Rapid Evolution of Diversity in the Root Nodule Bacteria ...Rapid Evolution of Diversity in the Root Nodule Bacteria of Biserrula pelecinus L. Kemanthi Gayathri Nandasena This thesis

Rapid Evolution of Diversity in the Root

Nodule Bacteria of Biserrula pelecinus L.

Kemanthi Gayathri Nandasena

This thesis is presented for the degree of

Doctor of Philosophy

of

Murdoch University

2004

I declare that this thesis is my own account of my research and

contain as its main content work which has not been submitted for a

degree at any tertiary education institution.

……………………………………………….

Kemanthi Gayathri Nandasena

………..To my beloved parents

With much love………….

TABLE OF CONTENTS

PUBLICATIONS ARISING FROM THIS THESIS……………………………………………………I

ABSTRACT……………………………………………………………….………………………………II

ACKNOWLEDGMENTS………………………………………………………………………...……..VI

1. LITERATURE REVIEW.................................................................................................... 1

1.1 THE LEGUME-RHIZOBIA RELATIONSHIP – A BENEFICIAL SYMBIOSIS TO AGRICULTURE................ 1 1.1.1 LEGUMES AND THEIR IMPORTANCE TO AGRICULTURE ......................................................1 1.1.2 RHIZOBIA ...........................................................................................................................2 1.1.3 GENUS MESORHIZOBIUM ....................................................................................................5 1.2 SYMBIOTIC INTERACTION – A MOLECULAR DIALOGUE..................................................................... 6 1.2.1 RECOGNITION ....................................................................................................................6 1.2.2 RHIZOBIAL NODULATION GENES .......................................................................................9 1.2.3 MOLECULAR BASIS OF HOST SPECIFICITY........................................................................11 1.2.4 INFECTION AND NODULE DEVELOPMENT.........................................................................13 1.2.5 MOLECULAR BASIS OF NITROGEN FIXATION ...................................................................14 1.3 SYMBIOTIC PROMISCUITY – A CURE AND A CURSE FOR LEGUME PRODUCTIVITY ......................... 16 1.4 MOBILE GENETIC ELEMENTS – A CHALLENGE TO CONCEPTS OF RHIZOBIAL EVOLUTION........... 20 1.4.1 MECHANISMS DRIVING BACTERIAL EVOLUTION .............................................................20 1.4.2 INFLUENCE OF MOBILE GENETIC ELEMENTS ON EVOLUTION OF RHIZOBIAL DIVERSITY .21 1.5 RHIZOBIAL DIVERSITY AND COMPETITION – A THREAT TO AGRICULTURE................................... 25 1.5.1 RHIZOBIAL DIVERSITY AND DYNAMICS ...........................................................................25 1.5.2 METHODS USED TO INVESTIGATE RHIZOBIAL DIVERSITY................................................27 1.5.3 RHIZOBIAL COMPETITION AND ITS SIGNIFICANCE ...........................................................29 1.6 STUDY AT HAND.................................................................................................................................. 32 1.6.1 BACKGROUND..................................................................................................................32 1.6.2 AIMS OF THIS THESIS........................................................................................................33

2. GENETIC DIVERSITY AMONG RNB ISOLATED FROM BISERRULA PELECINUS L. SIX YEARS AFTER INTRODUCTION AND INOCULATION WITH WSM1271 ............. 35

2.1 INTRODUCTION................................................................................................................................... 35 AIMS.............................................................................................................................................37 2.2 MATERIALS AND METHODS ............................................................................................................... 37 2.2.1 FIELD SITE AND COLLECTION OF NODULES FROM B. PELECINUS .....................................37 2.2.2 ISOLATION OF ROOT NODULE BACTERIA .........................................................................38 2.2.3 MOLECULAR FINGERPRINTING WITH PRIMER RPO1........................................................40 2.2.4 MOLECULAR FINGERPRINTING WITH PRIMER ERIC ........................................................41 2.2.5 AUTHENTICATION OF ISOLATES.......................................................................................41 2.2.6 SEQUENCING THE 16S RRNA GENE.................................................................................43 2.3 RESULTS.............................................................................................................................................. 44 2.3.1 ISOLATION OF RNB .........................................................................................................44 2.3.2 GENETIC DIVERSITY INDICATED THROUGH MOLECULAR FINGERPRINTING ....................45 2.3.3 GENETIC DIVERSITY DISTINGUISHED THROUGH 16S RRNA GENE BASED PHYLOGENY..47 2.4 DISCUSSION ........................................................................................................................................ 55

3. PHENOTYPIC DIVERSITY AMONG RNB ISOLATED FROM BISERRULA PELECINUS L. SIX YEARS AFTER INTRODUCTION AND INOCULATION WITH WSM1271 ......... 61

3.1 INTRODUCTION................................................................................................................................... 61 AIMS.............................................................................................................................................63 3.2 MATERIAL AND METHODS ................................................................................................................ 64 3.2.1 EFFECTIVENESS TESTS .....................................................................................................64 3.2.2 CARBOHYDRATE UTILISATION.........................................................................................67 3.2.3 ANTIBIOTIC RESISTANCE .................................................................................................69 3.2.4 PH RANGE ........................................................................................................................70 3.2.5 HOST RANGE EXPERIMENTS.............................................................................................70 3.3 RESULTS.............................................................................................................................................. 73 3.3.1 EFFECTIVENESS ...............................................................................................................73 3.3.2 CARBOHYDRATE UTILISATION.........................................................................................77 3.3.3 ANTIBIOTIC RESISTANCE .................................................................................................85 3.3.4 PH RANGE ........................................................................................................................86 3.3.5 HOST RANGE ....................................................................................................................86 3.4 DISCUSSION ........................................................................................................................................ 89

4. EVIDENCE FOR GENE TRANSFER FROM WSM1271 TO OTHER SOIL BACTERIA IN SITU ............................................................................................................................... 95

4.1 INTRODUCTION................................................................................................................................... 95 AIM...............................................................................................................................................97 4.2 MATERIAL AND METHODS ................................................................................................................ 97 4.2.1 GENOMIC DNA EXTRACTION ..........................................................................................97 4.2.2 PRIMERS...........................................................................................................................99 4.2.3 SEQUENCING OF NIFH ....................................................................................................100 4.2.4 SEQUENCING OF NODA...................................................................................................101 4.2.5 SEQUENCING OF INTS .....................................................................................................101 4.2.6 ECKHARDT GEL ELECTROPHORESIS...............................................................................102 4.2.7 SOUTHERN HYBRIDIZATION OF MOBILIZED PLASMIDS ..................................................103 4.3 RESULTS............................................................................................................................................ 104 4.3.1 SEQUENCING OF NIFH ....................................................................................................104 4.3.2 SEQUENCING OF NODA...................................................................................................109 4.3.3 SEQUENCING OF INTS .....................................................................................................112 4.3.4 ECKHARDT GEL ELECTROPHORESIS...............................................................................115 4.3.5 SOUTHERN HYBRIDIZATION OF MOBILIZED PLASMIDS ..................................................115 4.4 DISCUSSION ...................................................................................................................................... 117

5. GENERAL DISCUSSION............................................................................................. 121

5.1 MECHANISMS DRIVING RAPID EVOLUTION OF RHIZOBIAL DIVERSITY IN AGRICULTURAL SOILS... 121 5.2 RAPID EVOLUTION OF NODULATING OPPORTUNISTS MAY THREATEN LEGUME PRODUCTIVITY 126 5.3 INSIGHT TO THE DEVELOPMENT OF RHIZOBIAL PROMISCUITY .................................................... 130 5.4 GENERAL CONCLUSIONS.................................................................................................................. 134

BIBLIOGRAPHY………………..……………………………………………………………………..137

APPENDIX……...…………..…………………………………………………………………………..191

Publications I

Publications arising from this thesis

Nandasena, K. G., G. W. O'Hara, R. P. Tiwari, R. J. Yates, and J. G.

Howieson. 2001. Phylogenetic relationships of three bacterial strains isolated

from the pasture legume Biserrula pelecinus L. International Journal of

Systematic and Evolutionary Microbiology 51:1983-1986.

Nandasena, K. G., G. W. O'Hara, R. P. Tiwari, R. J. Yates, B. D.

Kishinevsky, and J. G. Howieson. 2004. Symbiotic relationships and root

nodule ultrastructure of the pasture legume Biserrula pelecinus L.-a new

legume in agriculture. Soil Biology and Biochemistry 36:1309-1317.

Abstract II

Abstract

Biserrula pelecinus L. has been introduced to Australia from the

Mediterranean region, in the last decade due to many attractive agronomic

features. This deep rooted, hard seeded, acid tolerant and insect resistant

legume species provides high quality food for cattle and sheep, and grows well

under the harsh edaphic and environmental conditions of Australia. In 1994, B.

pelecinus was introduced to a site in Northam, Western Australia where there

were no native rhizobia capable of nodulating this legume. The introduced

plants were inoculated with a single inoculant strain of Mesorhizobium sp.,

WSM1271. This study investigated whether a diversity of rhizobia emerged over

time. A second objective was to investigate the possible mechanisms involved

in the diversification of rhizobia able to nodulate B. pelecinus.

Eighty eight isolates of rhizobia were obtained from nodules on B.

pelecinus growing at the Northam site in August 2000, six years after

introduction. These plants were self-regenerating offspring from the original

seeds sown. Molecular fingerprinting PCR with RPO1 and ERIC primers

revealed that seven strains (novel isolates) had banding patterns distinct from

WSM1271 while 81 strains had similar banding patterns to WSM1271. A 1400

bp internal fragment of the 16S rRNA gene was amplified and sequenced for

four of the novel isolates (N17, N18, N45 and N87) and WSM1271. The

phylogenetic tree developed using these sequences clustered the novel isolates

in Mesorhizobium. There were >6 nucleotide mismatches between three of the

novel isolates (N17, N18, N87) and WSM1271 while there were 23 nucleotide

mismatches between N45 and WSM1271.

Abstract III

When B. pelecinus cv. Casbah was inoculated with the novel isolates,

five (N17, N18, N39, N46 and N87) yielded <40% of the shoot dry weight of the

plants inoculated with the original inoculant (WSM1271). Novel isolates N15

and N45 were completely ineffective on B. pelecinus cv. Casbah.

Physiological experiments to test the ability of the novel isolates and

WSM1271 to grow on 14 different carbon sources (N acetyl glucosamine,

arabinose, arbutine, dulcitol, β-gentiobiose, lactose, maltose, melibiose, D-

raffinose, saccharose, L-sorbose, D-tagatose, trehalose and D-turanose) as the

sole source of carbon, intrinsic resistance to eight different antibiotics

(ampicillin, chloramphenicol, gentamicin, kanamycin, nalidixic acid,

spectinomycin, streptomycin and tetracycline) and pH tolerance (pH 4.5, 5.0,

7.0, 9.0) revealed that the novel isolates had significantly different carbon

source utilization patterns to WSM1271. However, pH tolerance and intrinsic

resistance to antibiotics were similar between the novel isolates and WSM1271

except for streptomycin (100 μg/ml). Novel isolates N17, N18, N46 and N87

were susceptible for this antibiotic while the other novel isolates and WSM1271

were resistant.

Host range experiments were performed for the novel isolates N17, N18,

N45, N87, WSM1271 and two other root nodule bacteria (RNB) previously

isolated from B. pelecinus growing in the Mediterranean region (WSM1284 and

WSM1497) for twenty one legumes (Amorpha fruticosa, Astragalus adsurgens,

Astragalus membranaceus, Astragalus sinicus, Biserrula pelecinus cv Casbah,

Dorycnium hirsutum, Dorycnium rectum, Glycyrrhiza uralensis, Hedysarum

spinosissimum, Leucaena leucocephala, Lotus corniculatus, Lotus edulis, Lotus

glaber, Lotus maroccanus, Lotus ornithopodioides, Lotus parviflorus, Lotus

Abstract IV

pedunculatus, Lotus peregrinus, Lotus subbiflorus, Macroptilium atropurpureum,

and Ornithopus sativus). Only isolate N17 have the same host range as

WSM1271 in that they both nodulated B. pelecinus and A. membranaceus,

while the other three novel isolates, WSM1284 and WSM1497 had a broader

host range than WSM1271. Three isolates N18, N45 and N87 formed small

white nodules on M. atropurpureum, in addition to nodulating the above hosts.

Isolates N18 and N45 also nodulated A. adsurgens while N45 was the only

isolate to nodulate L. edulis. Isolate N87 was the only isolate to nodulate A.

fruticosa. WSM1497 nodulated A. adsurgens, A. membranaceus, B. pelecinus

and L. corniculatus while WSM1284 was a promiscuous strain that nodulated

16 host species out of the 21 tested.

A 710 bp internal region of nifH, a 567 bp internal region of nodA and a

1044 bp internal region of intS were sequenced for N17, N18, N45, N87 and

WSM1271. The sequence comparison showed that the sequences of the above

three genes of the four novel isolates were identical to that of WSM1271.

Eckhardt gel electrophoresis revealed that WSM1271, three other RNB

isolates from B. pelecinus from the Mediterranean region and isolate N18 each

have a plasmid of approximately 500 kb while N17, N45 and N87 are plasmid

free. Probing of the plasmid DNA from the Eckhardt gel with nifH and nodA

probes indicated that these two genes were not located on the plasmid.

Furthermore, the results of this study demonstrated that 92% of the

nodules on B. pelecinus growing in the Northam site six years after the

introduction of this plant were occupied by the inoculant strain and the N2

fixation efficiency of the progeny strains of WSM1271 remain similar to the

mother culture. This study also showed that the carbon source utilization

Abstract V

pattern, intrinsic antibiotic resistance and pH range of the progeny strains of

WSM1271 remain relatively similar, except for few variations in carbon source

utilization patterns.

This thesis clearly demonstrated that phenotypicaly, genetically and

phylogenetically diverse strains capable nodulating B. pelecinus evolved

through symbiotic gene transfer from the inoculant strain to other soil bacteria

within six years. The presence of intS, and the evidence of gene transfer

between these Mesorhizobium strains indicates that transfer of symbiotic genes

may have occurred via a symbiosis island present in WSM1271.

Acknowledgments VI

Acknowledgements

I would like to take this opportunity to show my gratitude to many people

who have helped me and supported me during my PhD.

Firstly, to my three great supervisors: I sincerely thank my principal

supervisor Dr. Graham O’Hara for all the hearty discussions and valuable

advice, correcting endless amounts of drafts of my thesis, and the

encouragement and confidence you have given me at times when I was feeling

weak. I wish to extend my gratitude to Dr. Ravi Tiwari for the superb guidance

you have given me to do my experiments promptly and precisely, your

remarkable patience in tolerating me and the million questions I ask. I am

forever grateful to A/Prof. John Howieson for providing me a brilliant project and

a great facility to do my PhD. Your enthusiasm in the project and the stimulating

discussions steer me through to the end. I felt very lucky to have not just one

but three wonderful souls and brilliant minds as supervisors.

I also wish to acknowledge Prof. Mike Dilworth for letting me pinch his

valuable time to ask the sudden questions that I come up with and your

guidance in solving problems. I greatly appreciate all the assistance and advice

given to me by Dr. Wayne Reeve.

I am very grateful for Prof. Tony Tate, all the staff at the School of

Biological Sciences and Biotechnology for providing me with the IPRS

scholarship and to the Murdoch University for providing me with the MURS

scholarship.

My sincere thanks go to Dr. Mike Calver for helping me with the

statistical analysis and to Dr. David Berryman for providing the facility to use the

Acknowledgments VII

equipment in State Agricultural Biotechnology Centre and for assistance in

using various computer programmes.

I wish to thank Prof Clive Ronson for generously providing me M. loti

strain R7A and for allowing me valuable time for discussions during

conferences and Prof. Peter Van Berkum for providing all the type strains of

rhizobia.

My heartfelt gratitude goes to all the members of Centre for Rhizobium

Studies. You all have given me a home away from home and I wouldn’t have

been able to make it to the end without all the care and support you all have

given me. Special thanks to Julie Ardley, Regina Carr and Beau Fenner for

helping me with various tasks at work and for Ron Yates for putting a smile on

my face all the time.

I extend my thanks to my friends and colleagues, Dr. Anabel Vivas, Dr

Yvonne Cheng and Dr. Lambert Brau for proofreading my thesis.

Special thanks go to my dearest friend Ertug Sezmis for always being

there for me, looking after me and putting up with me during this difficult time. I

am very grateful to Dincer Eren for all the joy and happiness you have given me

to relieve my stress and it was always a great feeling to know that I can always

count on you for anything, even at a time when you were thousands of miles

away. I also wish to thank Jason Terpolilli for assisting me with my English and

for your great friendship.

Many thanks go to my darling friends Juliana Hamzah, Tobias Schoep,

Jamie Coote, Sandra Nogueira, Nick Evangelinos, Kim Smith, Navid

Moheimaani, Giovanni Garau, Mike Baker and Aleck Nikoloski. You all have

made my stay in Australia a very enjoyable and a memorable one. My

Acknowledgments VIII

appreciation also goes to Ganesha Ekanayaka for your support and concern

even from a far way land.

Finally I would like to thank my parents for always being there for me.

Your love, support and encouragement have been invaluable to me.

Chapter 1

1

1. Literature review

1.1 The legume-rhizobia relationship – a beneficial symbiosis

to agriculture

1.1.1 Legumes and their importance to agriculture

Plants commonly known as legumes belong to the plant family

Leguminosae which contains approximately 18 000 species distributed in three

subfamilies, Mimosoideae, Caesalpinioideae, and Papilionoideae (Royal

Botanic Garden, Kew, 2003). The members of the Leguminosae have a

worldwide distribution and have been used by mankind since antiquity as a

source of food and forage (Hadri et al., 1998; Howieson et al., 2000b). Many

legumes have the ability to form nitrogen (N2) fixing root nodules with soil

bacteria, collectively called rhizobia (Sprent, 2001) and thus contribute to the

biological fixation of N2. The symbiotic association between rhizobia and

legumes plays a significant role in world agricultural productivity by annually

converting approximately 120 million tonnes of atmospheric nitrogen into

ammonia (Freiberg et al., 1997) thereby saving $US 6.8 billion expenditure on

nitrogenous fertilizer (Herridge & Rose, 2000).

Legumes and their rhizobia are often introduced to agricultural

ecosystems to improve soil fertility and farming systems flexibility (Brockwell &

Bottomley, 1995; Sessitsch et al., 2002). Economically important species of the

Leguminosae include grain legumes (pulses and oil seeds) and pasture

legumes. Whilst grain legumes provide high protein food for humans, both,

grain and pasture legume species provide high quality feed for cattle and sheep

Chapter 1

2

(Minson et al., 1993; Baker & Dynes, 1999; Howieson, 1999; Francis, 1999),

increase soil nitrogen (Unkovich et al., 1995), improve the structure of soil

(porosity, aggregate stability, water retention; Greenland 1971), provide a

disease break (Meagher & Rooney, 1966; King et al., 1982; Mayfield & Clare,

1984; Reeves & Ewing, 1993), and assist in weed control (Reeves & Smith,

1975; Thorn & Perry, 1987; Latta & Carter, 1998). Additionally, deep-rooted

pasture legume species can assist in reducing rising water tables in areas

prone to secondary salinity (Howieson et al., 2000b).

Only a small fraction of legumes from the large diversity that exist on

earth have been systematically sampled for their symbionts (Young, 1996;

Sprent, 2001). Therefore, the present rhizobial systematics is based upon these

relatively few isolates and it is likely to change with new discoveries of nodule

bacteria from ongoing legume exploration as it has in the last few years with the

discovery of rhizobia in β-Proteobacteria (Chen et al., 2001; Moulin et al., 2001;

Vandamme et al., 2002). Brief descriptions of the genera and species of

rhizobia identified to date are given in the following section.

1.1.2 Rhizobia

Root nodule bacteria (RNB) are facultative microsymbionts (Provorov,

1998) that can infect roots of some, but not all, legumes and transform

atmospheric N2 into forms usable by the plant (Phillips, 1999; Herridge et al.,

2001; Sessitsch et al., 2002). RNB are Gram negative, motile, rods that are

pleomorphic under adverse growth conditions (Jordan, 1984). They usually

accumulate granules of poly-β-hydroxybutyrate when carbon is in excess and

Chapter 1

3

are aerobic, possessing a respiratory type of metabolism with oxygen as the

terminal electron acceptor (Jordan, 1984).

Currently there are 44 accepted species of RNB distributed in 12 genera

and they are mainly in the class α-Proteobacteria (Fig 1.1 from Sawada et al.,

2003). Recently, nodulation of legumes by members of the β-Proteobacteria

have also been reported (Chen et al., 2001; Moulin et al., 2001; Vandamme et

al., 2002). As illustrated in Fig 1.1, RNB are intermingled with other bacterial

genera that do not contain legume symbionts. Therefore, RNB are considered

to have a polyphyletic origin (Young, 1996).

The RNB in the α-Proteobacteria are contained in five families:

Rhizobiaceae (including the genera Allorhizobium, Rhizobium and

Sinorhizobium), Phyllobacteriaceae (including the genus Mesorhizobium),

Bradyrhizobiaceae (including the genus Bradyrhizobium), Hyphomicrobiaceae

(including the genera Azorhizobium and Devosia) and Methylobacteriaceae

(including Methylobacterium) as defined by their 16S rDNA sequence analysis

(Garrity et al., 2003; Sawada et al., 2003). The root nodulating β-Proteobacteria

are contained in two genera: Burkholderia and Wautersia in the family

Burkholderiaceae (Garrity et al., 2003; Sawada et al., 2003). The RNB

investigated in this thesis belong to Mesorhizobium and therefore a brief

description of this genus is given in the following section.

Chapter 1

4

Fig 1.1 Phylogenetic tree constructed with the 16S rRNA gene sequences

Taken from Sawada et al., (2003)

Chapter 1

5

1.1.3 Genus Mesorhizobium

Jarvis et al., (1997) described the genus Mesorhizobium (rhizobia

phylogenetically intermediate between the genera Bradyrhizobium and

Rhizobium) to include RNB that had considerable phenotypic and genotypic

differences to the other RNB genera. The members of the Mesorhizobium are

clearly distinct in their DNA homology (Crow et al., 1981) and phylogeny based

on small subunit rRNA sequences (Willems & Collins, 1993; Yanagi &

Yamasato, 1993; Young & Haukka, 1996). The other characteristics of

Mesorhizobium spp. as described by Jarvis et al., (1997) are:

• Cells are Gram-negative, aerobic, non-spore-forming rods, motile,

usually with one polar or subpolar flagellum.

• Cells may contain poly-β-hydroxybutyrate inclusion bodies.

• Growth on yeast mannitol agar produces colonies that are 2-4 mm in

diameter after incubation for 3-7 days at 28ºC.

• All species assimilate glucose, rhamnose and sucrose with the

production of acidic end products.

• The guanine-plus-cytosine contents of the DNAs are 59 to 64 mol% (as

determined by the thermal denaturation method).

• At the molecular level the members of this genus can be recognized by

their fatty acid profiles and 16S rRNA gene sequence.

There are eight species described under this genus at present (Garrity et

al., 2003; Sawada et al., 2003): M. amorphae (Wang et al., 1999c), M.

chacoense (Velázquez et al., 2001), M. ciceri (Nour et al., 1994), M. huakuii

(Chen et al., 1991), M. loti (Jarvis et al., 1982), M. mediterraneum (Nour et al.,

Chapter 1

6

1995), M. plurifarium (de Lajudie et al., 1998) and M. tianshanense (Chen et al.,

1995).

A previous study has shown that RNB isolated from Biserrula pelecinus,

the host-legumes used in this study, growing in the Mediterranean region

belong to Mesorhizobium based on a polyphasic taxonomic approach that

included morphological and physiological characteristics, plasmid profiles,

symbiotic performance and 16S rRNA gene sequencing (Nandasena et al.,

2001).

1.2 Symbiotic interaction – a molecular dialogue

A successful symbiotic interaction requires compatibility between the

RNB and the legume at many different stages starting from initial recognition,

through successful differentiation to nitrogen fixation (Long & Ehrhardt, 1989).

Some important features of these stages are reviewed in sequential order in this

section.

1.2.1 Recognition

Recognition between prokaryotic cells and eukaryotic organisms is an

essential component of symbiosis and pathogenesis. In the legume-rhizobia

interaction, symbiotic nitrogen fixation takes place in a symbiosome, an

organelle inside the root nodules (Hadri et al., 1998). The fixation of

atmospheric N2 is the end-point of a long developmental programme which

begins with a molecular recognition system that allows the entry of rhizobia into

root cells. Therefore, the initial recognition between compatible partners is

crucial for the successful development of a symbiotic nodule and it seems

Chapter 1

7

logical that surface interactions between the two partners may be involved in

this complicated recognition process (Long & Ehrhardt, 1989).

The plant rhizosphere is generally colonized by a diversity of soil bacteria

due to the secretion of large amounts of organic matter by the plant roots

(Barran & Bromfield, 1997; Perret et al., 2000). Very few of these rhizosphere

organisms penetrate intracellularly; hence there must be sophisticated

signalling mechanisms that permit the exclusive entry of specific bacteria. The

communication and molecular recognition between the plant host and rhizobia

is directed by a signal exchange between the two partners. The chemical

mediators involved in the molecular dialogue include flavonoids, Nod factors,

surface polysaccharides and extracelular proteins (Broughton et al., 2000;

Perret et al., 2000; Rélić et al., 1994).

Lectins are plant proteins believed to bind to bacterial surface

determinants and play a role in this symbiotic dialogue (Long & Ehrhardt, 1989).

Díaz et al., (1989) introduced a lectin gene from P. sativum into the genome of

Trifolium repens and demonstrated that the transgenic T. repens roots were

able to nodulate well with R. leguminosarum biovar viciae (RNB from P.

sativum).

Symbiotic and pathogenic bacteria commonly use TTSS machinery to

communicate with eukaryotes (Saad et al., 2005). Rhizobial proteins secreted

via the type III secretion system (TTSS) play a role in nodulation and are termed

nodulation outer proteins (nops; Marie et al., 2003). Nops are known to

influence nodulation and nodule number on the legume host as well as

effectiveness of the nodules (Viprey et al., 1998; Krishnan, 2002; Marie et al.,

Chapter 1

8

2003; Ausmees, 2004). Exopolysaccharides (EPS) produced by rhizobia also

influence root nodule symbiosis (Becker & Pühler, 1998).

Rhizobia produce a morphogenic signal called a ‘Nod-factor’ in response

to specific plant released inducer signals which in most cases studied to date

are flavonoids (Fisher & Long, 1992). Betaines, erythronic or tetronic acids are

also known to be produced by some legumes as inducers for symbiotic

interaction (Gagnon & Ibrahim, 1998).

Nod-factors are lipo-chito-oligosaccharides with an N-acetylglucosamine

backbone (Downie, 1998). They can be ‘decorated’ with other chemical groups

depending on the RNB species. A more comprehensive description of Nod-

factors and their role in determining host specificity will be discussed later. The

type of Nod-factor produced may vary between different species of RNB

(Downie, 1998) and Nod-factors play a significant role in host-range

determination because they behave as the “keys to opening the legume doors”

(Broughton et al., 2000; Parniske & Downie, 2003). For example, the Nod-factor

produced by S. meliloti is responsible for the nodulation of alfalfa by this

species, but not vetch or pea (Lerouge et al., 1990).

The “locks” on the legumes for these rhizobial Nod-factors were identified

recently as a special class of receptor kinases (Limpens et al., 2003; Madsen et

al., 2003; Radutoiu et al., 2003). Kinases are molecular switches that regulate

enzyme or signalling pathways by adding phosphate groups to other proteins

(Parniske & Downie, 2003). The genes NFR1, NFR5 (both in Lotus japonicus;

Madsen et al., 2003; Radutoiu et al., 2003) and LYK (in Pisum sativum;

Limpens et al., 2003) code kinases with extracellular LysM motifs. LysM motifs

typically bind to polymers containing N-acetylglucosamine (Amon et al., 1998;

Chapter 1

9

Bateman & Bycroft, 2000) indicating that these kinases play a major role in

recognition by binding to the Nod-factors (Parniske & Downie, 2003). Another

molecule that is believed to act in harmony with the other receptors and play an

important role in the recognition is SYMRK (symbiosis receptor-like kinase)

which is a receptor kinase that lacks a LysM motif (Endre, 2002; Stracke et al.,

2002).

The initial recognition between RNB and legumes takes place at several

levels involving different types of molecules and is a complex process. Many

plant and RNB genes work together in this process. The RNB genes involved in

nodulation are discussed below.

1.2.2 Rhizobial nodulation genes

Many of the rhizobial genes involved in nodulation or regulation of

nodulation are commonly termed as nod genes. Genes required for nodulation

can be located in rhizobia on a plasmid (Hynes & MacGregor 1990; Brom et al.,

1992; Barnett et al., 2001; Finan et al., 2001), on the chromosome (Kaneko et

al., 2000) or on a mobile symbiosis island which is integrated into the

chromosome (Sullivan et al., 2002). Many studies have been undertaken to

reveal the functions and regulation of nodulation genes (Downie, 1998;

Schlaman et al., 1998), Yet, they are not fully understood, due in part to the

involvement of many plant and bacterial genes in the nodulation process and

their inconsistent patterns of occurrence in different individuals (eg. strain

specific genes).

The rhizobial genes involved in nodulation are divided into five different

categories based on their functions. (i) regulatory genes, (ii) genes involved in

Chapter 1

10

biosynthesis and modification of Nod-factors, (iii) genes involved in Nod-factor

secretion, (iv) genes involved in protein secretion and (v) genes with undefined

functions (Downie, 1998). Over fifty different nodulation genes have been

discovered to date and their respective assigned functions were given by

Downie (1998).

The nodA, nodB, nodC, nodD, nodI and nodJ are the common nod

genes and they are present in all rhizobia studied to date. Other nod genes are

only present in certain groups, species or strains of rhizobia. For example nodX

is only present in R. leguminosarum bv. viciae strain TOM (Firmin et al., 1993).

Some of the nodulation genes (for example nodA, nodB and nodC) are present

in a single copy whilst paralogous sequences are found elsewhere in the

genome for nodD, nodM, nodP, nodQ and nodT (Surin & Downie, 1988;

Schwedock & Long, 1989; Baev et al., 1991; Rivilla & Downie, 1994).

Nodulation genes are commonly clustered together in a small region of the

genome and are organized in operons which can be conserved among certain

rhizobia (Downie, 1998). The physical organization of the nodulation genes can

vary between rhizobial genera and species. For example in R. leguminosarum

and in S. meliloti, nodA, nodB and nodC are located on one operon in the given

order (Downie, 1998) while in M. loti, nodA and nodC are located together in

one operon and nodB is separated and found downstream of the operon

(Sullivan, et al., 2002). By contrast, in rhizobia nodulating Austragalus sinicus,

nodBC are separated from nodA (Zhang et al., 2000).

Chapter 1

11

1.2.3 Molecular basis of host specificity

An intensive signal exchange between the plant and the RNB initiates

legume nodulation. Many plant and RNB derived molecules take part in this

process and the specificity in the symbiotic interaction is thus controlled at many

levels. The first level is at the type of NodD protein present in the RNB,

secondly by the type of the flavonoid produced by the legume host, thirdly by

the type of Nod-box in the promoter region of nodulation genes and fourthly by

the type(s) of Nod-factor produced by the RNB. The functions of nod genes and

their role in determining host specificity are elaborated below.

The DNA sequence of the nodD gene differs considerably for the

rhizobial species (Downie, 1994). Thus it can be assumed that different species

produce different NodD proteins which respond to different types of plant

flavonoids. NodD1 of the broad host range Rhizobium sp. strain NGR234

recognises a wide range of flavonoids and transfer of the nodD1 of strain

NGR234 to other restricted host range RNB has been shown to extend the host

range (Bender et al., 1988). Thus the initial level in symbiotic specificity is

controlled by nodD.

In the presence of flavonoid inducers, the bacterial NodD or SyrM

proteins regulate the initial infection by activating the transcription of other nod

genes (Roche et al., 1996; Downie, 1998;). SyrM is a nodulation-regulatory

locus with sequence similarity to nodD proteins identified in S. meliloti (Barnett

& Long, 1990; Schlaman et al., 1992). NodD and SyrM proteins act as both

plant signal sensors and transcriptional activators (Perret et al., 2000). These

two proteins belong to the LysR family of DNA binding proteins which have a

typical helix-turn-helix motif and act as transcriptional activators (Schell, 1993).

Chapter 1

12

NodD and SyrM proteins trigger the transcription of the nodABC operon

in RNB by binding to the Nod-box in the promoter region of this operon.

Rhizobium sp. strain NGR234, which can nodulate a broad range of legumes,

contains 19 different homologous sequences for Nod-box, thereby providing

many possibilities for fine-tuning nod gene expression (Perret et al., 2000). The

Nod-box sequence plays a key role in the control of symbiotic specificity (Perret

et al., 2000). However, there are symbiotic genes that do not have Nod-boxes.

