may be related to the more oligotrophic nature of the aquatic
environments from which the latter were isolated. Of 37 carbon substrates tested, M.
flocculans TFBT grew only on yeast extract, peptone and tryptone Takeda et al.,
2004). M. subterranea FaiI4T grew on yeast extract, tryptone, casein hydrolysate,
xylose and acetate (Kanso & Patel, 2003). M. aerophila and M. aerilata (isolated
from atmospheric samples) are also reported to be unable to assimilate any of the
substrates present in the API 20NE and ID 32GN strips (Weon et al., 2010). The
soil-borne M. guangxiensis 25BT utilises a slightly different set of carbon sources
from the Listia angolensis and Lupinus texensis strains. This strain uses arabitol, (+)-
D-glucose, myo-inositol, (+)-maltose, (+)-D-mannitol, (+)-melezitose, (+)-melibiose,
peptone, (+)-D-sorbitol and (+)-D-xylose, but not (+)-cellobiose, ethanol, (+)-D-
fructose, glycerol or α-L-rhamnose (Zhang et al., 2009).
The carbon substrate utilisation patterns of the Listia angolensis and Lupinus
texensis strains, in minimal media and on the GN2 and PM microplates, demonstrate
evidence of their adaptation to a soil or rhizosphere environment. They are able to
metabolise arabinose, glucose, mannose, rhamnose, xylose and galacturonic acid,
which are present in legume and other plant root mucilages (Knee et al., 2001).
WSM3693T and the Lut isolates grow on p-hydroxybenzoate, one of the aromatic
compounds obtained from the breakdown of lignin, an important plant-derived
component of land-based biomass (Harwood & Parales, 1996). The utilisation of
some substrates may also have symbiotic implications. Catabolism of inositol and
rhamnose has been shown to be important to rhizobial competitiveness in some
symbioses (Jiang et al., 2001; Oresnik et al., 1998). Cellobiose and pectin, along
Chapter 4
185
with arabinose, glucose, rhamnose, xylose and galacturonic acid, are components of
plant cell walls (Delmer & Amor, 1995; Mort & Grover, 1988). Penetration of the
plant cell wall occurs as part of the rhizobial infection process, and the ability to
degrade pectin may play a role in this process. Symbiotic induction of a pectate lyase
has been demonstrated for Rhizobium etli (Fauvart et al., 2009). Rhizobia also
require the ability to utilise dicarboxylic acids, as the dicarboxylates fumaric,
succinic and malic acid are primary carbon sources for bacteroids within nodules
(Lodwig & Poole, 2003). In contrast to M. flocculans TFBT and M. subterranea
FaiI4T, the Listia angolensis and Lupinus texensis strains are able to grow on
succinate in minimal media.
In pea nodules, Rhizobium leguminosarum bacteroids become symbiotic
auxotrophs and are reliant on the legume host for the supply of the branched chain
amino acids leucine, isoleucine and valine (Prell et al., 2009). It is therefore
interesting that these amino acids were not metabolised as either carbon or nitrogen
sources in the PM and GN2 microplates. These are preliminary results that need to be
confirmed, but may reflect differences in the functioning of the pea nodule, as
compared with the structurally different nodules of Listia and Lupinus species
(Chapter 3). Utilisation of at least one of these branched chain amino acids as a
carbon source has been demonstrated for strains of Mesorhizobium loti, Rhizobium
leguminosarum, Rhizobium tropici, Ensifer fredii, Ensifer meliloti, Ensifer saheli and
Ensifer terangae, but not for Azorhizobium caulinodans (de Lajudie et al., 1994). On
Biolog PM microplates, E. meliloti 1021 utilises leucine, but not isoleucine and
valine, as carbon sources, and all three amino acids as nitrogen sources (Biondi et al.,
2009).
Chapter 4
186
Metabolic versatility varies among different rhizobial taxa. A recent paper by
Biondi et al. (2009) used Biolog PM plates to analyse the metabolic capacity of E.
meliloti strains and allows direct comparisons to be made between E. meliloti strain
Sm1021 and WSM3557T and Lut6
T. Sm1021 utilised a far wider range of substrates
(84 of 190 carbon sources, 84 of 95 nitrogen sources, 55 of 59 phosphorus sources
and 29 of 35 sulfur sources (Biondi et al., 2009)) than did either WSM3557T or
Lut6T (Section 4.3.2.5.2; Table 1, Appendix II). The Microvirga rhizobial strains
described in this study thus appear to have a relatively narrow range of carbon
substrate utilisation. Interestingly, the carbon substrate utilisation pattern of the
Microvirga rhizobia also differs from that of the pigmented Methylobacterium
rhizobia associated with Listia spp., which on GN2 plates oxidise very few
carbohydrates and all 24 carboxylic acid sources (Jaftha et al., 2002).
The requirement for supplementary growth factors varies in Microvirga
species. Yeast extract is an absolute requirement for growth of M. subterranea
FaiI4T, Lut5 and Lut6
T in minimal media, while the Listia angolensis strains require
either yeast extract or the complex vitamin mix detailed in Egli and Auling (2005). In
contrast, M. flocculans TFBT and M. guangxiensis 25B
T require no supplements for
growth in minimal media. Vitamin requirements vary amongst rhizobial strains;
biotin, thiamine and calcium pantothenate are essential for the growth of many fast-
growing species (Graham, 1963), but not required for some strains of slow-growing
rhizobia (Stowers & Elkan, 1984). The Listia angolensis and Lupinus texensis strains
appear to have the most stringent growth factor requirements of any rhizobial
species. Interestingly, methylobacteria require no supplements for growth in minimal
Chapter 4
187
media (Green, 1992); this holds true also for the pigmented Listia methylobacteria (J.
Ardley, unpublished data).
4.4.4 Biochemical characteristics of Listia angolensis and Lupinus
texensis strains
The results obtained for the Listia angolensis and Lupinus texensis strains from
the API 20E strips and other biochemical tests are consistent with results obtained for
other species of Microvirga (Table 4.8). All strains of bacteria in this clade gave
negative results for indole production, glucose fermentation and arginine
dihydrolase, but were positive for catalase. Hydrolysis of gelatin and starch,
reduction of nitrate to nitrite, production of oxidase and urease and weak production
of acetoin and tryptophan deaminase are distinguishing phenotypic features for the
different species. The Listia angolensis and Lupinus texensis strains and M.
guangxiensis 25BT did not produce acid from growth on D-glucose or D-mannitol
(Zhang et al., 2009; this study). M. subterranea FaiI4T is reported to show weak
production of acid from D-glucose, although it is unable to grow on this substrate
(Kanso & Patel, 2003).
4.4.5 Host range
Previous reports have indicated that both the Listia angolensis and Lupinus
texensis strains have a narrow host range (Andam & Parker, 2007; Yates et al.,
2007). The results in this study confirm this finding, as WSM3557T, WSM3693
T,
Lut5 and Lut6T were able to elicit nodules on some other, usually promiscuous,
hosts; but such nodulation was usually only occasional and always ineffective. The
host range of the Lupinus texensis strains appears to be narrower than that of the
Listia angolensis strains. Lut6T is unable to nodulate other native North American
Chapter 4
188
Lupinus spp. (Andam & Parker, 2007), while WSM3557T, although unable to
nodulate Listia bainesii and Listia heterophylla, is able to nodulate, and in some
cases fix nitrogen, with other Lotononis s. l. species (Chapter 2). Furthermore,
WSM3557T and WSM3693
T are able to form ineffective nodules on Lupinus texensis
(Matt Parker, unpublished data), whereas Lut5 and Lut6T are unable to nodulate
Listia angolensis. This is an interesting finding, considering the specificity of L.
texensis. It also suggests that the molecular basis for specificity in these symbioses is
not limited to Nod factors, given that the nodA phylogenetic tree (Figure 2, Appendix
VIII) places the nodA of Lut6T in a separate group to that of WSM3557
T and
WSM3693T.
4.5 Summary, emended description of Microvirga and
description of Microvirga lotononidis sp. nov., Microvirga
zambiensis sp. nov. and Microvirga lupini sp. nov.
In summary, on the basis of 16S rRNA and concatenated housekeeping gene
sequence identity, the Listia angolensis strains WSM3557T and WSM3693
T and the
Lupinus texensis strains Lut5 and Lut6T belong to the genus Microvirga. The ability
of these strains to nodulate and form N2-fixing symbioses with species of leguminous
plants distinguishes them from previously reported species of Microvirga. Additional
genotypic, phenotypic, and chemotaxonomic data support the classification of these
strains as three new species within the genus Microvirga. The names M. lotononidis
sp. nov., M. zambiensis sp. nov. and M. lupini sp. nov. are proposed, with the isolates
WSM3557T, WSM3693
T and Lut6
T representing the respective type strains.
Chapter 4
189
4.5.1 Emended description of Microvirga (Kanso & Patel, 2003 emend.
Zhang et al, 2009, emend. Weon et al, 2010)
The description remains as given by Kanso & Patel (2003), Zhang et al.
(2009) and Weon et al. (2010), with the following modifications. Some strains are
capable of nodulation and symbiotic nitrogen fixation with legumes. The type
species is Microvirga subterranea.
4.5.2 Description of Microvirga lotononidis sp. nov.
Microvirga lotononidis (lo.to.no'ni.dis. N.L. gen. n. lotononidis, of Lotononis,
a taxon of leguminous plants, referring to the isolation source of the first strains,
nodules of Listia angolensis, a species in the Lotononis s. l. clade.
Cells are strictly aerobic, asporogenous, Gram-negative rods (0.4-0.5 x 1.0-2.2
µm), motile with one or more polar flagella. Grows well on YMA, ½ lupin agar, TY
agar and nutrient agar. On ½ LA after three days at 28 ºC, colonies are light pink,
convex, smooth, mucilaginous and circular, with entire margins, 0.5-1.5 mm in
diameter. Grows from 15-44/45 ºC; optimum temperature for the type strain is 41 ºC
and mean generation time at this temperature is 1.6 h. Best growth is at pH 7.0-8.5
(range 5.5-9.5), and 0.0-1.0 % (w/v) NaCl (range 0-2.0 % (w/v)). Yeast extract or the
vitamin mix detailed in Egli and Auling (2005) is an absolute requirement for growth
in minimal media. The main cellular fatty acids are 18:1 ω7c and 19:0 cyclo ω8c.
Positive for catalase and urease and weakly positive for tryptophan deaminase and
acetoin production. Oxidase, β-galactosidase, arginine dihydrolase, lysine
decarboxylase, ornithine decarboxylase, indole and hydrogen sulphide production are
negative, as is utilisation of citrate. Gelatin and starch are not hydrolysed. Nitrite is
produced from nitrate. Acid is produced from growth on L-arabinose but not from
Chapter 4
190
growth on α-D-glucose or D-mannitol. Resistant to gentamicin and some strains are
partially resistant to ampicillin, chloramphenicol, kanamycin and spectinomycin.
Sensitive to nalidixic acid, rifampicin, streptomycin and tetracycline. Assimilates L-
arabinose, D-cellobiose, D-fructose, α-D-glucose, glycerol, D-mannitol, acetate,
succinate and glutamate. The G + C content of the type strain is 62.8-63.0 %.
The type strain WSM3557T (= LMG26455
T, = HAMBI3237
T) and other strains
were isolated from N2-fixing nodules of Listia angolensis originally collected in
Zambia.
4.5.3 Description of Microvirga zambiensis sp. nov.
Microvirga zambiensis (zam.bi.en'sis. N.L. fem. adj. zambiensis, of or
belonging to Zambia, from where the type strain was isolated).
Cells are strictly aerobic, asporogenous, Gram-negative rods (0.4-0.5 x 1.0-
2.2 µm), motile with one or more polar flagella. Grows well on YMA, ½ lupin agar,
TY agar and nutrient agar. On ½ LA after three days at 28ºC, colonies are cream
coloured, convex, smooth, mucilaginous and circular, with entire margins, 0.5-1.5
mm in diameter. Grows from 15-38 ºC; optimum temperature is 35 ºC and mean
generation time at this temperature is 1.7 h. Best growth is at pH 7.0-8.5 (range 6.0-
9.5) and 0.0-0.5 % (w/v) NaCl (range 0-1.5 % (w/v)). Yeast extract or the vitamin
mix detailed in Egli and Auling (2005) is an absolute requirement for growth in
minimal media. The main cellular fatty acids are 18:1 ω7c and 19:0 cyclo ω8c.
Positive for catalase and urease and weakly positive for acetoin production. Oxidase,
β-galactosidase, arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase,
tryptophan deaminase, indole and hydrogen sulphide production are negative, as is
Chapter 4
191
utilisation of citrate. Gelatin and starch are not hydrolysed. Nitrite is produced from
nitrate. Acid is produced from growth on L-arabinose but not from growth on α-D-
glucose or D-mannitol. Resistant to gentamicin. Sensitive to ampicillin,
chloramphenicol, kanamycin nalidixic acid, rifampicin, spectinomycin, streptomycin
and tetracycline. Assimilates L-arabinose, D-cellobiose, D-fructose, α-D-glucose,
glycerol, D-mannitol, acetate, succinate, p-hydroxybenzoate and glutamate. The G +
C content of the type strain is 62.6 %.
The type strain WSM3693T (= LMG26454
T, = HAMBI3238
T) was isolated
from N2-fixing nodules of Listia angolensis originally collected in Zambia.
4.5.4 Description of Microvirga lupini sp. nov.
Microvirga lupini (lu.pi'ni. L. n. lupinus, a lupine and also a botanical generic
name (Lupinus); L. gen. n. lupini, of Lupinus, isolated from Lupinus texensis.
Cells are strictly aerobic, asporogenous, Gram-negative non-motile rods (0.4-
0.5 x 1.0-2.2 µm). Grows well on YMA, ½ lupin agar, TY agar and nutrient agar. On
½ LA after three days at 28 ºC, colonies are pale orange, convex, smooth and
circular, with entire margins, 0.5-1.5 mm in diameter. Grows from 10-43 ºC;
optimum temperature is 39 ºC and mean generation time at this temperature is 1.8 h.