The products of the common nod genes (nodABC) together with

products of host-specific nod genes (eg. nodFE) produce Nod-factors (Dénarié

et al, 1996, van Rhijn & Vanderleyden, 1995). One of the well studied levels of

host specificity involves the type of Nod-factor produced by RNB. The common

nod genes nodA, nodB and nodC are responsible for the synthesis of the Nod-

factor core (Section 1.2.2.). Although these genes are common to all RNB, their

sequences can still vary between RNB species and this has been shown to

influence host specificity (Roche et al., 1996). For example, the type of N-acyl

substitution transferred into the oligosaccharide backbone of Nod-factor is

determined by nodA (Ritsema et al., 1996), and a Nod-factor acylated with

vaccenic acid instead of C16:2 is produced when the nodA of S. meliloti is

replaced with the nodA of R. tropici (Debellé et al., 1988). NodC is also believed

to influence host specificity as it is involved in the determination of the length of

the Nod-factor backbone (Perret et al., 2000).

The Nod-factor core carries other chemical substituents. The genes

coding for these different types of chemical substituents are specific to the

various RNB species and are therefore partly responsible for the determination

of host specificity (hsn genes). For instance, R. leguminosarum bv. trifolii loses

Chapter 1

13

the ability to nodulate its original host Trifolium repens and gains the ability to

nodulate Medicago sativa when nodEFGHPQ of S. meliloti is transferred

(Debellé et al., 1988). The chemical substituents can be fatty acids (nodEF) or

they could result from 6-0 glycosylation (noeC, nodZ, nolK), sulfation (nodH,

noeE), acetylation (nodL, nodX, nolL), N methylation (nodS, nolO),

carbamoylation (nodU) or 2-0 methylation (noeI). Apart from these qualitative

issues, the amounts of Nod-factor produced by RNB are also known to play a

part in determining the host range (Perret et al., 2000).

Interestingly, R. etli and M. loti are known to produce identical Nod-

factors and yet these two species have very different host ranges (Cardenas et

al., 1995). Furthermore, both R. tropici and R. etli effectively nodulate P.

vulgaris (Poupot et al., 1993; 1995) but these two RNB produce two different

types of Nod factors. Therefore, there is no strict correlation between the types

of Nod-factor produced and the host range. Thus Nod-factors alone can not be

used to determine host specificity (Perret et al., 2000).

Molecular recognition between the plant and the microbe induce

developmental changes in both partners, as described in the next section.

1.2.4 Infection and nodule development

In root nodule development, infection and nodule organogenesis have

been shown to coincide (Hadri et al., 1998). In legumes where the infection

occurs via root hairs, the initial signal exchange between the plant and RNB

triggers a rapid developmental switch in the root hairs (Hadri et al., 1998).

Infection is initiated by the attachement of rhizobia onto the root hair which is

followed by the root hair deformation (Kijne et al., 1992). The root hair curls

Chapter 1

14

instead of growing straight and trap rhizobia in a pocket. The rhizobia then

grow into an intracellular ‘infection thread’ which is of plant origin (Turgeon &

Bauer, 1985; Kijne, 1992). Concomitant to infection, root cortical cells

differentiate to form nodule primordia from which the nodule develops (Hadri et

al., 1998). The infection thread containing the proliferating rhizobia grows

towards the nodule primordium situated in the inner cortex of the root

(Bakhuizen, 1988). Rhizobia are then released into the cytoplasm of the host

cells, surrounded by the peribacteroid membrane (Newcomb, 1981; Kijne,

1992). Here, the rhizobia may differentiate into bacteroids their endosymbiotic

form depending on the type of nodule. Bacteroids surrounded by the

peribacteroid membrane are the primary unit of N2 fixation, termed a

symbiosome (Kijne, 1992, Roth & Stacey 1989). The function within these

symbiosomes is described in the following section.

1.2.5 Molecular basis of nitrogen fixation

Symbiotic nitrogen fixation is the process in which root nodule bacteria

are able to reduce atmospheric nitrogen (N2) into ammonia. The biochemistry

and the molecular basis of this process have been studied extensively (Dilworth

& Glenn, 1991; Leigh, 2002). Nitrogenase is a key catalyst in N2 fixation, yet, the

description of the entire process remains incomplete.

Molybdenum nitrogenase (Mo nitrogenase) is the most common type of

nitrogenase found in RNB (Fisher & Newton, 2002). This enzyme is a complex

of two distinct metalloproteins, neither active without the other, termed the

MoFe protein (or dinitrogenase or component I) and the Fe protein (or

dinitrogen reductase or component II; Peters et al., 1995; Howard & Rees,

Chapter 1

15

1996). The MoFe protein is a α2β2 heterotetramer containing two different

metalloclusters - the P-cluster and the iron-molybdenum cofactor (FeMo-Co).

Each individual αβ-dimer containing FeMo-Co and a P-cluster is considered as

a functional unit of nitrogen fixation (Benton et al., 2002). The Fe protein is a

homodimer containing two MgATP-binding sites and a single [4Fe-4S] cluster

(Benton et al., 2002).

An anaerobic environment and adenosine triphosphate (MgATP) are two

requirements that must be met for nitrogenase to catalyze substrate reduction

(Bulen et al., 1965; Carnahan & Castle, 1963). Biological nitrogen fixation is a

high energy consumption reaction and can be described as below.

N2 + 8H+ + 8e- + 16MgATP 2NH3 + H2 16MgADP + 16Pi

Two interconnecting processers namely, the Fe-protein cycle and the MoFe-

protein cycle operate in the sequential delivery of electrons to MoFe protein and

then to the substrate for its reduction. A numerical model for the above process

was given by Lowe & Thorneley (1984).

The products of nif (nitrogen fixation) and fix genes are involved in the

structural development of nitrogenase and its regulation in rhizobia (Rubio &

Ludden, 2002). Most of the nitrogen fixation genes are located in operons but

the genes contributing to one operon vary between the different species of

rhizobia studied to date (Kaminski et al., 1998). All of the nif and fix genes

identified so far are known to occur in a single copy except for nifH which

occurs in multiple copies in some species. For example R. etli has three

identical copies of nifH and A. caulinodans has two copies of this gene with six

nucleotide differences between them (Kaminski et al., 1998).

Chapter 1

16

Ten nif genes are related to nitrogenase structure while two are

responsible for the regulation of N2 fixation. nifD and nifK are involved in the

structural development of component I of nitrogenase while nifH is responsible

for the development of component II (Rubio & Ludden, 2002). Furthermore, nifE

and nifN are connected to the biosynthesis of FeMo-Cofactor (Aguilar et al.,

1987) while nifB plays a role in its assembly (Paustain et al., 1989). Cystein

desulphurase activity which releases sulphur necessary for the metallocluster

formation is governed by nifS (Zheng et al., 1993). The function of nifW is not

yet very clear but it is believed to participate in the O2 protection of the FeMo

protein (Kim & Burgess, 1996). The nifA codes for a specific transcriptional

activator of the nif operons and the fixABCX operon (Hill et al., 1996) while nifX

plays a part in the negative regulation of N2 fixation genes (Gosink et al., 1990).

1.3 Symbiotic promiscuity – a cure and a curse for legume

productivity

As early as the late 19 century, it was known that RNB isolated from

some legumes were not restricted to their host of isolation and could nodulate

other legume spp. (Perret et al., 2000). Traditionally, legumes and rhizobia were

categorized into cross-inoculation groups (groups of plants within which the root

nodule organisms are mutually interchangeable; Allen & Allen, 1981). The

classifications based on cross-inoculation groups became less meaningful with

the expansion of molecular studies investigating the symbiotic specificity

between the plant and the microbe (Eardly et al., 1995; Martínez-Romero &

Chapter 1

17

Caballero-Mellado, 1996; Michiels et al., 1998; Pueppke & Broughton, 1999;

Perret et al., 2000).

The specificity between legume hosts and RNB can range from the

highly specific, i.e. where only a single species of RNB nodulate a given legume

host (e.g. Galega orientalis Lindström et al., 1983; Cicer arietinum Nour et al.,

1994a,b, 1995; Phaseolus vulgaris Martínez-Romero, 2003), to being very

promiscuous (e.g. Phaseolus vulgaris Michiels et al., 1998). It has been

demonstrated that both the legume host and RNB may play a role in highly

specific legume-rhizobia interaction (Sadowsky & Graham, 1998). For example,

the successful nodulation of Pisum sativum cv. Afghanistan can be achieved

only by R. leguminosarum bv. viceae strain TOM and a few other European R.

leguminosarum bv. viceae strains, and not by any other R. leguminosarum bv.

viceae strains (Lie, 1978). Subsequently it was shown that this conditioning for

restricted nodulation by European R. leguminosarum bv. viceae was governed

by a single recessive gene, sym-2, found in pea cultivar Afghanistan (Holl,

1975; Lie, 1984).

Symbiotic promiscuities can be described in two forms.

1. The promiscuity of RNB (broad host-range RNB): Promiscuous RNB

strains enter into symbiosis with a range of different host plants (Perret et

al., 2000). For example Rhizobium sp. strain NGR234 is very

promiscuous and can enter into symbiosis with legumes belonging to 112

genera representing the three sub families of Leguminosae (Pueppke &

Broughton, 1999).

2. The promiscuity of the host plant: A single legume may be nodulated by

a range of RNB belonging to different species (Bromfield & Barran, 1990;

Chapter 1

18

Laguerre et al., 1993; Eardly et al., 1995; Ezura et al., 2000). For

example Phaseolus vulgaris was considered as a non-selective host by

Michiels et al., (1998) as this plant can be nodulated by RNB belonging

to many different species distributed in at least three genera,

Bradyrhizobium, Rhizobium and Sinorhizobium (Bromfield & Barran,

1990; Amarger et al., 1997; Aguilar et al., 2001; Martínez-Romero,

2002).

The legumes that can form nodules with a broad range of RNB are

frequently referred as ‘promiscuous legumes’ (Allen & Allen, 1981; Trinick &

Hadobas 1989; Bromfield & Barran, 1990; Howieson & Ballard, 2004).

Developments in understanding the evolution of RNB through lateral transfer of

symbiotic genes (Young & Wexler, 1988; Souza et al., 1992; Sullivan et al.,

1995; Wernegreen et al., 1997) and the recent advances made in the

understanding of the molecular basis of symbiotic interactions (Perret et al.,

2000; Parniske & Downie, 2003), necessitate a clarification for what is meant by

a ‘broad range of RNB'. Does a broad range of RNB mean RNB with different

chromosomal backgrounds irrespective of the Nod-factors produced by these

strains? Or does it mean a collection of strains that produce different Nod-

factors, irrespective of their chromosomal background? Or both?

It is known that a complicated signal exchange between the plant and the

microbe initiates the nodulation process and the molecular signal produced by

the microbe (Nod-factor) physically associates with the legume roots in a lock

and key mechanism to initiate nodulation (Parniske & Downie, 2003). Many

different types of Nod-factors have been identified to date (Downie, 1994,

1998). Therefore, the definition of a truly promiscuous legume should be related

Chapter 1

19

to the amount of different Nod-factors it can interact with, rather than the

chromosomal diversity of RNB able to nodulate the legume.

Promiscuity in RNB has been considered as a valuable trait for elite

inoculants selected for commercial use (Howieson et al., 2000a). Broad host-

range RNB can be particularly beneficial when selecting an inoculant to

facilitate optimal N2 fixation, for several legume genera. Thus strain WSM1455

(R. leguminosarum bv viceae) is used commercially in Australia as an inoculant

for Pisum sativum and this strain is also highly effective on a wide range of

Vicia, Lathyrus and Lens spp. (Howieson, 1999; Howieson et al., 2000a).

However, ineffective nodulation by promiscuous RNB that are indigenous or

resident in agricultural soils can reduce the benefits of legume inoculation to

agriculture (Demezas & Bottomley, 1984; Barran & Bromfield 1997; Ballard &

Charman, 2000; Denton et al., 2002).

Promiscuous legumes species may face reduced productivity due to

nodulation by a range of ineffective or less effective RNB (Hungria & Vargas,

2000; Trinick & Hadobas, 1989). Contrast to this, the promiscuity of a legume

may be beneficial in legume breeding programs if the aim is to breed for

commercial legume species with the ability to form effective nodules with many

different soil rhizobia (Abaidoo et al., 2000; Sessitsch et al., 2002; Howieson &

Ballard, 2004). Symbiotic promiscuity (both legume and RNB) may thus be both

a cure and a curse in agriculture.

Chapter 1

20

1.4 Mobile genetic elements – a challenge to concepts of

rhizobial evolution

1.4.1 Mechanisms driving bacterial evolution

Prokaryotes are the most widely distributed organisms in the biosphere

and it has been estimated that the Eubacteria and Archaea together comprise

over one billion species (Dykhuizen, 1998) indicating a rapid evolution. They

have diversified and speciated to exploit a broad array of environments from

superheated hydrothermal vents to highly alkaline pools or even Antarctic ice

floes (Lawrence, 2001). The pertinent questions here are what enabled bacteria

to gain such a high frequency of diversification and what are the mechanisms

governing this phenomenon leading to rapid bacterial evolution?

Bacteria habitually reproduce by binary fission (they are haploid) and

their DNA is vertically transmitted from parent to progeny cells. If reproduction

was the only means for bacterial evolution, then it would be limited to creation of

new genes following accumulation of mutations over time (Brown et al., 2001).

However, this mechanism for evolution would be slow as it is only by chance

that a new gene with a practical function would evolve as a result of an

accumulation of mutations. Indeed, this may be the only method by which a

novel gene with a new biological function i.e. ability to oxidise a new substrate,

would arise (Ochman & Moran, 2001). Yet, this slow rate of evolution does not

reconcile well with the vast number of bacterial species found on earth. What

mechanism then is responsible for the development of the massive number of

Chapter 1

21

bacterial species? It is now considered that the phenomenon of lateral transfer

of DNA accounts for some of the diversity formed in bacteria (Bushman, 2002).

The discovery of lateral transfer of DNA among bacteria revolutionised

the concepts behind bacterial evolution and speciation (de la Cruz & Davies,

2000; Ochman et al., 2000; Dutta & Pan, 2002; Jain et al., 2002; Lawrence,

2002). DNA can transfer from one organism to another and be stably

incorporated into the genome of the recipient, changing its genetic composition

permanently (Bushman, 2002). This process is termed lateral transfer of DNA

and may also be referred to as horizontal transfer of DNA. Conjugation,

transformation and transduction are the mechanisms that mediate gene transfer

(Haker & Kaper, 2002).

Genes, or in most cases sets of genes (operons), can be gained or lost

rapidly between closely related (homologous recombination) or between

unrelated lineages conferring the recipient complex and novel abilities which

can subsequently allow them to exploit new ecological niches (Sullivan et al.,

1995; Preston et al., 1998; Ochman et al., 2000; Ochman & Moran, 2001).

1.4.2 Influence of mobile genetic elements on evolution of rhizobial

diversity

The phylogenetic incongruence observed between different loci (Young

& Wexler, 1988; Normand & Bousquet, 1989; Dolbert et al., 1994; Ueda et al.,

1995; Young & Haukka, 1996; Souza & Eguiarte, 1997; Haukka et al., 1998;

Zhang et al., 2000; Laguerre et al., 2001; Suominen et al., 2001; Moulin et al.,

2004), the mosaic composition of individual genomes and plasmids (Lawrence

et al., 1991; Sullivan et al., 2002; González et al., 2003) and the linkage

Chapter 1

22

equilibrium inferred from multilocus enzyme electrophoresis (Souza et al., 1992;

Maynard Smith et al., 1993), provide evidence for recombination within RNB

genera and species. The key elements involved in genomic plasticity of rhizobia

are transmissible plasmids and gene islands.

1.4.2.1 Plasmids

Plasmids are one of the most widely studied mobile genetic elements

(Bushman, 2002). They are extrachromosomal, circular DNA elements which

can replicate independently of the chromosome and are maintained at a

characteristic stable number from generation to generation (Lawrence, 1995).

Transmissible plasmids play an important role in bacterial evolution due to

several distinctive characteristics: They might be lost and gained in populations,

their copy number can be rapidly changed and they are believed to undergo

higher mutation rates due to the common occurrence of reiterated DNA (Modi &

Adams, 1991; Wernegreen, et al., 1997).

Antibiotic resistance, colicin production, as well as symbiotic nitrogen

fixation are some of the well known characteristics whose genes are carried on

plasmids (Bushman, 2002). In most examples the information carried on

plasmid DNA is not essential for the survival of the organism but can be useful

to exploit new ecological niches. However, it is relevant that the 1.6 Mb mega

plasmid pSymb of S. meliloti codes for arginine-tRNA which is essential for

normal growth (Weidner et al., 2002).

Numerous plasmids with varying functions are found in many genera and

species of RNB (Farrand, 1998; Mercado-Blanco & Toro, 1996; Brom et al.,

2002). Some RNB species carry most of the genes essential for nodulation

Chapter 1

23

(including host specificity genes) and N2 fixation on a plasmid called the sym-

plasmid (Johnston et al., 1978; Brewin et al.,1980; Hooykaas et al., 1981;

Kondorosi et al., 1982; Lamb et al., 1982; Young & Wexler, 1988; Wang et al.,

1999c). Therefore, the presence or absence of this plasmid may influence the

ecological niche (i.e. the nodule on a legume root) a strain may exploit. The

rhizobial sym-plasmid can be one transmissible plasmid of particular ecological

significance (Wernegreen, et al., 1997).

Interestingly, non-symbiotic plasmids, commonly referred to as cryptic

plasmids, also play a role in influencing the legume-rhizobium interaction and

N2 fixation at some level (Hynes & McGregor, 1990). Nodulation ability and

competitiveness have been shown to be related to the presence of cryptic

plasmids in M. loti (Pankhurst et al., 1986), R. tropici (Pardo et al., 1994) and S.

meliloti (Bromfield et al., 1985; Toro & Olivares, 1986; Sanjuán & Olivares,

1989). It has also been demonstrated, albeit more infrequently, that

effectiveness of N2 fixation can be related to the presence of cryptic plasmids

(Thurman, et al., 1985; Pankhurst et al., 1986; Barbour & Elkan, 1989; Hynes &

McGregor, 1990; Baldani et al., 1992; Brom et al., 1992; Kuykendall et al.,

1994; Velázquez et al., 1995). Cryptic plasmids are very stable and may be

abundant in cells (Weaver et al., 1990; Mercado-Blanco & Olivares, 1993). The

transfer of cryptic plasmids between RNB strains in the rhizosphere has been

reported (Broughton et al., 1987; Schofield et al., 1987; Rao et al., 1994) and

thus plays an important role in rhizobial diversity and diversification.

Chapter 1

24

1.4.2.2 Genomic islands

Plasmid like DNA regions that are integrated into the chromosome have

been termed genomic islands (Kaper & Hacker, 1999). Genomic islands can

confer a variety of functions on the host genome and like plasmids, can extend

their capacity to adapt into new environments. These attributes include

resistance, degradation, metabolism, pathogenicity, secretion and symbiosis

(Kaper & Hacker, 1999). There are several types of genomic islands that have

been recognised to date. Genomic islands that confer an advantage to the

respective bacteria for survival in an ecological niche are named ‘fitness islands’

(Kaper & Hacker, 1999). However, if that niche is a given host (human, animal

or plant) and the result is an infection that is detrimental to the host and whose

functions can be linked to the island, such islands are named ‘pathogenicity

islands’ (PAIs) (Kaper & Hacker, 1999).

Sullivan & Ronson (1998) described a genomic island present in

Mesorhizobium loti (strain R7A) that can confer nitrogen fixation ability to

nonsymbiotic bacteria. This they termed a ‘symbiosis island’ and it exhibits

similarities to PAIs such as the symbiosis island integrates into a tRNA gene

and carry genes coding for mobility and factors such as integrases,

transposases (Sullivan et al., 2002) similar to PAIs (Kaper & Hacker, 1999).

Further details on the characteristics of PAIs and other gene islands were given

by Kaper & Hacker, (1999). All the genes required for Nod-factor synthesis,

nitrogen fixation in rhizobial symbiosis and island transfer are known to be

carried on the symbiosis island (Sullivan et al., 2002). Kaneko et al., (2000)

identified a symbiosis island in MAFF303099, a strain they considered to be M.

loti that has since been re-classified as M. huakuii (Tumer et al., 2002).

Chapter 1

25

MAFF303099 is considered as M. loti in this thesis. A comprehensive

comparison of these two symbiosis islands found in the M. loti strains is given

by Sullivan et al., (2002). The presence of a symbiosis island in B. japonicum

strain USDA110 has also been revealed through sequence comparison

(Kaneko et al., 2002).

The two symbiosis islands of M. loti integrate into a phenylalanine tRNA

gene on the chromosome in a process mediated by a P4-type integrase

(Sullivan et al., 2002). Transfer genes of the symbiosis islands include a trb

operon and a cluster of potential tra genes, but they lack plasmid replication

genes suggesting that these islands are site-specific conjugative transposons

(Sullivan et al., 2002).

1.5 Rhizobial diversity and competition – a threat to agriculture

1.5.1 Rhizobial diversity and dynamics

The strains of RNB inhabiting a particular soil may be diverse in both

symbiotic as well as other phenotypic and genetic characters (Pinto et al.,

1974). The variation in the DNA sequences between strain types in a rhizobial

population is called genetic diversity (McInnes, 2002).

Since the early 20th century, researchers have been aware of the

presence of indigenous RNB strains in agricultural soils that limit legume

nodulation by inoculant strains (Baldwin & Fred, 1929; Dunham & Baldwin,

1931). The density of indigenous RNB populations able to nodulate a particular

legume species can vary from <10 – 107 g-1 soil (Bottomley, 1992; Vincent,

1974). Cropping history may impact on the size of this indigenous RNB

Chapter 1

26

population (Triplett & Sadowsky, 1992; Brockwell & Bottomley, 1995). Wang et

al., (1999a) observed that Mesorhizobium strains nodulating Leucaena are no

longer observed after cropping Phaseolus vulgaris (bean). The number of RNB

species able to nodulate a certain host appears greater in the presence of the

host (Weaver et al., 1972; Kuykendall et al., 1982; Woomer et al., 1988).

However, diversity of RNB in many agricultural soils may be restricted to intra-

specific diversity due to the monoculture of a legume species over a long period

of time (Howieson & Ballard, 2004). Contrary to this, soils of undisturbed natural

environments may contain a wide range of legume species that host a diversity

of RNB species. Odee et al., (2002) isolated RNB belonging to four genera;

(Rhizobium, Sinorhizobium, Mesorhizobium and Bradyrhizobium), from a site in

Kibwezi savanna, Kenya, where there was no history of domesticated legumes.

Rhizobial diversity as measured in a particular soil may be influenced by

the method used to isolate RNB. Diversity measured by trap host only

resembles the diversity of RNB able to nodulate particular trap hosts and not

the diversity of RNB residing in that soil. Methods have been developed to

isolate RNB directly from the soil (Gault & Schwinghamer, 1993; Kinkle et al.,

1994; Tong & Sadowsky, 1994; Bromfield et al., 1995; Soberon-Chavez &

Najera 1988). The genetic diversity is also greatly influenced by the method

used to discriminate between strains. The discriminatory power of individual

strain typing methods varies and this can give rise to different diversity

assessments for the same field site tested (Schwinghamer & Dudman, 1980;

Barnet, 1991; Bottomley, 1992). At present there is a substantial array of

techniques used for detecting and describing rhizobial diversity and they are

discussed in the next section.

Chapter 1

27

1.5.2 Methods used to investigate rhizobial diversity

Prior to the molecular era, rhizobial diversity studies were mainly based

on phenotypic characters such as host range, comparative growth in culture,

serological relatedness, bacteriocin production, intrinsic antibiotic resistance

and bacteriophage resistance (Schwinghamer & Dudman, 1980). Later, other

methods including substrate utilization, protein profiling, Multilocus enzyme

electrophoresis (MLEE) and FAME became prominent in rhizobial diversity

studies (Graham et al., 1995; van Rossum et al., 1995). Although these

phenotypic methods provided a valuable insight into rhizobial population

structure and strain diversity, they had some limitations, particularly low

discriminatory power compared to molecular methods (Jenkins & Bottomley

1985, Mullen & Wollum 1989; Barnet, 1991; Bottomley, 1992). There was also

often a poor correlation between strain groupings (Kleczkowski & Thornton,

1944; Roughley et al., 1992; van Rossum et al., 1995) which may be due to the

instability of strain characters over time (Lindström et al., 1990).

At present there are a large number of genotypic methods used for

rhizobial diversity studies and the most common methods comprise:

a) Plasmid profiling (Broughton et al., 1987; Young & Wexler, 1988;

Laguerre et al., 1992; Louvrier et al., 1996; Wernegreen et al., 1997)

b) Restriction Fragment Length Polymorphism (RFLP) (Schofield et al.,

1987; Young & Wexler, 1988; Laguerre et al., 1993; Bromfield et al.,

1995; Kishinevsky et al., 1996; Lafay & Burdon, 1998; Vinuesa et al.,

1998; Saleena et al., 2001; Odee et al.. 2002)

Chapter 1

28

c) Polymerase Chain Reaction based techniques (PCR) (de Bruijn,

1992; Richardson et al., 1995; Louvrier et al., 1996; Laguerre et al.,

1997; Gao et al., 2001)

Genotypic methods generally have high discriminatory power and the

majority of these methods are rapid compared to most phenotypic methods

(Handley et al., 1998). However, it is important to note some of their limitations.

Reproducibility of some genotypic methods, especially RAPD PCR and other

related PCR based techniques (BOX PCR, ERIC PCR, Rep PCR and RPO1

PCR) are reported to be low, and known to be highly dependent on the DNA

extraction protocol, colony age, source of reagents, concentration and purity,

and thermal cycling conditions (Welsh & McClelland, 1990; Coutinho et al.,

1993; Hengen, 1994; Kay et al., 1994; Richardson et al., 1995; Laguerre et al.,

1996; Schneider & de Bruijn, 1996; Sato et al., 1999; Vachot et al., 1999).

These disadvantages can be overcome by rigorously standardising the protocol,

using many repeats, replicates and including appropriate controls (Farber,

1996).

Many studies have assessed the diversity of RNB strains nodulating a

particular legume species or the diversity of RNB that exist in a particular soil

(Brunell et al., 1998; Kishinevsky et al., 2002; Lafay, 1998; Laguerre et al.,

1994,1996, 1997, 1998; Wang et al., 1999c; Young & Cheng, 1998; Zhang et

al., 2001a). The significance and the economic importance of rhizobial diversity

are discussed in the following section.

Chapter 1

29

1.5.3 Rhizobial competition and its significance

Agricultural soils often contain established populations of RNB and many

common, cultivated legume species achieve nodulation without inoculation

(Thies et al., 1991a; Mpepereki et al., 1996, 2000; Wang et al., 1999c; Ballard &

Charman, 2000; Sessitsch et al., 2002). This may be due the worldwide

distribution of rhizobia of certain plant species and their establishment over a

long period of time (Brockwell & Bottomley, 1995; Ballard & Charman, 2000).

Although nodulated, these legumes may fix nitrogen poorly (Keyser & Li, 1992;

Ballard & Charman, 2000; Denton et al., 2002). Soybean (Glycine max) has

been domesticated in China since the 11 century B.C. and is readily nodulated

without inoculation (Keyser & Li, 1992). However, in USA nitrogen fixation in

soybean by natural RNB is often poor (Zdor & Pueppke, 1988). As a

consequence, it is a common agricultural practice to inoculate legumes with

superior inoculant strains (qualities of a superior inoculant are given by

Brockwell et al., 1982) to promote nitrogen fixation and increase crop yield

(Thies et al., 1991b; Howieson & Ballard, 2004). Legume inoculation is

particularly important when introducing a legume species to a new region

(Brockwell & Bottomley, 1995).

A positive inoculation response with high nodule occupancy of the

legume by the inoculant strain has been reported where the legume has been

grown for the first time in soils deficient in compatible indigenous RNB (Bell &

Nutman, 1971; Roughley et al., 1976; Bromfield & Ayanaba, 1980; Brockwell et

al., 1987; Somasegaran et al., 1988; Slattery & Coventry, 1993). However, in

many agricultural soils, well established indigenous RNB populations present an

aggressive competition for nodulation, even in the year of inoculation (Jonson et

Chapter 1

30

al., 1965; Holland, 1970; Boonkerd et al., 1978; Noel & Brill, 1980; Bromfield et

al., 1986; Bohlool et al., 1992). Often, the inoculant may dominate the first

growing season (Vlassak & Vanderleyden, 1997) but there are many reports

showing the progressive displacement of the inoculant by indigenous RNB in

the subsequent years (Parker et al., 1977; Brockwell et al., 1982; Dowling &

Broughton, 1986; Streeter, 1994; Hebb et al., 1998). Thus the indigenous (or

naturalized) RNB present a competition barrier to the successful establishment

of an inoculant. Occupation of nodules by indigenous RNB to the exclusion of

the inoculant has been reported, even when the levels of inoculant far exceed

the level of indigenous RNB (Weaver & Frederick, 1974a,b). Elimination of the

inoculation response has been shown in the presence of as few as 50

indigenous RNB per g of soil (Thies et al., 1991b). Indigenous RNB are well

adapted to their niche (Triplett & Sadowsky, 1992) but often have inferior

nitrogen fixation capacity (Ballard & Charman, 2000; Denton et al., 2002). The

inability of the superior inoculant to nodulate and enhance legume productivity

due to competition by indigenous soil RNB populations is referred to as the

Rhizobium competition problem (Triplett & Sadowsky, 1992).

Clearly, competition through ineffective nodulation reduces the benefits

of nitrogen fixation to agriculture (Holland, 1970; Sessitsch et al., 2002). This

has been a significant problem in many parts of the world including large parts

of southern Australia (Ballard & Charman, 2000; Denton et al., 2002). For

example, the ineffectiveness of the natural RNB populations on subterranean

(Trifolium subterraneum) and crimson clover (Trifolium incarnatum L.) in

Richmond River district of New South Wales, Australia, demands the successful

establishment of effective inoculant strains (Pinto et al., 1974). Antagonistic

Chapter 1

31

effects on the growth of Trifolium spp. have been reported when the plant was

nodulated by more than one strain of Rhizobium leguminosarum bv. trifolii

(Ames-Gottfred & Christie, 1989). Similarly Demezas & Bottomley (1984) noted

suboptimal growth of Trifolium spp. even when 50% of the nodules were

occupied by superior inoculant strains. Furthermore, blocking of nodulation is

also known to exist (Martínez-Romero et al., 1998).

Alarmingly, the few rhizobial species that have proliferated in southern

Australia have been shown to now face a competitive rhizobial environment for

the nodulation of their host legume (McInnes, 2000). One must then ask the

question that if the rhizobial introduction to southern Australia has been

managed well (by releasing elite genotypes of a few species) how has

competition for nodulation arisen and what are the mechanisms for this

phenomenon? Many studies provide anecdotal evidence of horizontal transfer

of DNA containing symbiotic genes in field populations isolated from cultivated

hosts (Young & Wexler, 1988; Laguerre et al., 1992; Louvrier et al., 1996) and

recently from rhizobial inoculants to native or naturalised bacterial (non-

rhizobial) populations (Sullivan et al., 1995). Where the recipient organism

acquires the ability to nodulate, an ineffective symbiosis may arise. The

recipient organism may already have the benefit of excellent adaptation to the

soil niche and hence become more competitive than the introduced inoculant.

The development of biodiversity in southern Australia amongst those few

rhizobial species for introduced legumes is an intriguing field of study for

contemporary rhizobiology (Howieson & Ballard, 2004). With the aid of

molecular typing methodologies (Thies et al., 2001) there is little doubt that the

range of strains found nodulating legumes of Mediterranean origin in southern

Chapter 1

32

Australia is far greater than the number of strains ever released as inoculants.

How did this intra-specific biodiversity of RNB arise after the introduction of

exotic legumes? Answering this question is the essence of this thesis and is

described below.

1.6 Study at hand

1.6.1 Background

An opportunity to observe the development of rhizobial biodiversity after

the introduction of an exotic legume and its RNB arose with the introduction of

the pasture legume B. pelecinus from the Mediterranean basin to Western

Australia (WA) and its commercial adoption in 1995 (Fig 1.2). B. pelecinus is a

monospecific genus nodulated by a particular Mesorhizobium sp (Nandasena et

al., 2001) and is a new legume to agriculture (Howieson et al. 1995).

Preliminary studies indicated that indigenous rhizobial populations in WA soils

were incapable of nodulating B. pelecinus (Howieson et al. 1995). This species

is having a substantial impact on agricultural productivity in the acidic and sandy

soils of New South Wales and Western Australia where its deep-rooted nature

is providing a valuable tool in reducing the development of dry-land salinity (Loi

et al., 1999). To maximise the value of B. pelecinus in farming systems, it is

imperative that the nitrogen-fixing symbiosis between this new species and its

rhizobia is maintained at the highest level of efficiency.

As part of the agronomic investigation of B. pelecinus, a single rhizobial

strain (WSM1271) previously isolated from root nodules of B. pelecinus growing

in Sardinia was introduced to a field in Northam, Western Australia as inoculant

Chapter 1

33

for surface sterilized seeds of B. pelecinus (Howieson et al. 1995). This

provided a unique opportunity to study the development of rhizobial diversity in

situ as there had been no substantial study of rhizobial populations able to

nodulate this species.