Best growth is at pH 7.0-8.5 (range 5.5-9.5) and 0.0-0.5 % (w/v) NaCl (range 0-1.5
% (w/v)). Yeast extract is an absolute requirement for growth in minimal media. The
main cellular fatty acids are 18:1 ω7c and summed feature 2 (16:1 iso I / 14:0 3 OH /
unknown 10.938). Positive for catalase and urease and weakly positive for
tryptophan deaminase and acetoin production. Oxidase, β-galactosidase, arginine
dihydrolase, lysine decarboxylase, ornithine decarboxylase, indole and hydrogen
Chapter 4
192
sulphide production are negative, as is utilisation of citrate. Gelatin and starch are not
hydrolysed. Nitrite is not produced from nitrate. Acid is produced from growth on L-
arabinose but not from growth on α-D-glucose or D-mannitol. Partially resistant to
ampicillin, chloramphenicol, gentamicin and streptomycin and sensitive to
kanamycin, nalidixic acid, rifampicin, spectinomycin and tetracycline. Assimilates
L-arabinose, D-cellobiose, D-fructose, α-D-glucose, D-mannitol, acetate, succinate,
glutamate, ethanol and p-hydroxybenzoate. The G + C content of the type strain is
61.9 %.
The type strain Lut6T (= LMG26460
T; = HAMBI3236
T) and other strains
were isolated from N2-fixing nodules of Lupinus texensis collected in Texas, USA.
Chapter 5
193
CHAPTER 5
General discussion
Chapter 5
194
Chapter 5
195
5.1 Symbiotic relationships within Lotononis s. l.
The nitrogen-fixing symbiosis between the southern African crotalarioid
legume Listia bainesii (formerly Lotononis bainesii (Boatwright et al., 2011) and
strains of pigmented Methylobacterium sp. has long been known to be a model of
symbiotic specificity, both for the plant and the microsymbiont (Broughton et al.,
1986; Norris, 1958; Pueppke & Broughton, 1999). Until recently, however, this
symbiosis had been examined in isolation and outside the context of the symbiotic
relationships and biogeography of the Lotononis s. l. clade (comprising the genera
Leobordea, Listia and Lotononis s. str. (Boatwright et al., 2011)) in its southern
African centre of origin. Studies undertaken by the Centre for Rhizobium Studies
(Yates et al., 2007; R. Yates, unpublished data) partially addressed this issue by
showing that all examined Listia species were effectively nodulated by pigmented
Methylobacterium sp. strains, except for the more tropically distributed Listia
angolensis, which forms N2-fixing nodules with a novel lineage of rhizobia. No
previous study, however, has examined the symbiotic relationships between the
different Lotononis s. l. taxa and their cognate rhizobia. The work presented here
sought to assess the nodulation and N2-fixation abilities of Lotononis s. l.- associated
rhizobia on host species that were representative of the taxonomic diversity of the
Lotononis s. l. clade. To determine whether symbiotic specificity in the genus Listia
was confined to L. bainesii, three Listia host species (L. angolensis, L. bainesii and
L. heterophylla) were also inoculated with type strains, or well-characterised strains,
of diverse rhizobia. In addition, this work sought to analyse the phylogeny of the
chromosomal and symbiotic genes of Lotononis s. l.-associated rhizobia, to
investigate infection, nodule initiation and/or nodule morphology in Lotononis s. l.
Chapter 5
196
species and to validly name and characterise the L. angolensis rhizobia, identified in
this study as novel species of Microvirga.
The results of these studies demonstrate three salient features, summarised in
Figure 5.1, of the symbioses between Lotononis s. l. species and their cognate
rhizobia. Firstly, the rhizobia isolated from nodules of Lotononis s. l. species are
remarkably diverse. Authenticated rhizobial strains able to nodulate Lotononis s. l.
hosts have been identified as belonging to the genera Bradyrhizobium, Ensifer,
Mesorhizobium, Methylobacterium and Microvirga.
Chap
ter
5
197
Fig
ure
5.1
. S
ym
bio
tic
and p
hylo
gen
etic
rel
atio
nsh
ips
of
Loto
nonis
s.
l. s
pec
ies
and a
ssoci
ated
rhiz
obia
. R
hiz
obia
l phylo
gen
y i
s bas
ed o
n t
he
16S
rR
NA
gen
e;
Loto
nonis
s.
l. p
hylo
gen
y i
s ac
cord
ing t
o v
an W
yk (
1991).
Fil
led c
ircl
es =
eff
ecti
ve
nodula
tion;
empty
cir
cles
= i
nef
fect
ive
nodula
tion;
bar
red c
ircl
es =
the
rhiz
obia
l st
rain
was
iso
late
d f
rom
this
host
, but
the
host
was
not
test
ed f
or
nodula
tion w
ith a
ny r
hiz
obia
; no c
ircl
e =
no n
odula
tion.
Leobord
ea b
olu
sii,
for
exam
ple
, is
eff
ecti
vel
y n
odula
ted b
y t
he
Bra
dyrh
izobiu
m a
nd M
icro
virg
a s
trai
ns,
and f
orm
s in
effe
ctiv
e nodule
s w
ith t
he
rem
ainin
g i
nocu
lant
stra
ins.
1 =
Lis
tia;
2 =
Leobord
ea;
3 =
Loto
nonis
s.
str.
Br.
= B
radyrh
izobiu
m;
E.
= E
nsi
fer;
Mes
. =
Meso
rhiz
obiu
m;
Mic
. =
Mic
rovi
rga;
Mtb
. =
Meth
ylobacte
rium
. *
Lis
tia h
ete
rophyll
a w
as o
nly
tes
ted w
ith W
SM
2598 a
nd W
SM
3557.
† T
hes
e host
spec
ies
wer
e not
inocu
late
d w
ith a
ny r
hiz
obia
. ‡
Thes
e host
spec
ies
wer
e
not
inocu
late
d w
ith W
SM
2598 o
r W
SM
3557.
Li.
an
go
len
sis
Li.
ba
ine
sii
Li.
he
tero
ph
yll
a*
Le
. lo
ng
iflo
ra
Le
. m
oll
is‡
Le
. ca
lyc
ina
†
Lo
. p
un
ge
ns
‡
Le
. p
laty
carp
a
Le
. st
ipu
losa
Le
. b
olu
sii
Le
. p
oly
cep
ha
la ‡
Lo
. cr
um
an
ina
Lo
. d
eli
cata
‡
Lo
. fa
lca
ta
Lo
. la
xa
‡
Br.
sp W
SM
25
96
Br.
sp W
SM
26
32
Br.
sp W
SM
27
83
Mic
. lo
ton
on
idis
WS
M3
55
7L
e.
foli
osa
†
Mtb
. n
od
ula
ns
WS
M2
66
7
Mtb
.sp
. W
SM
25
98
Me
s.
tia
nsh
an
en
se
WS
M2
62
4
E.
me
lilo
ti W
SM
26
53
E.
me
lilo
tiW
SM
30
40
1
2 3
Li.
an
go
len
sis
Li.
ba
ine
sii
Li.
he
tero
ph
yll
a*
Le
. lo
ng
iflo
ra
Le
. m
oll
is‡
Le
. ca
lyc
ina
†
Lo
. p
un
ge
ns
‡
Le
. p
laty
carp
a
Le
. st
ipu
losa
Le
. b
olu
sii
Le
. p
oly
cep
ha
la ‡
Lo
. cr
um
an
ina
Lo
. d
eli
cata
‡
Lo
. fa
lca
ta
Lo
. la
xa
‡
Br.
sp W
SM
25
96
Br.
sp W
SM
26
32
Br.
sp W
SM
27
83
Mic
. lo
ton
on
idis
WS
M3
55
7L
e.
foli
osa
†L
e.
foli
osa
†
Mtb
. n
od
ula
ns
WS
M2
66
7
Mtb
.sp
. W
SM
25
98
Me
s.
tia
nsh
an
en
se
WS
M2
62
4
E.
me
lilo
ti W
SM
26
53
E.
me
lilo
tiW
SM
30
40
1
2 3
Chapter 5
198
Secondly, different specificity groups exist within the Lotononis s. l. clade.
Leobordea and Lotononis s. str. appear to be more or less promiscuous and able to
nodulate (often ineffectively) with a range of Lotononis s. l. rhizobia. In contrast,
Listia species are more specific. This study has confirmed the extreme symbiotic
specificity of L. bainesii, which does not nodulate with any of the Lotononis s. l.
rhizobia, other than its cognate methylobacteria. Additionally, none of the diverse
rhizobial strains (including Azorhizobium, Bradyrhizobium, Burkholderia, Ensifer
and Rhizobium species and Methylobacterium nodulans) inoculated onto L. bainesii
and L. heterophylla was able to elicit nodules on these host plants. From these and
previous results (Norris, 1958; Yates et al., 2007; R. Yates, unpublished data) it
appears likely that this pattern of extreme symbiotic specificity applies to all the
Listia species that are effectively nodulated by these methylobacteria. The single
exception is L. angolensis, which can be nodulated by the Listia methylobacteria and
by Methylobacterium nodulans strains ORS2060 and WSM2667 (but not by any
other tested rhizobial strains), but forms effective symbioses only with the novel
Microvirga species.
Thirdly, the rhizobia associated with Lotononis s. l. species exhibit a wide
range of variability in both host range and effectiveness. The extremely narrow host
range of the Listia methylobacteria was confirmed. Methylobacterium strain
WSM2598 was highly effective on L. bainesii, but ineffective on L. angolensis and
only able only to form (ineffective) nodules on the most promiscuous species of the
remaining Lotononis s. l. hosts. Somewhat surprisingly, of all the rhizobial isolates,
the Microvirga strain WSM3557 was effective over the widest range of Lotononis s.
l. host species. The remaining rhizobial strains did not appear to be strongly
Chapter 5
199
associated with any taxonomic group of Lotononis s. l. and were generally poorly
effective on these hosts.
As legume hosts radiate from their centre of origin and adapt to different
climatic and edaphic conditions, what happens to their associations with their
microsymbionts? The results obtained for the Lotononis s. l. clade raise interesting
questions about this process and the development of symbiotic specificity that has
occurred in the genus Listia.
5.1.1 Habitat as a driver of rhizobial diversity
It is not surprising that rhizobia isolated from Lotononis s. l. hosts should be
taxonomically diverse. As South Africa is the centre of origin of these legumes, their
microsymbionts would also be expected to have a wide range of genotypes, in
accordance with gene centre theory (Andronov et al., 2003). Numerous previous
studies have demonstrated the genotypic diversity of rhizobia isolated from wild
legumes in a given geographical area (Han et al., 2009; Lorite et al., 2010; Odee et
al., 2002; Rasolomampianina et al., 2005; Sylla et al., 2002; Zhao et al., 2010). In
this regard, topo-edaphic heterogeneity, seen for example in the Cape Floristic
Region, may not only be a driver of diversification and speciation of the flora of this
region (Cowling et al., 2009) but may also be a factor in the genotypic diversity of
the rhizobia associated with the Lotononis s. l. clade. As rhizobia are not obligate,
vertically transmitted symbionts (Young & Johnston, 1989), free-living root nodule
bacteria require saprophytic competence in the host plant’s preferred habitat if they
are to be available for selection by the host. There are characteristic differences in the
ability of various species and genera of rhizobia to tolerate acid or alkaline
conditions (Graham, 2008; Herridge, 2008; Zahran, 1999) and several studies on
Chapter 5
200
legumes nodulated by taxonomically diverse rhizobia have found a correlation
between eco-regions and/or edaphic factors and the microsymbiont genotype (Bala &
Giller, 2006; Diouf et al., 2007; Garau et al., 2005; Han et al., 2009; Lu et al., 2009).
In the case of the genus Listia, the waterlogged habitat favoured by these
plants (van Wyk, 1991) may be an important determining factor in their symbiotic
association with strains of Methylobacterium and Microvirga, two bacterial genera
not commonly known to contain rhizobial species. In addition, Listia species are
distinguished by their adventitious roots, which arise from stem nodes, and by
lupinoid nodules, which typically develop on the hypocotyl and tap root (Boatwright
et al., 2011; Yates et al., 2007; this study). Methylobacterium and Microvirga
rhizobia associated with Listia species may harbour traits that confer a competitive
advantage for saprophytic competence in waterlogged environments, or for
colonisation of the hypocotyl and rhizosphere in these conditions.
Members of the genus Methylobacterium have demonstrated saprophytic
competence in aquatic habitats, being ubiquitous in water, as well as soil and air
environments; numerous strains have been isolated from water samples (Gallego et
al., 2005; Gallego et al., 2006; Green, 1992; Hiraishi et al., 1995). Methylobacteria
are known to colonise plant surfaces (Madhaiyan et al., 2009b), although it is
interesting that a recent study on the ability of epiphytic methylobacteria to colonise
the Arabidopsis thaliana phyllosphere found that rhizobial Methylobacterium species
are not competitive in this environment (Knief et al., 2010). Microvirga species have
similarly been isolated from aquatic or potentially waterlogged environments (Kanso
& Patel, 2003; Takeda et al., 2004; Zhang et al., 2009). Additionally, the ability of
Chapter 5
201
all described Microvirga species (including the novel rhizobial Microvirga species
characterised in this study) to tolerate relatively high temperatures correlates well
with their isolation from thermal waters or subtropical regions (Kanso & Patel, 2003;
Takeda et al., 2004; Zhang et al., 2009) and with the isolation of rhizobial
Microvirga species from the tropically or sub-tropically distributed Listia angolensis
and Lupinus texensis (Andam & Parker, 2007; van Wyk, 1991; Yates et al., 2007).
Strikingly, the legume nodule isolates AC72a and ARRI 185 (genospecies AL) that
have now been identified as Microvirga strains (this study) have also come from
tropically or sub-tropically distributed host plants (Lafay & Burdon, 2007; Wolde-
Meskel et al., 2005). Graham (2008) has recommended that new rhizobial organisms
be examined for unusual traits that could influence their ecological performance.
Both the Methylobacterium and Microvirga microsymbionts are unusual and
uncommon species of rhizobia. They merit further study to determine which genes
and physiological factors contribute to saprophytic or competitive ability within the
host plant’s environment.