Fig 1.2. Biserrula pelecinus L. with purple flower

1.6.2 Aims of this thesis

• To investigate whether there is a diversity of strains nodulating the exotic

legume B. pelecinus six years after its introduction (to a field site in

regional WA), when inoculated with a single strain of Mesorhizobium sp.

strain WSM1271

• If genetic diversity is present, to investigate whether the diverse RNB fix

N2 as effectively as Mesorhizobium sp. strain WSM1271 on B. pelecinus

• To investigate how this diversity of strains capable of nodulating

B. pelecinus arose

Chapter 1

34

Chapter 2

35

2. Genetic diversity among RNB isolated from

Biserrula pelecinus L. six years after introduction

and inoculation with WSM1271

2.1 Introduction

The diversity of RNB in agricultural soils can vary greatly and may

depend on factors such as cropping history (Dughri & Bottomley, 1984; Cregan

& Keyser, 1988; Thurman & Bromfield, 1988; Wang et al., 1999c; Abaidoo et

al., 2000), soil type (Ham et al., 1971), soil acidity (Dughri & Bottomley, 1983),

salinity (Singleton & Bohlool, 1983) and application of lime and phosphate

(Dughri & Bottomley, 1983; Almendras & Bottomley, 1987). Agricultural soils

may host a diversity of RNB able to nodulate well established, cosmopolitan

agricultural legume species and therefore inoculation is not always necessary

(Diatloff & Langford, 1975; Wang et al., 1999c; Sessitsch et al., 2002;

Mpepereki et al., 2000).

However, the scenario for newly introduced legume species is different to

that of the cosmopolitan agricultural legumes. There is a degree of specificity in

all legume-rhizobium interactions (Section 1.2.3) and the resident soil

populations of RNB may not be able to nodulate an exotic legume (Parker,

1962; Diatloff & Brockwell, 1976). Therefore, there is often an imperative need

to inoculate when introducing a legume to a new region (Brockwell & Bottomley,

1995).

Chapter 2

36

Two factors contributing to nodulation failure and which limit rhizobial

growth and survival in soil are high temperature and moisture deficiency

(Hungria & Vargas, 2000). Therefore, having a diversity of rhizobia capable of

nodulating a legume species may be beneficial for the successful establishment

and survival of the legume in adverse agricultural soils, by providing an array of

strains from which to select for stress tolerance (Baraibar et al., 1999; Hungria

& Vargas, 2000). Yet, rhizobial diversity may be beneficial only if the strains are

highly effective on the legume host.

Unfortunately, many agricultural soils contain RNB with poor N2 fixation

capacity and this leads to declining legume productivity (Ballard and Charman,

2000; Denton et al., 2002; Sessitsch et al., 2002). Clearly, competition for

nodulation by these poorly effective rhizobia reduces the benefits of N2 fixation

to agriculture (Sessitsch et al., 2002). Understanding the genetic diversity of

RNB able to nodulate an introduced legume in a particular soil may therefore

provide information useful for the successful establishment and enhancement of

productivity of the legume host (Brockwell & Bottomley, 1995; Howieson, 1999;

Sessitsch et al., 2002).

Both genotypic and phenotypic methods are employed to determine

rhizobial diversity as discussed previously (Section 1.5.2.). Genotypic methods

can be more beneficial in diversity studies as they have a higher discriminatory

power, i.e. they can distinguish between two closely related strains, (Farber,

1996) unlike the traditional phenotypic methods (Mazurek, 1993; Swaminathan

& Matar, 1993; Tompkins, 1992; Versalovic et al., 1993). Furthermore, some

molecular fingerprinting methods such as ERIC PCR, RAPD PCR, REP PCR

Chapter 2

37

and RPO1 PCR are not labor intensive and results may be obtained rapidly

(Farber, 1996).

This chapter reports the search for genetic diversity of root nodule

occupants of B. pelecinus six years after introduction to a field site in Australia

and inoculation with the inoculant strain WSM1271.

Aims

This chapter has 2 aims.

1. To investigate whether there is genetic diversity among the nodule

occupants of B. pelecinus six years after introduction to an agricultural

soil devoid of naturalized RNB capable of nodulating the plant and initial

inoculation with a single inoculant strain (Mesorhizobium sp. strain

WSM1271).

2. If diversity exists, to investigate the phylogenetic relationships among the

diverse strains.

2.2 Materials and methods

2.2.1 Field site and collection of nodules from B. pelecinus

The field site was located at Northam (S 31° 30”, E 116° 50”), Western

Australia on a private farm at an altitude of 160 m. This region has a typical

Mediterranean environment with an annual mean maximum temperature of

25°C, annual mean minimum temperature of 11°C and an annual rainfall of 430

mm. The duplex soil consisted of 25-40 cm of brown sandy loam overlying a

clay subsoil.

Chapter 2

38

The experimental site was established in 1994 as an agronomic

investigation of B. pelecinus in which surface sterilized seeds were inoculated

with a peat culture of Mesorhizobium sp. strain WSM1271 prior to sowing

(Howieson et al., 1995). The site had pasture phases, consisting of annual

herbs and grasses in 1995, 1996, 1999 and 2000, and was carrying a wheat

crop (Triticum aestivum) during 1997 and 1998. The original plot was re-located

in August, 2000 by observing the distribution of B. pelecinus. Four plants of B.

pelecinus were collected every 2m from the centre of the plot for a distance of

10m going in each of the four main directions (North, East, West, South) within

the original plot (70 X 30 m2; Fig 2.1). A 150 m diameter area from the centre of

the plot was thoroughly searched for B. pelecinus. Two plants were found and

collected. Five nodules were picked per plant.

2.2.2 Isolation of root nodule bacteria

The nodules were surface sterilised by washing for 30 s in 70% (v/v)

ethanol followed by 1 min in 3% (v/v) sodium hypochlorite and finally six washes

with sterile DDi H2O. Following sterilisation the nodules were crushed and the

nodule extract was streaked onto ½LA medium (Howieson et al., 1988) under

aseptic conditions. Streaking was performed in a diluting manner by flaming the

loop after streaking in each direction (method 2; Somasegaran & Hoben 1994).

Plates were incubated at 28°C for 4-6 days. Single colonies with typical

morphology of RNB (Jordan, 1984) were picked from these plates to subculture

by re-streaking onto ½LA in the same manner as described above. All plates

with any fungal contaminants were discarded.

Chapter 2

39

Figure 2.1 An abstract map of the experimental plot at Northam showing the points of isolation of the 88 strains. Distance between two bars is 2m. Isolates N1 and N2 were collected outside of the plot area (140m south). Novel isolates that are genetically different to WSM1271, * re-isolates of WSM1271. Isolates from the same plant are given in same colour.

South

North

EastWest

R3,R4,*R5,R6,R7,R8,R9,R10,R11

R12,R13,R14, N15

N17, N18, N87,*R19,R20,R21,

R30,R31,R32,R33,R34,R35,*R36

R37,R38, N39,R40,R41,R42

N43,N44, NN45, N46,R47

R56

R62R61R60*R59R58R57

R16

R22,R23,R24,R25,R26,R27,R28,R29

*R48,R49,R50,R51,R52,R53,R54,R55

R66R65*R64R63

R74R73R72R71R70R69R68R67

R77R76R75

R78R79R80R81R82

R83*R84

R85R86R88

South

North

EastWest

R3,R4,*R5,R6,R7,R8,R9,R10,R11

R12,R13,R14, N15

N17, N18, N87,*R19,R20,R21,

R30,R31,R32,R33,R34,R35,*R36

R37,R38, N39,R40,R41,R42

N43,N44, NN45, N46,R47

R56

R62R61R60*R59R58R57

R16

R22,R23,R24,R25,R26,R27,R28,R29

*R48,R49,R50,R51,R52,R53,R54,R55

R66R65*R64R63

R74R73R72R71R70R69R68R67

R77R76R75

R78R79R80R81R82

R83*R84

R85R86R88

Chapter 2

40

2.2.3 Molecular fingerprinting with primer RPO1

The 88 isolates obtained (Table 2.1) were fingerprinted with the primer

RPO1 designed by Richardson et al., (1995). Cells from a culture grown on

½LA (not more than 10 days old), were concentrated in 0.89% (w/v) saline to an

OD600 of 6.0. Each reaction mixture for PCR contained 1 μL of concentrated

cells and 2.5U of Amplitaq® DNA polymerase (Perkin Elmer EC 2.7.7.7), 50 μM

of the primer, 1.5 mM MgCl2, 5X PCR Polymerisation buffer [67 mM Tris-HCL

(pH 8.8 at 25°C), 16 mM [NH4]2SO4, 0.45% Triton X-100, 0.2 mg/ml Gelatin, 0.2

mM dNTPs - Biotech International Ltd. Cat # PB-1], in a final volume of 20

μL.

The reaction mixture was held at 94°C for 5 min followed by 5 cycles at

94°C for 30 s, 50°C for 20 s, 72°C for 90 s and then 35 cycles at 94°C for 30 s,

50°C for 20 s and 72°C for 90 s and a final extension at 72°C for 5 min.

The amplified DNA fragments were analysed by agarose gel

electrophoresis. A Bio-Rad Sub-Cell GT Agarose gel electrophoresis system

was used. A 2% (w/v) agarose gel prepared in TBE (90 mM Tris-base, 90 mM

Boric acid, 2.5 mM EDTA, pH 8.3) was poured to a thickness of 8 mm. Prior to

electrophoresis, a gel-loading buffer (0.25% w/v bromophenol blue; 0.25% w/v

xylene cyanol FF; 40% w/v sucrose) was added to the DNA samples. The gel

tanks were buffered with TBE (0.04 M Tris-acetate; 0.001 M EDTA; pH 8.0).

The marker used was 1 kb Ladder (Cat. No. G5711, Promega). Electrophoresis

was carried out at 80 V for 3 h.

Chapter 2

41

After electrophoresis, the gel was stained in EtBr (0.5 μg/ml) for 30-60

min and destained in DDi H2O for 10-20 min. DNA bands were visualised under

UV light using the Geldocumentation system (BIORAD Gel Doc 2000)

At least three repeats were completed for each isolate to overcome the

difficulties in reproducibility faced with molecular fingerprinting PCR methods

(Farber, 1996; Perret & Broughton, 1998).

2.2.4 Molecular fingerprinting with primer ERIC

The 88 isolates were fingerprinted by a second primer pair, EricF and

EricR designed by de Bruijn (1992). The procedure was similar to the method

described for RPO1 (Section 2.2.3) except for the following changes. The initial

5 cycles with a lower annealing temperature were omitted. The annealing was

at 52°C for 1 min and extension at 65°C for 5 min, for 35 cycles. At least three

repeats were completed for each isolate.

2.2.5 Authentication of isolates

Isolates that were considered genetically different (see Section 2.3.2. for

definition) to WSM1271, and seven isolates that gave a similar PCR banding

pattern to WSM1271 were authenticated by observing their ability to nodulate B.

pelecinus as described in Section 3.2.5.

Chapter 2

42

Table 2.1 Origins of the isolates. All isolates were collected from separate nodules on roots of Biserrula pelecinus (S- south, N- north, E- east, W- west) Distance from the centre (m)

Plant collected

Isolate

2S P1 N3 2S P1 N4 2S P1 N5 2S P1 N6 2S P2 N7 2S P2 N8 2S P2 N9 2S P2 N10 2S P2 N11 2S P3 N12 2S P3 N13 2S P3 N14 2S P3 N15 4S P1 N16 8S P1 N17 8S P1 N18 8S P1 N87 8S P2 N19 8S P2 N20 8S P2 N21

10S P1 N22 10S P1 N23 10S P1 N24 10S P1 N25 10S P2 N26 10S P3 N27 10S P3 N28 10S P3 N29 2N P1 N30 2N P1 N31 2N P2 N32 2N P2 N33 2N P2 N34 2N P2 N35 2N P2 N36 2N P3 N37 2N P3 N38 2N P3 N39 2N P4 N40 2N P4 N41 2N P4 N42 4N P1 N43 4N P2 N44 4N P3 N45

Distance from the centre (m)

Plant collected

Isolate

4N P4 N46 4N P4 N47 8N P1 N48 8N P1 N49 8N P1 N50 8N P2 N51 8N P2 N52 8N P2 N53 8N P2 N54 8N P2 N55

10N P1 N56 2E P1 N57 2E P1 N58 2E P2 N59 2E P2 N60 2E P3 N61 2E P4 N62 4E P1 N63 4E P1 N64 4E P2 N65 4E P2 N66 6E P1 N67 6E P1 N68 6E P1 N69 6E P1 N70 6E P2 N71 6E P2 N72 6E P2 N73 6E P2 N74 8E P1 N75 8E P2 N76 8E P3 N77 2W P1 N78 2W P2 N79 2W P2 N80 2W P2 N81 2W P2 N82 6W P1 N83 6W P2 N84 8W P1 N85 8W P2 N86 8W P3 N88

140S P1 N1 140S P2 N2

Chapter 2

43

2.2.6 Sequencing the 16S rRNA gene

PCR conditions: An internal fragment of 1400 bp (internal fragment) of the

16S rRNA gene was amplified and sequenced for WSM1271 and four isolates

randomly picked out of seven that gave distinctive molecular fingerprinting PCR

banding patterns (N17, N18, N45, N87). The reaction mixture was the same as

described for molecular fingerprinting except primers 20F and 1540R (Yanagi &

Yamasato, 1993) were used in a final volume of 100 μl. The reaction mixture

was held at 94°C for 5 min followed by 30 cycles at 94°C for 30 s, 55°C for 30 s,

72°C for 30 s and a final extension at 72°C for 7 min.

Purification of PCR product: PCR products were purified using the

BRESASPINTM PCR Purification kit (BT-2000-100) according to the

manufacture’s instructions with 70 μl of amplified product being eluted into 50 μl

of elution buffer.

Sequencing: All DNA sequencing was carried out as described by the

manufacturer (Applied Biosystems) using ABI PRISMTM dye terminator cycle

sequencing ready reaction kit and automated sequencer (ABI Model 377A).

Four forward primers (20F, 420F, 800F and 1100F) and four reverse primers

(1540R, 1190R, 820R and 520R), as designed by Yanagi & Yamasato (1993),

were used. Half reactions were done and the extension products were purified

using an ethanol precipitation protocol provided by Perkin Elmer (Protocol P/N

402078; http://www.perkinelmer.com).

Chapter 2

44

Sequence analysis: Analytical software and databases were accessed

through Bionavigator (http//:www.bionavigator.com). Initially, a BLAST (Altschul

et al., 1990) search service provided by the National Centre for Biotechnological

Information (NCBI) was carried out to find the close relationships through

sequence similarity. Next, the 16S rRNA sequences of the RNB species within

the α-Proteobacteria were retrieved from the GenBank using the ENTREZ

facility provided by NCBI. The sequences were aligned using the ClustalW

program in the Wisconsin package of the Genetics Computer Group (Madison,

WI, USA). The DNAdist programme was used to produce a Kimura 2 parameter

(Kimura, 1980) distance matrix for the aligned sequences. This algorithm was

selected as it gives different values to transitions and transversions and

therefore does not underestimate the true distance between distantly related

species. NEIGHBOUR was used to construct a phylogenetic tree through the

neighbour-joining method (Saitou & Nei, 1987) using the distance data.

2.3 Results

2.3.1 Isolation of RNB

Although 400 isolates were possible from the 400 nodules collected from

B. pelecinus growing in August, 2000 at the field site in Northam, Western

Australia, many cultures had fungal contaminants. Therefore, only the 88 pure

cultures obtained from 88 different nodules were selected for further study.

These 88 isolates (Table 2.1) produced 2-4 mm diameter, circular, convex,

semitranslucent, mucilaginous, white colonies within 4-6 days on ½LA medium

incubated at 28 °C.

Chapter 2

45

2.3.2 Genetic diversity indicated through molecular fingerprinting

All the repeats of the molecular fingerprints of the isolates were

considered for each isolate and it was quite common to observe two or three

PCR bands missing or two-three additional bands between repeats of the same

strain. Molecular fingerprinting PCR methods are known to have some

difficulties in their reproducibility (Farber, 1996; Perret & Broughton, 1998).

Therefore, the overall visual banding pattern was considered for each isolate

and in this study, isolates having similar banding patterns to the pattern of

WSM1271 were considered as genetically similar isolates to WSM1271. All

other isolates were considered as genetically diverse isolates.

Fingerprinting PCR with the primer RPO1 resulted in 81 isolates

displaying a similar banding pattern to that of the inoculant strain, WSM1271

(Appendix 1). Seven isolates (N15, N17, N18, N39, N45, N46 and N87)

produced distinctive banding patterns with this primer (Fig. 2.2).

Similarly, fingerprinting PCR with the ERIC primers resulted in the same

81 isolates displaying a similar banding pattern to WSM1271 (Appendix 1). The

same seven isolates that had distinct PCR banding patterns with RPO1 also

gave distinct PCR banding patterns to that of WSM1271 with the ERIC primers

(Fig. 2.3). However, of these seven, two (N17 and N18) showed a similar

pattern to each other using the ERIC primers. The seven isolates with distinct

PCR banding patterns with both the primers will be referred to as ‘novel

isolates’ (N15, N17, N18, N39, N45, N46 and N87) hereafter. Novel isolates

will be in bold characters throughout this thesis.

Chapter 2

46

Figure 2.2 Agarose gel showing the fingerprinting PCR banding pattern of isolates with primer RPO1.

Figure 2.3 Agarose gel showing the fingerprinting PCR banding pattern of isolates with primer ERIC.

Mar

ker

WS

M12

71

N5

N19

N

36

N48

N

59

N64

N

84

WS

M12

71

N15

N

17

N18

N

39

N45

N

46

N87

N

egat

ive

cont

rol

Mar

ker

WS

M12

71

N5

N19

N

36

N48

N

59

N64

N

84

WS

M12

71

N15

N

17

N18

N

39

N45

N

46

N87

N

egat

ive

cont

rol

Re-isolates Novel isolates

Re-isolates Novel isolates

Chapter 2

47

Out of the 81 isolates with similar PCR banding patterns to WSM1271,

seven (N5, N19, N36, N48, N59, N64 and N84) were randomly selected for

further studies undertaken in chapter 3, and they will be referred to as ‘re-

isolates’ hereafter.

The seven novel isolates, the seven re-isolates and strain WSM1271

nodulated B. pelecinus cv. Casbah in the authentication experiment.

2.3.3 Genetic diversity distinguished through 16S rRNA gene based

phylogeny

An internal fragment of 1440 bp of the 16S rRNA gene was amplified and

sequenced for the original inoculant strain WSM1271 and for four of the novel

isolates (N17, N18, N45 & N87). These sequences were submitted to GenBank

and their accession numbers are AY601513 (WSM1271), AY601514 (N18),

AY601515 (N45), AY601516 (N17), AY601517 (N87). The sequences of these

four novel isolates and WSM1271 were aligned using the software Gene Tool-

Lite, Double Twist, Inc (Fig. 2.4). Two of the novel isolates, N18 and N87, had

identical 16S rRNA gene sequences that differed by six nucleotide mismatches

from the 16S rRNA gene sequence of the inoculant strain WSM1271. Novel

isolate N17 had one nucleotide mismatch with N18 and N87 and consequently

seven mismatches with WSM1271. Novel isolate N45 had a markedly different

16S rRNA sequence to the other novel isolates sequenced having 23 nucleotide

mismatches with N18 and N87, 24 nucleotide mismatches with N17 and 29

nucleotide mismatches with WSM1271.

Chapter 2

48

The 16S rRNA gene sequence of the inoculant strain WSM1271 was

also compared with the sequences of other strains isolated from B. pelecinus in

the Mediterranean region (WSM1283, WSM1284, WSM1497; Nandasena et al.,

2001). Strain WSM1271 had identical sequences to both strains WSM1284 and

WSM1497 while it had one nucleotide mismatch with strain WSM1283 in a 1440

nucleotide fragment of this gene (Appendix 3).

The 16S rRNA gene sequences of the novel isolates and WSM1271

were compared with the type strains of the other RNB genera within the α-

Proteobacteria and the type strains of the other species of Mesorhizobium. The

similarity percentages are given in Table 2.2. The novel isolates and WSM1271

had < 89.8% similarity to Bradyrhizobium japonicum, < 91.8% to Azorhizobium

caulinodans, < 94% to R. leguminosarum bv. phaseoli and < 95.6% to

Sinorhizobium meliloti. The novel isolates and WSM1271 had > 97% sequence

similarity with all the type strains of Mesorhizobium. WSM1271 had the highest

sequence similarity to M. ciceri (99.8%) and the least similarity within this genus

to M. huakuii. Three novel isolates (N17, N18 and N87) displayed a similar

sequence similarity pattern to that of WSM1271 when compared with the type

strains of Mesorhizobium. Interestingly novel isolate N45 showed the highest

sequence similarity to M. amorphae (99.5%) and the least similarity within this

genus to M. loti (97.4).

Chapter 2

49

WSM1271 AGTTTGATCCTGGCTCAGAACGAACGCTGGCGGCAGGCTTAACACATGCAAGTCGAG 57 N17 AGTTTGATCCTGGCTCAGAACGAACGCTGGCGGCAGGCTTAACACATGCAAGTCGAG 57 N18 AGTTTGATCCTGGCTCAGAACGAACGCTGGCGGCAGGCTTAACACATGCAAGTCGAG 57 N87 AGTTTGATCCTGGCTCAGAACGAACGCTGGCGGCAGGCTTAACACATGCAAGTCGAG 57 N45 AGTTTGATCCTGGCTCAGAACGAACGCTGGCGGCAGGCTTAACACATGCAAGTCGAG 57 WSM1271 CGCCCCGCAAGGGGAGCGGCAGACGGGTGAGTAACGCGTGGGAATCTACCCATCTCT 114 N17 CGCCCCGCAAGGGGAGCGGCAGACGGGTGAGTAACGCGTGGGAATCTACCCATCTCT 114 N18 CGCCCCGCAAGGGGAGCGGCAGACGGGTGAGTAACGCGTGGGAATCTACCCATCTCT 114 N87 CGCCCCGCAAGGGGAGCGGCAGACGGGTGAGTAACGCGTGGGAATCTACCCATCTCT 114 N45 CGCCCCGCAAGGGGAGCGGCAGACGGGTGAGTAACGCGTGGGAATCTACCCATCTCT 114 WSM1271 ACGGAACAACTCCGGGAAACTGGAGCTAATACCGTATACGTCCTTCGGGAGAAAGAT 171 N17 ACGGAACAACTCCGGGAAACTGGAGCTAATACCGTATACGTCCTTTTGGAGAAAGAT 171 N18 ACGGAACAACTCCGGGAAACTGGAGCTAATACCGTATACGTCCTTTTGGAGAAAGAT 171 N87 ACGGAACAACTCCGGGAAACTGGAGCTAATACCGTATACGTCCTTTTGGAGAAAGAT 171 N45 ACGGAACAACTCCGGGAAACTGGAGCTAATACCGTATACGTCCTTACGGAGAAAGAT 171 WSM1271 TTATCGGAGATGGATGAGCCCGCGTTGGATTAGCTAGTTGGTGGGGTAATGGCCTAC 228 N17 TTATCGGAGATGGATGAGCCCGCGTTGGATTAGCTAGTTGGTGGGGTAATGGCCTAC 228 N18 TTATCGGAGATGGATGAGCCCGCGTTGGATTAGCTAGTTGGTGGGGTAATGGCCTAC 228 N87 TTATCGGAGATGGATGAGCCCGCGTTGGATTAGCTAGTTGGTGGGGTAATGGCCTAC 228 N45 TTATCGGAGATGGATGAGCCCGCGTTGGATTAGCTAGTTGGTGGGGTAATGGCCTAC 228 WSM1271 CAAGGCGACGATCCATAGCTGGTCTGAGAGGATGATCAGCCACATTGGGACTGAGAC 285 N17 CAAGGCGACGATCCATAGCTGGTCTGAGAGGATGATCAGCCACACTGGGACTGAGAC 285 N18 CAAGGCGACGATCCATAGCTGGTCTGAGAGGATGATCAGCCACACTGGGACTGAGAC 285 N87 CAAGGCGACGATCCATAGCTGGTCTGAGAGGATGATCAGCCACACTGGGACTGAGAC 285 N45 CAAGGCGACGATCCATAGCTGGTCTGAGAGGATGATCAGCCACACTGGGACTGAGAC 285 WSM1271 ACGGCCCAAACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGAAAGC 342 N17 ACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGCAAGC 342 N18 ACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGCAAGC 342 N87 ACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGCAAGC 342 N45 ACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGCAAGC 342 WSM1271 CTGATCCAGCCATGCCGCGTGAGTGATGAAGGCCCTAGGGTTGTAAAGCTCTTTCAA 399 N17 CTGATCCAGCCATGCCGCGTGAGTGATGAAGGCCCTAGGGTTGTAAAGCTCTTTCAA 399 N18 CTGATCCAGCCATGCCGCGTGAGTGATGAAGGCCCTAGGGTTGTAAAGCTCTTTCAA 399 N87 CTGATCCAGCCATGCCGCGTGAGTGATGAAGGCCCTAGGGTTGTAAAGCTCTTTCAA 399 N45 CTGATCCAGCCATGCCGCGTGAGTGATGAAGGCCCTAGGGTTGTAAAGCTCTTTCAA 399 WSM1271 CGGTGAAGATAATGACGGTAACCGTAGAAGAAGCCCCGGCTAACTTCGTGCCAGCAG 456 N17 CGGTGAAGATAATGACGGTAACCGTAGAAGAAGCCCCGGCTAACTTCGTGCCAGCAG 456 N18 CGGTGAAGATAATGACGGTAACCGTAGAAGAAGCCCCGGCTAACTTCGTGCCAGCAG 456 N87 CGGTGAAGATAATGACGGTAACCGTAGAAGAAGCCCCGGCTAACTTCGTGCCAGCAG 456 N45 CGGTGAAGATAATGACGGTAACCGTAGAAGAAGCCCCGGCTAACTTCGTGCCAGCAG 456 WSM1271 CCGCGGTAATACGAAGGGGGCTAGCGTTGTTCGGAATTACTGGGCGTAAAGCGCACG 513 N17 CCGCGGTAATACGAAGGGGGCTAGCGTTGTTCGGAATTACTGGGCGTAAAGCGCACG 513 N18 CCGCGGTAATACGAAGGGGGCTAGCGTTGTTCGGAATTACTGGGCGTAAAGCGCACG 513 N87 CCGCGGTAATACGAAGGGGGCTAGCGTTGTTCGGAATTACTGGGCGTAAAGCGCACG 513 N45 CCGCGGTAATACGAAGGGGGCTAGCGTTGTTCGGAATTACTGGGCGTAAAGCGCACG 513 WSM1271 TAGGCGGATTGTTAAGTTAGGGGTGAAATCCCAGGGCTCAACCCTGGAACTGCCTTT 570 N17 TAGGCGGATTGTTAAGTTAGGGGTGAAATCCCAGGGCTCAACCCTGGAACTGCCTTT 570 N18 TAGGCGGATTGTTAAGTTAGGGGTGAAATCCCAGGGCTCAACCCTGGAACTGCCTTT 570 N87 TAGGCGGATTGTTAAGTTAGGGGTGAAATCCCAGGGCTCAACCCTGGAACTGCCTTT 570 N45 TAGGCGGATACTTAAGTCAGGGGTGAAATCCCGGGGCTCAACCCCGGAACTGCCTTT 570 WSM1271 AATACTGGCAATCTCGAGTCCGAGA- GAGGTGAGTGGAATTCCGAGTGTAGAGGTGA 626 N17 AATACTGGCAATCTCGAGTCCGAGA- GAGGTGAGTGGAATTCCGAGTGTAGAGGTGA 626 N18 AATACTGGCAATCTCGAGTCCGAGA- GAGGTGAGTGGAATTCCGAGTGTAGAGGTGA 626 N87 AATACTGGCAATCTCGAGTCCGAGA- GAGGTGAGTGGAATTCCGAGTGTAGAGGTGA 626 N45 GATACTGGGTATCTCGAGTCCG -GAAGAGGTGAGTGGAATTCCGAGTGTAGAGGTGA 626 WSM1271 AATTCGTAGATATTCGGAGGAACACCAGTGGCGAAGGCGGCTCACTGGCTC- GGTAC 682 N17 AATTCGTAGATATTCGGAGGAACACCAGTGGCGAAGGCGGCTCACTGGCTC- GGTAC 682 N18 AATTCGTAGATATTCGGAGGAACACCAGTGGCGAAGGCGGCTCACTGGCTC- GGTAC 682 N87 AATTCGTAGATATTCGGAGGAACACCAGTGGCGAAGGCGGCTCACTGGCTC- GGTAC 682 N45 AATTCGTAGATATTCGGAGGAACACCAGTGGCGAAGGCGGCTCACTGG -TCCGGTAC 682

Chapter 2

50

WSM1271 TGACGCTGAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCA 739 N17 TGACGCTGAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCA 739 N18 TGACGCTGAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCA 739 N87 TGACGCTGAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCA 739 N45 TGACGCTGAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCA 739 WSM1271 CGCCGTAAACTATGAGA-GCTAGCCGTCGGCAAGTTTACTTGTCGGTGGCGCAGCTA 795 N17 CGCCGTAAACTATGAGA-GCTAGCCGTCGGCAAGTTTACTTGTCGGTGGCGCAGCTA 795 N18 CGCCGTAAACTATGAGA-GCTAGCCGTCGGCAAGTTTACTTGTCGGTGGCGCAGCTA 795 N87 CGCCGTAAACTATGAGA-GCTAGCCGTCGGCAAGTTTACTTGTCGGTGGCGCAGCTA 795 N45 CGCCGTAAACGATG-GAAGCTAGCCGTTGGCAAGTTTACTTGTCGGTGGCGCAGCTA 795 WSM1271 ACGCATTAAGCTCTCC- GCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAAGGAAT 851 N17 ACGCATTAAGCTCTCC- GCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAAGGAAT 851 N18 ACGCATTAAGCTCTCC- GCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAAGGAAT 851 N87 ACGCATTAAGCTCTCC- GCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAAGGAAT 851 N45 ACGCATTAAGCT- TCCCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAAGGAAT 851 WSM1271 TGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGCAGA 908 N17 TGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGCAGA 908 N18 TGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGCAGA 908 N87 TGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGCAGA 908 N45 TGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGCAGA 908 WSM1271 ACCTTACCAGCCCTTGACATCCCGGTCGCGGTTTCCAGAGATGGATACCTTCAGTTC 965 N17 ACCTTACCAGCCCTTGACATCCCGGTCGCGGTTTCCAGAGATGGATACCTTCAGTTC 965 N18 ACCTTACCAGCCCTTGACATCCCGGTCGCGGTTTCCAGAGATGGATACCTTCAGTTC 965 N87 ACCTTACCAGCCCTTGACATCCCGGTCGCGGTTTCCAGAGATGGATACCTTCAGTTC 965 N45 ACCTTACCAGCCCTTGACATCCCGGTCGCGGTTTCCAGAGATGGAAACCTTCAGTTC 965 WSM1271 GGCTGGACCGGTGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTG 1022 N17 GGCTGGACCGGTGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTG 1022 N18 GGCTGGACCGGTGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTG 1022 N87 GGCTGGACCGGTGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTG 1022 N45 GGCTGGACCGGTGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTG 1022 WSM1271 GGTTAAGTCCCGCAACGAGCGCAACCCTCGCCCTTAGTTGCCAGCATTAAGTTGGGC 1079 N17 GGTTAAGTCCCGCAACGAGCGCAACCCTCGCCCTTAGTTGCCAGCATTAAGTTGGGC 1079 N18 GGTTAAGTCCCGCAACGAGCGCAACCCTCGCCCTTAGTTGCCAGCATTAAGTTGGGC 1079 N87 GGTTAAGTCCCGCAACGAGCGCAACCCTCGCCCTTAGTTGCCAGCATTAAGTTGGGC 1079 N45 GGTTAAGTCCCGCAACGAGCGCAACCCTCGCCCTTAGTTGCCAGCATTCAGTTGGGC 1079 WSM1271 ACTCTAAGGGGACTGCCGGTGATAAGCCGAGAGGAAGGTGGGGATGACGTCAAGTCC 1136 N17 ACTCTAAGGGGACTGCCGGTGATAAGCCGAGAGGAAGGTGGGGATGACGTCAAGTCC 1136 N18 ACTCTAAGGGGACTGCCGGTGATAAGCCGAGAGGAAGGTGGGGATGACGTCAAGTCC 1136 N87 ACTCTAAGGGGACTGCCGGTGATAAGCCGAGAGGAAGGTGGGGATGACGTCAAGTCC 1136 N45 ACTCTAAGGGGACTGCCGGTGATAAGCCGAGAGGAAGGTGGGGATGACGTCAAGTCC 1136 WSM1271 TCATGGCCCTTACGGGCTGGGCTACACACGTGCTACAATGGTGGTGACAGTGGGCAG 1193 N17 TCATGGCCCTTACGGGCTGGGCTACACACGTGCTACAATGGTGGTGACAGTGGGCAG 1193 N18 TCATGGCCCTTACGGGCTGGGCTACACACGTGCTACAATGGTGGTGACAGTGGGCAG 1193 N87 TCATGGCCCTTACGGGCTGGGCTACACACGTGCTACAATGGTGGTGACAGTGGGCAG 1193 N45 TCATGGCCCTTACGGGCTGGGCTACACACGTGCTACAATGGTGGTGACAGTGGGCAG 1193 WSM1271 CGAGACCGCGAGGTCGAGCTAATCTCCAAAAACCATCTCAGTTCGGATTGCACTCTG 1250 N17 CGAAACCGCGAGGTCGAGCTAATCTCCAAAAGCCATCTCAGTTCGGATTGCACTCTG 1250 N18 CGAGACCGCGAGGTCGAGCTAATCTCCAAAAGCCATCTCAGTTCGGATTGCACTCTG 1250 N87 CGAGACCGCGAGGTCGAGCTAATCTCCAAAAGCCATCTCAGTTCGGATTGCACTCTG 1250 N45 CGAGACCGCGAGGTCGAGCTAATCTCCAAAAGCCATCTCAGTTCGGATTGCACTCTG 1250 WSM1271 CAACTCGAGTGCATGAAGTTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTG 1307 N17 CAACTCGAGTGCATGAAGTTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTG 1307 N18 CAACTCGAGTGCATGAAGTTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTG 1307 N87 CAACTCGAGTGCATGAAGTTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTG 1307 N45 CAACTCGAGTGCATGAAGTTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTG 1307 WSM1271 AATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTTGGTTTTACC 1364 N17 AATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTTGGTTTTACC 1364 N18 AATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTTGGTTTTACC 1364 N87 AATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTTGGTTTTACC 1364 N45 A - TACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTTGGTTTTACC 1364

Chapter 2

51

WSM1271 CGAAGGCGCTGTGCTAACCGCAAGGAGGCAGGCGACCACGGTAGGGTCAGCGACTGG 1421 N17 CGAAGGCGCTGTGCTAACCGCAAGGAGGCAGGCGACCACGGTAGGGTCAGCGACTGG 1421 N18 CGAAGGCGCTGTGCTAACCGCAAGGAGGCAGGCGACCACGGTAGGGTCAGCGACTGG 1421 N87 CGAAGGCGCTGTGCTAACCGCAAGGAGGCAGGCGACCACGGTAGGGTCAGCGACTGG 1421 N45 CGAAGGCGCTGTGCTAACCGCAAGGAGGCAGGCGACCACGGTAGGGTCAGCGACTGG 1421 WSM1271 GGTGAAGTCGTAACAAG - T 1440 N17 GGTGAAGTCGTAACAAGGT 1440 N18 GGTGAAGTCGTAACAAGGT 1440 N87 GGTGAAGTCGTAACAAGGT 1440 N45 GGTGAAGTCGTAACAAGGT 1440

Figure 2.4 16S rRNA gene sequence alignment of strain WSM1271 and the novel isolates N17, N18, N87 and N45. Regions with nucleotide mismatches are highlighted in grey, nucleotide mismatches unique to N45 are in red and all other nucleotide mismatches are in blue

Chapter 2

52

Table 2.2 16S rRNA gene sequence similarity of WSM1271, N17, N18, N87 and N45 against the type strains of some of the RNB in α-Proteobacteria and the species of Mesorhizobium

Organism WSM1271 N17, N18, N87 N45

Bradyrhizobium japonicum

X66024 89.4% 89.4% 89.8%

Azorhizobium caulinodans

X67221 91.4% 91.6% 91.8%

R. leguminosarum bv.

phaseoli U29388 94.0% 93.9% 93.9%

Sinorhizobium meliloti

X67222 95.6% 95.5% 95.1%

Mesorhizobium loti

X67229 98.7% 98.5% 97.4%

M. ciceri

U07934 99.8% 99.4% 98.3%

M. tianshanense

AF041447 99.0% 98.7% 99.2%

M. mediterraneum

L38825 98.7% 98.6% 99.3%

M. amorphae

AF041442 98.7% 98.7% 99.5%

M. huakuii

D12797 97.1% 97.3% 98.3%

M. plurifarium

Y14158 97.3% 97.4% 98.3%

Chapter 2

53

The retrieved 16S rRNA gene sequences of the type strains of the RNB

within α-Proteobacteria from GenBank were aligned using ClustalW (Appendix

3) and the Kimura distance values (Kimura, 1980) were calculated for all these

strains and also the previously sequenced strains from B. pelecinus (WSM1283,

WSM1284, WSM1497; Nandasena et al., 2001) as described in methods

(Section 2.2.6) and are given in the distance matrix in (Apendix 4).

A unique sequence pattern could be observed for the novel isolates in

the nucleotide positions 164 and 165 in the alignment given (Appendix 3). In

these two nucleotide positions, two thymine bases (T) were observed instead of

a cytosine (C) and a guanine (G) respectively, as is normal for all other

members of Mesorhizobium. Interestingly these very same two nucleotide

positions of novel isolate N45 also have a unique pattern with adenine (A) and a

cytosine (C) respectively.

Based on the Kimura distance values, a phylogenetic tree was

developed using the Neighbor joining method (Fig. 2.5). Novel isolates N17,

N18 and N87 clustered together, but separately from other members of the

Mesorhizobium and RNB from B. pelecinus (Fig. 2.5). Novel isolate N45

clustered close to M. huakuii and M. amorphae.

Chapter 2

54

Figure 2.5 Phylogenetic tree showing relationships of four novel isolates (N17, N18, N45, N87) and WSM1271 within some members of RNB in α-Proteobacteria based upon aligned sequences of the small subunit rRNA gene (16S rRNA). Kimura distances were derived from the aligned sequences to construct an unrooted tree using the neighbour-joining method (T-type strains) A – Agrobacterium, B – Bradyrhizobium, M – Mesorhizobium, R – Rhizobium, S - Sinorhizobium

0.1

R. hainanensis (U71078, 166)

R. mongolense (U89817, USDA1844)

R. gallicum (U86343, R602sp)

R. etli (U28916, USDA9032) T

R. leguminosarum bv. phaseoli (U29388, USDA2671)

R. leguminosarum bv. trifolii (U31074, T24)

R. leguminosarum bv. viciae (U29386, USDA2370) T

R. tropici (D12798, IAM14206)

A. rhizogenes (X67224, LMG152)

R.huautlense (AF025852, SO2)

R. galegae (X67226, LMG6214) T

A. vitis (X67225, LMG8750) T

A. tumefaciens (X67223, LMG196)

A. rubi (X67228, LMG156) T

Sinorhizobium meliloti (X67222, LMG6133) T

S. terangae (X68387, LMG6463)

S. saheli (X68390, LMG7837) T

S. fredii (X67231, LMG6217) T

S. xinjiangensis (D12796, IAM14142) T

Azorhizobium caulinodans (X67221, LMG6465) T

B. elkanii (U3500, USDA76) T

B. japonicum (X66024, LMG6138)

M. tianshanense (AF041447, USDA3592) T

M. mediterraneum (L38825, UPMCa36) T

N45 M. amorphae (AF041442, ACC19665) T

M. plurifarium (Y14158, LMG11892)

M. huakuii (D12797, IAM14158) T

N17

N18 N87

WSM1284 WSM1271

M. ciceri (U07934, UPM-Ca7) T

WSM1497

WSM1283

M. loti (X67229, LMG6125) T

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55

2.4 Discussion

The results obtained by molecular fingerprinting PCR and 16S rRNA

gene sequencing show that some plants of B. pelecinus growing at the Northam

site were nodulated by RNB genetically and phylogenetically different to the

original inoculant WSM1271, six years after introduction and inoculation.

The phylogeny revealed by the 16S rRNA gene sequences in this study

clearly clustered these genetically different RNB (novel isolates) separately to

the original inoculant WSM1271 (Fig. 2.5). There were at least six nucleotide

mismatches in a sequence length of 1440 bases between the novel isolates and

WSM1271 (Fig. 2.4). These results imply that the novel isolates may not be

diversified representatives of the original inoculant, as the 16S rRNA gene is

considered to evolve very slowly over time (making it a good molecular

chronometer) and six years seems too short a time period to observe significant

nucleotide changes in this gene (Woese, 1987).

Preliminary sequencing of the 16S rRNA gene from three strains isolated

from B. pelecinus growing in the Mediterranean region, WSM1283 (Morocco),

WSM1284 (Sardinia) and WSM1497 (Mykonos, Greece), resulted with

nucleotide ambiguities (non-specific bases) for this gene and therefore showed

only 98% - 99% similarity among these strains (Nandasena et al., 2001). Re-

sequencing of a 1440 bp internal fragment of the 16S rRNA gene for the above

three strains with optimised conditions resulted in unambiguous sequences that

showed 100% sequence similarity between the three strains (Appendix 3).

These three strains may have been separated for thousands of years as they

were isolated from native settings with minimum human activity and in

geographically different locations (Morocco, Sardinia and Greece). B. pelecinus

Chapter 2

56

is not cultivated as an agricultural legume sp. in the Mediterranean region.

Therefore, the identical 16S rRNA gene sequences observed for these three

strains and their long separation from a common ancestor further supports the

notion that the 16S rRNA gene indeed evolves very slowly over time.

Although the 16S rRNA sequencing method has been regarded as the

undisputed standard for determining phylogenetic relationships (Woese, 1987;

Graham et al, 1991; Young, et al., 1991; Eardly, et al., 1992; Yanagi &

Yamasato, 1993; Murray & Scrleifer, 1994; Van Berkum, et al., 1996; Kataoka

et al, 1997; Oren et al, 1997; Lindström et al.,1998; Young, 1998), this is not a

method without limitations. Existence of interoperon variation (Clayton, et al.,

1995) and lateral transfer and recombination in the 16S rRNA gene (Sneath,

1993; Eardly et al.,1996) are two problems impacting the reliability of this gene

in inferring phylogenies. Therefore, phenotypic studies described in chapter 3

were undertaken to further clarify whether these genetically different isolates

(N17, N18, N45 and N87) are strains diversified from the original inoculant

which has undergone mutation and recombination within itself, or whether they

are other resident soil bacteria.

Molecular fingerprinting PCR with the primers RPO1 (Richardson et al.,

1995) and ERIC (de Bruijn, 1992) provided a rapid and inexpensive means of

identification of genetic diversity in RNB strains in this study. The nif-directed

primer RPO1 is reported to be useful for fingerprinting rhizobia because of its

ability to give distinct banding patterns even at strain level (Richardson et al.,

1995). The primer sequence of RPO1 contains the highly conserved nif

promoter consensus element at the 3’ end and therefore, theoretically, it can be

expected to amplify DNA regions that carry nif genes (Richardson et al., 1995).

Chapter 2

57

However, whether the RPO1 primer binds only to the nif promoter region or to

other nif promoter like regions has not been experimentally tested and therefore

this method is not reliable for making firm conclusions regarding diversity of

symbiotic regions between novel isolates.

Enterobacterial Repetitive Intergenic Consensus elements (ERIC

elements) are small repetitive units of 126 bp containing a conserved central

inverted repeat of 40 bp in enterobacteria (Hulton et al., 1991; Versalovic, et al.,

1991). ERIC primers were developed complementary to the inverted repeat,

and have been demonstrated to generate complex amplification patterns not

only in the members of Enterobacteriaceae but also from many other

eubacterial genera (Versalovic, et al., 1991; de Burjun, 1992). Although many

eubacterial genera produce strain specific complex PCR banding patterns with

ERIC primers, as was observed for novel isolates in this study, whether these

PCR products result from the annealing of the primers to ERIC elements or

whether they anneal to other ERIC-like sequences are not known for many

eubacterial genera. Niemann et al., (1999) have provided evidence that in

members of Sinorhizobium, the ERIC primers can bind to a DNA region that

putatively encodes the C-terminal part of a protein displaying similarity to 2-

hydroxyacid dehydrogenase. Furthermore, circumstantial evidence based on

the fact that in various organisms the ERIC fingerprint patterns could change

depending on the PCR temperature used for (annealing and extension) suggest

that ERIC fingerprints do not always result from the amplification from ERIC

elements (Gillings & Holley, 1997). Therefore, similar sized PCR products could

result from two strains but the products may be due to the amplification of

different genomic regions in these two separate strains. Hence, it is not logical

Chapter 2

58

to use ERIC-PCR for phylogenetic studies although it was suggested previously

by de Bruijn, (1992).

However, it can be confidently said that two strains are genetically

different at some degree to each other if they give different banding patterns

when an identical PCR cycling condition is used for both strains in molecular

fingerprinting. Hence, molecular fingerprinting PCR provided a good basis to

distinguish novel isolates from WSM1271. Yet, the vise-versa of the above is

not always true. i.e. two strains having similar molecular fingerprints are not

always exactly the same strain.

RPO1 and ERIC based PCR methods have been used to identify nodule

occupants relative to the inoculant strains (McInnes, 2002; Denton et al., 2002;

Nandasena et al., 2004; Yates et al., 2004). A bacterial strain has been defined

as an isolate or group of isolates that can be distinguished by using either (or

both) phenotypic or genotypic characteristics (Tenover et al., 1995). This

definition of a bacterial strain is very shallow and the demarcation between two

individuals that are considered as two different strains is not clear. Therefore, in

this study, re-isolates were not considered as genetically diverse strains to

WSM1271 even though they may have genetic variation to some degree.

Approximately 8% of the nodule occupants of B. pelecinus growing at

Northam had genetic differences to WSM1271 based on molecular

fingerprinting PCR and 16S rRNA gene sequencing. However, this percentage

may be an underestimate of the diversity of bacteria present in Northam field

capable of nodulating B. pelecinus due to the screening method used in this

study. Only the colonies displaying typical characteristics of the known fast and

moderately fast growing rhizobia were selected. Moreover, it is also possible

Chapter 2

59

that there were genetically diverse rhizobial strains present on the plates

discarded due to fungal contamination.

An intriguing observation in this study was that three novel isolates N17,

N18 and N87 were all isolated from separate nodules on roots of the same

plant. Furthermore, two other novel isolates (N45 and N46) were isolated from

nodules on two different plants which were only a few centimetres apart. At

present it is not clear whether these observations are mere coincidence or

whether the isolates are from “hot spots” in the Northam field where an

enhanced level of diversity had occurred. An extensive study involving a large

collection of nodules from many different points, combined with testing the biotic

and abiotic factors of the soil in the collection spots may provide insight to

understanding the factors that influence gene transfer between rhizobia in

agricultural soils.

Phenotypic experiments were performed to identify any changes that

might have taken place in re-isolates and also to investigate whether the

genetically diverse strains described in this chapter (novel isolates) have

phenotypic differences. These experiments are described in the following

chapter.

Chapter 2

60

Chapter 3

61

3. Phenotypic diversity among RNB isolated from

Biserrula pelecinus L. six years after introduction and

inoculation with WSM1271

3.1 Introduction

The emergence of diversity among the nodule occupants of exotic legume

species after introduction to new regions presents a challenge to agricultural

productivity. When these diverse strains are highly competitive for nodulation, but

not effective in N2 fixation, the benefits of legume introduction are lost or

substantially reduced. This has been the scenario following the introduction of

soybeans (Glycine max) in North America (Keyser & Li, 1992, Herridge & Rose,

2000), alfalfa (Medicago sativa), annual medics (Medicago spp.) and annual

clovers (Trifolium spp.) in southern Australia (Brockwell, 2001; Denton et al., 2002),

and common bean (Phaseolus vulgaris) in Latin America and Africa (Hungria &

Vargas, 2000). The gradual replacement of the inoculant by established strains of

indigenous RNB with poor N2 fixation has been an intractable constraint to legume

productivity in many agricultural systems for decades (Dowling & Broughton, 1986;

Triplett & Sadowsky, 1992; Denton at al., 2000). Therefore, in this context it is

important to investigate whether the genetically different RNB (novel isolates)

isolated from B. pelecinus, as described in the previous chapter, can fix N2 on

B. pelecinus as effectively as the original inoculant strain WSM1271.

Chapter 3

62

Despite improvements in inoculant technology and selection of strains with

greater N2 fixation capacity, inoculation does not always result in increased crop

yield (Boonkerd et al., 1978; Sparrow & Ham, 1983; Torres et al., 1987; Thies et

al., 1991a). Could decreased N2 fixation also result from the loss of N2 fixation

efficiency in the inoculant strain? A secondary objective of this chapter was

therefore to test whether the re-isolates (see definition in Section 2.3.2) could fix N2

as effectively as the mother culture of strain WSM1271.

Both, genetic and phenotypic data are used in distinguishing strains and

therefore play an important role in studies of bacterial diversity (Farber, 1996).

RNB strains differ in their ability to utilise carbon sources (Graham et al., 1991). In

this chapter data are reported on the ability of the novel isolates to utilise 14

different carbon compounds as sole carbon source, their intrinsic resistance to

eight antibiotics and their pH tolerance.

A list of the assigned standard hosts for RNB has been published (Graham

et al., 1991). In initial investigations of B. pelecinus, experiments revealed that

RNB isolated from B. pelecinus growing in the Mediterranean region were unable

to nodulate the following Australian agricultural legume species; Medicago

polymorpha, Ornithopus compressus, Trifolium subterraneum, Vicia bangalensis,

Lupinus angustifolius (Howieson, et al., 1995). The host range of isolates from

Northam was assessed using twenty different legumes that are known to be

nodulated by the members of Mesorhizobium, as well as some of the legumes

listed by Graham et al., (1991), selected on the basis of ease of obtaining the

seeds, their cost and quarantine considerations in Western Australia. Previous

Chapter 3

63

work has demonstrated that Strain WSM1284, isolated from B. pelecinus growing

in the Mediterranean region, nodulates Lotus corniculatus, L. ornithopodioides,

L. pedunculatus and Dorycnium hirsutum (Nandasena et al., 2004). Therefore, nine

available species of Lotus and two available species of Dorycnium were chosen to

investigate the host range of isolates. Earlier work has also revealed that two RNB

strains isolated from Hedysarum spinosissimum were capable of nodulating B.

pelecinus (Nandasena et al., 2004). Hence H. spinosissimum was also used for the

host range experiment.

Aims


1. To investigate the N2 fixation effectiveness of the novel isolates on

B. pelecinus cv Casbah

2. To investigate physiological properties of the novel isolates and to compare

them with WSM1271.

3. To investigate the host range of the novel isolates N17, N18, N45 and N87.

4. To investigate whether re-isolates have altered physiological and symbiotic

characters six years after introduction of Mesorhizobium sp. strain

WSM1271 to the soil.

Chapter 3

64

3.2 Material and Methods

3.2.1 Effectiveness tests

Experimental design: The seven novel isolates (N15, N17, N18, N39, N45, N46

and N87), seven selected re-isolates (N5, N19, N36, N48, N59, N64 and N84) and

WSM1271 were tested for their effectiveness on B. pelecinus cv Casbah. This

cultivar was selected as it forms an effective symbiosis with WSM1271 and is the

most widely used commercial cultivar of this species (Legume crop inoculation

groups-2004; BIO-CARE TECHNOLOGY PTY. LIMITED, RMB 1084, Pacific

Highway Somersby, NSW 2250, Australia). Three replicates were used for each

treatment. Added N (+N) and N free (-N) treatments were used as controls. A two

factorial experimental design was used with the free draining pot method of

Howieson, et al., (1995).

Preparation of the pots: Plants were grown in free-draining pots containing

1.5 kg steam treated 1:1 river sand: yellow sand mix. Polyvinyl chloride tubes (2.5

cm diameter, 25 cm length) were inserted into the sand for supply of water and

nutrients. The tubes were closed with lids. The pots, lids and tubes were surface

sterilised by soaking in 3% (v/v) sodium hypochloride solution for 5 days followed

by rinsing with sterile DDi H2O. To prevent rapid drainage, the bottom of each pot

was lined with sterile absorbent paper. Each pot of soil was flushed three times

with boiling water to remove inorganic nitrogen.

Chapter 3

65

Preparation of seeds: Seeds of B. pelecinus cv Casbah were scarified using

number 0.01 sand paper and then surface sterilised for 30 s in 70% (v/v) ethanol,

followed by 1 min in 3% (v/v) sodium hypochloride and finally washed six times

with sterile DDi H2O (Howieson, et al., 1995). Sterilised seeds were then spread on

the surface of 1.5% (w/v) agar/water plates with an ethanol sterilised spatula. A

small volume (0.5 ml) of sterile DDi H2O was added to each plate to facilitate

germination. Plates containing seeds were wrapped in aluminium foil and

incubated at 28°C for 24 h.

Preparation of inocula: Starter cultures were grown in McCartney bottles (30

ml) containing 5 ml of ½ LA medium incubated at 28°C to stationary phase. An

appropriate amount of each starter culture was used to inoculate 500 ml

Erlenmeyer flasks containing 100 ml of ½ LA medium to an OD600 0.1. Inoculated

flasks were incubated at 28°C on a gyratory shaker (200 rpm) for three days.

Sowing: Each pot was sown with four germinated seeds of B. pelecinus cv.

Casbah. The germinated seeds were sown at a depth of 1 cm using sterile, fine

forceps. Each seed was each inoculated with 1 ml of the appropriate inoculum. The

seeds in the +N treatment were each supplied with 1 ml of sterile 10% (w/v) KNO3

and the seeds in the N free treatment were given 1 ml of sterile water. The seeds

were then covered with sand, and the surface of each pot was completely covered

with sterile polythene beads to a depth of 1.5 cm.

Chapter 3

66

Glasshouse conditions, nutrients and watering: The glasshouse was

maintained with a maximum day time temperature of 21°C ±2°C. DDi H2O was

used at all times. Water and the nutrient solution (Howieson et al., 1995) were

given alternately using a Turborl 50 ml dispenser. Nutrient solutions and water

were autoclaved at 121°C for 20 min and cooled to room temperature before use.

The rubber tube attached to the dispenser was sterilised regularly by pumping 70%

(v/v) ethanol followed by a wash with sterile water to avoid algal growth. Pots were

watered to saturation every second day. Twenty ml of nutrient solution was given

when the plants were 1-2 weeks old and subsequently it was increased to 40 ml. A

volume of 5 ml of 10% (w/v) KNO3 was added to the +N treatment pots weekly.

Harvesting and shoot dry weight measurement: Plants were harvested

after eight weeks by washing away the soil with running water. The soil was

discarded in soil bins to avoid contamination. Nodule colour, number and position

on the roots were noted for each treatment. Shoots were removed, oven dried at

70°C for 48 h then weighed.

Statistical analysis: Means and standard errors were calculated for all data

(Appendix 5). ANOVA was used to test the hypothesis that shoot dry weights were

similar across all strains followed by least significant difference tests (LSD) to

determine which strains were significantly different. The software used was

Statistica for Macintosh, version 4.1. Data conformed to all fundamental

assumptions of ANOVA except equal variances and this could not be corrected by

Chapter 3

67

data transformation. Therefore significance was set at 0.01 (Tabachinck & Fidell,

1996).

3.2.2 Carbohydrate utilisation

Carbon sources: Growth of strains was assessed on 14 sources of carbon: N

acetyl glucosamine (99%, Sigma, MO, USA), arabinose (99%, Sigma, MO, USA),

arbutine (98%, Sigma, MO, USA), dulcitol (99.85% The British drughouse Ltd.

Poole, UK), β-gentiobiose (99% Fluka Chelie, Buchs, Switzerland), lactose (99.8%,

APS Chemicals, NSW, Australia), maltose (99.5%, BDH Laborotary supplies,

Poole, UK), melibiose (98%, Sigma, MO, USA), D-raffinose (98%, Aldrich Chem.

Co. WI, USA), L-sorbose, (98%, Sigma, MO, USA), Sucrose (99.85%, APS

Chemicals, NSW, Australia), D-tagatose (98%, Sigma, MO, USA), trehalose (99%,

Aldrich Chem. Co. WI, USA) and D-turanose (98%, Aldrich Chem. Co. WI, USA).

These carbon sources were selected on the basis of their ability to distinguish

biserrula RNB from other rhizobial genera (Nandasena et al., 2001).

Bacterial strains: Strain WSM1271, seven re-isolates (N5, N19, N36, N48, N59,

N64 and N84) and six novel isolates (N17, N18, N39, 45, N46, N87) were used.

Isolate N15 failed to grow on Minimal Salt Medium (MSM; mannitol as the carbon

source; Carson et al., 1992) and therefore was not tested for its carbon source

utilisation pattern, antibiotic resistance or pH range. The type strains of Rhizobium

(R. leguminosarum strain USDA2370), Sinorhizobium (S. meliloti strain

Chapter 3

68

USDA1002) and Mesorhizobium (M. loti strain NZP2213) with known carbon

source utilisation patterns were used as control strains.

Preparation of media: All glassware was acid washed by soaking over night in

10% (v/v) hydrochloric acid followed by a wash with detergent, rinsing with water

and two washes with DDi water to remove nutrients adhered to the glass. The final

concentration of each carbon source was 1 mM. Each carbon source was filter

sterilised using 0.2 µm millipore filters and an appropriate volume of each carbon

source was added separately to autoclaved carbon free MSM (Carson et al., 1992)

cooled to room temperature. One ml of MSM medium containing separate carbon

sources was dispensed into transparent 5 ml plastic tubes. There were three

replicates for media with each carbon source.

Inoculation and observation of growth on different carbon sources:

McCartney bottles (30 ml) containing 5 ml of MSM medium with mannitol as the

sole carbon source were initially inoculated with a loop-full of cells from a ½LA

plate (Howieson et al., 1988) and grown for seven days (Mesorhizobium sp. strains

used in this study grew slowly in the minimal medium) on a gyratory shaker (200

rpm) at 28°C. One ml of culture from each strain was then centrifuged for 3 min at

3000 rpm. Supernatant was discarded and the pellets were resuspended in 1 ml of

sterile 0.89% (w/v) NaCl. This step was repeated to wash the cell pellets

thoroughly in order to remove any exopolysaccharides or media from the starter

culture. An appropriate volume of each suspension of these cells was used to

Chapter 3

69

inoculate the 5 ml tubes containing 1 ml of the MSM + carbon source to obtain a

starting OD600 of 0.05. These tubes were then placed on racks and incubated for

10 days on a gyratory shaker (200 rpm) at 28°C. Growth was determined by

measuring the OD600 of the cultures.

3.2.3 Antibiotic resistance

Antibiotics used: Eight antibiotics were assessed at the following

concentrations: ampicillin (50µg/ml), chloramphenicol (40 µg/ml), gentamicin (40

µg/ml), kanamycin (50 µg/ml), nalidixic acid (50 µg/ml), spectinomycin (50 µg/ml),

streptomycin (100 µg/ml) and tetracycline (20 µg/ml).

Bacterial strains: Same strains used for carbon source utilisation experiment

were used (Section 3.2.2).

Media and inoculation: Starter cultures were grown in MSM medium as

described in Section 3.2.2, sub-cultured into McCartney bottles containing 5 ml of

MSM medium with a starting OD600 of 0.1, then grown for 3 d on a gyratory shaker

(200 rpm) at 28°C. One ml of culture was then washed as described for carbon

source utilisation (Section 3.2.2) and serial dilutions made. Ten μl from each of the

following cell dilutions: 10-1, 10-3, 10-5 and 10-7 were plated on to ½LA plates. Four

different strains were spotted onto each plate as shown in Fig 3.1. Plates were

incubated at 28°C for four days.

Chapter 3

70

Fig 3.1 Layout on the Petri dish showing strain positions and spots for each dilution of cell culture inoculated

3.2.4 pH range

Bacterial strains: Strain WSM1271, seven re-isolates and six novel isolates

(N17, N18, N39, 45, N46, N87) were used.

Media and inoculation: Growth of the strains at pH 4.5, pH 5.0, pH 7.0 and pH

9.0 was assessed on ½LA plates with the following buffers to maintain the pH of

the medium. Homopipes (10 mM; Ballen et al., 1998) for pH 4.5 and pH 5.0, Hepes

(10 mM) for pH 7.0 and Trizma (10 mM) for pH 9.0. Growth conditions and

inoculation were the same as described for antibiotic resistance (Section 3.2.3).

3.2.5 Host range experiments

Plants and bacterial strains: The original inoculant WSM1271, four novel

isolates: N17, N18, N45, N87 and two other RNB isolated from B. pelecinus

growing in the Mediterranean region: WSM1284 and WSM1497 were used.

10-1

●10-5●

10-3●

10-7●

Strain 2

Strain 4

Strain 3

10-1

●10-5●

10-3●

10-7●

Strain 2

Strain 4

Strain 3

Chapter 3

71

Plants, their source and additional information are given in Table 3.1

Table 3.1 Legumes used for the cross-inoculation experiment Host plants

Seed source Reference

Amorpha fruticosa BARC, USDA Wang et al. (1999c)

Astragalus adsurgens BARC, USDA Wei et al., (2003)

Laguerre et al. (1997) Gao et al., (2001) Astragalus membranaceus Phoenix seeds Wang & Chen, (1996)

Laguerre et al. (1997) Astragalus sinicus BARC, USDA Chen et al., (1991)

Zhang et al., (2000) Biserrula pelecinus cv Casbah DAWA, GRC Nandasena et al., (2001)

Nandasena et al., (2004) Dorycnium hirsutum DAWA, GRC

Dorycnium rectum DAWA, GRC

Glycyrrhiza uralensis Phoenix seeds Chen et al., (1995)

Hedysarum spinosissimum DAWA, GRC Kishinevsky et al. (2002)

Leucaena leucocephala Phoenix seeds De Lajudie et al., (1994)

Moreira et al., (1993) Jarvis et al. (1982) De Lajudie et al., (1998) Lotus corniculatus DAWA, GRC Jarvis et al. (1982)

Lotus edulis DAWA, GRC

Lotus glaber DAWA, GRC

Lotus maroccanus DAWA, GRC Jarvis et al. (1986)

Lotus ornithopodioides DAWA, GRC

Lotus parviflorus DAWA, GRC

Lotus pedunculatus DAWA, GRC Jarvis et al. (1982)

Lotus peregrinus DAWA, GRC

Lotus subbiflorus DAWA, GRC

Macroptilium atropurpureum DAWA, GRC Trinick & Hadobas, (1989)

Odee et al. (2002) Jarvis et al. (1982) Ornithopus sativus DAWA, GRC Jarvis et al. (1982)

Department of Agriculture Western Australia, Genetic Resource Centre (DAWA, GRC), Beltsville Agricultural Research Center, United States Department of Agriculture, Beltsville, Md., U.S.A. (BARC, USDA), Phoenix seeds, Tasmania, Australia

Chapter 3

72

Experimental design: A cross nodulation experiment was conducted under

quarantine permitted, temperature controlled, axenic glasshouse conditions using a

closed vial growth system. This system appears to reduce contamination in

comparison to the free-draining pot method (Howieson et al. 1995), especially

when dealing with known promiscuous legume hosts. The closed vial system

consisted of a screw topped polycarbonate vial (500ml) containing 200g of mixed

sand medium (1 part washed river sand to 1 part yellow sand) and autoclaved at

121°C for 20 min. Fourty milliliters of autoclaved nutrient solution (Howieson et al.,

1995) devoid of nitrogen were then applied to each vial.

The experiment was a split plot design with one strain of root-nodule

bacteria as the main treatment ans two species of legume as the sub treatment.

The legume seed was surface sterilized as described by Howieson, et al., (1995).

Two germinated seedlings of two species were placed into a single vial then

inoculated with one putative rhizobial strain (approximately 1 X 107 cells ml-1).

Inocula were made as described in (Section 3.2.1.). There were three replicates

for each strain, as well as uninoculated controls. Vials were arranged in

completely randomised blocks in a naturally lit glasshouse maintained at 20°C ±

2°C. After eight weeks, roots were carefully exhumed from the vials and washed

free of sand. Nodule colour, number and position on the roots were noted for each

treatment which had nodulation. The remaining plant material and the soil were

autoclaved before disposal.

Chapter 3

73

3.3 Results

3.3.1 Effectiveness

The one way ANOVA indicated that the interaction between host plant and

inoculation treatments was significant (F(1,17) = 30.856, P < 0.01; Appendix 5)

according to their shoot dry weight measurements. The N-free control had poorly

grown stunted plants with yellow leaves while the N-fed plants were large, well

grown with dark green leaves. Nodules were not observed on the N-free and N-fed

control plants. A significant difference (P<0.01) in the shoot dry weight was

observed between the N-free and N-fed treatments. These observations were

made eight weeks after sowing and inoculation.

B. pelecinus cv Casbah inoculated with WSM1271 were large green plants

(Fig 3.2) with bifurcate, pink nodules (Fig 3.3) on both main and lateral roots.

Similarly, the plants inoculated with the seven re-isolates were large green plants

(Fig 3.2) with bifurcate, pink nodules on both main and lateral roots. There was no

difference (p>0.01) between the shoot dry weights of B. pelecinus cv Casbah

inoculated with WSM1271 and the shoot dry weights of the B. pelecinus cv Casbah

inoculated with the re-isolates (Fig 3.5.).

Plants inoculated with five of the novel isolates (N17, N18, N39, N46, N87)

were light green (Fig 3.2) and formed bifurcate, pink nodules on both main and

lateral roots. These strains were poorly effective as they fixed less N2 than

WSM1271 (p<0.01) but produced a greater top dry weight (p<0.01) than the N-free

control.

Chapter 3

74

+N-N

N45

N18

N19

1271

+N-N

N45

N18

N19

1271

Fig

3.2

Pla

nt g

row

th re

spon

ses

of B

. pel

ecin

us c

v. C

asba

h to

the

diffe

rent

inoc

ulan

t tre

atm

ent.

Add

ed N

-con

trol (

+N),

inoc

ulan

t stra

in W

SM

1271

, re-

isol

ate

N19

, poo

rly e

ffect

ive

nove

l iso

late

N18

, ine

ffect

ive

nove

l iso

late

N45

and

N-fr

ee

cont

rol (

-N) e

ight

wee

ks a

fter s

awin

g.