Environmental pressures may drive the selection of saprophytically
competent rhizobia, but do host plants provide a similar selective pressure for
particular phylogenies of rhizobial nod genes? Evidence for this varies and can be
examined in the context of the Lotononis s. l.-rhizobia symbioses.
5.1.2 Lotononis s. l. rhizobia have diverse nodA sequences
The nod genes encode Nod factors that are required by most rhizobia for
infection and nodulation of the legume host and are important in determining host
range and specificity (Dénarié et al., 1996; Perret et al., 2000). The nodA gene is a
useful symbiotic marker as it encodes a product that is a host-specific determinant of
Chapter 5
202
the transfer of fatty acids in Nod factor biosynthesis (Dénarié et al., 1996; Haukka et
al., 1998; Roche et al., 1996) and nodA phylogeny can give an indication of the Nod
factor structure (Moulin et al., 2000; Moulin et al., 2004). Previous studies of
legume-rhizobia symbiotic relationships have found a correlation between nod gene
phylogeny and host range (Ba et al., 2002; Dobert et al., 1994; Haukka et al., 1998;
Laguerre et al., 2001; Lu et al., 2009); in other studies, however, this association is
not as strong and nod gene phylogeny is more closely aligned with the rhizobial
chromosomal background than with legume taxonomy (Han et al., 2010; Lorite et
al., 2010; Zhao et al., 2010).
The nodA genes of rhizobia associated with the Lotononis s. l. clade are
polyphyletic and their phylogenies appear to correlate more with those of the
rhizobial 16S rRNA genes than with the taxonomy of the host plants, as the
polyphyletic nodA lineages of the Lotononis s. l. rhizobia are associated with
taxonomically diverse legumes (Figure 2.11). The high nodA sequence similarity
seen in strains within genera (and within the pigmented Methylobacterium and
Methylobacterium nodulans species) is consistent with horizontal gene transfer
(HGT) and supports the view of Wernegreen & Riley (1999) that HGT occurs
within, but not between, genetic subdivisions. That HGT does occur between
rhizobial genera has been shown in studies by Andam et al. (2007), Barcellos et al.
(2007) and Cummings et al. (2009), which provide evidence for symbiotic gene
transfer from Burkholderia to Cupriavidus strains, from a Bradyrhizobium strain to
an indigenous Ensifer strain, and from an Ensifer strain to a novel rhizobial
Agrobacterium strain, respectively. Presumably, however, these are comparatively
rare events due to the mechanistic barriers that constrain HGT between
Chapter 5
203
phylogenetically distant species (Lawrence & Hendrickson, 2003), whereas HGT
between related rhizobia is more common. Physical proximity also appears to be
important, as the main trends in gene transfer are observed among species from
different taxa inhabiting the same habitat (Kloesges et al., 2011).
In this regard, it is significant that aquatic environments (which, in
comparison to soil, are oligotrophic) support fewer bacterial taxa than soil, and
saturated soils have both fewer taxa and a more uneven distribution than unsaturated
soils (Horner-Devine et al., 2004; Zhou et al., 2002). The implications for the Listia-
rhizobia symbioses are that their waterlogged habitat may reduce the diversity of the
rhizobial pool available for plant selection and act as an additional barrier to the
transfer of symbiotic genes across bacterial taxa.
5.1.3 Specificity and effectiveness within Lotononis s. l.
Previous studies of wild legumes nodulated by diverse rhizobia have noted
the generally promiscuous nature of these relationships and the preference of
endemic rhizobial populations of a certain geographical area for a wide spectrum of
hosts (Han et al., 2010; Zahran, 2001). There appears to be a natural variation in the
symbiotic infectivity and effectiveness of rhizobial isolates within wild host species
(Burdon et al., 1999; Odee et al., 2002; Thrall et al., 2000). Similarly, the variations
in response to an inoculant strain (i.e. no nodulation, ineffective nodulation or
effective nodulation) that were seen in individual plants within species of Leobordea
and Lotononis s. str. (where seeds from wild plants, rather than from a particular
accession were used) have been found in other studies (Wilkinson et al., 1996). This
is likely to be related to seed provenance and variability in the host genotype, leading
to differing symbiotic compatibilities with rhizobial partners.
Chapter 5
204
In contrast, all Listia species, other than L. angolensis, form effective N2-
fixing associations with all tested strains of their cognate, pigmented methylobacteria
(Eagles & Date, 1999; Yates et al., 2007; R. Yates, unpublished data). It is
remarkable that this effectiveness extends across a range of plant accessions and over
80 isolates collected across a broad geographical area in South Africa and Zimbabwe
and is in direct contrast to the variability in effectiveness seen in individual plants
within species of Leobordea and Lotononis s. str. Clearly, despite the variability in
host genotypes (five Listia species are known to nodulate with pigmented
methylobacteria (R. Yates, unpublished data); additionally, L. bainesii is highly
allogamous (Real et al., 2004)), effective symbiosis with pigmented methylobacteria
is strongly selected for in this group of legumes. The results confirm Sprent’s
(2008b) observations that specificity is associated with effectiveness, but show that
this close mutual relationship is not confined to temperate areas, but also found in
legumes from warmer latitudes.
Hosts are presumably under selection pressure to maximise symbiont
effectiveness (Douglas, 1998). It has been experimentally shown that legumes are
able to selectively favour the most beneficial RNB by imposing sanctions on non-
fixing rhizobia (Kiers et al., 2008). In mutualisms, however, there is a potential for
conflict of interest between the symbiont and the host (Herre et al., 1999). Factors
that are suggested to align these interests are: vertical transmission of symbionts,
genotypic uniformity of symbionts within individual hosts, spatial structure of
populations leading to repeated interactions between would-be mutualists, and
restricted options outside the relationship for both partners; conversely, horizontal
transmission, multiple symbiont genotypes and varied options decrease symbiotic
Chapter 5
205
stability (Herre et al., 1999). The legume-rhizobia symbiosis falls into the latter
category (Kiers et al., 2008). In the case of Listia species, however, the waterlogged
habitat of the host plants may potentially reduce the diversity of rhizobial partners
available for host selection. In such a case, the selection pressures may favour the co-
evolution of the host and microsymbiont towards a more effective symbiosis and the
development of specificity, as seen in the Listia-Methylobacterium symbiosis. Thrall
et al. (2000) have noted that Acacia species with more limited distributions or tighter
ecological requirements have a greater degree of specificity than widespread species.
An analysis of the rhizobial diversity present in habitats favoured by Listia species
would confirm whether or not the host plants face a restricted choice of rhizobial
partner. The reduced specificity seen in L. angolensis may be a function of the
putative expansion of L. angolensis into more tropical habitats and symbiont
replacement of Methylobacterium species by less symbiotically adapted Microvirga
strains. Molecular clock analyses of the divergence of L. angolensis from other Listia
species and of the genus Listia from the Lotononis s. l. clade could help answer these
questions.
5.1.4 Models of legume-rhizobia symbiosis
From the data presented here and in previous studies, there appear to be
several models of the relationships between legume hosts, rhizobial chromosomal
backgrounds and nodulation genes:
1) Legumes are generally promiscuous and able to associate with a wide
range of different rhizobial chromosomal backgrounds and nod gene lineages, as
seen in species of Leobordea and Lotononis s. str. in this study and in Lathyrus,
Lotus and Sophora species (Han et al., 2010; Zhao et al., 2010)
Chapter 5
206
2) Legumes are associated with both a particular rhizobial chromosomal
background and a nod gene lineage. This model is seen in species in the Vicieae tribe
that are nodulated by Rhizobium leguminosarum bv vicieae strains containing a
distinct nodD clade (Mutch & Young, 2004) and in European Genisteae
bradyrhizobia, which all group within the nodA gene clade II (Kalita et al., 2006;
Moulin et al., 2004; Stepkowski et al., 2007).
3) Legumes associate with rhizobia of different chromosomal backgrounds,
but harbouring similar nod genes. This infers horizontal transfer of the symbiotic
genes. Rogel et al. (2011) have used the term “symbiovar” to distinguish
symbiotically distinct subgroups within a single rhizobial species. Examples include
the symbioses between Acacia tortilis and Phaseolus vulgaris and their diverse
rhizobia (Ba et al., 2002; Laguerre et al., 2001).
4) Occasionally, a subset of legumes within a taxon is specifically nodulated
by rhizobia that are both chromosomally distinct and harbour a different lineage of
nod genes from the rhizobia that nodulate other hosts within that legume taxon. This
has been observed in Listia (this study) and in species of Crotalaria (Renier et al.,
2008), Lupinus (Andam & Parker, 2007), Rhynchosia (Garau et al., 2009) and
Sesbania (Cummings et al., 2009).
This suggests that the different symbiotic models are functions of the weight
assigned to selective pressures exerted by the environment and the host plant on
rhizobia. Further research on the microsymbionts of wild legumes in their centres of
diversity, especially in environments that exert a strong selective pressure on the
rhizobia, could help to shed light on the evolution of these symbiotic relationships.
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5.2 Infection and nodule organogenesis in Listia spp.
The initial infection process in Listia species is remarkable, in that epidermal
cells on the hypocotyl and root, unlike other studied legumes (Bhuvaneswari et al.,
1980; Tang et al., 1992), retain their potential for infection, regardless of age. This
study also indicates that rhizobial infection and nodulation in Listia species, as in
other genistoid legumes, does not involve root hair curling (RHC) and development
of infection threads (ITs). It has confirmed the value of nodule morphology and
structure as a marker for legume taxonomy, as lupinoid nodules are synapomorphic
for the genus Listia. Nodules from Crotalarieae species are also confirmed to contain
uniformly infected central tissue with no uninfected interstitial cells.
Root hair infection is considered to be an advanced feature that provides the
legume with a tighter and more selective control of bacterial passage through the
epidermis (Madsen et al., 2010), while epidermal entry has been linked with more
promiscuous nodulation (Boogerd & van Rossum, 1997; Sprent, 2007). The effective
and highly specific symbiosis of L. bainesii with its cognate methylobacterial
rhizobia shows, however, that epidermal entry does not preclude specificity.
Most of the research on the molecular mechanisms that govern specificity and
signalling pathways has concentrated on legumes that are infected via root hair
curling, including the model plants Lotus japonicus and Medicago truncatula. The
choice of these two species has been justified partly because they exhibit different
developmental systems (determinate and indeterminate nodules, respectively)
(Maunoury et al., 2008). The mechanics of infection and nodule organogenesis in
these model legumes are similar, however, whereas bacterial entry and nodule
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organogenesis in the epidermally infected dalbergioid and genistoid legumes are
structurally different to the processes seen in IT legumes. There are several
commonalities, but also several points of departure, in the nodulation signalling
pathways of IT and non-IT legumes. The key symbiotic genes SymRK and CCaMK
(Oldroyd & Downie, 2008) have also been isolated and characterised in Lupinus
species and Arachis hypogea, respectively (Mahé et al., 2011; Sinharoy & DasGupta,
2009). Nod factors are required for induction of meristematic activity in cortical
cells, but not for rhizobial colonisation of the root cortex in the dalbergioid legume
Arachis (Ibáñez & Fabra, 2011); additionally, Nod-factor-independent infection and
nodulation occurs in some Aeschynomene species nodulated by specific strains of
Bradyrhizobium (Giraud et al., 2007; Miché et al., 2010).
Deciphering the signalling pathways and the molecular basis of specificity in
non-IT symbioses is important for several reasons. Firstly, it provides a greater
understanding of the development and evolution of N2-fixing symbioses within
legumes. Secondly, determining the means by which legume hosts exclude non-
effective strains of rhizobia could arguably aid the maintenance of effective
symbioses in agricultural systems, where competition for nodulation by ineffective
strains is a constraint to N2 fixation (Howieson et al., 2008). Finally, a greater
understanding of the nodulation pathway in legumes may offer the possibility of
extending N2-fixing symbiosis to cereal and other non-legume crops. Charpentier &
Oldroyd (2010) have suggested that the comparatively primitive crack entry
nodulation pathway is a more realistic target for transfer to cereals than the root hair
infection process. In this case, knowledge of the molecular mechanisms that govern
Chapter 5
209
specificity, infection and nodule organogenesis in non-IT legumes will be a vital tool
in the development of strategies to effect this transfer.
5.3 Characterisation of novel rhizobial species of Microvirga
An important outcome of this study has been the naming and description of
three novel rhizobial species of Microvirga, a genus not previously known to contain
strains capable of symbiotic nitrogen fixation with legumes, or to be associated with
plants. This extends the range of bacterial chromosomal backgrounds that are able to
support the processes of infection, nodulation and nitrogen fixation with a legume
host. With the addition of the novel rhizobial species, there are now eight described
species in the genus Microvirga. The rhizobial strains AC72a (Wolde-Meskel et al.,
2005) and ARRI 185 (Lafay & Burdon, 2007) also belong within this clade, as do
several other as-yet-uncharacterised legume nodule isolates, based on the 16S rRNA
gene sequences deposited in the GenBank database (accession numbers EU618028,
GQ922067, HM042680). This points to the existence of additional potentially novel
rhizobial species within the genus Microvirga.
It is interesting to compare the rhizobial Microvirga with the described non-
rhizobial species. The substrate utilisation patterns of the rhizobial Microvirga strains
differ markedly from those of the aquatic Microvirga flocculans and Microvirga
subterranea (Kanso & Patel, 2003; Takeda et al., 2004) and provide evidence of the
adaptation of the rhizobial strains to the more nutrient-rich soil or rhizosphere
environment. Rhizobial Microvirga utilise a wider range of carbon substrates, and in
particular dicarboxylic acids, whereas M. flocculans and M. subterranea are unable
to grow on these substrates. Utilisation of dicarboxylic acids, along with the
acquisition of nodulation and nitrogen fixation genes, would appear to be a
Chapter 5
210
prerequisite for potentially rhizobial bacteria (Lodwig & Poole, 2003) and the limited
metabolic capacity of some Microvirga strains appears to preclude them from
attaining symbiotic capability.
As noted previously, described Microvirga species are able to tolerate
relatively high temperatures. Strains related to M. subterranea and to Microvirga sp.