Chapter 3

75

Fig 3.3 Nodules on B. pelecinus cv. Casbah inoculated with Mesorhizobium sp. strain WSM1271

Fig 3.4 Nodules on B. pelecinus cv. Casbah inoculated with novel isolate N45

Chapter 3

76

dd

d

ccc

c

c

bbb

b

b

a

b

b

b

0

0.05

0.1

0.15

0.2

0.25

0.3

(-)N

(+)N

WSN12

71 N15 N17 N18 N39 N45 N46 N87 N5N19 N36 N48 N59 N64 N84

Inoculant strain

Shoo

t dry

wei

ght (

g)

Fig 3.5 Shoot dry weights of B. pelecinus cv. Casbah inoculated with WSM1271, novel isolates (N15, N17, N18, N39, N45, N46, N87), re-isolates (N5, N19, N36, N48, N59, N64, N84), and uninoculated N-free control (-N) and uninoculated added N-control (+N). Different letters on the top of each column indicate significant differences at the level of 0.01, calculated following square-root transformations

Plants inoculated with N15 and N45 were similar in appearance to the N-

free control plants (Fig 3.2). The nodules formed by these two isolates on B.

pelecinus cv Casbah were white and smaller (Fig 3.4) than those formed by the

other five novel isolates and located only on lateral roots. There was no difference

(p>0.01) between the shoot dry weights of these two treatments and the N-free

control, and therefore these two strains were ineffective.

Chapter 3

77

3.3.2 Carbohydrate utilisation

Strains that failed to reach an OD600 of 0.2 (i.e. failed to complete at least

two doublings as they were initially inoculated at an OD600 of 0.05) within 10 days

of incubation in the presence of a particular carbon source were considered to be

unable to grow on that carbon source. Strains that achieved an OD600 between

0.2-0.4 were considered to have poor growth on that carbon source.

Carbon source utilisation pattern of WSM1271: Strain WSM1271 grew well on

N-acetyl glucosamine, arabinose, arbutine, melibiose, and D-tagatose as sole

carbon sources. This strain grew poorly on dulcitol, β-gentiobiose and lactose, and

did not grow on maltose, D-raffinose, L-sorbose, sucrose or trehalose as sole

carbon sources.

Carbon source utilisation pattern of re-isolates All the seven re-isolates

displayed similar carbon source utilisation patterns to that of WSM1271, with only a

very few differences (Table 3.2). All re-isolates grew well on arabinose, arbutine,

melibiose and D-tagatose while they failed to grow on maltose, L-sorbose, sucrose

and trehalose as sole carbon sources. All but N48 and N64 grew well on N-acetyl

glucosamine, while these two isolates grew poorly on this carbon source. All

isolates grew poorly on D-turanose, while all except N64 grew poorly on β-

gentiobiose and lactose. Isolate N64 failed to grow on β-gentiobiose and lactose.

Isolate N19 grew well on dulcitol while the other re-isolates grew poorly on this

Chapter 3

78

Table 3.2 Carbon source utilisation patterns of re-isolates, WSM1271, novel isolates, and type strains of Mesorhizobium, Sinorhizobium and Rhizobium

Meso – Mesorhizobium loti strain NZP2213 (type strain), Sino – Sinorhizobium meliloti strain USDA1002 (type strain), Rhizobium legumonosarum USDA2370 (type strain) 0 OD600 0.0-0.1; + OD600 0.1-0.3; ++ OD600 >0.3

Stra

in

N A

cety

l glu

cosa

min

e

Arab

inos

e

Arbu

tine

Dul

cito

l

β G

entio

bios

e

Lact

ose

Mal

tose

Mel

ibio

se

D-R

affin

ose

L-So

rbos

e

Sucr

ose

D-T

agat

ose

Treh

alos

e

D-T

uran

ose

N5 ++ ++ ++ + + + 0 ++ 0 0 0 ++ 0 +

N19 ++ ++ ++ ++ + + 0 ++ 0 0 0 ++ 0 +

N36 ++ ++ ++ + + + 0 ++ 0 0 0 ++ 0 +

N48 + ++ ++ + + + 0 ++ 0 0 0 ++ 0 +

N59 ++ ++ ++ + + + 0 ++ 0 0 0 ++ 0 +

N64 + ++ ++ + 0 0 0 ++ 0 0 0 ++ 0 +

N84 ++ ++ ++ + + + 0 ++ + 0 0 ++ 0 +

WSM1271 ++ ++ ++ + + + 0 ++ 0 0 0 ++ 0 +

N17 ++ ++ ++ 0 ++ ++ + ++ 0 0 + ++ ++ ++

N18 0 ++ 0 0 + 0 0 ++ 0 0 0 0 0 0

N39 ++ ++ ++ + + + 0 ++ + 0 + ++ 0 +

N45 ++ ++ ++ 0 ++ ++ ++ ++ 0 0 ++ ++ ++ ++

N46 + ++ ++ 0 ++ ++ + ++ 0 0 ++ ++ + +

N87 + ++ ++ 0 + ++ ++ ++ + 0 ++ ++ ++ ++

Meso + ++ ++ 0 + ++ ++ + + ++ ++ ++ ++ ++

Sino ++ + ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++

Rhiz ++ ++ ++ ++ ++ ++ ++ ++ ++ 0 ++ ++ ++ ++

Re-

isol

ates

N

ovel

-isol

ates

Chapter 3

79

N Acetyl glucosamine

0.000

0.200

0.400

0.600

0.800

1.000

1.200

1.400

1.600

N5N19

N36 N48N59 N64

N84

WSM1271

N17 N18N39 N45

N46 N87 NZMEL

LEG

Arabinose

0.000

0.200

0.400

0.600

0.800

1.000

1.200

1.400

1.600

N5N19 N36 N48 N59 N64 N84

WSM1271 N17 N18 N39 N45 N46 N87 NZ

MELLE

G

Arbutine

0.000

0.200

0.400

0.600

0.800

1.000

1.200

1.400

1.600

N5N19 N36 N48 N59 N64 N84

WSM1271 N17 N18 N39 N45 N46 N87 NZ

MELLE

G

a

b

c

OD

600

OD

600

OD

600

Chapter 3

80

Dulcitol

0.000

0.200

0.400

0.600

0.800

1.000

1.200

1.400

1.600

N5N19 N36 N48 N59 N64 N84

WSM1271 N17 N18 N39 N45 N46 N87 NZ

MELLE

G

β-Gentibiose

0.000

0.200

0.400

0.600

0.800

1.000

1.200

1.400

1.600

N5N19 N36 N48 N59 N64 N84

WSM1271 N17 N18 N39 N45 N46 N87 NZ

MELLE

G

Lactose

0.000

0.200

0.400

0.600

0.800

1.000

1.200

1.400

1.600

N5N19 N36 N48 N59 N64 N84

WSM1271 N17 N18 N39 N45 N46 N87 NZ

MELLE

G

d

e

f

OD

600

OD

600

OD

600

Chapter 3

81

Maltose

0.000

0.200

0.400

0.600

0.800

1.000

1.200

1.400

1.600

N5N19 N36 N48 N59 N64 N84

WSM1271 N17 N18 N39 N45 N46 N87 NZ

MELLE

G

Melibiose

0.000

0.200

0.400

0.600

0.800

1.000

1.200

1.400

1.600

N5N19 N36 N48 N59 N64 N84

WSM1271 N17 N18 N39 N45 N46 N87 NZ

MELLE

G

D-Raffinose

0.000

0.200

0.400

0.600

0.800

1.000

1.200

1.400

1.600

N5N19

N36 N48 N59N64 N84

WSM1271 N17

N18 N39 N45N46 N87 NZ

MELLE

G

g

h

i

OD

600

OD

600

OD

600

Chapter 3

82

Sucrose

0.000

0.200

0.400

0.600

0.800

1.000

1.200

1.400

1.600

N5N19 N36 N48 N59 N64 N84

WSM1271 N17 N18 N39 N45 N46 N87 NZ

MELLE

G

D-Tagatose

0.000

0.200

0.400

0.600

0.800

1.000

1.200

1.400

1.600

N5N19 N36 N48 N59 N64 N84

WSM1271 N17 N18 N39 N45 N46 N87 NZ

MELLE

G

j

k

l

OD

600

OD

600

OD

600

L-Sorbose

0.000

0.200

0.400

0.600

0.800

1.000

1.200

1.400

1.600

N5N19 N36 N48 N59 N64 N84

WSM12

71 N17 N18 N39 N45 N46 N87 NZMEL

LEG

L-Sorbose

0.000

0.200

0.400

0.600

0.800

1.000

1.200

1.400

1.600

N5N19 N36 N48 N59 N64 N84

WSM12

71 N17 N18 N39 N45 N46 N87 NZMEL

LEG

Chapter 3

83

Fig 3.6 (a-n)Growth of re-isolates (N5, N19, N36, N48, N59, N64, N84), WSM1271, novel isolates (N17, N18, N39, N45, N87, N87) and the control strains NZ- (M. loti type strain NZP2213), MEL- (S. meliloti type strain USDA1002) and LEG- (R. leguminosarum type strain USDA2370) on a – N acetyl glucosamine, b – arabinose, c – arbutine, d – dulcitol, e – β-gentiobiose, f – lactose, g – maltose, h – melibiose, i – D-raffinose, j – L-sorbose, k – sucrose, l – D-tagatose, m – trehalose, n – D-turanose, as the sole carbon source for growth after 10 days.

Carbon source control (with no carbon source)

Trehalose

0.000

0.200

0.400

0.600

0.800

1.000

1.200

1.400

1.600

N5N19 N36 N48 N59 N64 N84

WSM1271 N17 N18 N39 N45 N46 N87 NZ

MELLE

G

D-Turanose

0.000

0.200

0.400

0.600

0.800

1.000

1.200

1.400

1.600

N5N19 N36 N48 N59 N64 N84

WSM1271 N17 N18 N39 N45 N46 N87 NZ

MELLE

G

m

n

OD

600

OD

600

Chapter 3

84

carbon source. (Fig d). N84 was the only re-isolate to grow on D-raffinose and it

showed poor growth on this carbon source.

Carbon source utilisation pattern of novel isolates: The six novel isolates

tested had carbon source utilisation patterns distinct from WSM1271. No difference

(p>0.01) in growth level (all strains had OD600 increase above 0.4) was observed

between the novel isolates and WSM1271 for arabinose (Fig b) and melibiose (Fig

h). All novel isolates failed to grow on L-sorbose, similar to WSM1271. The most

prominent differences in carbon source utilisation between the novel isolates and

WSM1271 are as follows. All novel isolates grew on β-gentibiose while all but N18

grew on N-acetyl glucosamine, arbutine, lactose, sucrose, D-tagatose and D-

turanose. In fact, N18 failed to grow on 11 (N-acetyl glucosamine, arbutine,

dulcitol, lactose, maltose, D-raffinose, L-sorbose, sucrose, D-tagatose, trehalose

and D-turanose) of the carbon sources tested (Fig d,f,g,i,j,k,l,m,n). N18 and N39

were the only novel isolates unable to grow on maltose and trehalose. While N39

and N87 were the only novel isolates able to grow on D-raffinose, N39 was the

only isolate able to grow on dulcitol. None of the novel isolates shared identical

carbon source utilisation patterns to each other.

Carbon source utilisation pattern of control species: The three control

strains had good growth on arbutine, β-gentibiose, lactose, maltose, sucrose, D-

tagatose, trahalose and D-turanose. However, variation in growth was observed

between these control strains for the other carbon sources. Strain NZP2213 had

Chapter 3

85

poor growth on N-acetyl glucosamine, β-gentibiose, melibiose, and D-raffinose

while strains USDA1002 and USDA2370 grew well on these two carbon sources.

Furthermore, strain USDA1002 grew poorly on arabinose while the other two

control strains grew well on this substance. Strain USDA2370 could not utilize L-

sorbose (similar to WSM1271) while the other two control strains grew well on L-

sorbose.

3.3.3 Antibiotic resistance

Strain WSM1271 was not resistant to ampicillin (50 µg/ml), chloramphenicol

(40 µg/ml), spectinomycin (50 µg/ml) and tetracycline (20 µg/ml) while it was

resistant to gentamicin (40 µg/ml), kanamycin (50 µg/ml), nalidixic acid (50 µg/ml)

and streptomycin (100 µg/ml) (Appendix 6). The seven re-isolates had identical

antibiotic resistance patterns to WSM1271 (Appendix 6).

The novel isolates also shared identical antibiotic resistance patterns for all

the antibiotics tested except streptomycin (100 µg/ml) (Appendix 7). Novel isolates

N17, N18, N46 and N87 were not resistant to streptomycin (100 µg/ml) while the

other novel isolates were resistant to this antibiotic. Novel isolate N45 displayed

very slight growth at the 10-1 dilution indicating, that there may have been a few

mutant cells that gained antibiotic resistance while the majority remained

susceptible for streptomycin (100 µg/ml). Novel isolates and re-isolates displayed

similar antibiotic resistance patterns except for streptomycin (100 µg/ml).

Chapter 3

86

The three control species NZP2213 (M. loti), USDA1002 (S. meliloti) and

USDA2370 (R. leguminosarum) were susceptible to chloramphenicol (40 µg/ml)

and tetracycline (20 µg/ml) (Appendix 7). Strains NZP2213 and USDA2370 had

low resistance to nalidixic acid (50 µg/ml; as some colonies appeared in the 10-1

and 10-3 dilutions) and good resistance to ampicillin (50 µg/ml) and streptomycin

(100 µg/ml) while strain NZP2213 was fully susceptible to these three antibiotics

(Appendix 7). Furthermore, strains NZP2213 and USDA1002 were resistant to

gentamicin (40 µg/ml) and kanamycin (50 µg/ml) while strain USDA2370 was

susceptible to these two antibiotics (Appendix 7). Strain USDA1002 was the only

control species able to grow on spectinomycin (50 µg/ml) (Appendix 7).

3.3.4 pH range

WSM1271, the seven novel isolates and the seven re-isolates did not grow

at pH 4.5 while they all grew at pH 5.0, 7.0 and 9.0. However, novel isolate N45

displayed growth only at the 10-1 dilution for pH 9.0 (Appendix 8).

3.3.5 Host range

WSM1271 nodulated B. pelecinus and Astragalus membranaceus. Among

the novel isolates, only isolate N17 exhibited the same host-range as WSM1271

while the other three isolates had broader host-ranges. Isolates N18, N45 and N87

formed small white nodules on M. atropurpureum, in addition to nodulating B.

pelecinus, and A. membranaceus. Isolates N18 and N45 also nodulated A.

Chapter 3

87

adsurgens while N45 was the only isolate to nodulate L. edulis. Isolate N87 was

the only isolate to nodulate A. fruticosa. The original inoculant WSM1271 and the

four novel isolates failed to nodulate the following species: Amorpha fruticosa,

Astragalus adsurgens, A. sinicus, Dorycnium rectum, Glycine uralensis,

Hedysarum spinosissimum, Leucaena leucocephala, Lotus corniculatus, L. glaber,

L. hisipidus, L. maroccanus, L. ornithopodioides, L. pedunculatus, L. peregrinus, L.

subbiflorus, Ornithopus sativus and Trifolium lupinaster (Table 3.3).

The two other RNB isolated from B. pelecinus growing in the Mediterranean

also had different host ranges to WSM1271. In addition to B. pelecinus and A.

membranaceus, WSM1497 nodulated L. corniculatus and A. adsurgens. Strain

WSM1284 was very promiscuous and nodulated 15 other host species: Astragalus

adsurgens, A. membranaceus, D. rectum, D. hirsutum, G. uralensis, Leucaena

leucocephala, Lotus corniculatus, L. edulis, L. glaber, L. maroccanus, L.

ornithopodioides, L. pedunculatus, L. peregrinus, L. subbiflorus and Ornithopus

sativus. None of the RNB from B. pelecinus growing in the Mediterranean region

nodulated A. fruticosa, A. sinicus, the two cultivars of Cicer, H. spinosissimum, L.

parviflorus, M. atropurpureum or T. lupinaster, (Table 3.3).

Chapter 3

88

Table 3.3 Host range of Mesorhizobium sp. strains WSM1497, WSM1284, WSM1271 and the novel isolates N17, N18, N45 and N87

Host plant

WSM1497

WSM1284

WSM1271

N17 N18 N45 N87

Amorpha fruticosa - - - - - - + Astragalus adsurgens + + - - + + -

Astragalus membranaceus + + + + + + + Astragalus sinicus - - - - - - -

Biserrula pelecinus cv Casbah + + + + + + + Dorycnium hirsutum - + - - - - -

Dorycnium rectum - + - - - - -

Glycyrrhiza uralensis - + - - - - -

Hedysarum spinosissimum - - - - - - -

Leucaena leucocephala - + - - - - -

Lotus corniculatus + + - - - - -

Lotus edulis - + - - - - -

Lotus glaber - + - - - - -

Lotus parviflorus - - - - - - -

Lotus maroccanus - + - - - - -

Lotus ornithopodioides - + - - - - -

Lotus pedunculatus - + - - - - -

Lotus peregrinus - + - - - + -

Lotus subbiflorus - + - - - - -

Macroptilium atropurpureum - - - - + + + Ornithopus sativus - + - - - - -

+ Nodules present, - nodules absent

Chapter 3

89

3.4 Discussion

The key significance of this study was the observation of poorly effective

and ineffective strains capable of nodulating the exotic legume B. pelecinus six

years after its introduction to Western Australia. Plants nodulated by all seven

novel isolates yielded less than 40% of the dry weight of plants inoculated with

WSM1271 (P<0.01, Fig. 3.5) and in fact two novel isolates (N15 and N45) were

completely ineffective on B. pelecinus.

N2 fixation is governed by nif genes and fix genes (Section 1.2.5.). The

results of this study imply that these novel isolates may have different nif genes

and fix genes to those of WSM1271, or that these genes are regulated differently in

these isolates and in the case of N15 and N45, the nif and fix genes may even be

absent.

Successful establishment of an exotic legume often depends on the survival

of its inoculant RNB, as naturalised strains are often unable to nodulate or fix N2

effectively (Brockwell & Bottomley, 1995; Howieson, 1999). Therefore, it is quite

critical to establish whether an inoculant strain retains its ability to nodulate and fix

N2. The results of this study show that the re-isolates from B. pelecinus at Northam

formed nodules that were similar to those of WSM1271 and other RNB isolated

from B. pelecinus growing in the Mediterranean basin (Nandasena et al., 2004),

and they were able to fix N2 as efficiently as WSM1271. Therefore, it is clearly

seen that the original inoculant WSM1271 has maintained its ability to nodulate

and fix N2 over a period of six years. This could be an advantage for the successful

establishment of B. pelecinus.

Chapter 3

90

Although 92% of the nodules on B. pelecinus at Northam were occupied by

WSM1271 (Chapter 2), the novel isolates could be competitive for nodulation of B.

pelecinus as they were able to occupy nodules in the presence of the original

inoculant WSM1271. Future research on investigating the level of competitiveness

of these novel isolates to nodulate B. pelecinus may provide valuable agronomic

information for the successful management of this legume species.

Carbon metabolism requires a number of genes (Lin, 1987) and the

biochemical pathways used to utilise a particular carbon source vary between

bacteria. For example, S. meliloti has the L-arabinose pathway leading to α–

ketoglutarate rather than to glycolaldehyde and pyruvate which is reported for B.

japonicum (Duncan & Fraenkel, 1979). A further complication is that there are

sometimes alternate pathways for the utilisation of a single carbon source (De

Smet et al., 2000; Wolf et al., 2003) in a single strain. Reports on the biochemical

pathways used by the members of the genus Mesorhizobium for the utilisation of

the carbon sources used in this study are scarce. Therefore, it is difficult to

speculate why a certain isolate can or cannot utilize a particular carbon source.

The seven re-isolates and WSM1271 had quite similar carbon source

utilisation patterns. Interestingly, differences were observed between some re-

isolates and WSM1271 for the utilisation of N-acetyl glucosamine, dulcitol, β-

gentibiose, lactose and D-raffinose. The molecular fingerprinting methods used in

Chapter two (Richardson et al., 1995; de Bruijn, 1992) amplified the genome in a

few directed locations which resulted in some very small (<250 bp) and some very

large (>5000 bp) DNA fragments. The resolution of agarose gel electrophoresis is

not powerful enough to detect single gene transfers, transfer of a small amount of

Chapter 3

91

genetic material or any mutations occurring on individual genes which are all

possible means to alter the utilisation of a carbon source (Lin, 1987). Although

these re-isolates displayed very similar molecular fingerprints to WSM1271

(Chapter 2), as discussed previously, it is possible that some genetic changes may

have occurred over six years in some of the re-isolates in the field.

Another intriguing observation is the inability of WSM1271 and all of the re-

isolates to utilize the three disaccharides maltose (Fig 3.6g), sucrose (Fig 3.6k) and

trehalose (Fig 3.6m). Maltose consists of two α-D-glucose molecules with the

alpha bond at carbon one of one molecule attached to the oxygen at carbon four of

the second molecule while trehalose has two α-D-glucose molecules connected

through carbon number one of both molecules. Sucrose is a disaccharide

composed of glucose and fructose. In S. meliloti, a single transport system is used

for the uptake of maltose, sucrose and trehalose (Glenn & Dilworth, 1981; Willis &

Walker, 1999). The fact that three of the RNB isolated from B. pelecinus growing in

the Mediterranean region are able to utilize the above three disaccharides

(Nandasena et al., 2001) while strain WSM1271 is unable to utilize these

compounds indicates that the biserrula RNB may have an uptake system similar to

that observed for S. meliloti and that mutations are present on the genes involved

in this uptake system in strain WSM1271 or these genes are absent in this strain.

However molecular and biochemical experiments are needed to draw firm

conclusions in this regard.

According to the description given for the genus Mesorhizobium by Jarvis et

al., (1997), all species included in this genus assimilate sucrose. However, some

strains of M. tianshanense are not able to utilize sucrose (Chen et al., 1995). The

Chapter 3

92

ability to utilize maltose or trehalose is not known for all the species of

Mesorhizobium.

Significant differences were observed between the novel isolates and

WSM1271 for the utilisation of many carbon sources in this study. These results

confirm that the novel isolates are not diverging representatives of WSM1271 as

they were genetically, phylogenetically and phenotypically distinct strains from

WSM1271.

Howieson et al., (1995) reported that the RNB isolated from B. pelecinus

were unable to grow at pH 4.0, but showed pronounced growth at pH 5.0, up to pH

8.0. The results of this study are in agreement with the above observations and this

study further demonstrates that RNB isolated from B. pelecinus are also capable of

growth up to pH 9.0 while they are not able to grow at pH 4.5.

A. fruticosa (Wang, et al., 1999c), A. adsurgens (Laguerre et al. 1997; Gao

et al., 2001; Wei et al., 2003), A. membranaceus (Wang & Chen, 1996; Laguerre et

al. 1997) and M. atropurpureum (Jarvis et al. 1982; Trinick & Hadobas, 1989) are

known to be nodulated by chromosomally diverse RNB and therefore appear to be

promiscuous hosts. One could assume therefore, that the ability of the novel

isolates to nodulate these hosts may be due to the promiscuity of the plant.

However, whether these chromosomally diverse strains able to nodulate the above

legumes also harbour diverse symbiotic genes is as yet unknown. Not all four

novel isolates nodulated these legume hosts, implying that the genes of the

isolates played an important role in selecting the legume hosts. These results also

suggest that the symbiotic genes of the four novel isolates may be different to each

other. Similarly, it is not possible to comment on whether the ability of N45 to

Chapter 3

93

nodulate L. edulis is due to the promiscuity of the plant due to the scarcity of

studies reported on the nodulation of this species.

A previous study has shown that the indigenous soil RNB populations of

south west Australia were incapable of nodulating B. pelecinus (Howieson et al.,

1995). Yet, the results obtained in Chapter 2 and 3 show that genetically and

phenotypically diverse rhizobia have nodulated B. pelecinus six years after

introduction and inoculation with a single strain. So the pertinent question here is

how did these novel isolates emerge? This question is addressed in the following

chapter.

Chapter 3

94

Chapter 4

95

4. Evidence for gene transfer from WSM1271 to other

soil bacteria in situ

4.1 Introduction

When legumes are introduced to agricultural soils, sown in monoculture

and inoculated with a high density of a selected RNB strain, the indigenous

rhizobial population may be subjected to a great selection pressure (Jenkins &

Bottomley, 1985; Bromfield et al., 1986; Demezas & Bottomley 1986a,b). As a

result some indigenous RNB may gain the ability to nodulate the new host

(Sullivan et al., 1995).

Lateral transfer of DNA is a key phenomenon driving the rapid evolution

of prokaryotes (de la Cruz & Davies, 2000; Ochman et al., 2000; Bushman,

2002; Dutta & Pan, 2002; Jain et al., 2002; Lawrence, 2002). The incongruence

observed between the phylogenies based on symbiotic and housekeeping

genes (Haukka et al., 1998; Laguerre et al., 2001; Suominen et al., 2001;

Toledob et al., 2004; Moulin et al., 2004), and the observation of the distribution

of identical sym-plasmids in diverse chromosomal backgrounds of RNB (Young

& Wexler, 1988; Souza & Eguiarte, 1997; Wernegreen et al., 1997; Wernegreen

& Riley, 1999), suggests that lateral transfer of symbiotic genes have taken

place between RNB strains. Furthermore, Sullivan et al., (1995) have

demonstrated that nodulating strains can arise through transfer of chromosomal

symbiotic genes from M. loti strains to other bacteria.

Whether there has been transfer of symbiotic genes between WSM1271

and the novel isolates from B. pelecinus was initially investigated by sequencing

Chapter 4

96

two symbiotic genes, nifH and nodA of WSM1271 and the novel isolates and

sequence comparison.

nifH and nodA were used in this study as these genes have been

sequenced for the majority of RNB identified so far (Haukka et al., 1998; Zhang

et al., 2000; Laguerre et al., 2001; Suominen et al., 2001; Moulin et al., 2004)

and therefore many homologous sequences are available to find conserved

regions to facilitate the development of primers for the strains used in this study.

They are both relatively small genes (< 900 bp). nifH was chosen as it encodes

structural genes of component II of nitrogenase, the enzyme involved in N2

fixation, and is present in all RNB (Rubio & Ludden, 2002). nodA was selected

as it is a common nod gene found in all RNB and plays a critical role in

determining the structure of the Nod-factor, which in turn is responsible for

determining the host range of a strain as was discussed previously

(Section1.2.3.).

The gene intS codes for a phage P4 like integrase that is responsible for

the excision and integration of the symbiosis island of M. loti (Sullivan et al.,

2002). One kb internal fragment of this gene was sequenced for WSM1271, the

novel isolates and the three RNB isolated from B. pelecinus growing in the

Mediterranean region (WSM1283, WSM1284 and WSM1497) to investigate the

possibility of the presence of a symbiosis island (Sullivan & Ronson, 1998) in

WSM1271.

This chapter reports evidence for symbiotic gene transfer between the

inoculant WSM1271 and other resident soil bacteria.

Chapter 4

97

Aim


5. To investigate whether there has been a transfer of symbiotic genes from

WSM1271 to the novel isolates.

6. To investigate the location of nodA and nifH genes in WSM1271, four

novel isolates (N17, N18, N45, N87) and three RNB strains isolated from

B. pelecinus growing in the Mediterranean (WSM1283, WSM1284,

WSM1497) region

4.2 Material and Methods

Bacterial strains used for sequencing nifH, nodA and intS: WSM1271,

WSM1283, WSM1284, WSM1497, N17, N18, N45, N87. Sequencing of nodA

was not attempted for WSM1283.

Bacterial strains investigated for localization of symbiotic genes: All

of the strains listed above were investigated for the localization of their

symbiotic genes. R. leguminosarum strain VF39 was included as positive

control as this strain has a sym-plasmid and five other plasmids (Zhang et al.,

2001b). M. loti strain R7A was used as a negative control as plasmids are

absent from this strain (Sullivan et al., 1995).

4.2.1 Genomic DNA extraction

The phenol-chloroform genomic DNA extraction protocol described by

Reeve et al., (1997) was used with modifications. For each strain 5 ml of TY

broth (Beringer, 1974), contained in McCartney bottles (capacity 30 ml), was

Chapter 4

98

inoculated with a loop-full of bacterial cells and the culture was grown to

stationary phase (4 days) at 28°C on a gyratory shaker (200 rpm). The culture

was subcultured (1:50) into fresh TY and grown overnight at 28°C on a gyratory

shaker (200 rpm). Ten ml of overnight grown culture was centrifuged at 2000 g

(Beckman Avanti JA-25) for 2 min at 20°C. The supernatant was discarded and

the pellet was resuspended in 4 ml of 70% (v/v) ethanol by vortexing vigorously.

The suspension was centrifuged at 2000 g for 2 min. The supernatant was

discarded and the pellet was resuspended in 10 ml of TES buffer (30 mM Tris-

chloride; 50 mM NaCl; 5 mM EDTA; pH 8.0) by vigorous vortexing. The

resuspended cells were centrifuged at 2000 g for 2 min and the pellet was

resuspended in 4 ml TES buffer. A volume of 800 μl of freshly prepared

lysozyme solution (10 mg/ml in TES buffer) was mixed with these cells and

incubated at 37°C for 30 min. A 550 μl aliquot of SDS (10% w/v) and 200 μl of

Proteinase K (6 mg/ml in TES, Sigma, MO, USA) were added and incubated at

45°C for 1 h. After 1 h another 200 μl aliquot of Proteinase K (6mg/ml) was

added and the mixture incubated at 55°C for 1 h. An equal volume of

phenol:chloroform:isoamylalcohol (25:24:1 v/v) was then added and the mixture

was mixed gently by inverting the tubes 4-5 times. The two phases were

separated by centrifugation at 3000 g for 10 min at 20°C. The upper aqueous

phase was transferred into a new 10 ml centrifuge tube. Two further extractions

were performed for the upper phase, first using

phenol:chloroform:isoamylalcohol and then chloroform:isoamylalcohol (24:1

v/v). The upper phase was transferred to 1.5 ml Eppendorf tube. An equal

volume of isopropanol was added into each Eppendorf tube and the tubes were

then incubated at 4°C for 1 h. These tubes were centrifuged at 21 000 g

Chapter 4

99

(Eppendorf centrifuge 5417C) for 10 min at 20°C. The supernatant was

discarded and the pellet was washed with 500 ul of 70 % (v/v) ethanol. Tubes

were air dried and the DNA pellet was resuspended in 500 μl of TE buffer (10

mM Tris; 1 mM EDTA; pH 8.0) prior to storage at -20°C.

4.2.2 Primers

Table 4.1 Details of the PCR primers used in this study

Primer Sequence 5’ 3’ Reference

nifH-1 AAGTGCGTGGAGTCCGGTGG Eardly et al., (1992)

nifH-2 GTTCGGCAAGCATCTGCTCG Eardly et al., (1992)

nifH-midF AGCCGAACACCGCCCGAATG This work

nifH-midR CTCGGGGACGTGGTTTGCG This work

nodA-KF GGTTATGCTGGGAAAATGAGTTGC This work

nodA-KR CATAGCTCTGGACCGTTCC This work

nodA-midF CGGGGTGCGTCGGGATCTTG This work

nodA-midR CCGAACCGTGCCGAAAGCGAATGG This work

msi001-KF GTGCAGCCTACCGGATCTCG This work

msi001-KR CCAGATAATCGGCCCACCAT This work

msi001-MF CGATATGCAATCGCAACCGC This work

msi001-MR CGCCCTCAGCAAGCCTCCCAA This work

Chapter 4

100

Sequences for nifH, nodA and intS of Mesorhizobium spp. were retrieved

from GenBank and aligned using Gene Tool Lite (Doubletwist, Inc). Each primer

was constructed from an internal conserved region of the targeted gene based

on sequence alignments with orthologous genes. The Gene Tool Lite was used

to select regions with melting temperatures between 50°C and 60°C for the

primers and to avoid regions which would form primer dimers or hair pin loops.

4.2.3 Sequencing of nifH

Amplification of nifH: The PCR reaction mixture contained 20 μl of 5X PCR

Polymerisation buffer [67 mM Tris-HCL (pH 8.8 at 25°C), 16 mM [NH4]2SO4,

0.45% Triton X-100, 0.2 mg/ml Gelatin, 0.2 mM dNTPs - Biotech International

Ltd. Cat # PB-1] , 1.5 mM of MgCl2, 50 μM of the primers nifH-1 and nifH-2

(Table 4.1), 2.5 U of Tth Plus* DNA polymerase (Biotech International Ltd.),

100 ng of template DNA and autoclaved miliQ water to make up to a total

volume of 100 μl of reaction mixture. Thermocycler conditions were as follows:

94°C for 5 min, denaturation at 94°C for 30 s, annealing at 57°C for 30 s and

extention at 72°C for 30 s for 25 cycles followed by a last extention at 72°C for 7

min.

Sequencing of nifH PCR product: Approximately 800 bp of the nifH gene

was amplified and then each primer was used separately to sequence the DNA.

The sequencing methodology has been described in Section 2.2.6. The

sequences generated by the two primers were aligned using Gene Tool Lite.

Another two primers, nifH-midF and nifH-midR (Table 4.1) were developed from

Chapter 4

101

the middle overlapping region of the sequences obtained by nifH-1 and nifH-2.

All of the above four primers were used to obtain a double stranded DNA

sequence of the gene.

4.2.4 Sequencing of nodA

Amplification of nodA: The PCR mixture described in Section 4.2.3 was

used to amplify an internal fragment of nodA using the primers nodA-KF and

nodA-KR (Table 4.1). The cycling conditions were as follows: 94°C for 5 min, 5

cycles of denaturation at 94°C for 30 s, annealing at 52°C for 30 s and

extension at 72°C for 1 min, 30 cycles with the annealing temperature increased

to 55°C and a final extension at 72°C for 7 min.

Sequencing of nodA PCR product: A 570 bp intergenic fragment of the

nodA gene was sequenced as described in Section 4.2.3. The primers, nodA-

midF and nodA-midR (Table 4.1) were developed in the same way as described

for nifH-midF and nifH-midR in Section 4.2.3.

4.2.5 Sequencing of intS

Amplification of intS: The PCR mixture described in Section 4.2.3 was used

with the primers msi001-KF and msi001-KR (Table 4.1). The thermocycler

conditions (Section 4.2.4) were modified to remove the 5 initial cycles and set

the annealing temperature in the remaining cycle to 62°C.

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Sequencing of intS PCR product: A 1040 bp intergenic fragment of the

intS gene was sequenced as described in Section 4.2.3. The two primers,

msi001-MF and msi001-MR (Table 4.1) were developed in the same way as

described for nifH-midF and nifH-midR in Section 4.2.3.

4.2.6 Eckhardt gel electrophoresis

Growth conditions: A modified ‘in gel lysis’ method from Hynes et al., (1985)

was used to visualize plasmids from RNB. A loop full of bacterial cells was

initially inoculated into 5 ml HP medium (Hynes et al., 1985) contained in

McCartney bottles and grown to an OD600 1.0 on a gyratory shaker (200 rpm) at

28°C. These cells were sub cultured into TY broth (Beringer, 1974) at a starting

OD600 0.05 and grown for 8-10 h on a gyratory shaker (200 rpm) at 28°C until

approximately OD600 0.2 – 0.35 was reached.

Preparation of Eckhardt gel: A 0.7 % (w/v) agarose gel was made in TBE

(90 mM Tris-base, 90 mM Boric acid, 2.5 mM EDTA; pH 8.3) with 10% (w/v)

SDS, and poured to a thickness of 5 mm. A single comb set up was used.

Cell lysis and agarose gel electrophoresis: A volume of 200 μl of

bacterial culture was mixed with 1 ml of 0.3% (w/v) Sarkosyl made in TBE in an

Eppendorf tube. The Eppendorf tube was placed on ice for 10 min and then

centrifuged at 20, 800 g for 5 min at 4°C in a microfuge. The supernatant was

discarded and the pellet gently resuspended in 25 μl of lysis solution [0.2 mg/ml

lysozyme, sucrose 10% (w/v), an Eppendorf tube in TBE]. An aliquot of 20 μl

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was loaded into the well and electrophoresis was carried out at 5 - 10 V for 30

min and then at 50 V for 12 - 15 h.

Gel visualization: The gel was stained in 0.5 μg/ ml ethydium bromide (EtBr)

for 40 min and then destained in distilled water for 20 min in the cold room. The

bands were visualised with UV and image captured with GelDoc -2000

documentation system (BioRad).

4.2.7 Southern hybridization of mobilized plasmids

Preparation of probes: A nifH probe was developed by amplifying the nifH

gene of WSM1271 as described in Section 4.2.3., and the amplified gene

product was gel purified using MinEluteTM gel extraction kit (QIAGEN) according

to manufacturer’s instruction. Re-amplification of the same gene was performed

using 1 μl of the purified gene product. This step prevented the amplification of

other ambiguous products. The amplified gene was purified using QIAquickTM

PCR purification kit (QIAGEN) according to manufacturer’s instruction and the

amount of DNA in the PCR product was estimated by the ethidium bromide dot

quantification method (Ausubel et al., 1992). DIG labeling was performed by

mixing 100 ng of the gene product and 4 μl of DIG High Prime labeling mixture

(Cat. No. 1585606, Roche Diagnostics, VIC, Australia) in a final volume of 20 μl

and incubating at 37°C overnight. The labelled probe was purified using

MinEluteTM PCR purification kit (QIAGEN) according to the manufacturer’s

instruction.

The nodA probe was developed in the same way as the nifH probe and

the PCR conditions for gene amplification have been described in Section 4.2.4.

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A nodC probe was prepared from S. medicae strain WSM419 and this

served as a positive control in the hybridisation to detect the nodC of the control

strain R. leguminosarum bv. viciae strain VF39. The PCR mixture and cycling

conditions were as described in Section 2.2.6 except that the primers 251F and

566R (Ueda et al., 1995) were used. Probe preparation was performed as

described for nifH.

Southern hybridization: The Eckhardt gel was denatured, blotted and UV-

cross-linked onto a nylon membrane (Boehringer Mannheim). Prehybridization

and hybridization was performed at 42°C. A probe mixture containing 7 μl of

each of the 3 probes (nifH, nodA and nodC) was used with 20 ml of

hybridization buffer for hybridization. High stringency washing was done at

68°C. The chemiluminescent substrate CSPD (Roche) was used to detect the

hybridized digoxigenin-labelled probe (Tiwari et al., 1996).

4.3 Results

4.3.1 Sequencing of nifH

The four novel isolates (N17, N18, N45, N87) had identical nifH

sequences to that of WSM1271 in a 710 bp internal fragment of nifH (Fig 4.1).

By contrast the other three RNB isolates from B. pelecinus growing in the

Mediterranean region (WSM1497, WSM1283 and WSM1284) had only 99.4%,

99.3% and 93% sequence similarity to WSM1271 respectively.

nifH sequences retrieved from GenBank for all the available RNB

species within the α-Proteobacteria were compared with that of WSM1271.

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105

Interestingly, nifH of R. gallicum had the highest sequence similarity (89.7%) to

WSM1271 while all the available members of Mesorhizobium had <87.5%

sequence similarity with WSM1271 for this gene (Table 4.2). Strain WSM1271

was least similar to B. japonicum having only 77.2% sequence similarity for this

gene.

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N17 AATAATAAGCTTATCCTGATCGTCGGCTGCGACCCCAAAGCGGATTCCACCCGCCTGA 58 N18 AATAATAAGCTTATCCTGATCGTCGGCTGCGACCCCAAAGCGGATTCCACCCGCCTGA 58 N45 AATAATAAGCTTATCCTGATCGTCGGCTGCGACCCCAAAGCGGATTCCACCCGCCTGA 58 N87 AATAATAAGCTTATCCTGATCGTCGGCTGCGACCCCAAAGCGGATTCCACCCGCCTGA 58 WSM1271 AATAATAAGCTTATCCTGATCGTCGGCTGCGACCCCAAAGCGGATTCCACCCGCCTGA 58 WSM1283 AATAATAAGCTTATCCTGATCGTCGGCTGCGACCCCAAAGCGGATTCCACCCGCCTGA 58 WSM1284 AATAATAAGCTTATCCTGATCGTCGGCTGCGACCCCAAAGCCGACTCCACCCGCCTGA 58 WSM1497 AATAATAAGCTTATCCTGATCGTCGGCTGCGACCCCAAAGCGGATTCCACCCGCCTGA 58 N17 TCCTGAACTCGAAGGCTCAGGATACTGTCCTGCATCTCGCGGCACAGGAAGGTTCGGT 116 N18 TCCTGAACTCGAAGGCTCAGGATACTGTCCTGCATCTCGCGGCACAGGAAGGTTCGGT 116 N45 TCCTGAACTCGAAGGCTCAGGATACTGTCCTGCATCTCGCGGCACAGGAAGGTTCGGT 116 N87 TCCTGAACTCGAAGGCTCAGGATACTGTCCTGCATCTCGCGGCACAGGAAGGTTCGGT 116 WSM1271 TCCTGAACTCGAAGGCTCAGGATACTGTCCTGCATCTCGCGGCACAGGAAGGTTCGGT 116 WSM1283 TCCTGAACTCGAAGGCTCAGGATACTGTCCTGCATCTCGCGGCACAGGAAGGTTCGGT 116 WSM1284 TCCTGAACTCGAAGGCTCAGGATACTGTCCTGCATCTGGCGGCACAGGAAGGTTCGGT 116 WSM1497 TCCTGAACTCGAAGGCTCAGGATACTGTCCTGCATCTCGCGGCACAGGAAGGTTCGGT 116 N17 GGAAGACCTCGAGCTTCAGGACGTGCTTAAGGTAGGCTACAGAGGCATCAAGTGCGTG 174 N18 GGAAGACCTCGAGCTTCAGGACGTGCTTAAGGTAGGCTACAGAGGCATCAAGTGCGTG 174 N45 GGAAGACCTCGAGCTTCAGGACGTGCTTAAGGTAGGCTACAGAGGCATCAAGTGCGTG 174 N87 GGAAGACCTCGAGCTTCAGGACGTGCTTAAGGTAGGCTACAGAGGCATCAAGTGCGTG 174 WSM1271 GGAAGACCTCGAGCTTCAGGACGTGCTTAAGGTAGGCTACAGAGGCATCAAGTGCGTG 174 WSM1283 GGAAGACCTCGAGCTTCAGGACGTGCTCAAGGTAGGCTACAGAGGCATCAAGTGCGTG 174 WSM1284 GGAAGATCTCGAACTGCAGGACGTGCTCAAGATTGGCTACAGAGGCATCAAATGCGTG 174 WSM1497 GGAAGACCTCGAGCTTCAGGACGTGCTCAAGGTAGGCTACAGAGGCATCAAGTGCGTG 174 N17 GAGTCCGGCGGCCCGGAGCCGGGTGTGGGCTGCGCCGGCCGCGGCGTCATCACCTCAA 232 N18 GAGTCCGGCGGCCCGGAGCCGGGTGTGGGCTGCGCCGGCCGCGGCGTCATCACCTCAA 232 N45 GAGTCCGGCGGCCCGGAGCCGGGTGTGGGCTGCGCCGGCCGCGGCGTCATCACCTCAA 232 N87 GAGTCCGGCGGCCCGGAGCCGGGTGTGGGCTGCGCCGGCCGCGGCGTCATCACCTCAA 232 WSM1271 GAGTCCGGCGGCCCGGAGCCGGGTGTGGGCTGCGCCGGCCGCGGCGTCATCACCTCAA 232 WSM1283 GAGTCCGGCGGCCCGGAGCCGGGTGTAGGCTGCGCCGGCCGCGGCGTCATCACCTCAA 232 WSM1284 GAGTCCGGCGGCCCGGAGCCGGGTGTTGGCTGCGCGGGCCGAGGCGTCATCACATCAA 232 WSM1497 GAGTCCGGCGGCCCGGAGCCGGGTGTAGGCTGCGCCGGCCGCGGCGTCATCACCTCAA 232 N17 TCAATTTCCTTGAGGAGAACGGCGCCTACGACGATGTCGACTATGTCTCCTACGACGT 290 N18 TCAATTTCCTTGAGGAGAACGGCGCCTACGACGATGTCGACTATGTCTCCTACGACGT 290 N45 TCAATTTCCTTGAGGAGAACGGCGCCTACGACGATGTCGACTATGTCTCCTACGACGT 290 N87 TCAATTTCCTTGAGGAGAACGGCGCCTACGACGATGTCGACTATGTCTCCTACGACGT 290 WSM1271 TCAATTTCCTTGAGGAGAACGGCGCCTACGACGATGTCGACTATGTCTCCTACGACGT 290 WSM1283 TCAATTTCCTTGAGGAGAACGGCGCCTACGACGATGTCGACTATGTCTCCTACGACGT 290 WSM1284 TCAACTTCCTCGAGGAGAACGGCGCCTATGACGATGTCGACTATGTCTCCTACGACGT 290 WSM1497 TCAATTTCCTTGAGGAGAACGGCGCCTACGACGATGTCGACTATGTCTCCTACGACGT 290 N17 GCTCGGGGACGTGGTTTGCGGCGGTTTCGCGATGCCGATCCGCGAGGGCAAGGCGCAG 348 N18 GCTCGGGGACGTGGTTTGCGGCGGTTTCGCGATGCCGATCCGCGAGGGCAAGGCGCAG 348 N45 GCTCGGGGACGTGGTTTGCGGCGGTTTCGCGATGCCGATCCGCGAGGGCAAGGCGCAG 348 N87 GCTCGGGGACGTGGTTTGCGGCGGTTTCGCGATGCCGATCCGCGAGGGCAAGGCGCAG 348 WSM1271 GCTCGGGGACGTGGTTTGCGGCGGTTTCGCGATGCCGATCCGCGAGGGCAAGGCGCAG 348 WSM1283 GCTCGGGGACGTGGTTTGCGGCGGTTTCGCGATGCCGATCCGCGAGGGCAAGGCGCAG 348 WSM1284 GCTCGGCGACGTGGTTTGCGGCGGTTTCGCGATGCCGATCCGCGAGGGCAAGGCGCAG 348 WSM1497 GCTCGGGGACGTGGTTTGCGGCGGTTTCGCGATGCCGATCCGCGAGGGCAAGGCGCAG 348 N17 GAAATCTATATCGTCATGTCCGGGGAGATGATGGCGCTCTATGCCGCCAATAATATCG 406 N18 GAAATCTATATCGTCATGTCCGGGGAGATGATGGCGCTCTATGCCGCCAATAATATCG 406 N45 GAAATCTATATCGTCATGTCCGGGGAGATGATGGCGCTCTATGCCGCCAATAATATCG 406 N87 GAAATCTATATCGTCATGTCCGGGGAGATGATGGCGCTCTATGCCGCCAATAATATCG 406 WSM1271 GAAATCTATATCGTCATGTCCGGGGAGATGATGGCGCTCTATGCCGCCAATAATATCG 406 WSM1283 GAAATCTATATCGTCATGTCCGGGGAGATGATGGCGCTCTATGCCGCTAATAATATCG 406 WSM1284 GAAATCTACATCGTCATGTCCGGGGAGATGATGGCGCTCTATGCCGCCAACAACATCG 406 WSM1497 GAAATCTATATCGTCATGTCCGGGGAGATGATGGCGCTCTATGCCGCCAATAATATCG 406

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N17 CCAAGGGCATCCTGAAATATGCACATTCGGGCGGTGTTCGGCTCGGCGGACTGATCTG 464 N18 CCAAGGGCATCCTGAAATATGCACATTCGGGCGGTGTTCGGCTCGGCGGACTGATCTG 464 N45 CCAAGGGCATCCTGAAATATGCACATTCGGGCGGTGTTCGGCTCGGCGGACTGATCTG 464 N87 CCAAGGGCATCCTGAAATATGCACATTCGGGCGGTGTTCGGCTCGGCGGACTGATCTG 464 WSM1271 CCAAGGGCATCCTGAAATATGCACATTCGGGCGGTGTTCGGCTCGGCGGACTGATCTG 464 WSM1283 CCAAGGGCATCCTGAAATATGCACATTCGGGCGGCGTTCGGCTCGGCGGACTGATCTG 464 WSM1284 CCAAGGGCATCCTGAAATATGCCCATTCGGGGGGCGTGCGGCTCGGAGGCCTGATCTG 464 WSM1497 CCAAGGGCATCCTGAAATATGCACATTCGGGCGGTGTTCGGCTCGGCGGACTGATCTG 464 N17 TAACGAGCGTCAGACCGACCGTGAGCTTGACCTGGCCGAAGCACTGGCTTCCAGGCTC 522 N18 TAACGAGCGTCAGACCGACCGTGAGCTTGACCTGGCCGAAGCACTGGCTTCCAGGCTC 522 N45 TAACGAGCGTCAGACCGACCGTGAGCTTGACCTGGCCGAAGCACTGGCTTCCAGGCTC 522 N87 TAACGAGCGTCAGACCGACCGTGAGCTTGACCTGGCCGAAGCACTGGCTTCCAGGCTC 522 WSM1271 TAACGAGCGTCAGACCGACCGTGAGCTTGACCTGGCCGAAGCACTGGCTTCCAGGCTC 522 WSM1283 TAACGAGCGTCAGACCGACCGTGAGCTTGACCTGGCCGAAGCACTGGCTTCCAGGCTC 522 WSM1284 CAACGAACGTCAAACCGACCGTGAGCTCGACCTTGCTGAAGCCCTGGCTTCCAGGCTC 522 WSM1497 TAACGAGCGTCAGACCGACCGTGAGCTTGACCTGGCCGAAGCACTGGCTTCCAGGCTC 522 N17 AACTCCAAGCTCATCCACTTCGTGCCGCGCGACAACATCGTCCAGCACGCCGAGCTCA 580 N18 AACTCCAAGCTCATCCACTTCGTGCCGCGCGACAACATCGTCCAGCACGCCGAGCTCA 580 N45 AACTCCAAGCTCATCCACTTCGTGCCGCGCGACAACATCGTCCAGCACGCCGAGCTCA 580 N87 AACTCCAAGCTCATCCACTTCGTGCCGCGCGACAACATCGTCCAGCACGCCGAGCTCA 580 WSM1271 AACTCCAAGCTCATCCACTTCGTGCCGCGCGACAACATCGTCCAGCACGCCGAGCTCA 580 WSM1283 AACTCCAAGCTCATCCACTTCGTGCCGCGCGACAACATCGTCCAGCACGCCGAGCTCA 580 WSM1284 AATTCCAAGCTCATCCACTTCGTGCCGCGCGACAACATCGTTCAGCACGCCGAGCTCA 580 WSM1497 AACTCCAAGCTCATCCACTTCGTGCCGCGCGACAACATCGTCCAGCACGCCGAGCTCA 580 N17 GAAAGATGTCAGTGATCCAATATGCGCCCGACTCCAAGCAGGCCGGAGAATACCGCGC 638 N18 GAAAGATGTCAGTGATCCAATATGCGCCCGACTCCAAGCAGGCCGGAGAATACCGCGC 638 N45 GAAAGATGTCAGTGATCCAATATGCGCCCGACTCCAAGCAGGCCGGAGAATACCGCGC 638 N87 GAAAGATGTCAGTGATCCAATATGCGCCCGACTCCAAGCAGGCCGGAGAATACCGCGC 638 WSM1271 GAAAGATGTCAGTGATCCAATATGCGCCCGACTCCAAGCAGGCCGGAGAATACCGCGC 638 WSM1283 GAAAGATGTCAGTGATCCAATATGCGCCCGACTCCAAGCAGGCCGGAGAATACCGCGC 638 WSM1284 GGAAGATGTCAGTTATCCAGTACGCGCCGGATTCCAAGCAGGCCGGGGAGTACCGCGC 638 WSM1497 GAAAGATGTCAGTGATCCAATATGCGCCCGACTCCAAGCAGGCCGGAGAATACCGCGC 638 N17 GCTGGCTGAGAAGATACATGCGAATTCTGGCCAGGGTACCATCCCGACCCCGATCACC 696 N18 GCTGGCTGAGAAGATACATGCGAATTCTGGCCAGGGTACCATCCCGACCCCGATCACC 696 N45 GCTGGCTGAGAAGATACATGCGAATTCTGGCCAGGGTACCATCCCGACCCCGATCACC 696 N87 GCTGGCTGAGAAGATACATGCGAATTCTGGCCAGGGTACCATCCCGACCCCGATCACC 696 WSM1271 GCTGGCTGAGAAGATACATGCGAATTCTGGCCAGGGTACCATCCCGACCCCGATCACC 696 WSM1283 GCTGGCTGAGAAGATCCATGCGAATTCTGGCCAGGGTACCATCCCGACCCCGATCACC 696 WSM1284 GCTGGCCGAGAAGATCCATGCCAATTCTGGCCAGGGCACCATCCCGACCCCGATCACC 696 WSM1497 GCTTGCTGAGAAGATCCATGCGAATTCTGGCCAGGGTACCATCCCGACCCCGATCACC 696 N17 ATGGAGGAGCTCGA 710 N18 ATGGAGGAGCTCGA 710 N45 ATGGAGGAGCTCGA 710 N87 ATGGAGGAGCTCGA 710 WSM1271 ATGGAGGAGCTCGA 710 WSM1283 ATGGAGGAGCTCGA 710 WSM1284 ATGGAGGAGCTCGA 710 WSM1497 ATGGAGGAGCTCGA 710 Figure 4.1 nifH sequence alignment of WSM1271, the novel isolates (N17, N18, N87, N45) and RNB isolated from B. pelecinus growing in the Mediterranean region (WSM1283, WSM1284, WSM1497). Nucleotide mismatches are in red.

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Table 4.2 nifH sequence similarity of WSM1271, N17, N18, N87, N45, WSM1283, WSM1284 and WSM1497 against the type strains of some genera of RNB in α-Proteobacteria and some species of Mesorhizobium

Organism

WSM1271, N17, N18, N45, N87

WSM1283 WSM1284 WSM1497

WSM1283

99.3% 100% 93.0% 99.6%

WSM1497

99.4% 99.6% 92.1% 100%

WSM1284

93.0% 93.0% 100% 92.1%

Mesorhizobium loti

ML0672114 86.9% 88.0% 89.0% 88.1%

M. ciceri

AY318755 83.8% 84.5% 88.4% 88.8%

M. mediterraneum

AJ457917 85.0% 85.7% 88.7% 89.3%

M. amorphae

AF484651 89.7% 89.9% 90.6% 89.9%


AE007235 83.8% 84.7% 83.8% 85.5%

R. leguminosarum

M55228 85.8% 86.0% 80.9% 81.9%


AP005941 76.1% 76.6% 74.7% 76.7%


AJ563960 71.1% 71.3% 71.1% 67.0%

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4.3.2 Sequencing of nodA

The four novel isolates (N17, N18, N45, N87) had 100% sequence

similarity with WSM1271 in a 567 bp internal fragment of nodA (Fig 4.2). A RNB

strain isolated from B. pelecinus growing in Greece, WSM1497, had only 97.5%

sequence similarity to WSM1271 which was isolated from B. pelecinus growing

in Sardinia. Strain WSM1284, also isolated from B. pelecinus growing in

Sardinia, produced an approximately 500 bp product when amplified with the

nodA-KF and nodA-KR. However, attempts to sequence nodA using the above

two primers for WSM1284 failed due to the presence of a lot of non-specific

bases in the sequence contigues. Further attempts were not made to sequence

this strain due to time and money constraints.

All the available sequences of nodA were retrieved from GenBank for

species in the family Rhizobiaceae and these were compared with that of

WSM1271 (Table 4.3). There were no other nodA sequences that gave >77%

sequence similarity to that of WSM1271. M. ciceri strain USDA3383 gave the

highest sequence similarity to WSM1271 (77.9%). Strain WSM1271 was least

similar to Azorhizobium caulinodans strain ORS590 having only 62.3%

sequence similarity for this gene (Table 4.3).

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N17 GGTTATGCTGGGAAAATGAGTTGCAACGGCATGAGCATGTCGAGCTCTCAGACTTCTT 58 N18 GGTTATGCTGGGAAAATGAGTTGCAACGGCATGAGCATGTCGAGCTCTCAGACTTCTT 58 N45 GGTTATGCTGGGAAAATGAGTTGCAACGGCATGAGCATGTCGAGCTCTCAGACTTCTT 58 N87 GGTTATGCTGGGAAAATGAGTTGCAACGGCATGAGCATGTCGAGCTCTCAGACTTCTT 58 WSM1271 GGTTATGCTGGGAAAATGAGTTGCAACGGCATGAGCATGTCGAGCTCTCAGACTTCTT 58 WSM1497 GGTTATGCGGGAAAATGAGTTGCAACTGCATGAGCATGTCGAGCTGCTCAGACTTCTT 58 N17 GCACAAAACCTATGGAGGGACTGGCACCTTCTCCGCAATGCGATTCGAAGGCGGGCGG 116 N18 GCACAAAACCTATGGAGGGACTGGCACCTTCTCCGCAATGCGATTCGAAGGCGGGCGG 116 N45 GCACAAAACCTATGGAGGGACTGGCACCTTCTCCGCAATGCGATTCGAAGGCGGGCGG 116 N87 GCACAAAACCTATGGAGGGACTGGCACCTTCTCCGCAATGCGATTCGAAGGCGGGCGG 116 WSM1271 GCACAAAACCTATGGAGGGACTGGCACCTTCTCCGCAATGCGATTCGAAGGCGGGCGG 116 WSM1497 GCACAAAACCTATGGAGGGACTGGCACCTTCTCCGCAATGCGATTCGAAGGCGGCCGC 116 N17 AGTTGGTTCGGCGCGAGGCCAGAGCTCCGAGCAATTGGTAGGGACACGCACGGTGTAG 174 N18 AGTTGGTTCGGCGCGAGGCCAGAGCTCCGAGCAATTGGTAGGGACACGCACGGTGTAG 174 N45 AGTTGGTTCGGCGCGAGGCCAGAGCTCCGAGCAATTGGTAGGGACACGCACGGTGTAG 174 N87 AGTTGGTTCGGCGCGAGGCCAGAGCTCCGAGCAATTGGTAGGGACACGCACGGTGTAG 174 WSM1271 AGTTGGTTCGGCGCGAGGCCAGAGCTCCGAGCAATTGGTAGGGACACGCACGGTGTAG 174 WSM1497 AGTTGGTTCGGCGCGAGGCCAGAGTTCCGCGCAATTGGTAGGGACACGCACGGTGTAG 174 N17 CGGCACATCTCGGTCTACTGCGCCGTTTCATCAAAGTTGGCGCAATCGACGTGCTGGT 232 N18 CGGCACATCTCGGTCTACTGCGCCGTTTCATCAAAGTTGGCGCAATCGACGTGCTGGT 232 N45 CGGCACATCTCGGTCTACTGCGCCGTTTCATCAAAGTTGGCGCAATCGACGTGCTGGT 232 N87 CGGCACATCTCGGTCTACTGCGCCGTTTCATCAAAGTTGGCGCAATCGACGTGCTGGT 232 WSM1271 CGGCACATCTCGGTCTACTGCGCCGTTTCATCAAAGTTGGCGCAATCGACGTGCTGGT 232 WSM1497 CGGCACATCTCGGTCTACTGCGCCGGTTCATCAAAGTTGGCGAAATCGACGTGCTGGT 232 N17 GGCCGAACTCGGATTGTACGGGGTGCGTCGGGATCTTGAGGGCCTCGGCATCAGCTTG 290 N18 GGCCGAACTCGGATTGTACGGGGTGCGTCGGGATCTTGAGGGCCTCGGCATCAGCTTG 290 N45 GGCCGAACTCGGATTGTACGGGGTGCGTCGGGATCTTGAGGGCCTCGGCATCAGCTTG 290 N87 GGCCGAACTCGGATTGTACGGGGTGCGTCGGGATCTTGAGGGCCTCGGCATCAGCTTG 290 WSM1271 GGCCGAACTCGGATTGTACGGGGTGCGTCGGGATCTTGAGGGCCTCGGCATCAGCTTG 290 WSM1497 GGCCGAACTCGGATTGTACGGGGTGCGTAGGGATCTTGAGGGCCTCGGCATCAGCTTG 290 N17 TCCCTGCGCAGTCTGTATCCGGCGCTGCAGCAGATGGGCGTTCCATTCGCTTTCGGCA 348 N18 TCCCTGCGCAGTCTGTATCCGGCGCTGCAGCAGATGGGCGTTCCATTCGCTTTCGGCA 348 N45 TCCCTGCGCAGTCTGTATCCGGCGCTGCAGCAGATGGGCGTTCCATTCGCTTTCGGCA 348 N87 TCCCTGCGCAGTCTGTATCCGGCGCTGCAGCAGATGGGCGTTCCATTCGCTTTCGGCA 348 WSM1271 TCCCTGCGCAGTCTGTATCCGGCGCTGCAGCAGATGGGCGTTCCATTCGCTTTCGGCA 348 WSM1497 TCCCTGCGCAGTCTGTATCCGGCGCTGCAGCAGATGGGCGTTCCATTCGCTTTCGGC- 348 N17 C-GGTTCGGCACGAAATGCGAAATCACATCCAGAGGCTCTGCAAGGTGGGGCTTGGGA 405 N18 C-GGTTCGGCACGAAATGCGAAATCACATCCAGAGGCTCTGCAAGGTGGGGCTTGGGA 405 N45 C-GGTTCGGCACGAAATGCGAAATCACATCCAGAGGCTCTGCAAGGTGGGGCTTGGGA 405 N87 C-GGTTCGGCACGAAATGCGAAATCACATCCAGAGGCTCTGCAAGGTGGGGCTTGGGA 405 WSM1271 C-GGTTCGGCACGAAATGCGAAATCACATCCAGAGGCTCTGCAAGGTGGGGCTTGGGA 405 WSM1497 CAGGTTCGGCACGAAATGCGAAATCACATCCAGAGGCTCTGCAAGGTGGGGCTTGGGA 405 N17 AGATTGTGCCGGACGTTCGCATCCGCTCAACCTTGGCAGACATGCATCCCGACCTGCC 463 N18 AGATTGTGCCGGACGTTCGCATCCGCTCAACCTTGGCAGACATGCATCCCGACCTGCC 463 N45 AGATTGTGCCGGACGTTCGCATCCGCTCAACCTTGGCAGACATGCATCCCGACCTGCC 463 N87 AGATTGTGCCGGACGTTCGCATCCGCTCAACCTTGGCAGACATGCATCCCGACCTGCC 463 WSM1271 AGATTGTGCCGGACGTTCGCATCCGCTCAACCTTGGCAGACATGCATCCCGACCTGCC 463 WSM1497 AGATTGTGCCGGACGTTCGCATCCGCTCGACCCTGGCAGACATGCATCCCGACCTGCC 463 N17 GCCCACGCGCTTGGAGGACGATCTCGTCTTTGTCTCGCCGATTGGGCGGTCGATTGAC 521 N18 GCCCACGCGCTTGGAGGACGATCTCGTCTTTGTCTCGCCGATTGGGCGGTCGATTGAC 521 N45 GCCCACGCGCTTGGAGGACGATCTCGTCTTTGTCTCGCCGATTGGGCGGTCGATTGAC 521 N87 GCCCACGCGCTTGGAGGACGATCTCGTCTTTGTCTCGCCGATTGGGCGGTCGATTGAC 521 WSM1271 GCCCACGCGCTTGGAGGACGATCTCGTCTTTGTCTCGCCGATTGGGCGGTCGATTGAC 521 WSM1497 GCCCACGCGCTTGGAGGACGATCTCCTCTTTGTCTCGCCGATTGGGCGGTCGATTGAC 521 N17 GAGTGGCCGTCCGGCACCTTCATCGAGCGGAACGGTCCAGAGCTAT 567 N18 GAGTGGCCGTCCGGCACCTTCATCGAGCGGAACGGTCCAGAGCTAT 567 N45 GAGTGGCCGTCCGGCACCTTCATCGAGCGGAACGGTCCAGAGCTAT 567 N87 GAGTGGCCGTCCGGCACCTTCATCGAGCGGAACGGTCCAGAGCTAT 567 WSM1271 GAGTGGCCGTCCGGCACCTTCATCGAGCGGAACGGTCCAGAGCTAT 567 WSM1497 GAGTGGCCGTCCGGCACCTTCATCGAGCGGAACGGTCCAGAGCTAT 567

Figure 4.2 nodA sequence alignment of WSM1271, the novel isolates (N17, N18, N87, N45) and RNB isolated from B. pelecinus growing in the Mediterranean region (WSM1497). Nucleotide mismatches are in red.

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Table 4.3 nodA sequence similarity of WSM1271, N17, N18, N87, N45, and WSM1497 against the type strains of some genera of RNB in α-Proteobacteria and some species of Mesorhizobium

Organism

WSM1271, N17, N18, N45, N87

WSM1497

WSM1497

97.5% 100%

Mesorhizobium loti (R7A)

AL672113 76.8% 75.9%

Mesorhizobium loti (MAFF)

AP003008 74.8% 73.7%

M. ciceri

AJ250140 77.0% 77.5%

M. mediterraneum

AJ250141 77.0% 77.5%

M. tianshanense

AJ250142 77.0% 77.0%

M. plurifarium

AJ302678 69.4% 69.0%


M11268 69.0% 68.2%

R. leguminosarum

X01650 67.8% 67.5%


AJ300252 70.3% 70.1%


AJ300261 62.3% 62.8%

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4.3.3 Sequencing of intS

The four novel isolates each had an identical intS sequence to that of

WSM1271 in a 1044 bp fragment of intS (Fig 4.3). The other three RNB isolates

from B. pelecinus, growing in the Mediterranean region, WSM1284, WSM1497

and WSM1283 had only 99.5%, 94.3% and 93.6% sequence similarity to

WSM1271 respectively. When a blast search was performed with the intS gene

sequence of WSM1271 at NCBI, there were no other homologous genes found

other than M. loti strains MAF303099 and R7A. These two strains gave 95%

and 93.5% sequence similarity for the intS gene of WSM1271.