AC72a have been isolated from Colorado Plateau soil crusts and the rhizosphere of
the endemic Thar Desert grass Lasiurus sindicus, respectively (Chowdhury et al.,
2009; Gundlapally & Garcia-Pichel, 2006); additionally, a Microvirga strain (TP1)
has been isolated from nodules of Tephrosia purpurea growing in the Thar Desert
(H. S. Gehlot, unpublished data). These deserts are subject to aridity, high
temperatures and high solar UV flux, indicating that Microvirga species may be well
adapted to such environmental stresses. Rhizobial Microvirga strains may therefore
have potential as inoculants in marginal agricultural areas.
Interesting questions can also be raised about the host range and
biogeography of rhizobial Microvirga strains, especially as compared with the
rhizobial Methylobacterium species. Nodulating Microvirga strains have a wider
geographical distribution and host range, whereas rhizobial Methylobacterium strains
appear to be confined to African crotalarioid legumes (Andam & Parker, 2007; Lafay
& Burdon, 2007; Sy et al., 2001; Wolde-Meskel et al., 2005; Yates et al., 2007). One
of the outstanding questions about the evolution of rhizobia is what genetic
predisposition is required for a potential bacterial recipient of nod and nif genes to
evolve into a rhizobium (Masson-Boivin et al., 2009). To this could be added, what
are the circumstances under which horizontal transfer of symbiotic genes and/or
Chapter 5
211
symbiont replacement is favoured? Willems (2006) has suggested that novel
rhizobial species are likely to be found amongst genera that have at least some plant-
associated species and are therefore probably more able to overcome plant defenses.
In this regard, Methylobacterium is a somewhat puzzling genus, as many strains are
epiphytes or endophytes of diverse plant hosts and capable of colonising both the
rhizosphere and phyllosphere (Andreote et al., 2009; Madhaiyan et al., 2009a;
2009b; Omer et al., 2004; Van Aken et al., 2004), yet the nodulating
methylobacterial taxa so far described are associated almost exclusively with African
crotalarioid legume hosts (Jaftha et al., 2002; Sy et al., 2001; Yates et al., 2007).
The genomes of eight Methylobacterium strains, including the Listia bainesii
symbiont Methylobacterium sp. 4-46 (Giraud & Fleischman, 2004), M. nodulans
ORS 2060 and strains of four non-symbiotic species have now been sequenced and
are available on the Integrated Microbial Genomes (IMG) database of the Joint
Genome Institute (JGI) (http://img.jgi.doe.gov/cgi-bin/w/main.cgi). The rhizobial
Microvirga strains Lut6 and WSM3557 have been entered in the JGI sequencing
program and the sequenced genomes of these microbes should soon be available (W.
Reeve, pers. comm. (http://genome.jgi.doe.gov/genome-projects/pages/projects.jsf)).
These sequences will provide platforms for intra- and interspecies genomic
comparisons in these genera and may shed further light on the traits required for
saprophytic competence in a given environment and the evolution of rhizobial
microsymbionts.
Chapter 5
212
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Appendix
249
Appendix
Appendix
250
Appen
dix
251
Ap
pen
dix
I
1 10 20 30 40 50 60 70
M4-46
ATGCGCTTCAAGGGCCTCGATCTCAATCTTCTCGTTGCGCTCGACGCTTTGATGAAAGAGAGAAATCTCA
Mn ORS2060
ATGCGCTTCAAGGGCCTCGATCTCAATCTCCTCATCGCGCTCGACGCTTTGATGAAAGAGCGAAACCTCA
Mes loti
ATGCGCTTCAAAGGCCTTGATCTGAATCTTCTCGTCGTGCTCGACGCACTGCTGACCGAGCGGACCCTCA
Brs NC92
ATGCGGTTCAAGGGCCTTGATCTAAATCTTCTTGTTGCGCTCGATGCTTTGATGACCGAACGCAATCTCA
Brj USDA 191
ATGCGTTTTAAGGGCCTTGATCTCAATCTCCTCGTTGCGCTCGACGCACTGATGACCGAACGCAAACTCA
Bre USDA 94
ATGCGGTTCAAGGGACTTGATCTAAACCTTCTCGTTGCGCTCGATGCTCTAATGACGGAGCGCAACCTCA
Rlp
ATGCGGTTCAAGGGCCTTGATCTGAATCTCCTCGTCGCACTCGACGCCCTGATGACCGAGCGTAATCTCA
NGR234
ATGCGTTTTAAGGGCCTTGATCTCAATCTCCTCGTTGCGCTCGACGCACTGATGACCGAACGCAAACTCA
71 80 90 100 110 120 130 140
M4-46
CCCGCGCGGCAAGCAGTCTCAACCTAAGCCAGCCCGCAATGAGCGCGGCTGTCGCGCGTTTGCGC
Mn ORS2060
CCCGCGCGGCATGCAGCATCAACCTGAGCCAACCCGCCATGAGCGCGGCCGTCGCGCGTTTGCG
Mes loti
CGGCGGCGGCAAGCAGCATCAACCTTAGTCAGCCGGCCATGAGCGCGGCCGTCGCCCGGCTGCGC
Brs NC92
CGGCGGCGGCACGCAAAATACACTTGAGCCAGCCGGCCATGAGTGCCGCCGTTGCCCGGCTACGC
Brj USDA 191
CGGCCGCTGCACGCAGCATCAACCTGAGCCAGCCGGCGATGAGCGCAGCCATCACCCGGCTTCGG
Bre USDA 94
CAGCGGCGGCGCGCCAAATTAACCTGAGCCAGCCCGCCATGAGCGCTGCGATCGCGCGGCTGCGC
Rlp
CGGCGGCAGCGCGCAGCATCAACTTGAGCCAACCGGCCATGAGCGCGGCCGTCGGCAGGTTACGC
NGR234
CGGCCGCTGCACGCAGCATCAACCTGAGCCAGCCGGCGATGAGCGCAGCCATCACCCGGCTTCGG
GTTGGATTCGGTCGGGCGTTA Forward primer f1
GATTCGGTCGGGCGTTACTCG Forward primer f2
Fig
ure
1.
Ali
gnm
ents
of
nodD
nucl
eoti
de
sequen
ces
from
rhiz
obia
l st
rain
s, s
how
ing f
orw
ard p
rim
er b
indin
g s
ites
. M
4-4
6 =
Meth
ylo
bacte
rium
sp.
4-4
6;
Mn =
Meth
ylo
bact
eri
um
nodula
ns;
Ml
= M
eso
rhiz
obiu
m l
oti
; B
rs =
Bra
dyrh
izobiu
m s
p.;
Brj
= B
radyrh
izobiu
m j
aponic
um
; B
re =
Bra
dyrh
izobiu
m e
lkanii
; R
lp =
Rhiz
obiu
m l
egum
inosa
rum
bv p
hase
oli
.
Appen
dix
252
1 10 20 30 40 50 60 70
M4-46
GTGCTGGCGGAATACGGCGTGCCGGCAACGTTCTGTGTCATTGGCGCGTACGCGGCAAGGGAGCCGCTGC
Mn ORS2060
GTGCTGGCTGAACACCGGGTGCCGGCGACGTTCTGCGTCATTGGCGCGTACGCAGCAAATGAGCCTGAAC
Bus STM678
GTGCTGGCGCAACACCGGGTGCCAGCGACTTTCTGCGTCATCGGCGCGTACGCAGCGGACCAGCCGAAGC
Brs LMTR 25
GTGCTCGCGGAACACCGAGTGCCCGCAACGTTCTTCGTCATCGGCGCGTACGCGGCGGACCAGCCGAAAC
Brs LMTR 21
GTGCTCGCGGAACACCGAGTGCCCGCAACGTTCTTCGTCATCGGCGCGTACGCGGCGGACCAGCCGAAAC
GCAAGACACAGTAACCGCGC Reverse primer r1
71 80 90 100 110 120 130
M4-46
TAATCCGGCGCATGATCTCAGATGGGCATGAGGTGGCAAACCACACCATGACTCACCCGGAT
Mn ORS2060
TAATCCAAAGAATGATCGCAGAAGGGCACGAAGTCGCAAATCACACGATGACTCATCCGGAC
Bus STM678
TGATCCGACGGATGATCGCAGAAGGACACGAGGTCGCAAACCACTCGATGACTCATCCGGAC
Brs LMTR 25
TAATCCGACGAATGATCGCAGAAGGGCACGAGGTCGCAAACCACACGATGACGCATCCGGAC
Brs LMTR 21
TAATCCGACGAATGATCGCAGAAGGGCACGAGGTCGCAAACCACACGATGACGCATCCGGAC
GTCTACCCGTACTCCACCGTT Reverse primer r2
Fig
ure
2.
Ali
gnm
ents
of
nodB
nucl
eoti
de
sequen
ces
from
rhiz
obia
l st
rain
s, s
how
ing r
ever
se p
rim
er b
indin
g s
ites
. M
4-4
6 =
Meth
ylobacte
rium
sp.
4-4
6;
Mn =
Meth
ylo
bact
eri
um
nodula
ns;
Bus
= B
urk
hold
eri
a s
p;
Brs
= B
radyrh
izobiu
m s
p.
Appendix
253
Appendix II
Figure 1. Gel electrophoresis showing molecular fingerprinting patterns of N2-fixing Li.
angolensis strains, generated with ERIC primers. a) Lane 1, 1 kb DNA marker; lane 2,
WSM3557; lane 3, WSM3673; lane 4, WSM3674; lane 5, WSM3675; lane 6, WSM3686;
lane 7. b) Lane 1, 1 kb DNA marker; lane 2, WSM3693.
Figure 2. Gel electrophoresis showing molecular fingerprinting patterns of isolates
generated with ERIC primers. Lanes 1, 7, 11 and 15: 1 kb DNA marker; lanes 2 – 6,
WSM2596 and reisolates from Lo. delicata and Le. polycephala; lanes 8 – 10, WSM2624
and reisolates from Le. mollis; lanes 12 – 14, WSM3040 and reisolates from Le.
polycephala.
Figure 1b. Gel electrophoresis showing molecular fingerprinting patterns of isolates
generated with ERIC primers. Lanes 1, 7, 13 and 19, 1 kb DNA marker; lanes 2 – 6,
WSM2632 and reisolates from Lo. delicata and Le. polycephala; lanes 8 – 12, WSM2667
and reisolates from Lo. delicata and Le. mollis; lanes 14 – 18, WSM2783 and reisolates from
Lo. delicata and Le. polycephala; lanes 20 – 25, WSM2653 and reisolates from Le. mollis
and Le. polycephala; lane 26, no DNA template.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 1510,000 bp
3000 bp
1000 bp
1 2 3 4 5 6 7 8 9 10 11 12 13 14 1510,000 bp
3000 bp
1000 bp
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
10,000 bp
3000 bp
1000 bp
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
10,000 bp
3000 bp
1000 bp
1 2 3 4 5 6
10.000 bp
3000 bp
1000 bp
a b
1 2 1 2 3 4 5 6
10.000 bp
3000 bp
1000 bp
a b
1 2
Appendix
254
Appendix III
L. angolensis
0
2
4
6
8
10
12
14
16
WSM2596 WSM2598 WSM2624 WSM2632 WSM2653 WSM2667 WSM2783 WSM3040 WSM3557 Uninoc N+
Inoculation treatments
Me
an
no
du
les
pe
r p
lan
t
0
0.01
0.02
0.03
0.04
0.05
0.06
Sh
oo
t d
ry
we
igh
t (
g/p
lan
t)
Figure 1. Symbiotic ability of rhizobia associated with Lotononis s. l. on Listia
angolensis, assessed by nodule number ( ) and dry weight of shoots ( ) of L.
angolensis plants harvested after ten weeks growth.
Figure 1. Symbiotic ability of rhizobia associated with Lotononis s. l. on Listia
angolensis, assessed by nodule number ( ) and dry weight of shoots ( ) of L.
angolensis plants harvested after ten weeks growth.
L. bainesii
0
2
4
6
8
10
12
14
WSM2596 WSM2598 WSM2624 WSM2632 WSM2653 WSM2667 WSM2783 WSM3040 WSM3557 Uninoc N+
Inoculation treatment
Me
an
no
du
les
pe
r p
lan
t
0.00
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
Sh
oo
t d
ry
we
igh
t (g
/pla
nt)
Figure 2. Symbiotic ability of rhizobia associated with Lotononis s. l. on Listia
bainesii, assessed by nodule number ( ) and dry weight of shoots ( ) of L.
bainesii plants harvested after ten weeks growth.
Figure 2. Symbiotic ability of rhizobia associated with Lotononis s. l. on Listia
bainesii, assessed by nodule number ( ) and dry weight of shoots ( ) of L.
bainesii plants harvested after ten weeks growth.
Appendix
255
L. bolusii
0
5
10
15
20
25
30
WSM2596 WSM2598 WSM2624 WSM2632 WSM2653 WSM2667 WSM2783 WSM3040 WSM3557 Uninoc N+
Inoculation treatment
Me
an
no
du
les
pe
r p
lan
t
0.00
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
Sh
oo
t d
ry
we
igh
t (g
/pla
nt)
Figure 3. Symbiotic ability of rhizobia associated with Lotononis s. l. on
Leobordea bolusii, assessed by nodule number ( ) and dry weight of
shoots ( ) of L. bolusii plants harvested after ten weeks growth.
Figure 3. Symbiotic ability of rhizobia associated with Lotononis s. l. on
Leobordea bolusii, assessed by nodule number ( ) and dry weight of
shoots ( ) of L. bolusii plants harvested after ten weeks growth.
L. longiflora
0
2
4
6
8
10
12
WSM2596 WSM2598 WSM2624 WSM2632 WSM2653 WSM2667 WSM2783 WSM3040 WSM3557 Uninoc N+
Inoculation treatment
Me
an
no
du
les
pe
r p
lan
t
0.000
0.002
0.004
0.006
0.008
0.010
0.012
Sh
oo
t d
ry
we
igh
t (
g/p
lan
t)
Figure 4. Symbiotic ability of rhizobia associated with Lotononis s. l. on
Leobordea longiflora, assessed by nodule number ( ) and dry weight of
shoots ( ) of L. longiflora plants harvested after ten weeks growth.