N17 CCGGATCTCGCCTATGGCGCCTCGCCTATCGTTTTGGCGGCAAGCAGAAGCTTCTGG 57 N18 CCGGATCTCGCCTATGGCGCCTCGCCTATCGTTTTGGCGGCAAGCAGAAGCTTCTGG 57 N45 CCGGATCTCGCCTATGGCGCCTCGCCTATCGTTTTGGCGGCAAGCAGAAGCTTCTGG 57 N87 CCGGATCTCGCCTATGGCGCCTCGCCTATCGTTTTGGCGGCAAGCAGAAGCTTCTGG 57 WSM1271 CCGGATCTCGCCTATGGCGCCTCGCCTATCGTTTTGGCGGCAAGCAGAAGCTTCTGG 57 WSM1283 CCGGATCTCGCNTATGGCGCCTCGCCTATCGTTTTGGCGGCAAGCAGAAGCTTCTGG 57 WSM1284 CCGGATCTCGCCTATGGCGCCTCGCCTATCGTTTTGGCGGCAAGCAGAAGCTTCTGG 57 WSM1497 CCGGATCTCGCNTATGGCGCCTCGCCTATCGTTTTGGCGGCAAGCAGAAGCTTCTGG 57 N17 CGTTGGGCAGCTATCCCCTCATCTCCCTCGCTGAGGCACGCGAGGCGCGCGATACCG 114 N18 CGTTGGGCAGCTATCCCCTCATCTCCCTCGCTGAGGCACGCGAGGCGCGCGATACCG 114 N45 CGTTGGGCAGCTATCCCCTCATCTCCCTCGCTGAGGCACGCGAGGCGCGCGATACCG 114 N87 CGTTGGGCAGCTATCCCCTCATCTCCCTCGCTGAGGCACGCGAGGCGCGCGATACCG 114 WSM1271 CGTTGGGCAGCTATCCCCTCATCTCCCTCGCTGAGGCACGCGAGGCGCGCGATACCG 114 WSM1283 CGTTGGGCAGCTATCCCCTCATCTCTCTCGCTGAGGCACGCGAGGCCCGCGATACCG 114 WSM1284 CGTTGGGCAGCTATCCCCTCATCTCCCTCGCTGAGGCACGCGAGGCGCGCGATACCG 114 WSM1497 CGTTGGGCAGCTATCCGCTCATCTCCCTGGCTGAGGCACGCGAGGCGCGCGATACCG 114 N17 CCAAACGTCTCCTCCTACGTGGCATCGACCCCGCACTGGAGCGTAAGTTGCAAAAGG 171 N18 CCAAACGTCTCCTCCTACGTGGCATCGACCCCGCACTGGAGCGTAAGTTGCAAAAGG 171 N45 CCAAACGTCTCCTCCTACGTGGCATCGACCCCGCACTGGAGCGTAAGTTGCAAAAGG 171 N87 CCAAACGTCTCCTCCTACGTGGCATCGACCCCGCACTGGAGCGTAAGTTGCAAAAGG 171 WSM1271 CCAAACGTCTCCTCCTACGTGGCATCGACCCCGCACTGGAGCGTAAGTTGCAAAAGG 171 WSM1283 CCAAACGTCTCCTCCTACGTGGCATCGACCCGGCACAGGAGCGCAAGTCTCAAAAGG 171 WSM1284 CCAAACGTCTCCTCCTACGTGGCATCGACCCCGCACTGGAGCGTAAGTTGCAAAAGG 171 WSM1497 CCAAACGTCTCCTCCTACGTGGCATCGACCCCGCCCAGGAGCGTAAGTCACAAAAGG 171 N17 CTTCGGCAGAGGACACCTTTCGATCAATCGCGGAGGATTATGTCGACAAGCTGAAGA 228 N18 CTTCGGCAGAGGACACCTTTCGATCAATCGCGGAGGATTATGTCGACAAGCTGAAGA 228 N45 CTTCGGCAGAGGACACCTTTCGATCAATCGCGGAGGATTATGTCGACAAGCTGAAGA 228 N87 CTTCGGCAGAGGACACCTTTCGATCAATCGCGGAGGATTATGTCGACAAGCTGAAGA 228 WSM1271 CTTCGGCAGAGGACACCTTTCGATCAATCGCGGAGGATTATGTCGACAAGCTGAAGA 228 WSM1283 CCCCGGCAGAGGACACCTTTCGCTCAATCGCAGAGGACTACGTCGACAAGCTGAAGA 228 WSM1284 CTTCGGCAGAGGACACCTTTCGATCAATCGCGGAGGATTATGTCGACAAGCTGAAGA 228 WSM1497 CTTCGGCAGAGGACACCTTTCGCTCAATCGCGGAGGACTATGTCGACAAGCTGAAGA 228 N17 AGGAGGGACGTGCCGACCGGACCATCACCAAGGTCAAATGGTTGCTCGACTTTGCCT 285 N18 AGGAGGGACGTGCCGACCGGACCATCACCAAGGTCAAATGGTTGCTCGACTTTGCCT 285 N45 AGGAGGGACGTGCCGACCGGACCATCACCAAGGTCAAATGGTTGCTCGACTTTGCCT 285 N87 AGGAGGGACGTGCCGACCGGACCATCACCAAGGTCAAATGGTTGCTCGACTTTGCCT 285 WSM1271 AGGAGGGACGTGCCGACCGGACCATCACCAAGGTCAAATGGTTGCTCGACTTTGCCT 285 WSM1283 ACGAGGGACGTGCCGACCGGACCATCACTAAAGTCAAATGGTTGCTAGACTTTGCCC 285 WSM1284 AGGAGGGACGTGCCGACCGGACCATCACCAAGGTCAAATGGTTGCTCGACTTTGCCT 285 WSM1497 AGGAGGGTCGTGCCGACCGGACCATCACCAAGGTCAAATGGTTGCTCGACTTTGCCC 285

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N17 ATCCAGCGTTTGGGGACAAATGCGTTCGGGAGATCGATCCGGTTACAATTCTTGCCG 342 N18 ATCCAGCGTTTGGGGACAAATGCGTTCGGGAGATCGATCCGGTTACAATTCTTGCCG 342 N45 ATCCAGCGTTTGGGGACAAATGCGTTCGGGAGATCGATCCGGTTACAATTCTTGCCG 342 N87 ATCCAGCGTTTGGGGACAAATGCGTTCGGGAGATCGATCCGGTTACAATTCTTGCCG 342 WSM1271 ATCCAGCGTTTGGGGACAAATGCGTTCGGGAGATCGATCCGGTTACAATTCTTGCCG 342 WSM1283 ATCCAACGTTTGGGGACAAAAGCGTTCGGGAGATTGATCCGGCCACAATTCTTGCCG 342 WSM1284 ATCCAACGTTTGGGGACAAATGCGTTCGGGAGATCGATCCGGTTACAATTCTTGCCG 342 WSM1497 ATCCAACGTTGGGGGACAAAAGCGTTCGGGAGATTGATCCGGCCACAATTCTTGCCG 342 N17 CTCTCCGCAGCGTGGAGGATCGCGGTCGATACGAGTCGGCCAGGCGACTGCGCTCCA 399 N18 CTCTCCGCAGCGTGGAGGATCGCGGTCGATACGAGTCGGCCAGGCGACTGCGCTCCA 399 N45 CTCTCCGCAGCGTGGAGGATCGCGGTCGATACGAGTCGGCCAGGCGACTGCGCTCCA 399 N87 CTCTCCGCAGCGTGGAGGATCGCGGTCGATACGAGTCGGCCAGGCGACTGCGCTCCA 399 WSM1271 CTCTCCGCAGCGTGGAGGATCGCGGTCGATACGAGTCGGCCAGGCGACTGCGCTCCA 399 WSM1283 CTCTCCGCAGCGTGGAGGTTCGCGGTCGATATGAGTCGGCCAGGCGATTGCGCTCCA 399 WSM1284 CTCTCCGCAGCGTGGAGGATCGCGGTCGATACGAGTCGGCCAGGCGACTGCGCTCCA 399 WSM1497 CTCTCCGCAGCGTGGAGGTTCGCGGTCGATACGAGTCGGCCAGGCGACTGCGCTCCA 399 N17 CGATTGGCAGCGTGTTTCGATATGCAATCGCAACCGCACGCGCCGAT-CTAGATCCG 456 N18 CGATTGGCAGCGTGTTTCGATATGCAATCGCAACCGCACGCGCCGAT-CTAGATCCG 456 N45 CGATTGGCAGCGTGTTTCGATATGCAATCGCAACCGCACGCGCCGAT-CTAGATCCG 456 N87 CGATTGGCAGCGTGTTTCGATATGCAATCGCAACCGCACGCGCCGAT-CTAGATCCG 456 WSM1271 CGATTGGCAGCGTGTTTCGATATGCAATCGCAACCGCACGCGCCGAT-CTAGATCCG 456 WSM1283 CCATCGGCAGCGTGTTTCGATATGCAATCGCAACCGCACGCGCCGATG-TAGATCCG 456 WSM1284 CGATTGGCAGCGTGTTTCGATATGCAATCGCAACCGCACGCGCCGAT-CTAGATCCG 456 WSM1497 CTATCGGCAGCGTGTTTCGATATGCAATCGCAACCGCACGCGCCGATAC-AGATCCG 456 N17 ACGGTCGCCCT-CAGGGGCGCACTCGTCGGTCCGACGGTCACGCCGCGTGCCGCGGT 513 N18 ACGGTCGCCCT-CAGGGGCGCACTCGTCGGTCCGACGGTCACGCCGCGTGCCGCGGT 513 N45 ACGGTCGCCCT-CAGGGGCGCACTCGTCGGTCCGACGGTCACGCCGCGTGCCGCGGT 513 N87 ACGGTCGCCCT-CAGGGGCGCACTCGTCGGTCCGACGGTCACGCCGCGTGCCGCGGT 513 WSM1271 ACGGTCGCCCT-CAGGGGCGCACTCGTCGGTCCGACGGTCACGCCGCGTGCCGCGGT 513 WSM1283 ACGATCGCCCTG-AGGGGCGCACTTGTCGGTCCGACGGTCACGCCGCGTGCCGCCGT 513 WSM1284 ACGGTCGCCCT-CAGGGGCGCACTCGTCGGTCCGACGGTCAAGCCGCGTGCCGCGGT 513 WSM1497 ACGATCGCCCTGC-GGGGAGCACTCGTCGGTCCAACGGTCACGCCGCGCGCCGCCGT 513 N17 TACCGATCCTATGGCCTTGGGAGGCTTGCTGAGGGCGATCAATGCCTTTGACGGACA 570 N18 TACCGATCCTATGGCCTTGGGAGGCTTGCTGAGGGCGATCAATGCCTTTGACGGACA 570 N45 TACCGATCCTATGGCCTTGGGAGGCTTGCTGAGGGCGATCAATGCCTTTGACGGACA 570 N87 TACCGATCCTATGGCCTTGGGAGGCTTGCTGAGGGCGATCAATGCCTTTGACGGACA 570 WSM1271 TACCGATCCTATGGCCTTGGGAGGCTTGCTGAGGGCGATCAATGCCTTTGACGGACA 570 WSM1283 TACCGAGCCTAGGGCCTTGGGAGGCTTGCTGAGGGCGATCAATGCATTTGACGGACA 570 WSM1284 TACCGATCCTATGGCCTTGGGAGGCTTGCTGAGGGCGATCAATGCCTTTGACGGACA 570 WSM1497 TACCGATCCTAAGGCCTTGGGAGGCTTGCTGAGGGCGATCAATGCATTTGACGGGCA 570 N17 GCCTACAACCCGAGCCGCCCTGAAG-CTGATGGCCCTGCTGTTTCCCCGCCCCGGCG 627 N18 GCCTACAACCCGAGCCGCCCTGAAG-CTGATGGCCCTGCTGTTTCCCCGCCCCGGCG 627 N45 GCCTACAACCCGAGCCGCCCTGAAG-CTGATGGCCCTGCTGTTTCCCCGCCCCGGCG 627 N87 GCCTACAACCCGAGCCGCCCTGAAG-CTGATGGCCCTGCTGTTTCCCCGCCCCGGCG 627 WSM1271 GCCTACAACCCGAGCCGCCCTGAAG-CTGATGGCCCTGCTGTTTCCCCGCCCCGGCG 627 WSM1283 GCCTACAACCCGAGCCGCCCTTAAG-CTGATGGCTCTGCTGTTTCCCCGCCCCGGCG 627 WSM1284 GCCTACAACCCGAGCCGCCCTGAAG-CTGATGGCCCTGCTGTTTCCCCGCCCCGGCG 627 WSM1497 GCCGACAACCCGAGCCGCCCT-AAGGCTGATGGCCCTGCTGTTTCCCCGCCCCGGCG 627 N17 AGTTGCGTGCGGCCGGATGGGACGAGTTCGATTTCGAAAGCTCGGTGTGGAGCATCC 684 N18 AGTTGCGTGCGGCCGGATGGGACGAGTTCGATTTCGAAAGCTCGGTGTGGAGCATCC 684 N45 AGTTGCGTGCGGCCGGATGGGACGAGTTCGATTTCGAAAGCTCGGTGTGGAGCATCC 684 N87 AGTTGCGTGCGGCCGGATGGGACGAGTTCGATTTCGAAAGCTCGGTGTGGAGCATCC 684 WSM1271 AGTTGCGTGCGGCCGGATGGGACGAGTTCGATTTCGAAAGCTCGGTGTGGAGCATCC 684 WSM1283 AGCTGCGCGCGGCCGGATGGGACGAGTTCGATTTCGAGAGATCCGTGTGGAGCATCC 684 WSM1284 AGTTGCGTGCAGCCGGATGGGACGAGTTCGATTTCGAAAGCTCGGTGTGGAGCATCC 684 WSM1497 AGCTGCGCGCGGCCGGATGGGACGAGTTCGATTTCGAAAGCTCGGTGTGGAGCATCC 684 N17 CTGAAGGGCGCATGAAGATGAGACGGCCGCACCGCGTACCACTTTCCAGGCAGGCCG 741 N18 CTGAAGGGCGCATGAAGATGAGACGGCCGCACCGCGTACCACTTTCCAGGCAGGCCG 741 N45 CTGAAGGGCGCATGAAGATGAGACGGCCGCACCGCGTACCACTTTCCAGGCAGGCCG 741 N87 CTGAAGGGCGCATGAAGATGAGACGGCCGCACCGCGTACCACTTTCCAGGCAGGCCG 741 WSM1271 CTGAAGGGCGCATGAAGATGAGACGGCCGCACCGCGTACCACTTTCCAGGCAGGCCG 741 WSM1283 CTGAACGACGCATGAAGATGCGACGGCCGCACCGCGTACCTCTTTCCAGGCAGGCCG 741 WSM1284 CTGAAGGGCGCATGAAGATGAGACGGCCGCACCGCGTACCACTTTCCAGGCAGGCCG 741 WSM1497 CTGAAGGGCGCATGAAAATGAGACGGCCGCACCGCGTACCTCTCTCCAGGCAGGCCG 741

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N17 TCGGGGTCCTAACCTCACTTAGACGAA-TC-TCTGGTGGTGG-ATCGCTGTTGTTTC 798 N18 TCGGGGTCCTAACCTCACTTAGACGAA-TC-TCTGGTGGTGG-ATCGCTGTTGTTTC 798 N45 TCGGGGTCCTAACCTCACTTAGACGAA-TC-TCTGGTGGTGG-ATCGCTGTTGTTTC 798 N87 TCGGGGTCCTAACCTCACTTAGACGAA-TC-TCTGGTGGTGG-ATCGCTGTTGTTTC 788 WSM1271 TCGGGGTCCTAACCTCACTTAGACGAA-TC-TCTGGTGGTGG-ATCGCTGTTGTTTC 798 WSM1283 TCAGCGTCCTGACCTCACTTAGA-GAAATC-TCTGGTGGTGG-ATCGCTTTTGTTTC 798 WSM1284 TCGGGGTCCTAACCTCACTTAGACGAA-TC-TCTGGTGGTGG-ATCGCTGTTGTTTC 798 WSM1497 TCGGGGTCCTGACCTCACTTAGA-GAA-ACCTCTGGTGGGGGCA-CGCTGTTGTTTC 798 N17 CTAGTGTTCGATCGGGCTTGCGTCCGATTTCCGACAACACGCTTAACGCGGCCCTTC 855 N18 CTAGTGTTCGATCGGGCTTGCGTCCGATTTCCGACAACACGCTTAACGCGGCCCTTC 855 N45 CTAGTGTTCGATCGGGCTTGCGTCCGATTTCCGACAACACGCTTAACGCGGCCCTTC 855 N87 CTAGTGTTCGATCGGGCTTGCGTCCGATTTCCGACAACACGCTTAACGCGGCCCTTC 855 WSM1271 CTAGTGTTCGATCGGGCTTGCGTCCGATTTCCGACAACACGCTTAACGCGGCCCTTC 855 WSM1283 CTAGTGTTCGATCCGGTTCGCGTCCGATTTCCGACAACACGCTCAACGCCGCCCTCC 855 WSM1284 CTAGTGTTCGATCGGGCTTGCGTCCGATTTCCGACAACACGCTTAACGCGGCCCTTC 855 WSM1497 CTAGTATTCGATCGGGCTCGCGTCCGATTTCCGACAACACGCTTAACGCTGCCCTTC 855 N17 GCCGTATGGGGTACGGCAAGGAAGAAGCTACCGCGCACGGTTTTCGAGCAACCGCAT 912 N18 GCCGTATGGGGTACGGCAAGGAAGAAGCTACCGCGCACGGTTTTCGAGCAACCGCAT 912 N45 GCCGTATGGGGTACGGCAAGGAAGAAGCTACCGCGCACGGTTTTCGAGCAACCGCAT 912 N87 GCCGTATGGGGTACGGCAAGGAAGAAGCTACCGCGCACGGTTTTCGAGCAACCGCAT 912 WSM1271 GCCGTATGGGGTACGGCAAGGAAGAAGCTACCGCGCACGGTTTTCGAGCAACCGCAT 912 WSM1283 GCCGTATGGGCTACGGCAAGGAAGAAGCTACCGCGCACGGTTTTCGGGCAACGGCAT 912 WSM1284 GCCGTATGGGGTACGGCAAGGAAGAAGCTACCGCGCACGGTTTTCGAGCAACCGCAT 912 WSM1497 GCCGTATTGGGTACGGCAAGGAAGAAGCCACCGCGCACGGTTTTCGAGCAACGGCAT 912 N17 CAACTCTGCTGAACGAATGCGGAAAG-TGGCATCCGGACGCCATAGAGCGGCAGTTG 969 N18 CAACTCTGCTGAACGAATGCGGAAAG-TGGCATCCGGACGCCATAGAGCGGCAGTTG 969 N45 CAACTCTGCTGAACGAATGCGGAAAG-TGGCATCCGGACGCCATAGAGCGGCAGTTG 969 N87 CAACTCTGCTGAACGAATGCGGAAAG-TGGCATCCGGACGCCATAGAGCGGCAGTTG 969 WSM1271 CAACTCTGCTGAACGAATGCGGAAAG-TGGCATCCGGACGCCATAGAGCGGCAGTTG 969 WSM1283 CAACTCTGCTAAACGAATGCGGAAAG-TGGCATCCCGACGCCATAGAGCGGCAGCTG 969 WSM1284 CAACTCTGCTGAACGAATGCGGAAAG-TGGCATCCGGACGCCATAGAGCGGCAGTTG 969 WSM1497 CAACTTTGCTAAACGAATGCGGAA-GATGGCATCCGGACGCCATAGAGCGGCAGCTG 969 N17 GCCCATGTTGAGAACAACGACGTTCGTCGCGCCTACGCCCGGGCAGAGCACTGGGAA 1026 N18 GCCCATGTTGAGAACAACGACGTTCGTCGCGCCTACGCCCGGGCAGAGCACTGGGAA 1026 N45 GCCCATGTTGAGAACAACGACGTTCGTCGCGCCTACGCCCGGGCAGAGCACTGGGAA 1026 N87 GCCCATGTTGAGAACAACGACGTTCGTCGCGCCTACGCCCGGGCAGAGCACTGGGAA 1026 WSM1271 GCCCATGTTGAGAACAACGACGTTCGTCGCGCCTACGCCCGGGCAGAGCACTGGGAA 1026 WSM1283 GCCCATGTTGAGAAGAACGACGTTCGTCGCGCCTACGCCCGGGCAGAGCACTGGGAA 1026 WSM1284 GCCCATGTTGAGAACAACGACGTTCGTCGCGCCTACGCCCGGGCAGAGCACTGGGAA 1026 WSM1497 GCCCATGTTGAGAACAACGACGTACGTCGCGCCTACGCCCGGGCAGAGTACTGGGAA 1026 N17 GAGCGCGTAAAGATGATG 1044 N18 GAGCGCGTAAAGATGATG 1044 N45 GAGCGCGTAAAGATGATG 1044 N87 GAGCGCGTAAAGATGATG 1044 WSM1271 GAGCGCGTAAAGATGATG 1044 WSM1283 GAGCGCGTCAAGATGATG 1044 WSM1284 GAGCGCGTAAAGATGATG 1044 WSM1497 GAGCGCGTAAAGATGATG 1044

Figure 4.3 intS sequence alignment of WSM1271, the novel isolates (N17, N18, N87, N45) and RNB isolated from B. pelecinus growing in the Mediterranean region (WSM1283, WSM1284, WSM1497). Nucleotide mismatches are in red.

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4.3.4 Eckhardt gel electrophoresis

Eckhardt gel electrophoresis was performed to mobilize the plasmid/s of

WSM1271 and the novel isolates N17, N18, N45 and N87. WSM1271 had a

single plasmid of approximately 500 kb. Novel isolate N18 had a single plasmid

of approximately 500 kb (Fig 4.4a) while the other three novel isolates did not

contain a plasmid. The Eckhardt gel procedure was repeated three times with

multiple replica of the same strain to confirm the absence of plasmids in the

novel isolates N17, N45 and N87. The plasmid of WSM1271 was slightly

smaller than the plasmids of other RNB isolated from B. pelecinus as well as

the plasmid of isolate N18 (Fig 4.4a).

The three RNB isolated from B. pelecinus from the Mediterranean region

(WSM1283, WSM1284 and WSM1497) also had a single plasmid of

approximately 500 kb (Fig 4.4a).

Strain VF39 and M. loti strain R7A served as the controls while strain

VF39 was also used in sizing the other plasmids present in this study. Strain

VF39 had 6 plasmids while M. loti strain R7A did not reveal the presence of any

plasmids.

4.3.5 Southern hybridization of mobilized plasmids

When the blotted plasmid DNA from the Eckhardt gel was hybridized to

nifH, nodA and nodC probes, only the symbiotic plasmid of strain VF39 gave a

positive signal indicating the bonding of the nodC probe (Fig 4.4b). However,

the wells in the gel containing the other strains (except WSM1284 and R7A)

also gave a positive signal indicating the bonding of nifH and nodA probes to

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the genomic DNA in the wells. This showed that the probe and the hybridization

method had worked successfully.

Fig 4.4 A) Eckhardt gel displaying plasmid profiles of WSM1271, WSM1283, WSM1284, WSM1497, N17, N18, N45, N87, M. loti strain R7A and R. leguminosarum strain VF39. B) Southern blot of the Eckhardt gel hybridized with a probe mixture consisting of nifH. nodA and nodC probes. Note the hybridization of the nodC probe to the 240 kb plasmid of strain VF39 and the hybridization of the nifH. nodA probes to the chromosomal DNA in the wells of the gel.

615kb

508kb338kb

240kb

WSM

1271

WSM

1283

WSM

1284

WSM

1497

N17

N45 R7A

VF39

WS

M12

71

WSM

1283

WS

M12

84

WSM

1497

N18

N87 R7A

VF3

9

615kb

508kb338kb

240kb

A

B

615kb

508kb338kb

240kb

WSM

1271

WSM

1283

WSM

1284

WSM

1497

N17

N45 R7A

VF39

WS

M12

71

WSM

1283

WS

M12

84

WSM

1497

N18

N87 R7A

VF3

9

615kb

508kb338kb

240kb

615kb

508kb338kb

240kb

WSM

1271

WSM

1283

WSM

1284

WSM

1497

N17

N45 R7A

VF39

WS

M12

71

WSM

1283

WS

M12

84

WSM

1497

N18

N87 R7A

VF3

9

615kb

508kb338kb

240kb

615kb

508kb338kb

240kb

WSM

1271

WSM

1283

WSM

1284

WSM

1497

N17

N45 R7A

VF39

WS

M12

71

WSM

1283

WS

M12

84

WSM

1497

N18

N87 R7A

VF3

9

WSM

1271

WSM

1283

WSM

1284

WSM

1497

N17

N45 R7A

VF39

WS

M12

71

WSM

1283

WS

M12

84

WSM

1497

N18

N87 R7A

VF3

9

615kb

508kb338kb

240kb

A

B

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4.4 Discussion

There was 100% sequence similarity between the novel isolates and

WSM1271 for the sequenced regions of nodA, nifH and intS. This finding,

together with the genetic and phenotypic diversity established in chapters 2 and

3 strongly indicates that the novel isolates have gained these two symbiotic

genes and the integrase gene from the original inoculant WSM1271.

Sequence results obtained in this study show that strains with identical or

nearly identical 16S rRNA sequence types contained nucleotide variations in

their nodA, nifH and intS gene sequences. There were four nucleotide

mismatches between WSM1271 and WSM1497, five nucleotide mismatches

between WSM1271 and WSM1283 and 48 bp mismatches between WSM1271

and WSM1284 for the nifH gene while identical sequences were present

between the above four strains in a 1440 bp internal fragment of their 16S rRNA

gene sequence (Fig 4.1).

Furthermore, there were 14 bp mismatches between WSM1271 and

WSM1497 for nodA. The failure of nodA probe developed from WSM1271 to

bind to genomic DNA of WSM1284 in the southern blot hybridization experiment

together with the difficulties faced in amplifying nodA of strain WSM1284

indicate that the nodA gene sequence of WSM1284 may be significantly

different (<75% similar at least) to that of WSM1271. The fact that the nodA

probe did not bind to genomic DNA of M. loti strain R7A and there was <75 %

sequence similarity between nodA of WSM1271 and M. loti strain R7A further

support the idea that the nodA of WSM1284 is significantly different.

The above results indicate that it is quite common to find some

nucleotide variation in symbiotic genes of rhizobial strains with identical 16S

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rRNA sequence types as has been reported in other studies (Laguerre et al.,

2001; Haukka et al., 1998; Wang et al., 1999b). Degeneracy of the genetic code

allows a considerable number of nucleotide changes in homologous genes

(Lewin, 1997). Homologous genes may evolve separately over time in two

different strains, after their inheritance from a common ancestor (Ridley, 1997).

The failure of the symbiotic probes to bind to the plasmids in WSM1271,

novel isolate N18 and the three strains isolated from B. pelecinus growing in the

Mediterranean region, confirms that these symbiotic genes are not carried on a

plasmid but on the chromosome in these strains. Binding of the nifH and nodA

probes to the chromosomal DNA in the wells of the Eckhardt gel further

confirms the presence of these genes on the chromosome. Yet, the symbiotic

gene sequence similarity strongly suggests a transfer of these symbiotic genes

from WSM1271 to the novel isolates.

Biserrula RNB belong to the genus Mesorhizobium and are closely

related to M. loti (Nandasena et al., 2001). Sullivan et al., (1998) has

demonstrated the presence of symbiotic genes of M. loti on a chromosomally

located symbiosis island and its excision and integration is mediated by the

phage P4 like integrase coded by the intS gene. When a BLAST search was

performed for the intS gene of M. loti there were no close hits from the

members from any other rhizobial genera other than the two strains of M. loti,

strain R7A (Sullivan et al., 2002) and MAF303099 (Kaneko et al., 2000).

However, intS gene is present on biserrula RNB and it has 93.2% sequence

similarity to that of M. loti strain R7A and 94.8% to MAF303099. Therefore, it

may be possible that the symbiotic genes of biserrula RNB are located on a

symbiosis island similar to the one described for M. loti (Sullivan et al., 1998,

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119

2002). However, further experiments are necessary to draw firm conclusions on

this respect and are discussed in more detail in chapter 5.

There were <96% sequence similarity between WSM1271 and two of the

other biserrula isolates, WSM1283 and WSM1497 for the intS gene inferring

that the biserrula RNB contain an integrase coding gene but its sequence varies

considerably within the RNB nodulating B. pelecinus in the Mediterranean

region.

Interestingly, WSM1284 only had three nucleotide mismatches to

WSM1271 for the intS gene, inferring the recent inheritance of this gene from a

common ancestor. Yet, nodA and nifH sequences vary significantly between

these two strains. These results indicate that some of the symbiotic genes and

the integrase gene may be inherited independently in some strains.

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Chapter 5

121

5. General discussion

The emergence of rhizobial biodiversity after the introduction of exotic

legumes and their respective rhizobia to new regions is a challenge for

contemporary rhizobiology (Howieson and Ballard, 2004). Two confronting

questions are:

1. What are the mechanisms leading to the evolution of rhizobial

diversity in agricultural soils following exotic legume introduction?

2. What are the consequences of the development of a diversity of

strains able to nodulate a newly introduced legume species?

This chapter will discuss the above questions in relation to the results of

the present study. In addition, implications from these results for agriculture are

presented together with insights for future research to address the challenge of

development of diversity for contemporary rhizobiology.

5.1 Mechanisms driving rapid evolution of rhizobial diversity in

agricultural soils

The rates at which rhizobial populations diversify, and the evolutionary

forces structuring the genetic divergence of rhizobial populations in Australian

soils remain largely unknown. Therefore, the recent introduction of B. pelecinus

produced a need and an exciting opportunity to investigate the mechanisms

driving rhizobial diversification in agricultural soils in Australia. Studies

attempting to understand the mechanisms governing bacterial diversification

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and population dynamics have surfaced in many disciplines of science since the

dawn of the molecular era (Bushman, 2002; Hacker & Kaper, 2002). Both the

polyphyletic origin of RNB (i.e. there is no branch of the evolutionary tree based

on 16S rRNA gene sequences that carries exclusively RNB; Martínez-Romero

& Caballero-Mellado, 1996; Young, 1996) and the vast variation present in the

chromosomal backgrounds of RNB carrying symbiotic genes (Young & Wexler,

1988; Normand & Bousquet, 1989; Dolbert et al., 1994; Ueda et al., 1995;

Young & Haukka, 1996; Souza & Eguiarte, 1997; Haukka et al., 1998; Zhang et

al., 2000; Laguerre et al., 2001; Suominen et al., 2001; Moulin et al., 2004)

indicate there has been a high level of lateral transfer of symbiotic genes

between rhizobia.

A primary objective of this thesis was to investigate whether diverse

strains emerged after the introduction of the exotic legume B. pelecinus,

inoculated with a single strain (Mesorhizobium sp. strain WSM1271) into a soil

devoid of RNB capable of nodulating this plant. The results clearly show that

both genetically and phenotypically diverse strains capable of nodulating B.

pelecinus emerged within six years of this legume being introduced. A second

aim of this project was to identify possible mechanisms involved in the evolution

of diversity. This study demonstrated that two symbiotic genes (nodA and nifH)

located on the chromosome of WSM1271 were transferred into four novel

isolates from B. pelecinus nodules that cluster in Mesorhizobium on the basis of

their 16S rRNA gene sequence. Furthermore, data shows that a gene coding

for the enzyme integrase (intS) was transferred from WSM1271 to the four

novel isolates.

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Conjugation, transformation and transduction are the well known

mechanisms for transfer of DNA between bacteria (Haker & Kaper, 2002).

Plasmids and gene islands appear to be the key mobile elements involved in

lateral transfer of DNA leading to rapid evolution of bacterial genomes

(Bushman, 2002). The symbiotic genes of the RNB genera Rhizobium and

Sinorhizobium are located on symbiotic plasmids and the transfer of these

plasmids between strains has been demonstrated in the laboratory (Hynes et

al., 1986; Martinez et al., 1987; Rogel et al., 2001) and shown to occur in

cultivated soils (Young and Wexler, 1988; Laguerre et al., 1992; Louvrier et al.,

1996) and in natural settings (soils that are not under intensive, human-

mediated selection; Wernegreen & Riley, 1999). In contrast, the symbiotic

genes are located on the chromosome in Bradyrhizobium and Mesorhizobium

(except for M. amorphae, Wang et al., 1999c and M. huakuii, Zhang et al.,

2000). Sullivan & Ronson (1998) demonstrated the presence of symbiotic

genes of M. loti strain R7A on a chromosomally located mobile genetic element

(gene island), named a ‘symbiosis island’. These authors also demonstrated the

mobility of this symbiosis island. The comparative sequence analysis of the

symbiosis island of M. loti strain R7A with the completely sequenced genome of

M. loti strain MAFF303099 revealed the presence of a symbiosis island in the

latter strain (Sullivan et al., 2002; Kaneko et al., 2000). Recently the presence of

a putative symbiosis island has been observed in the genome sequence of B.

japonicum strain USDA110 (Kaneko et al., 2002; Moulin et al., 2004). However

the mobility of the latter two symbiosis islands has not been demonstrated.

The identification of WSM1271 and the four novel isolates (N17, N18,

N45 and N87) as Mesorhizobium based on their 16S rRNA gene sequences

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(Chapter 2), the localization of symbiotic genes on their chromosomes and the

transfer of nodA and nifH from WSM1271 to the novel isolates (Chapter 4),

strongly indicate the presence of a symbiosis island in WSM1271. The

existence of a gene coding for integrase (intS), an enzyme involved in the

excision and integration of symbiosis islands (Sullivan et al., 2002), in

WSM1271 and the four novel isolates further strengthens the proposal that the

symbiotic genes of WSM1271 are on a mobile symbiosis island.