Figure 4. Symbiotic ability of rhizobia associated with Lotononis s. l. on
Leobordea longiflora, assessed by nodule number ( ) and dry weight of
shoots ( ) of L. longiflora plants harvested after ten weeks growth.
Appendix
256
L. platycarpa
0
1
2
3
4
5
6
7
8
9
WSM2596 WSM2598 WSM2624 WSM2632 WSM2653 WSM2667 WSM2783 WSM3040 WSM3557 Uninoc N+
Inoculation treatment
Me
an
no
du
les p
er p
lan
t
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
Sh
oo
t d
ry
weig
ht
(g/p
lan
t)
Figure 5a. Symbiotic ability of rhizobia associated with Lotononis s. l. on
Leobordea platycarpa, assessed by nodule number ( ) and dry weight of
shoots ( ) of L. platycarpa plants harvested after ten weeks growth.
Figure 5a. Symbiotic ability of rhizobia associated with Lotononis s. l. on
Leobordea platycarpa, assessed by nodule number ( ) and dry weight of
shoots ( ) of L. platycarpa plants harvested after ten weeks growth.
L. platycarpa nodulated WSM3557 minus N+
0
1
2
3
4
5
6
7
8
9
WSM2596 WSM2598 WSM2624 WSM2632 WSM2653 WSM2667 WSM2783 WSM3040 WSM3557 Uninoc
Inoculation treatment
Me
an
no
du
les
pe
r p
lan
t
0.000
0.005
0.010
0.015
0.020
0.025
0.030
0.035
Sh
oo
t d
ry
we
igh
t (
g/p
lan
t)
Figure 5b. Symbiotic ability of rhizobia associated with Lotononis s. l. on
Leobordea platycarpa, assessed by nodule number ( ) and dry weight of shoots
( ) of L. platycarpa plants harvested after ten weeks growth. The graph has
been rescaled by removing the N+ treatment to reveal the rhizobial inoculant
response. Non-nodulated plants were removed from the WSM3557 treatment
data.
Figure 5b. Symbiotic ability of rhizobia associated with Lotononis s. l. on
Leobordea platycarpa, assessed by nodule number ( ) and dry weight of shoots
( ) of L. platycarpa plants harvested after ten weeks growth. The graph has
been rescaled by removing the N+ treatment to reveal the rhizobial inoculant
response. Non-nodulated plants were removed from the WSM3557 treatment
data.
Appendix
257
L. stipulosa
0
5
10
15
20
25
30
35
40
45
WSM2596 WSM2598 WSM2624 WSM2632 WSM2653 WSM2667 WSM2783 WSM3040 WSM3557 N+
Inoculation treatment
Me
an
no
du
les
pe
r p
lan
t
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
Sh
oo
t d
ry
we
igh
t (g
/pla
nt)
Figure 6a. Symbiotic ability of rhizobia associated with Lotononis s. l. on
Leobordea stipulosa, assessed by nodule number ( ) and dry weight of
shoots ( ) of L. stipulosa plants harvested after ten weeks growth. No
uninoculated control is included due to seedling death.
Figure 6a. Symbiotic ability of rhizobia associated with Lotononis s. l. on
Leobordea stipulosa, assessed by nodule number ( ) and dry weight of
shoots ( ) of L. stipulosa plants harvested after ten weeks growth. No
uninoculated control is included due to seedling death.
L. stipulosa minus N+
0
5
10
15
20
25
30
35
40
45
WSM2596 WSM2598 WSM2624 WSM2632 WSM2653 WSM2667 WSM2783 WSM3040 WSM3557
Inoculation treatment
Me
an
no
du
les
pe
r p
lan
t
0.000
0.001
0.002
0.003
0.004
0.005
Sh
oo
t d
ry
we
igh
t (g
/pla
nt)
Figure 6b. Symbiotic ability of rhizobia associated with Lotononis s. l. on
Leobordea stipulosa, assessed by nodule number ( ) and dry weight of
shoots ( ) of L. stipulosa plants harvested after ten weeks growth. No
uninoculated control is included due to seedling death. The graph has been
rescaled by removing the N+ treatment to reveal the rhizobial inoculant
response.
Figure 6b. Symbiotic ability of rhizobia associated with Lotononis s. l. on
Leobordea stipulosa, assessed by nodule number ( ) and dry weight of
shoots ( ) of L. stipulosa plants harvested after ten weeks growth. No
uninoculated control is included due to seedling death. The graph has been
rescaled by removing the N+ treatment to reveal the rhizobial inoculant
response.
Appendix
258
L. crumanina
0.0
1.0
2.0
3.0
4.0
5.0
6.0
WSM2596 WSM2598 WSM2624 WSM2632 WSM2653 WSM2667 WSM2783 WSM3040 WSM3557 Uninoc N+
Inoculation treatment
Me
an
no
du
les
pe
r p
lan
t
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
Sh
oo
t d
ry
we
igh
t (g
/pla
nt
)
Figure 7a. Symbiotic ability of rhizobia associated with Lotononis s. l. on
Lotononis crumanina, assessed by nodule number ( ) and dry weight of
shoots ( ) of L. crumanina plants harvested after ten weeks growth.
Figure 7a. Symbiotic ability of rhizobia associated with Lotononis s. l. on
Lotononis crumanina, assessed by nodule number ( ) and dry weight of
shoots ( ) of L. crumanina plants harvested after ten weeks growth.
L. crumanina minus N+ control
0
1
2
3
4
5
6
WSM2596 WSM2598 WSM2624 WSM2632 WSM2653 WSM2667 WSM2783 WSM3040 WSM3557 Uninoc
Inoculation treatment
Me
an
no
du
les p
er p
lan
t
0.000
0.002
0.004
0.006
0.008
0.010
0.012
0.014
0.016
Sh
oo
t d
ry
weig
ht
(g/p
lan
t)
Figure 7b. Symbiotic ability of rhizobia associated with Lotononis s. l. on
Lotononis crumanina, assessed by nodule number ( ) and dry weight of
shoots ( ) of L. crumanina plants harvested after ten weeks growth. The
graph has been rescaled by removing the N+ treatment to reveal the rhizobial
inoculant response.
Figure 7b. Symbiotic ability of rhizobia associated with Lotononis s. l. on
Lotononis crumanina, assessed by nodule number ( ) and dry weight of
shoots ( ) of L. crumanina plants harvested after ten weeks growth. The
graph has been rescaled by removing the N+ treatment to reveal the rhizobial
inoculant response.
Appendix
259
L. falcata
0
1
2
3
4
5
6
7
8
9
WSM2596 WSM2598 WSM2624 WSM2632 WSM2653 WSM2667 WSM2783 WSM3040 WSM3557 Uninoc N+
Inoculation treatment
Mean
no
du
les p
er p
lan
t
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
Sh
oo
t d
ry w
eig
ht
(g/p
lan
t)
Figure 8a. Symbiotic ability of rhizobia associated with Lotononis s. l. on
Lotononis falcata, assessed by nodule number ( ) and dry weight of shoots
( ) of L. falcata plants harvested after ten weeks growth.
Figure 8a. Symbiotic ability of rhizobia associated with Lotononis s. l. on
Lotononis falcata, assessed by nodule number ( ) and dry weight of shoots
( ) of L. falcata plants harvested after ten weeks growth.
L. falcata minus N+
0
1
2
3
4
5
6
7
8
9
WSM2596 WSM2598 WSM2624 WSM2632 WSM2653 WSM2667 WSM2783 WSM3040 WSM3557 Uninoc
Inoculation treatment
Mean
no
du
les p
er p
lan
t
0.000
0.001
0.002
0.003
0.004
0.005
Sh
oo
t d
ry w
eig
ht
(g/p
lan
t)
Figure 8b. Symbiotic ability of rhizobia associated with Lotononis s. l. on
Lotononis falcata, assessed by nodule number ( ) and dry weight of shoots
( ) of L. falcata plants harvested after ten weeks growth. The graph has
been rescaled by removing the N+ treatment to reveal the rhizobial
inoculant response.
Figure 8b. Symbiotic ability of rhizobia associated with Lotononis s. l. on
Lotononis falcata, assessed by nodule number ( ) and dry weight of shoots
( ) of L. falcata plants harvested after ten weeks growth. The graph has
been rescaled by removing the N+ treatment to reveal the rhizobial
inoculant response.
Appendix
260
Appendix IV
Vitamin Stock (1000x) From Egli & Auling (2005)
Pyridoxine HCl (Vitamin B6) 100 mg l-1
Thiamine HCl (Vitamin B1) 50 mg l-1
Riboflavin (Vitamin B2) 50 mg l-1
Nicotinic acid (Niacin) (Vitamin B3) 50 mg l-1
D calcium pantothenate (Vitamin B5) 50 mg l-1
p aminobenzoic acid (Vitamin Bx) 50 mg l-1
Lipoic acid 50 mg l-1
Nicotinamide (Vitamin B3 amide) 50 mg l-1
Cobalamin (Vitamin B12) 50 mg l-1
Biotin (Vitamin B7) 20 mg l-1
Folic acid (Vitamin B9) 20 mg l-1
Appendix
261
Appendix V
Figure 1. Growth of novel rhizobial strains of Microvirga at varying concentrations of NaCl
(0.01 – 2.0% (w/v)). Results are shown for Listia angolensis strains only.
Appendix
262
Appen
dix
263
Ap
pen
dix
VI
Tab
le 1
. S
ubst
rate
uti
lisa
tion p
atte
rn o
f W
SM
3557
T a
nd L
ut6
T i
n B
iolo
g P
M p
late
s co
mpar
ed w
ith S
inorh
izobiu
m m
eli
loti
1021 (
Sm
1021;
dat
a fr
om
Bio
ndi
et
al.
(2009))
and c
om
par
ed w
ith u
tili
sati
on i
n G
N2 p
late
s an
d m
inim
al m
edia
(M
M).
Gre
en =
+ve;
yel
low
= w
eak +
ve;
red
= -
ve.
PM
Well
T
este
d C
om
po
un
dT
yp
e o
f co
mp
ou
nd
S.m
1021
WS
M3557
GN
2 W
SM
3557
MM
WS
M3557
Lu
t6G
N2 L
ut6
MM
Lu
t6
PM
1 C
arb
on S
ourc
es-1
A01
Negative C
ontr
ol
PM
1 C
arb
on S
ourc
es-2
A02
L-A
rabin
ose
C s
ourc
e;
C5 c
arb
ohydra
te
PM
1 C
arb
on S
ourc
es-3
A03
N-A
cety
l-D
-Glu
cosam
ine
PM
1 C
arb
on S
ourc
es-4
A04
D-S
accharic A
cid
C-S
ourc
e,
carb
oxylic
acid
PM
1 C
arb
on S
ourc
es-5
A05
Succin
ic A
cid
C-S
ourc
e,
carb
oxylic
acid
PM
1 C
arb
on S
ourc
es-6
A06
D-G
ala
cto
se
PM
1 C
arb
on S
ourc
es-7
A07
L-A
spart
ic A
cid
C-S
ourc
e,
am
ino a
cid
PM
1 C
arb
on S
ourc
es-8
A
08
L-P
rolin
e
PM
1 C
arb
on S
ourc
es-9
A
09
D-A
lanin
e
PM
1 C
arb
on S
ourc
es-1
0
A10
D-T
rehalo
se
PM
1 C
arb
on S
ourc
es-1
1
A11
D-M
annose
C s
ourc
e;
C6 c
arb
ohydra
te
PM
1 C
arb
on S
ourc
es-1
2
A12
Dulc
itol
PM
1 C
arb
on S
ourc
es-1
3
B01
D-S
erine
PM
1 C
arb
on S
ourc
es-1
4
B02
D-S
orb
itol
PM
1 C
arb
on S
ourc
es-1
5
B03
Gly
cero
l
PM
1 C
arb
on S
ourc
es-1
6
B04
L-F
ucose
PM
1 C
arb
on S
ourc
es-1
7
B05
D-G
lucuro
nic
Acid
PM
1 C
arb
on S
ourc
es-1
8
B06
D-G
luconic
Acid
PM
1 C
arb
on S
ourc
es-1
9
B07
D,L
-α-G
lycero
l-P
hosphate
PM
1 C
arb
on S
ourc
es-2
0
B08
D-X
ylo
se
C s
ourc
e;
C5 c
arb
ohydra
te
PM
1 C
arb
on S
ourc
es-2
1
B09
L-L
actic A
cid
C-S
ourc
e,
C3 c
arb
oxylic
acid
PM
1 C
arb
on S
ourc
es-2
2
B10
Form
ic A
cid
PM
1 C
arb
on S
ourc
es-2
3
B11
D-M
annitol
C-s
ourc
e,
C6 s
ugar
alc
ohol
PM
1 C
arb
on S
ourc
es-2
4
B12
L-G
luta
mic
Acid
C-S
ourc
e,
am
ino a
cid
264
Tab
le 1
. (c
ont)
.
PM
1 C
arb
on S
ourc
es-2
5
C01
D-G
lucose-6
-Phosphate
PM
1 C
arb
on S
ourc
es-2
6
C02
D-G
ala
cto
nic
Acid
-γ-L
acto
ne
PM
1 C
arb
on S
ourc
es-2
7
C03
D,L
-Malic
Acid
C-S
ourc
e,
carb
oxylic
acid
PM
1 C
arb
on S
ourc
es-2
8
C04
D-R
ibose
C s
ourc
e;
C5 c
arb
ohydra
te
PM
1 C
arb
on S
ourc
es-2
9
C05
Tw
een 2
0C
-Sourc
e,
fatt
y a
cid
PM
1 C
arb
on S
ourc
es-3
0
C06
L-R
ham
nose
C s
ourc
e;
C6 c
arb
ohydra
te (
meth
yl pento
se)
PM
1 C
arb
on S
ourc
es-3
1
C07
D-F
ructo
se
C s
ourc
e;
C6 c
arb
ohydra
te
PM
1 C
arb
on S
ourc
es-3
2
C08
Acetic A
cid
PM
1 C
arb
on S
ourc
es-3
3
C09
α-D
-Glu
cose
C s
ourc
e;
C6 c
arb
ohydra
te
PM
1 C
arb
on S
ourc
es-3
4
C10
Maltose
PM
1 C
arb
on S
ourc
es-3
5
C11
D-M
elib
iose
PM
1 C
arb
on S
ourc
es-3
6
C12
Thym
idin
e
PM
1 C
arb
on S
ourc
es-3
7
D01
L-A
spara
gin
e
PM
1 C
arb
on S
ourc
es-3
8
D02
D-A
spart
ic A
cid
PM
1 C
arb
on S
ourc
es-3
9
D03
D-G
lucosam
inic
Acid
PM
1 C
arb
on S
ourc
es-4
0
D04
1,2
-Pro
panedio
l
PM
1 C
arb
on S
ourc
es-4
1
D05
Tw
een 4
0
PM
1 C
arb
on S
ourc
es-4
2
D06
α-K
eto
-Glu
taric A
cid
C-S
ourc
e,
carb
oxylic
acid
PM
1 C
arb
on S
ourc
es-4
3
D07
α-K
eto
-Buty
ric A
cid
PM
1 C
arb
on S
ourc
es-4
4
D08
α-M
eth
yl-D
Gala
cto
sid
e
PM
1 C
arb
on S
ourc
es-4
5
D09
α-D
-Lacto
se
PM
1 C
arb
on S
ourc
es-4
6
D10
Lactu
lose
PM
1 C
arb
on S
ourc
es-4
7
D11
Sucro
se
PM
1 C
arb
on S
ourc
es-4
8
D12
Uridin
e
PM
1 C
arb
on S
ourc
es-4
9
E01
L-G
luta
min
e
PM
1 C
arb
on S
ourc
es-5
0
E02
M-T
art
aric A
cid
PM
1 C
arb
on S
ourc
es-5
1
E03
D-G
lucose-1
-Phosphate
PM
1 C
arb
on S
ourc
es-5
2
E04
D-F
ructo
se-6
-Phosphate
C s
ourc
e;
C6 c
arb
ohydra
te
Appen
dix
265
Tab
le 1
. (c
ont)
.
PM
1 C
arb
on S
ourc
es-5
3
E05
Tw
een 8
0
PM
1 C
arb
on S
ourc
es-5
4
E06
α-H
ydro
xy G
luta
ric A
cid
-γ-L
acto
ne
PM
1 C
arb
on S
ourc
es-5
5
E07
α-H
ydro
xy B
uty
ric A
cid
PM
1 C
arb
on S
ourc
es-5
6
E08
β-M
eth
yl-D
Glu
cosid
e
PM
1 C
arb
on S
ourc
es-5
7
E09
Adonitol (=
Rib
itol)
C5 a
lcohol
PM
1 C
arb
on S
ourc
es-5
8
E10
Maltotr
iose
PM
1 C
arb
on S
ourc
es-5
9
E11
2-D
eoxy A
denosin
e
PM
1 C
arb
on S
ourc
es-6
0
E12
Adenosin
e
PM
1 C
arb
on S
ourc
es-6
1
F01
Gly
cyl-L-A
spart
ic A
cid
PM
1 C
arb
on S
ourc
es-6
2
F02
Citric A
cid
PM
1 C
arb
on S
ourc
es-6
3
F03
m-I
nositol
C s
ourc
e;
C6 c
arb
ohydra
te (
poly
ol of
cyclo
hexane)
PM
1 C
arb
on S
ourc
es-6
4
F04
D-T
hre
onin
e
PM
1 C
arb
on S
ourc
es-6
5
F05
Fum
aric A
cid
C-S
ourc
e,
carb
oxylic
acid
PM
1 C
arb
on S
ourc
es-6
6
F06
Bro
mo S
uccin
ic A
cid
C-S
ourc
e,
carb
oxylic
acid
PM
1 C
arb
on S
ourc
es-6
7
F07
Pro
pio
nic
Acid
PM
1 C
arb
on S
ourc
es-6
8
F08
Mucic
Acid
PM
1 C
arb
on S
ourc
es-6
9
F09
Gly
colic
Acid
PM
1 C
arb
on S
ourc
es-7
0
F10
Gly
oxylic
Acid
PM
1 C
arb
on S
ourc
es-7
1
F11
D-C
ello
bio
se
C s
ourc
e;
C12 c
arb
ohydra
te
PM
1 C
arb
on S
ourc
es-7
2
F12
Inosin
e
PM
1 C
arb
on S
ourc
es-7
3
G01
Gly
cyl-LG
luta
mic
Acid
PM
1 C
arb
on S
ourc
es-7
4
G02
Tricarb
ally
lic A
cid
PM
1 C
arb
on S
ourc
es-7
5
G03
L-S
erine
C-S
ourc
e,
am
ino a
cid
PM
1 C
arb
on S
ourc
es-7
6
G04
L-T
hre
onin
e
PM
1 C
arb
on S
ourc
es-7
7
G05
L-A
lanin
e
PM
1 C
arb
on S
ourc
es-7
8
G06
L-A
lanyl-G
lycin
e
PM
1 C
arb
on S
ourc
es-7
9
G07
Aceto
acetic A
cid
PM
1 C
arb
on S
ourc
es-8
0
G08
N-A
cety
l-β
-DM
annosam
ine
266
Tab
le 1
. (c
ont)
.
PM
1 C
arb
on S
ourc
es-8
1
G09
Mono M
eth
yl S
uccin
ate
C-S
ourc
e,
carb
oxylic
acid
PM
1 C
arb
on S
ourc
es-8
2
G10
Meth
yl P
yru
vate
C-S
ourc
e,
carb
oxylic
acid
PM
1 C
arb
on S
ourc
es-8
3
G11
D-M
alic
Acid
C-S
ourc
e,
carb
oxylic
acid
PM
1 C
arb
on S
ourc
es-8
4
G12
L-M
alic
Acid
C-S
ourc
e,
carb
oxylic
acid
PM
1 C
arb
on S
ourc
es-8
5
H01
Gly
cyl-L-P
rolin
e
PM
1 C
arb
on S
ourc
es-8
6
H02
p-H
ydro
xy P
henyl A
cetic A
cid
PM
1 C
arb
on S
ourc
es-8
7
H03
m-H
ydro
xy P
henyl A
cetic A
cid
PM
1 C
arb
on S
ourc
es-8
8
H04
Tyra
min
e
PM
1 C
arb
on S
ourc
es-8
9
H05
D-P
sic
ose
C s
ourc
e;
C6 c
arb
ohydra
te
PM
1 C
arb
on S
ourc
es-9
0
H06
L-L
yxose
C s
ourc
e;
C5 c
arb
ohydra
te
PM
1 C
arb
on S
ourc
es-9
1
H07
Glu
curo
nam
ide
C-S
ourc
e,
C6 a
mid
e
PM
1 C
arb
on S
ourc
es-9
2
H08
Pyru
vic
Acid
C-S
ourc
e,
carb
oxylic
acid
PM
1 C
arb
on S
ourc
es-9
3
H09
Dih
ydro
xyaceto
ne
C-S
ourc
e,
C3 a
lcohol
PM
1 C
arb
on S
ourc
es-9
4
H10
D-G
ala
ctu
ronic
Acid
C-S
ourc
e,
C6 c
arb
oxylic
acid
PM
1 C
arb
on S
ourc
es-9
5
H11
Phenyle
thyla
min
e
PM
1 C
arb
on S
ourc
es-9
6
H12
2-A
min
oeth
anol
PM
2 C
arb
on S
ourc
es-2
A
01
Negative C
ontr
ol
PM
2 C
arb
on S
ourc
es-3
A
02
Chondro
itin
Sulfate
C
PM
2 C
arb
on S
ourc
es-4
A
03
α-C
yclo
dextr
in
PM
2 C
arb
on S
ourc
es-5
A
04
β-C
yclo
dextr
in
PM
2 C
arb
on S
ourc
es-6
A
05
γ-C
yclo
dextr
in
PM
2 C
arb
on S
ourc
es-7
A
06
Dextr
in
PM
2 C
arb
on S
ourc
es-8
A
07
Gela
tin
PM
2 C
arb
on S
ourc
es-9
A
08
Gly
cogen
PM
2 C
arb
on S
ourc
es-1
0
A09
Inulin
PM
2 C
arb
on S
ourc
es-1
1
A10
Lam
inarin
PM
2 C
arb
on S
ourc
es-1
2
A11
Mannan
PM
2 C
arb
on S
ourc
es-1
3
A12
Pectin
C-S
ourc
e,
poly
mer
Appen
dix
267
Tab
le 1
. (c
ont)
.
PM
2 C
arb
on S
ourc
es-1
4
B01
N-A
cety
l-D
Gala
cto
sam
ine
PM
2 C
arb
on S
ourc
es-1
5
B02
N-A
cety
l-N
eura
min
ic A
cid
PM
2 C
arb
on S
ourc
es-1
6
B03
β-D
-Allo
se
PM
2 C
arb
on S
ourc
es-1
7
B04
Am
ygdalin
PM
2 C
arb
on S
ourc
es-1
8
B05
D-A
rabin
ose
C s
ourc
e;
C5 c
arb
ohydra
te
PM
2 C
arb
on S
ourc
es-1
9
B06
D-A
rabitol
PM
2 C
arb
on S
ourc
es-2
0
B07
L-A
rabitol
PM
2 C
arb
on S
ourc
es-2
1
B08
Arb
utin (
glu
cosyla
ted h
ydro
quin
one)
C s
ourc
e;
C12 c
arb
ohydra
te;
gly
cosyla
ted h
ydro
quin
one
PM
2 C
arb
on S
ourc
es-2
2
B09
2-D
eoxy-D
-Rib
ose
C s
ourc
e;
C5 c
arb
ohydra
te
PM
2 C
arb
on S
ourc
es-2
3
B10
I-E
ryth
rito
l
PM
2 C
arb
on S
ourc
es-2
4
B11
D-F
ucose
PM
2 C
arb
on S
ourc
es-2
5
B12
3-0
-β-D
-Gala
cto
pyra
nosyl-D
Ara
bin
ose
PM
2 C
arb
on S
ourc
es-2
6
C01
Gentiobio
se
PM
2 C
arb
on S
ourc
es-2
7
C02
L-G
lucose
PM
2 C
arb
on S
ourc
es-2
8
C03
Lactito
l
PM
2 C
arb
on S
ourc
es-2
9
C04
D-M
ele
zitose
PM
2 C
arb
on S
ourc
es-3
0
C05
Maltitol
PM
2 C
arb
on S
ourc
es-3
1
C06
a-M
eth
yl-D
Glu
cosid
e
PM
2 C
arb
on S
ourc
es-3
2
C07
β-M
eth
yl-D
Gala
cto
sid
e
PM
2 C
arb
on S
ourc
es-3
3
C08
3-M
eth
yl G
lucose
PM
2 C
arb
on S
ourc
es-3
4
C09
β-M
eth
yl-D
Glu
curo
nic
Acid
PM
2 C
arb
on S
ourc
es-3
5
C10
α-M
eth
yl-D
Mannosid
e
PM
2 C
arb
on S
ourc
es-3
6
C11
β-M
eth
yl-D
Xylo
sid
e
PM
2 C
arb
on S
ourc
es-3
7
C12
Pala
tinose (
isom
altulo
se)
C s
ourc
e;
C12 c
arb
ohydra
te
PM
2 C
arb
on S
ourc
es-3
8
D01
D-R
aff
inose
PM
2 C
arb
on S
ourc
es-3
9
D02
Salic
in
PM
2 C
arb
on S
ourc
es-4
0
D03
Sedoheptu
losan
PM
2 C
arb
on S
ourc
es-4
1
D04
L-S
orb
ose
268
Tab
le 1
. (c
ont)
.
PM
2 C
arb
on S
ourc
es-4
2
D05
Sta
chyose
PM
2 C
arb
on S
ourc
es-4
3
D06
D-T
agato
se
C s
ourc
e;
C6 c
arb
ohydra
te
PM
2 C
arb
on S
ourc
es-4
4
D07
Tura
nose
PM
2 C
arb
on S
ourc
es-4
5
D08
Xylit
ol
PM
2 C
arb
on S
ourc
es-4
6
D09
N-A
cety
l-D
Glu
cosam
initol
PM
2 C
arb
on S
ourc
es-4
7
D10
γ-A
min
o B
uty
ric A
cid
PM
2 C
arb
on S
ourc
es-4
8
D11
δ-A
min
o V
ale
ric A
cid
PM
2 C
arb
on S
ourc
es-4
9
D12
Buty
ric A
cid
PM
2 C
arb
on S
ourc
es-5
0
E01
Capric A
cid
PM
2 C
arb
on S
ourc
es-5
1
E02
Capro
ic A
cid
PM
2 C
arb
on S
ourc
es-5
2
E03
Citra
conic
Acid
PM
2 C
arb
on S
ourc
es-5
3
E04
Citra
malic
Acid
PM
2 C
arb
on S
ourc
es-5
4
E05
D-G
lucosam
ine
C s
ourc
e;
C6 c
arb
ohydra
te (
am
ino s
ugar)
PM
2 C
arb
on S
ourc
es-5
5
E06
2-H
ydro
xy B
enzoic
Acid
PM
2 C
arb
on S
ourc
es-5
6
E07
4-H
ydro
xy B
enzoic
Acid
PM
2 C
arb
on S
ourc
es-5
7
E08
β-H
ydro
xy B
uty
ric A
cid
PM
2 C
arb
on S
ourc
es-5
8
E09
γ-H
ydro
xy B
uty
ric A
cid
PM
2 C
arb
on S
ourc
es-5
9
E10
a-K
eto
-Vale
ric A
cid
PM
2 C
arb
on S
ourc
es-6
0
E11
Itaconic
Acid
PM
2 C
arb
on S
ourc
es-6
1
E12
5-K
eto
-DG
luconic
Acid
C-S
ourc
e,
carb
oxylic
acid
PM
2 C
arb
on S
ourc
es-6
2
F01
D-L
actic A
cid
Meth
yl E
ste
r
PM
2 C
arb
on S
ourc
es-6
3
F02
Malo
nic
Acid
C-S
ourc
e,
carb
oxylic
acid
PM
2 C
arb
on S
ourc
es-6
4
F03
Melib
ionic
Acid
PM
2 C
arb
on S
ourc
es-6
5
F04
Oxalic
Acid
PM
2 C
arb
on S
ourc
es-6
6
F05
Oxalo
malic
Acid
C-S
ourc
e,
C6 c
arb
oxylic
acid
PM
2 C
arb
on S
ourc
es-6
7
F06
Quin
ic A
cid
PM
2 C
arb
on S
ourc
es-6
8
F07
D-R
ibono-1
,4-L
acto
ne
PM
2 C
arb
on S
ourc
es-6
9
F08
Sebacic
Acid
Appen
dix
269
Tab
le 1
. (c
ont)
.
PM
2 C
arb
on S
ourc
es-7
0
F09
Sorb
ic A
cid
PM
2 C
arb
on S
ourc
es-7
1
F10
Succin
am
ic A
cid
C-S
ourc
e,
C4 c
arb
oxylic
acid
PM
2 C
arb
on S
ourc
es-7
2
F11
D-T
art
aric A
cid
PM
2 C
arb
on S
ourc
es-7
3
F12
L-T
art
aric A
cid
PM
2 C
arb
on S
ourc
es-7
4
G01
Aceta
mid
e
PM
2 C
arb
on S
ourc
es-7
5
G02
L-A
lanin
am
ide
PM
2 C
arb
on S
ourc
es-7
6
G03
N-A
cety
l-LG
luta
mic
Acid
PM
2 C
arb
on S
ourc
es-7
7
G04
L-A
rgin
ine
PM
2 C
arb
on S
ourc
es-7
8
G05
Gly
cin
e
PM
2 C
arb
on S
ourc
es-7
9
G06
L-H
istidin
e
PM
2 C
arb
on S
ourc
es-8
0
G07
L-H
om
oserine
PM
2 C
arb
on S
ourc
es-8
1
G08
Hydro
xy-L
Pro
line
PM
2 C
arb
on S
ourc
es-8
2
G09
L-I
sole
ucin
e
PM
2 C
arb
on S
ourc
es-8
3
G10
L-L
eucin
e
PM
2 C
arb
on S
ourc
es-8
4
G11
L-L
ysin
e
PM
2 C
arb
on S
ourc
es-8
5
G12
L-M
eth
ionin
e
PM
2 C
arb
on S
ourc
es-8
6
H01
L-O
rnithin
e
PM
2 C
arb
on S
ourc
es-8
7
H02
L-P
henyla
lanin
e
PM
2 C
arb
on S
ourc
es-8
8
H03
L-P
yro
glu
tam
ic A
cid
PM
2 C
arb
on S
ourc
es-8
9
H04
L-V
alin
e
PM
2 C
arb
on S
ourc
es-9
0
H05
D,L
-Carn
itin
e
PM
2 C
arb
on S
ourc
es-9
1
H06
Sec-B
uty
lam
ine
PM
2 C
arb
on S
ourc
es-9
2
H07
D.L
-Octo
pam
ine
PM
2 C
arb
on S
ourc
es-9
3
H08
Putr
escin
e
PM
2 C
arb
on S
ourc
es-9
4
H09
Dih
ydro
xy A
ceto
ne
C-S
ourc
e,
alc
ohol (C
3 k
eto
se)
PM
2 C
arb
on S
ourc
es-9
5
H10
2,3
-Buta
nedio
l
PM
2 C
arb
on S
ourc
es-9
6
H11
2,3
-Buta
none
PM
2 C
arb
on S
ourc
es-9
7
H12
3-H
ydro
xy 2
-Buta
none
270
Tab
le 1
. (c
ont)
.
PM
3 N
itro
gen S
ourc
es
A
01
Negative C
ontr
ol
PM
3 N
itro
gen S
ourc
es
A
02
Am
monia
N-S
ourc
e,
inorg
anic
PM
3 N
itro
gen S
ourc
es
A
03
Nitrite
N-S
ourc
e,
inorg
anic
PM
3 N
itro
gen S
ourc
es
A
04
Nitra
teN
-Sourc
e,
inorg
anic
PM
3 N
itro
gen S
ourc
es
A
05
Ure
aN
-Sourc
e,
inorg
anic
PM
3 N
itro
gen S
ourc
es
A
06
Biu
ret
PM
3 N
itro
gen S
ourc
es
A
07
L-A
lanin
eN
-Sourc
e,
am
ino a
cid
PM
3 N
itro
gen S
ourc
es
A
08
L-A
rgin
ine
N-S
ourc
e,
am
ino a
cid
PM
3 N
itro
gen S
ourc
es
A
09
L-A
spara
gin
eN
-Sourc
e,
am
ino a
cid
PM
3 N
itro
gen S
ourc
es
A
10
L-A
spart
ic A
cid
N-S
ourc
e,
am
ino a
cid
PM
3 N
itro
gen S
ourc
es
A
11
L-C
yste
ine
N-S
ourc
e,
am
ino a
cid
PM
3 N
itro
gen S
ourc
es
A
12
L-G
luta
mic
Acid
N-S
ourc
e,
am
ino a
cid
PM
3 N
itro
gen S
ourc
es
B
01
L-G
luta
min
eN
-Sourc
e,
am
ino a
cid
PM
3 N
itro
gen S
ourc
es
B
02
Gly
cin
eN
-Sourc
e,
am
ino a
cid
PM
3 N
itro
gen S
ourc
es
B
03
L-H
istidin
eN
-Sourc
e,
am
ino a
cid
PM
3 N
itro
gen S
ourc
es
B
04
L-I
sole
ucin
eN
-Sourc
e,
am
ino a
cid
PM
3 N
itro
gen S
ourc
es
B
05
L-L
eucin
eN
-Sourc
e,
am
ino a
cid
PM
3 N
itro
gen S
ourc
es
B
06
L-L
ysin
eN
-Sourc
e,
am
ino a
cid
PM
3 N
itro
gen S
ourc
es
B
07
L-M
eth
ionin
eN
-Sourc
e,
am
ino a
cid
PM
3 N
itro
gen S
ourc
es
B
08
L-P
henyla
lanin
eN
-Sourc
e,
am
ino a
cid
PM
3 N
itro
gen S
ourc
es
B
09
L-P
rolin
eN
-Sourc
e,
am
ino a
cid
PM
3 N
itro
gen S
ourc
es
B
10
L-S
erine
N-S
ourc
e,
am
ino a
cid
PM
3 N
itro
gen S
ourc
es
B
11
L-T
hre
onin
eN
-Sourc
e,
am
ino a
cid
PM
3 N
itro
gen S
ourc
es
B
12
L-T
rypto
phan
N-S
ourc
e,
am
ino a
cid
PM
3 N
itro
gen S
ourc
es
C
01
L-T
yro
sin
eN
-Sourc
e,
am
ino a
cid
PM
3 N
itro
gen S
ourc
es
C
02
L-V
alin
eN
-Sourc
e,
am
ino a
cid
PM
3 N
itro
gen S
ourc
es
C
03
D-A
lanin
eN
-Sourc
e,
am
ino a
cid
PM
3 N
itro
gen S
ourc
es
C
04
D-A
spara
gin
eN
-Sourc
e,
am
ino a
cid
Appen
dix
271
Tab
le 1
. (c
ont)
.
PM
3 N
itro
gen S
ourc
es
C
05
D-A
spart
ic A
cid
N-S
ourc
e,
am
ino a
cid
PM
3 N
itro
gen S
ourc
es
C
06
D-G
luta
mic
Acid
N-S
ourc
e,
am
ino a
cid
PM
3 N
itro
gen S
ourc
es
C
07
D-L
ysin
eN
-Sourc
e,
am
ino a
cid
PM
3 N
itro
gen S
ourc
es
C
08
D-S
erine
N-S
ourc
e,
am
ino a
cid
PM
3 N
itro
gen S
ourc
es
C
09
D-V
alin
eN
-Sourc
e,
am
ino a
cid
PM
3 N
itro
gen S
ourc
es
C
10
L-C
itru
lline
N-S
ourc
e,
am
ino a
cid
PM
3 N
itro
gen S
ourc
es
C
11
L-H
om
oserine
N-S
ourc
e,
am
ino a
cid
PM
3 N
itro
gen S
ourc
es
C
12
L-O
rnithin
eN
-Sourc
e,
am
ino a
cid
PM
3 N
itro
gen S
ourc
es
D
01
N-A
cety
l-D
,LG
luta
mic
Acid
N-S
ourc
e,
am
ino a
cid
PM
3 N
itro
gen S
ourc
es
D
02
N-P
hth
alo
yl-LG
luta
mic
Acid
N-S
ourc
e,
am
ino a
cid
PM
3 N
itro
gen S
ourc
es
D
03
L-P
yro
glu
tam
ic A
cid
N-S
ourc
e,
am
ino a
cid
PM
3 N
itro
gen S
ourc
es
D
04
Hydro
xyla
min
e
PM
3 N
itro
gen S
ourc
es
D
05
Meth
yla
min
e
PM
3 N
itro
gen S
ourc
es
D
06
N-A
myla
min
e
PM
3 N
itro
gen S
ourc
es
D
07
N-B
uty
lam
ine
PM
3 N
itro
gen S
ourc
es
D
08
Eth
yla
min
e
PM
3 N
itro
gen S
ourc
es
D
09
Eth
anola
min
e
PM
3 N
itro
gen S
ourc
es
D
10
Eth
yle
nedia
min
e
PM
3 N
itro
gen S
ourc
es
D
11
Putr
escin
e
PM
3 N
itro
gen S
ourc
es
D
12
Agm
atine
PM
3 N
itro
gen S
ourc
es
E
01
His
tam
ine
PM
3 N
itro
gen S
ourc
es
E
02
β-P
henyle
thyla
min
e
PM
3 N
itro
gen S
ourc
es
E
03
Tyra
min
e
PM
3 N
itro
gen S
ourc
es
E
04
Aceta
mid
e
PM
3 N
itro
gen S
ourc
es
E
05
Form
am
ide
PM
3 N
itro
gen S
ourc
es
E
06
Glu
curo
nam
ide
PM
3 N
itro
gen S
ourc
es
E
07
D,L
-Lacta
mid
e
PM
3 N
itro
gen S
ourc
es
E
08
D-G
lucosam
ine
N-S
ourc
e,
oth
er
272
Tab
le 1
. (c
ont)
.
PM
3 N
itro
gen S
ourc
es
E
09
D-G
ala
cto
sam
ine
N-S
ourc
e,
oth
er
PM
3 N
itro
gen S
ourc
es
E
10
D-M
annosam
ine
N-S
ourc
e,
oth
er
PM
3 N
itro
gen S
ourc
es
E
11
N-A
cety
l-D
Glu
cosam
ine
N-S
ourc
e,
oth
er
PM
3 N
itro
gen S
ourc
es
E
12
N-A
cety
l-D
Gala
cto
sam
ine
PM
3 N
itro
gen S
ourc
es
F
01
N-A
cety
l-D
Mannosam
ine
PM
3 N
itro
gen S
ourc
es
F
02
Adenin
e
PM
3 N
itro
gen S
ourc
es
F
03
Adenosin
e
PM
3 N
itro
gen S
ourc
es
F
04
Cytidin
e
PM
3 N
itro
gen S
ourc
es
F
05
Cyto
sin
e
PM
3 N
itro
gen S
ourc
es
F
06
Guanin
e
PM
3 N
itro
gen S
ourc
es
F
07
Guanosin
e
PM
3 N
itro
gen S
ourc
es
F
08
Thym
ine
PM
3 N
itro
gen S
ourc
es
F
09
Thym
idin
e
PM
3 N
itro
gen S
ourc
es
F
10
Ura
cil
PM
3 N
itro
gen S
ourc
es
F
11
Uridin
e
PM
3 N
itro
gen S
ourc
es
F
12
Inosin
e
PM
3 N
itro
gen S
ourc
es
G
01
Xanth
ine
N-S
ourc
e,
oth
er
PM
3 N
itro
gen S
ourc
es
G
02
Xanth
osin
e
PM
3 N
itro
gen S
ourc
es
G
03
Uric A
cid
PM
3 N
itro
gen S
ourc
es
G
04
Allo
xan
N-S
ourc
e,
oth
er
PM
3 N
itro
gen S
ourc
es
G
05
Alla
nto
inN
-Sourc
e,
oth
er
PM
3 N
itro
gen S
ourc
es
G
06
Para
banic
Acid
PM
3 N
itro
gen S
ourc
es
G
07
D,L
-α-A
min
o-N
Buty
ric A
cid
PM
3 N
itro
gen S
ourc
es
G
08
γ-A
min
o-N
Buty
ric A
cid
N-S
ourc
e,
oth
er
PM
3 N
itro
gen S
ourc
es
G
09
ε-A
min
o-N
Capro
ic A
cid
PM
3 N
itro
gen S
ourc
es
G
10
D,L
-α-A
min
o-C
apry
lic A
cid
N-S
ourc
e,
oth
er
PM
3 N
itro
gen S
ourc
es
G
11
δ-A
min
o-N
Vale
ric A
cid
N-S
ourc
e,
oth
er
PM
3 N
itro
gen S
ourc
es
G
12
α-A
min
o-N
Vale
ric A
cid
Appen
dix
273
Tab
le 1
. (c
ont)
.
PM
3 N
itro
gen S
ourc
es
H
01
Ala
-Asp
N-S
ourc
e,
peptide
PM
3 N
itro
gen S
ourc
es
H
02
Ala
-Gln
N-S
ourc
e,
peptide
PM
3 N
itro
gen S
ourc
es
H
03
Ala
-Glu
N-S
ourc
e,
peptide
PM
3 N
itro
gen S
ourc
es
H
04
Ala
-Gly
N-S
ourc
e,
peptide
PM
3 N
itro
gen S
ourc
es
H
05
Ala
-His
N-S
ourc
e,
peptide
PM
3 N
itro
gen S
ourc
es
H
06
Ala
-Leu
N-S
ourc
e,
peptide
PM
3 N
itro
gen S
ourc
es
H
07
Ala
-Thr
N-S
ourc
e,
peptide
PM
3 N
itro
gen S
ourc
es
H
08
Gly
-Asn
N-S
ourc
e,
peptide
PM
3 N
itro
gen S
ourc
es
H
09
Gly
-Gln
N-S
ourc
e,
peptide
PM
3 N
itro
gen S
ourc
es
H
10
Gly
-Glu
N-S
ourc
e,
peptide
PM
3 N
itro
gen S
ourc
es
H
11
Gly
-Met
N-S
ourc
e,
peptide
PM
3 N
itro
gen S
ourc
es
H
12
Met-
Ala
N-S
ourc
e,
peptide
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
A
01
Negative C
ontr
ol
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
A
02
Phosphate
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
A
03
Pyro
phosphate
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
A
04
Trim
eta
phosphate
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
A
05
Tripoly
phosphate
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
A
06
Trieth
yl P
hosphate
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
A
07
Hypophosphite
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
A
08
Adenosin
e-
2’-m
onophosphate
P-S
ourc
e,
org
anic
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
A
09
Adenosin
e-
3’-m
onophosphate
P-S
ourc
e,
org
anic
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
A
10
Adenosin
e-
5’-m
onophosphate
P-S
ourc
e,
org
anic
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
A
11
Adenosin
e-
2’,3’-cyclic
monophosphate
P-S
ourc
e,
org
anic
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
A
12
Adenosin
e-
3’,5’-cyclic
monophosphate
P-S
ourc
e,
org
anic
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
B
01
Thio
phosphate
P-S
ourc
e,
inorg
anic
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
B
02
Dithio
phosphate
P-S
ourc
e,
inorg
anic
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
B
03
D,L
-α-G
lycero
l P
hosphate
P-S
ourc
e,
org
anic
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
B
04
β-G
lycero
l P
hosphate
P-S
ourc
e,
org
anic
274
Tab
le 1
. (c
ont)
.
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
B
05
Carb
am
yl P
hosphate
P-S
ourc
e,
org
anic
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
B
06
D-2
-Phospho-G
lyceric A
cid
P-S
ourc
e,
org
anic
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
B
07
D-3
-Phospho-G
lyceric A
cid
P-S
ourc
e,
org
anic
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
B
08
Guanosin
e-
2’-m
onophaphate
P-S
ourc
e,
org
anic
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
B
09
Guanosin
e-
3’-m
onophosphate
P-S
ourc
e,
org
anic
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
B
10
Guanosin
e-
5’-m
onophosphate
P-S
ourc
e,
org
anic
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
B
11
Guanosin
e-
2’,3’-cyclic
monophosphate
P-S
ourc
e,
org
anic
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
B
12
Guanosin
e-
3’,5’-cyclic
monophosphate
P-S
ourc
e,
org
anic
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
C
01
Phosphoenol P
yru
vate
P-S
ourc
e,
org
anic
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
C
02
Phospho-G
lycolic
Acid
P-S
ourc
e,
org
anic
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
C
03
D-G
lucose-1
-Phosphate
P-S
ourc
e,
org
anic
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
C
04
D-G
lucose-6
-Phosphate
P-S
ourc
e,
org
anic
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
C
05
2-D
eoxy-D
Glu
cose 6
-Phosphate
P-S
ourc
e,
org
anic
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
C
06
D-G
lucosam
ine-6
-Phosphate
P-S
ourc
e,
org
anic
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
C
07
6-P
hospho-G
luconic
Acid
P-S
ourc
e,
org
anic
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
C
08
Cytidin
e-
2’-m
onophosphate
P-S
ourc
e,
org
anic
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
C
09
Cytidin
e-
3’-m
onophosphate
P-S
ourc
e,
org
anic
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
C
10
Cytidin
e-
5’-m
onophosphate
P-S
ourc
e,
org
anic
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
C
11
Cytidin
e-
2’,3’-cyclic
monophosphate
P-S
ourc
e,
org
anic
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
C
12
Cytidin
e-
3’,5’-cyclic
monophosphate
P-S
ourc
e,
org
anic
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
D
01
D-M
annose-1
-Phosphate
P-S
ourc
e,
org
anic
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
D
02
D-M
annose-6
-Phosphate
P-S
ourc
e,
org
anic
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
D
03
Cyste
am
ine-S
Phosphate
P-S
ourc
e,
org
anic
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
D
04
Phospho-L
Arg
inin
eP
-Sourc
e,
org
anic
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
D
05
o-P
hospho-D
Serine
P-S
ourc
e,
org
anic
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
D
06
o-P
hospho-L
Serine
P-S
ourc
e,
org
anic
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
D
07
o-P
hospho-L
Thre
onin
eP
-Sourc
e,
org
anic
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
D
08
Uridin
e-
2’-m
onophosphate
P-S
ourc
e,
org
anic
Appen
dix
275
Tab
le 1
. (c
ont)
.
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
D
09
Uridin
e-
3’-m
onophosphate
P-S
ourc
e,
org
anic
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
D
10
Uridin
e-
5’-m
onophosphate
P-S
ourc
e,
org
anic
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
D
11
Uridin
e-
2’,3’-cyclic
monophosphate
P-S
ourc
e,
org
anic
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
D
12
Uridin
e-
3’,5’-cyclic
monophosphate
P-S
ourc
e,
org
anic
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
E
01
O-P
hospho-D
Tyro
sin
eP
-Sourc
e,
org
anic
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
E
02
O-P
hospho-L
Tyro
sin
eP
-Sourc
e,
org
anic
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
E
03
Phosphocre
atine
P-S
ourc
e,
org
anic
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
E
04
Phosphory
l C
holin
eP
-Sourc
e,
org
anic
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
E
05
O-P
hosphory
l-E
thanola
min
eP
-Sourc
e,
org
anic
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
E
06
Phosphono A
cetic A
cid
P-S
ourc
e,
org
anic
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
E
07
2-A
min
oeth
yl P
hosphonic
Acid
P-S
ourc
e,
org
anic
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
E
08
Meth
yle
ne D
iphosphonic
Acid
P-S
ourc
e,
org
anic
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
E
09
Thym
idin
e-
3’-m
onophosphate
P-S
ourc
e,
org
anic
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
E
10
Thym
idin
e-
5’-m
onophosphate
P-S
ourc
e,
org
anic
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
E
11
Inositol H
exaphosphate
P-S
ourc
e,
org
anic
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
E
12
Thym
idin
e 3
’,5’-cyclic
monophosphate
P-S
ourc
e,
org
anic
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
F
01
Negative C
ontr
ol
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
F
02
Sulfate
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
F
03
Thio
sulfate
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
F
04
Tetr
ath
ionate
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
F
05
Thio
phosphate
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
F
06
Dithio
phosphate
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
F
07
L-C
yste
ine
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
F
08
D-C
yste
ine
S-S
ourc
e,
org
anic
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
F
09
L-C
yste
inyl-G
lycin
e
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
F
10
L-C
yste
ic A
cid
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
F
11
Cyste
am
ine
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
F
12
L-C
yste
ine S
ulfin
ic A
cid
276
Tab
le 1
. (c
ont)
.
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
G
01
N-A
cety
l-LC
yste
ine
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
G
02
S-M
eth
yl-LC
yste
ine
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
G
03
Cysta
thio
nin
e
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
G
04
Lanth
ionin
e
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
G
05
Glu
tath
ione
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
G
06
D,L
-Eth
ionin
e
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
G
07
L-M
eth
ionin
e
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
G
08
D-M
eth
ionin
e
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
G
09
Gly
cyl-LM
eth
ionin
e
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
G
10
N-A
cety
l-D
,LM
eth
ionin
e
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
G
11
L-
Meth
ionin
e S
ulfoxid
e
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
G
12
L-M
eth
ionin
e S
ulfoxid
e
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
H
01
L-D
jenkolic
Acid
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
H
02
Thio
ure
a
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
H
03
1-T
hio
-β-D
Glu
cose
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
H
04
D,L
-Lip
oam
ide
S-S
ourc
e,
org
anic
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
H
05
Tauro
cholic
Acid
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
H
06
Taurine
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
H
07
Hypota
urine
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
H
08
p-A
min
o B
enzene S
ulfonic
Acid
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
H
09
Buta
ne S
ulfonic
Acid
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
H
10
2-H
ydro
xyeth
ane S
ulfonic
Acid
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
H
11
Meth
ane S
ulfonic
Acid
PM
4 P
hosphoru
s a
nd S
ulfur
Sourc
es
H
12
Tetr
am
eth
yle
ne S
ulfone
Appendix
277
Appendix VII
Figure 1. Authentication of WSM3693 and Lut5 reisolated from harvested nodules: Gel
image of rep-PCR using ERIC primers. a) Lane 1: 1 kbp DNA ladder; lane 2: no template;
lane 3: A.s N1; lane 4: A.s N2; lane 5: I.f N1; lane 6: I.f N2; lane 7: I.f N3; lane 8: I.f N4;
lane 9: WSM3693 –80 stock; lane 10: L.a N1; lane 11: L.a N2; lane 12 L.a N3; lane 13: 1
kbp DNA ladder. b) Lane 1: 100 bp DNA ladder; lane 2: WSM3693 –80 stock; lane 3: P.v
N1; lane 4: P.v N2; lane 5: V.u N1; lane 6: V.u N2; lane 7: V.u N3; lane 8: V.u N4; lane 9:
V.u N5; lane 10: V.u N6; lane 11: V.u N7; lane 12: 100 bp DNA ladder; b) Lane 1: 100 bp
DNA ladder; lane 2: Lut5 –80 stock; lane 3: A.s N1; lane 4: 100 bp DNA ladder.
A.s = Acacia saligna; I.f = Indigofera frutescens; L.a = Listia angolensis; P.v = Phaseolus
vulgaris; V.u = Vigna unguiculata; N = nodule
1,500 bp
1,500 bp
800 bp
800 bp
b c
1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4
1 2 3 4 5 6 7 8 9 10 11 12 13 10,000 bp
3,000 bp
1,500 bp
a
Appendix
278
Appendix VIII
Figure 1. Bayesian tree for concatenated sequences of dnaK, gyrB, recA, rpoB (2427 bp)
from seven Microvirga strains and eleven Alphaproteobacterial reference taxa. Posterior
probabilities are listed above branches. Scale bar for branch lengths shows 0.05 substitutions
per site. (Matt Parker, unpublished data).
Appendix
279
Figure 2. Bayesian tree for nodA sequences (594 bp) from three symbiotic Microvirga
strains and 29 proteobacterial reference taxa. The posterior probability was 1.0 for 23 of the
29 internal branches of the tree; for the six other branches, the posterior probability is listed
on the tree. Scale bar for branch lengths shows 0.05 substitutions per site. (Matt Parker,
unpublished data).
Appendix
280
Figure 3. Bayesian tree for concatenated sequences of nifD and nifH (879 bp) from five
Microvirga strains and 17 Alphaproteobacterial reference taxa. Posterior probabilities are
listed above branches. Scale bar for branch lengths shows 0.05 substitutions per site. (Matt
Parker, unpublished data).
Appendix
281
Appendix IX
Table 1. % G+C and DNA:DNA hybridization results. (Anne Willems and Sofie De Meyer,
unpublished data.)
Lut 5 Lut 6T
WSM
3557T
WSM
3693T
% GC
Lut5 - 61.9
Lut6T
97 - 61.9
WSM3557T
28.5±1.5 34.5±0.5 - 62.9± 0.1
WSM3693T
20 25 33.5±0.5 - 62.6
Microvirga flocculans TFBT
19 26 26 64.0*
*From Takeda et al. (2004).
Appen
dix
282
Tab
le 2
. C
ellu
lar
fatt
y a
cid c
om
posi
tions*
. (A
nne
Wil
lem
s an
d S
ofi
e D
e M
eyer
, unpubli
shed
dat
a.)
FA
ME
on
TS
A m
ediu
m
Lut5
Lut6
T
WS
M
3557
T
WS
M
3693
T
Mic
rovirga
floccula
ns
LM
G 2
5472
T
Mic
rovirga
subte
rrane
a
LM
G 2
5504
T
Chela
tococcus
asaccharo
vora
ns
LM
G 2
5503
T
11 m
eth
yl 18:1
ω7c
1.1
12:0
0
Tr
1.2
7
14:0
0
1.6
3
1.1
1
Tr
15:0
3O
H
Tr
1.0
8
Tr
15:1
ω8c
Tr
T
r
16:0
0
4.2
5
2.4
8
8.7
7
7.8
5
9.8
11.5
10.4
16:0
3O
H
Tr
17:0
0
Tr
Tr
1.0
4
1.3
Tr
17:0
CY
CLO
1.8
2
2.1
3
1.4
17:1
ω8c
Tr
18:0
0
1.3
6
2.5
5
2.7
1
2.2
4
5
4.5
4.3
18:0
3O
H
1.0
0
1.0
7
1.2
4
T
r 3.1
2.3
18:1
ω7c
68.9
4
69.7
1
52.5
8
53.0
0
59.8
63.1
14.5
19:0
10 m
eth
yl
1.4
8
1.8
5
Tr
T
r
19:0
CY
CLO
ω8c
1.3
2
1.2
6
17.6
5
17.2
5
6.6
6.3
56.7
20:1
ω7c
2.2
20:2
ω6,9
c
Tr
Unknow
n 1
4.9
59
Tr
Tr
1
T
r
Sum
med F
eatu
re 1
T
r T
r
Sum
med F
eatu
re 2
16.0
6
15.4
1
5.6
0
11.2
9
11.4
8.3
4.7
Sum
med F
eatu
re 3
3.5
6
2.2
9
4.1
7
3.8
5
3
3.4
* S
um
med
Fea
ture
1 c
om
pri
ses
15:1
iso
H o
r I
/ 13:0
3O
H;
Sum
med
Fea
ture
2 c
om
pri
ses
16:1
iso
I, 14:0
3O
H /
unknow
n 1
0.9
38;
S
um
med
Fea
ture
3 c
om
pri
ses
16:1
w7c
/ 15:0
iso
2O
H;
Tr
= T
race
am
ounts
(le
ss t
han
1%
)