Gene islands are generally known to integrate into a tRNA gene (Kaper

& Hacker, 1999). Sullivan & Ronson (1998) demonstrated the integration of the

symbiosis island of M. loti strain R7A into a phe-tRNA gene. Furthermore,

sequence similarity studies suggest that the putative symbiosis island present

on B. japonicum strain USDA110 may integrate within a val-tRNA gene (Moulin

et al., 2004). Therefore, future experiments identifying the integration site of the

proposed symbiosis island in WSM1271 and the four novel isolates may provide

further evidence for the existence of such a mobile element in RNB that

nodulate B. pelecinus. The intS is known to be on one end of the symbiosis

islands (Sullivan et al., 2002) and this gene can be marked with an antibiotic

marker by using a targeted single crossover insertion technique (Ravi Tiwari,

Pers:comm). This method will facilitate cloning of intS and flanking DNA. DNA

sequencing and analysis will reveal the genes flanking intS. If the integration

site of intS is a tRNA gene, then firm conclusions can be made regarding the

presence of a symbiosis island in WSM1271.

Although mechanisms of plasmid movements between bacteria are well

described (Bushman, 2002), little is known about the transfer of symbiosis

islands due to their recent discovery. Sullivan et al., (2002) have suggested that

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the symbiosis island in M. loti strain R7A is unlikely to replicate as a plasmid

due to the lack of highly conserved repABC genes and these authors have

proposed that the island excises in a circle and transfers via a conjugative

transfer mechanism due to the presence of the trbBCDEJLFGI operon. Many

other genes thought to be involved in the island transfer were also reported by

the above authors. Southern hybridization-based methods can be performed to

investigate whether island transfer genes similar to the ones described for M.

loti strain R7A (Sullivan et al., 2002) are present in WSM1271. Targeted gene

inactivation and analysis of the resultant mutants may lead to the identification

of genes involved in island transfer in WSM1271.

Zhang et al., (2000) have demonstrated that the nodulation genes of M.

huakuii strains that nodulate Astragalus sinicus are conserved despite the

chromosomal diversity of these strains. Similarly, alignment of the nodA

sequences available in NCBI for M. ciceri strain USDA3383 and M.

mediterraneum strain USDA3392, two species that nodulate Cicer arietinum,

shows only two nucleotide mismatches suggesting that nodA is conserved

between these two species. These two examples provide evidence for the

transfer of symbiotic genes among the members of Mesorhizobium. The

indication from this study of the presence of a mobile symbiosis island in

Mesorhizobium sp. strain WSM1271, together with other reports (Sullivan &

Ronson, 1998; Kaneko et al., 2000), reveals how lateral transfer of symbiosis

islands may mediate rapid diversification of Mesorhizobium strains in

agricultural soils. It is also interesting to note that both the donor and the

recipient RNB in this study belong to Mesorhizobium similar to the previous

study (Sullivan et al., 1996). These observations indicate that the mechanism

Chapter 5

126

for symbiosis island transfer may function in certain chromosomal backgrounds

of RNB and not in others.

If the mechanisms involved in the transfer of symbiosis islands are

known, then it may be possible to create genetically stable inoculants for B.

pelecinus by inactivating the genes governing the island transfer in the inoculant

strain. The evolution of poorly effective or ineffective strains nodulating B.

pelecinus via symbiotic gene transfer from an effective inoculant could thus be

reduced.

5.2 Rapid evolution of nodulating opportunists may threaten

legume productivity

Australian agriculture is heavily dependent upon legume N2 fixation, more

so than agriculture in most other countries (Howieson et al., 2000b).

Importantly, this dependence appears likely to remain. Exotic pasture legumes,

particularly perennials, are forecast to become prominent in southern Australian

agriculture to overcome the development of secondary salinity (Cocks, 2001).

However, if the symbiosis is not highly effective, the benefits of symbiotic N2

fixation are lost, or at the very least considerably reduced. Many studies have

reported the presence of ineffective or less effective strains that are capable of

nodulating various legume species (Dowling & Broughton, 1986; Thies et al.,

1991a,b; Triplett & Sadowsky, 1992; Ballard & Charman, 2000; Denton et al.,

2002). Sullivan et al., (1995) have demonstrated the evolution of nodulating

strains of M. loti through the transfer of symbiotic genes from an inoculant strain

to non-symbiotic rhizobia. Yet the effectiveness of these resulting strains is not

Chapter 5

127

known. The results of the present study clearly demonstrated the evolution of

ineffective and less effective RNB capable of nodulating B. pelecinus through

the lateral transfer of symbiotic genes from the inoculant to other soil bacteria in

situ. One question is: what are the possible causes for the inability of N15 and

N45 to fix N2 on B. pelecinus?

Symbiotic N2 fixation is a complex process that involves several

biochemical pathways and many genes (Kaminski et al., 1998; Fisher &

Newton, 2000; Rubio & Ludden, 2000). Therefore, mutations on symbiotic

genes or incomplete transfer of the symbiotic genes are two possibilities for the

Fix- phenotype of N15 and N45. Although mutations causing the Fix- phenotype

were not investigated in this study, there are many examples of this occurring in

other RNB. The fixABCX operon codes for products involved in electron

transport to nitrogenase, the key enzyme in N2 fixation, and these gene

products participate in redox processes in microaerobic or aerobic diazotrophs

(Kaminski et al., 1998). Mutations to any of the genes on the fixABCX operon

had detrimental effects on nitrogen fixation in S. meliloti (Dusha et al., 1987;

Earl et al., 1987). Furthermore, fixNOQP genes code for a bacteroid terminal

oxidase with high affinity for oxygen (Kaminski et al., 1998) while the fixGHIS

operon codes for products involved in a membrane-located cation pump (Kahn

et al., 1989). Mutations to either of the above two operons resulted in a Fix-

phenotype in B. japonicum strain 110spc4 (Fischer, 1994 and1996; Preisig et

al., 1996).

Similarly there is no unequivocal evidence for partial transfer of

symbiosis islands but the mosaic nature present on the two M. loti symbiosis

islands sequenced so far (Sullivan et al., 2002) may indicate partial transfer. In

Chapter 5

128

M. loti strain R7A, the N2 fixation genes are located on operons that are spread

across the 500 kb symbiosis island (Sullivan et al., 2002). There are more than

375 kb between the fixIHGPQON operon and the fixABCX operon in this island.

The two symbiosis islands identified so far in M. loti share only 248 kb in

common while the highly conserved collinear DNA regions of the two islands

are known to be interrupted by multiple deletions and insertions of up to 168 kb

(Sullivan et al., 2002). The above observations may indicate that these two

symbiosis islands have evolved independently after inheritance from a common

ancestor. Alternatively, partial transfer of the symbiosis island may be a

possibility. Uchiumi et al., (2004) have recently demonstrated through global

transcriptional profiling that the genes within the symbiosis island of M. loti

strain MAFF303099 are collectively expressed during symbiosis with Lotus

japonicus, indicating that the symbiosis island acts as an entity during

symbiosis. Therefore, a possibility for the absence of N2 fixation by novel

isolates N15 and N45 may be related to partial transfer of N2 fixation genes from

WSM1271. Whether partial transfer of the symbiotic island has occurred can be

investigated by performing a Southern hybridization for the restriction digested

genomic DNA of novel isolates using cosmid clones developed from the

symbiotic region of WSM1271 as probes.

Non-nitrogen fixing nodules were observed on pea and vetch when these

plants were inoculated with S. meliloti transconjugants containing their own

symbiotic plasmid as well as the symbiotic plasmid of R. leguminosarum

(Hooykaas et al., 1982). Therefore, another possibility for the absence of N2

fixation by novel isolates N15 and N45 may be related to functional

incompatibility of resident symbiotic genes (Rogel et al., 2001).

Chapter 5

129

Mutations to the genes involved in TTSS mechinary have been shown to

strongly alter the host range of the broad host-range Rhizobium strain NGR234

(Viprey et al., 1998). Furthermore, non-nitrogen fixing nodules have been

observed on Crotalaria juncea inoculated with the NGR234 lacking TTSS-

dependent protiein secretion (Marie et al., 2003). It is possible that mutations to

nodulation outer proteins (nop) or genes involved in TTSS machinery may lead

to the inability of N15 and N45 to fix N2 in B. pelecinus.

A second question is, what are the possible causes for the reduced

efficiency of isolates N17, N18, N39, N46, and N87 to fix N2 on B. pelecinus?

The reasons are difficult to identify and may relate to differential regulation of

symbiotic genes in different chromosomal backgrounds. N2 fixation can also be

related to the presence of cryptic plasmids (Thurman, et al., 1985; Pankhurst et

al., 1986; Hynes & McGregor, 1990; Selbitschka & Lotz, 1991; Baldani et al.,

1992; Brom et al., 1992; Kuykendall et al., 1994). For example, significantly

higher nitrogenase activity has been observed for cryptic plasmid cured S. fredii

mutants than for the wild type strain which carries three cryptic plasmids

(USDA206; Barbour & Elkan, 1989). Similarly, S. meliloti strain SAF22 with

three cryptic plasmids was significantly less effective in N2 fixation on alfalfa

(Medicago sativa) than other S. meliloti strains with fewer cryptic plasmids

(Velázquez et al., 1995). Furthermore, the N2 fixation of pea (Pisum sativum)

inoculated with R. leguminosarum bv. viciae was severely inhibited by the

presence of derivatives of the broad host range plasmid RP4 in these strains

(O’Connell et al., 1998), indicating that in the above examples the presence of

cryptic plasmids has a negative effect on N2 fixation. In contrast, Martínez et al.,

(1987) reported that when the plasmids of R. phaseoli strain CFN299 were

Chapter 5

130

transferred to Agrobacterium tumefaciens only the transconjugants carrying

both the sym-plasmid and the cryptic plasmids of R. phaseoli were more

effective in N2 fixation. Thus the cryptic plasmids displayed a positive effect in

N2 fixation.

The combined Eckhardt gel electrophoresis and Southern hybridization

experiments in this study demonstrated the presence of an approximately 500

kb cryptic plasmid in the four RNB strains isolated from B. pelecinus growing in

the Mediterranean region, including WSM1271. The novel isolates N17 and N87

did not contain any plasmid. Therefore, a further possibility for the decreased N2

fixation observed for these two novel isolates could be related to the absence of

the cryptic plasmid. Future research involving plasmid cured WSM1271 and

effectiveness tests may contribute to the understanding of the role of this cryptic

plasmid in N2 fixation in B. pelecinus.

Of the four novel isolates tested, only N18 possessed a plasmid.

However, this plasmid was bigger than that of WSM1271, indicating that this

plasmid is different to the plasmid present in WSM1271, and may be a reason

for the reduced efficiency of N18.

5.3 Insight to the development of rhizobial promiscuity

An intriguing observation from this research was the fact that the four

novel isolates studied here (N17, N18, N45 and N87) had distinct host ranges

even though transfer of two symbiotic genes (nodA and nifH) from WSM1271

into these novel isolates was evident. Previously, Sullivan et al., (1996)

demonstrated the transfer of a symbiosis island from M. loti strain R7A to other

non-symbiotic rhizobia. However, whether the recipients of the symbiosis island

Chapter 5

131

had similar or different host ranges to R7A is unknown. What reasons are there

for the four novel isolates to have dissimilar host ranges to WSM1271?

The specificity of the legume-rhizobia interaction is governed at different

levels with many plant and microbial genes and their products involved in the

recognition and nodulation process (Downie, 1998; Hadri & Bisseling, 1998;

Schlaman et al., 1998). The initial level of interaction is governed by the type(s)

of nodD present in a rhizobial strain (Downie, 1994; Schlaman et al., 1998).

Transfer of nodD1 of Rhizobium sp. strain MPIK3030 (a derivative of the broad

host-range strain NGR234) into S. meliloti has extended the host range of S.

meliloti to nodulation of Macroptilium atropurpureum (Horvath et al., 1987). The

transfer of nodD1 of Rhizobium sp. strain NGR234 into the narrow host-range

R. trifolii strain ANU843 extended the nodulation ability of the recipient strain to

new host plants including some tropical legumes such as Vigna unguiculata,

Glycine ussuriensis, Leucaena leucocephala and M. atropurpureum (Bassam et

al., 1988) and also to the nonlegume Parasponia andersoni (Bender et al.,

1988). Furthermore, paralogous nodD genes have been reported for a number

of RNB (R. leguminosarum bv. phaseoli, Davis & Johnston, 1990; B. japonicum,

Göttfert et al., 1992; R. tropici, van Rhijn et al., 1993; Rhizobium sp. strain

NGR234, Perret et al., 2000). Therefore, one possibility for the distinct host

ranges evident for these novel isolates may be due to the pre-existence of

paralogous nodD genes in these strains. Amplification of DNA from the novel

isolates, using primers developed from the conserved regions of orthologus and

paralogous nodD genes to produce probes for Southern hybridization of

restriction digested genomic DNA of the isolates, may reveal the presence of

paralogous nodD genes (van Rhijn et al., 1993). If nodD paralogs are present

Chapter 5

132

then selective gene inactivation techniques may assist the understanding of the

involvement of paralogous nodD genes in the nodulation of B. pelecinus by the

novel isolates.

The complement of nod genes carried by a given RNB strain determines

the structural variation of the Nod-factors, which, in turn, determines the range

of legume hosts this strain can nodulate (Downie, 1994, 1998). A single strain

can produce a diversity of Nod-factors and the number of Nod-factors produced

by a strain may be proportional to the number of different hosts it can nodulate

(Downie, 1994). Therefore that the novel isolates may originally have been

symbiotic strains that had their own individual set of symbiotic genes. The

acquisition of symbiotic genes from WSM1271 would then have extended their

host range to nodulation of B. pelecinus. Development of random clone libraries

(Tiwari et al., 1996) from the individual novel isolates and mobilizing whole

libraries to WSM1271 then using these transconjugants as inocula for hosts

nodulated by the novel isolates may identify clones carrying genes required for

nodulation of a particular host. DNA sequencing may further facilitate the

identification of the genes involved in the nodulation of a specific host.

Cryptic plasmids in a rhizobial strain are also known to influence

nodulation and competitiveness for nodule occupation (Pankhurst et al., 1986;

Toro & Olivares, 1986; Pardo et al., 1994; Hartmann et al., 1998). Bromfield et

al., (1985) demonstrated that the cryptic plasmid pTA2 of S. meliloti enhances

the competitiveness of this strain to nodulate Medicago sativa cv. Apollo.

Furthermore, Sanjuán & Olivares, (1989) have identified a plasmid

(pRmeGR4b) that does not carry the common symbiotic genes that influences

nodulation efficiency of S. meliloti strain GR4 on M. sativa. In the present study,

Chapter 5

133

only novel isolate N18 was shown to carry a plasmid and some of the plasmid-

borne genes may be responsible for its distinctive host range. Testing the host

range of plasmid-cured strains of N18 may reveal the influence of this cryptic

plasmid on the host range of N18.

The chromosomal background of a rhizobial strain may also influence

nodulation (Roest et al., 1997; van Brussel et al., 1982). Another possibility for

the individual host ranges observed by the novel isolates may therefore be their

background chromosomal diversity. Although novel isolates N18 and N87 have

identical 16S rRNA gene sequences and N17 differs from them in only one

nucleotide, both molecular fingerprinting (ERIC and RPO1) and results from the

physiological experiments indicate these three isolates have considerably

different chromosomes. Strain N45 was clearly distinguishable from the other

three novel isolates, not only with molecular fingerprinting and physiological

results, but also with 16S rRNA gene sequence results.

Another intriguing observation from this study is the ability of strain

WSM1284, isolated from B. pelecinus growing in Sardinia, to nodulate 16 hosts

out of the 21 tested. There were 60 nucleotide mismatches for intS between

WSM1284 and WSM1497 (isolated from B. pelecinus in Greece) and 69

nucleotide mismatches between WSM1284 and WSM1283 (isolated from B.

pelecinus in Morocco) indicating that the intS evolves quite rapidly and thus

vary significantly in the DNA sequence in RNB strains that belong to the same

phylogenetic group (Nandasena et al., 2001). WSM1284 and WSM1271 were

collected from locations that are approximately 60 km apart and interestingly,

there were only three nucleotide mismatches between WSM1284 and

WSM1271 for intS indicating the recent inheritance of intS from a common

Chapter 5

134

ancestor by the above two strains. This raises the question, was intS the only

gene that was inherited from a common ancestor between WSM1271 and

WSM1284 or was there other genes that were inherited in a similar manner?

The failure to sequence the nodA of WSM1284 due to the presence of many

ambiguous bases suggests the possibility of having more than one nodA in

WSM1284. The above observations indicate that the ability of the broad host-

range strain WSM1284 to nodulate B. pelecinus may be due to acquisition of

genes responsible for the nodulation of B. pelecinus from a common ancestor

for WSM1284 and WSM1271.

5.4 General conclusions

This study clearly demonstrated that genetically and phenotypically

diverse strains capable of nodulating B. pelecinus arose through symbiotic gene

transfer from the original inoculant (Mesorhizobium sp. strain WSM1271) to

other soil rhizobia. Furthermore, the evidence presented here showed this

transfer occurred in situ, within six years, in a soil previously devoid of RNB

capable of nodulating this plant. A third significant finding was that some of the

recipient organisms were either ineffective or less effective in N2 fixation on B.

pelecinus. The results strongly suggest that the symbiotic genes of WSM1271

are on a chromosomally located symbiosis island.

This is the first reported evidence for evolution of ineffective strains of

Mesorhizobium sp. through lateral transfer of symbiotic genes from an inoculant

strain to other soil bacteria. However, despite this evolution of ineffective

strains, 92% of the nodules on B. pelecinus growing six years after introduction

and inoculation with WSM1271 were occupied by the inoculant strain.

Chapter 5

135

Furthermore, this original inoculant appears to have retained its N2 fixation

efficiency over a six year period.

Future research leading from this study may shed light on the

understanding of the development of promiscuity in Mesorhizobium and may

facilitate the management of maximum N2 fixation from newly introduced

legume species with specific inocula.

Chapter 5

136

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Appendix 1 – Agarose gels showing the fingerprinting pattern of isolates with primer RPO1 191

M C N1 N2 N3 N4 N5 N6 N7 N8 N9 N10 N11 N12 N13 N14 N16 N15 N19 N17 N18 N20 N21 N22 C M M C N23 N24 N25 N26 N27 N28 N29 N30 N31 N32 N33 N34 N35 N36 N37 N38 N39 N40 N41 N42 N43 N44 C M M C N45 N46 N47 N48 N49 N50 N51 N52 N53 N54 N55 N56 N57 N58 N59 N60 N61 N62 N63 N64 N65 N66 C M M C N67 N68 N69 N70 N71 N72 N73 N74 N75 N76 N77 N78 N79 N80 N81 N82 N83 N84 N85 N87 N86 N87 C

Appendix 2 - Agarose gels showing the fingerprinting pattern of isolates with primer ERIC 192

M C N1 N2 N3 N4 N5 N6 N7 N8 N9 N10 N11 N12 N13 N14 N15 N16 N17 N18 N19 N20 N21 N22 C M M C N23 N24 N25 N26 N27 N28 N29 N30 N31 N32 N33 N34 N35 N36 N37 N38 N39 N40 N41 N42 N43 N44 C M M C N45 N46 N47 N48 N49 N50 N51 N52 N53 N54 N55 N56 N57 N58 N59 N60 N61 N62 N63 N64 N65 N66 C M M C N67 N68 N69 N70 N71 N72 N73 N74 N75 N76 N77 N78 N79 N80 N81 N82 N83 N84 N85 N86 N87 N88 C M

Appendix 3 – 16S rRNA gene sequence alignment of some RNB in α-proteobacteria 196

1 11 21 31 41 51 61 71 81 91 consensus -------------------GAACGAACGCTGGCGGCAGGCTTAACACATGCAAGTCGAGC--GCCCCGCAAG--GGGAGCGGCAGACGGGTGAGTAACGC S.fredii ...................-................................................................................ S.xinjiang .................................................................................................... S.saheli ...................-............................................GTG....T..AC........................ S.teranga ...................-............................................GTA....T..AC........................ S.meliloti ...................-................................................................................ R.galegae ...................-.............................................T........A......................... Hautalense ..........CCTGGCTCA................................................................................. A.tumefaci ...................-......................................A....................T.................... A.rubi ...................-......................................A.....T..........A...T.................... A.vitis ...................-.............................................T........A......................... R.etli .................CAC................................................................................ Phaseoli ...................-................................................................................ Viceae ..................A................................................................................. Trifolii CAGAGTTGATCCTGGCTCA................................................-................................ A.rhizogen ...................-................................................................................ tropicii_D .................................................................................................... Mongolense ...................-................................................................................ Gallicum AGAGTTTGATCCTGGCTCA................................................................................. Hinansis AGAGTTTGATCCTGGCTCA.......................................................A......................... WSM1271 ..AGTTTGATCCTGGCTCA................................................................................. WSM1497 ..AGTTTGATCCTGGCTCA................................................................................. WSM1284 ..AGTTTGATCCTGGCTCA................................................................................. WSM1283 ..AGTTTGATCCTGGCTCA................................................................................. N18 ..AGTTTGATCCTGGCTCA................................................................................. N87 ..AGTTTGATCCTGGCTCA................................................................................. N17 ..AGTTTGATCCTGGCTCA................................................................................. M.ciceri ...................-................................................................................ M.loti ...................-.............................................T........A......................... M.tianshan .GAGTTTGATCCTGGCTCA................................................................................. M.meditere ...................-................................................................................ N45 ..AGTTTGATCCTGGCTCA................................................................................. M.plurifar ...................-................................................................................ M.huakuii .................................................................................................... M.amorphae .GAGTTTGATCCTGGCTCA................................................................................. B.elkani ..AGTTTGATCCTGGCTCA..G....................................T.GG..ATA....TAT.TC....................... B.japonicum ...................-.G......................................GG..GTA....TAC.TC....................... Azorhizobi ...................-.G..................C.........................A....T............................ 101 111 121 131 141 151 161 171 181 191 consensus GTGGGAATCTACCCATCTCTACGGAACAACTCCGGGAAACTGGAGCTAATACCGTATACGTCCTTCGGGAGAAAGATTTATCGG GATGGATGAGCCCGC S.fredii ..............T.T.........T...G.A........T.T.............GA.C........G..............GA.A............ S.xinjiang ..............T.T.........T...G.A........T.T.............GA.C........G..............GA.A............ S.saheli ..............T.T.........T...G.A........T.T.............GA.C........G..............GA.A............ S.teranga ..............T.T.........T...G.A........T.T................C....TT..G..............A..A............ S.meliloti ..............T.T.........T...G.A........T.T.............GA.C........G..............GA.A............ R.galegae .................C..........................................C........G..............G............... Hautalense .................C..........................................C........G..............G............... A.tumefaci .............GTG.C..G.....T.G...............AT........C.....C...A....G..............G.TAT........... A.rubi ...............A.C..G.....T.G...T...........AT........C.....C...A....G..............G............... A.vitis .............GTA.C........T.G...............AT..............C........G..............G.TAT........... R.etli .......CG.....T.TA........T...G.A........T.T.............GT.C....T...G..............TA.A....CG...... Phaseoli ..............T.GA........T...G.A........T.T.............GT.........................TC.A............ Viceae ..............T.GA........T...G.A........T.T.............GT.........................TC.A............ Trifolii ..............T.GA........T...G.A........T.T.............GT.........................TC.A............ A.rhizogen ..............T.T.........T...G.A........T.T.............GT.........................GA.A............ tropicii_D .............TT.TG........T...G.A........T.T.............GT.........................CA.GA........... Mongolense .......CG.....T.TA........T...G.A........T.T.............GT.C........G..............TA.G....CG...... Gallicum .......CG.....T.TA........T...G.A........T.T.............GT.C........G..............TA.G....CG...... Hinansis ......CAA.....T.TA........T...G.A........T.T.............GT.C........G..............TA.A....CG...... WSM1271 ....................................................................................A............... WSM1497 ....................................................................................A............... WSM1284 ....................................................................................A............... WSM1283 ....................................................................................A............... N18 .................................................................TT.................A............... N87 .................................................................TT.................A............... N17 .................................................................TT.................A............... M.ciceri ....................................................................................A............... M.loti ....................................................................................A............... M.tianshan .........................G..........................................................A............... M.meditere ....................................................................................A............... N45 .................................................................AC.................A............... M.plurifar ....................................................................................A............... M.huakuii ....................................................................................A............... M.amorphae ....................................................................................A............... B.elkani .......CG....TT.TGG.T..........GA........TC...........G...A.C....AC..G.............CC..AA...CG...... B.japonicu .......CG....TT.TGG.T.........A.A........T.T..........G...A.C....AC..G.............CC..AA...CG...... Azorhizobi .....G..G.G....ATGG.G.....T...C.A........T.GAT........C..GT.C........G.............CCAT.....C.A.....

201 211 221 231 241 251 261 271 281 291 consensus GTTGGATTAGCTAGTTGGTGGGGTAAAGGCCTACCAAGGCGACGATCCATAGCTGGTCTGAGAGGATGATCAGCCACATTGGGACTGAGACACGGCCCAA S.fredii .................................................................................................... S.xinjiang .................................................................................................... S.saheli .................................................................................................... S.teranga .................................................................................................... S.meliloti .................................................................................................... R.galegae .................................................................................................... Hautalense .................................................................................................... A.tumefaci .................................................................................................... A.rubi .................................................................................................... A.vitis .................................................................................................... R.etli .................................................................................................... Phaseoli .................................................................................................... Viceae ........................................................................................A........... Trifolii .................................................................................................... A.rhizogen .................................................................................................... tropicii_D .................................................................................................... Mongolense .................................................................................................... Gallicum .................................................................................................... Hinansis .................................................................................................... WSM1271 ..........................T......................................................................... WSM1497 ..........................T......................................................................... WSM1284 ..........................T......................................................................... WSM1283 ..........................T......................................................................... N18 ..........................T...................................................C....................G N87 ..........................T...................................................C....................G N17 ..........................T...................................................C....................G M.ciceri ..........................T......................................................................... M.loti ..........................T......................................................................... M.tianshan ..........................T......................................................................... M.meditere ..........................T......................................................................... N45 ..........................T...................................................C....................G M.plurifar ..........................T...................................................C....................G M.huakuii ..........................T...................................................C....................G M.amorphae ..........................T...................................................C....................G B.elkani ..CT................A.....T...TC...............AG................................................... B.japonicu ..CT................A.....T...TC...............AG................................................... Azorhizobi ..CT................A.........TC...............AG.............................C....................G 301 311 321 331 341 351 361 371 381 391 consensus ACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGC-AAGCCTGATCCAGCCATGCCGCGTGAGTGATGAAGGCCCTAGGGTTGTAAAGCTC S.fredii .................................................................................................... S.xinjiang .................................................................................................... S.saheli .................................................................................................... S.teranga .................................................................................................... S.meliloti .................................................................................................... R.galegae .................................................................................T.T....A........... Hautalense ...................................................................................T................ A.tumefaci ..........................................N........................................T................ A.rubi ...................................................................................T................ A.vitis .................................................................................T.T....A........... R.etli ..........................................C......................................................... Phaseoli ..........................................C......................................................... Viceae ..........................................C.C............A.......................................... Trifolii .................................................................................................... A.rhizogen .................................................................................................... tropicii_D .................................................................................................... Mongolense ..........................................C......................................................... Gallicum .................................................................................................... Hinansis .....................................................................................T.............. WSM1271 ...........................................A........................................................ WSM1497 ...........................................A........................................................ WSM1284 ...........................................A........................................................ WSM1283 ...........................................A........................................................ N18 .................................................................................................... N87 .................................................................................................... N17 .................................................................................................... M.ciceri ........................................N..A........................................................ M.loti ...........................................A........................................................ M.tianshan ...........................................A........................................................ M.meditere .................................................................................................... N45 .................................................................................................... M.plurifar ...........................................A........................................................ M.huakuii .................................................................................................... M.amorphae ...........................................A........................................................ B.elkani .................................................................................................... B.japonicu .........................................G.....C.................................................... Azorhizobi ...................................................................................T................


401 411 421 431 441 451 461 471 481 491 consensus TTTCACCGGTGAAGATAATGACGGTAACCGGAGAAGAAGCCCCG--GCTAACTTCGTGCCAGCAGCCGCGGTAATACGAAGGGGGCTAGCGTTGTTCGGA S.fredii .................................................................................................... S.xinjiang .................................................................................................... S.saheli .................................................................................................... S.teranga .................................................................................................... S.meliloti .................................................................................................... R.galegae .........A...C............T......................................................................... Hautalense .........A................T......................................................................... A.tumefaci .........A................T......................................................................... A.rubi .........A................T......................................................................... A.vitis ........A.................GT........................................................................ R.etli .........A................T......................................................................... Phaseoli .........A................T.....................................C................................... Viceae .........A................T......................................................................... Trifolii .........A................T......................................................................... A.rhizogen .........A................T......................................................................... tropicii_D .........A................T......................................................................... Mongolense .........A................T.................GG...................................................... Gallicum .........A................T......................................................................... Hinansis .........A................T......................................................................... WSM1271 .....A........................T..................................................................... WSM1497 .....A........................T..................................................................... WSM1284 .....A........................T..................................................................... WSM1283 .....A........................T..................................................................... N18 .....A........................T..................................................................... N87 .....A........................T..................................................................... N17 .....A........................T..................................................................... M.ciceri .....A........................T..................................................................... M.loti .....A........................T..................................................................... M.tianshan .....A........................T..................................................................... M.meditere .....A........................T..................................................................... N45 .....A........................T..................................................................... M.plurifar .....A........................T..................................................................... M.huakuii .....A........................T..................................................................... M.amorphae .....A........................T..................................................................... B.elkani ...TGTGC.G................C.GCA....T..........................................................C..... B.japonicu ...TGTGC.G................C.GCA....T..........................................................C..... Azorhizobi ....G.................................................................................A.......C..... 501 511 521 531 541 551 561 571 581 591 consensus ATTACTGGGCGTAAAGCGCACGTAGGCGGAT TTAAGTCAGGGGTGAAATCCCAGGGCTCAACCCTGGAACTGCCTTTGATACTGG ATCT GAGTCC S.fredii ..............................CAT.....................G...........C....................GTG...A...... S.xinjiang ..............................CAT.....................G...........C....................GTG...A...... S.saheli ..............................CAT.....................G...........C....................GTG...A...... S.teranga ..............................CAT.....................G...........C....................GTG...A...... S.meliloti ...............................TG......G.......................................C.......CA....A...... R.galegae ...............................AT.......................A.......T......................GT....T....AT Hautalense ...............................AT.......................A.......T......................GT....T....AT A.tumefaci ...............................AT.......................A.......T......................GT....T....AT A.rubi ...............................AT.......................A.......T......................GT....T....AT A.vitis ...............................AA.....................GCA.......TGC....................TT....T....AT R.etli ...............................CGA.C..................................................TCG....G....AT Phaseoli ...............................CGA.C................................T.................TCG....G....AT Viceae ...............................CGA.C..................................................TCG....G....AT Trifolii ...............................CGA.C..................................................TCG....G....AT A.rhizogen ...............................CGA.C..................................................TCG....G....AT tropicii_D ...............................CGA.C..................................................TCG....G....AT Mongolense ..............................CAT.......................A.......T......................GTG...G....AT Gallicum ..............................CAT.......................A.......T......................GTG...G....AT Hinansis ...............................CG..............................................A.......GT....C...... WSM1271 ...............................TG......T.......................................A.......CA....C...... WSM1497 ...............................TG......T.......................................A.......CA....C...... WSM1284 ...............................TG......T.......................................A.......CA....C...... WSM1283 ...............................TG......T.......................................A.......CA....C...... N18 ...............................TG......T.......................................A.......CA....C...... N87 ...............................TG......T.......................................A.......CA....C...... N17 ...............................TG......T.......................................A.......CA....C...... M.ciceri ...............................TG......T.................N.....................A.......CA....C...... M.loti ...............................TG......T.......................................A.......CA....C...... M.tianshan ...............................AT.....................G...........C....................GT....C...... M.meditere ...............................AT.....................G..C........C....................GT....C...... N45 ...............................AC.....................G...........C....................GT....C...... M.plurifar ...............................AC.....................G...........C....................GT....G...... M.huakuii ...............................AC.....................G...........C....................GT....C...... M.amorphae ...............................AC.....................G...........C....................GT....C...... B.elkani ..C.............G.TG.........G.CT....................TG.A.......T.CA..................AAG....T....T. B.japonicu ..C.............G.TG.........G.CT....................TG.A.......T.CA..................AGG....T....T. Azorhizobi ..C............................CG.................G..TG.A.......T.CA........C..........CG....T....T.

601 611 621 631 641 651 661 671 681 691 consensus GGAAGAGGTGAGTGGAATTCCGAGTGTAGAGGTGAAATTCGTAGATATTCGGAGGAACACCAGTGGCGAAGGCGGCTCACTGGTCCGGTACTGACGCTGA S.fredii .................................................................................................... S.xinjiang .................................................................................................... S.saheli ........................................................................................A........... S.teranga ........................................................................................A........... S.meliloti A....................................................................................T..A........... R.galegae .........A.........G...............................C.........................T........AT............ Hautalense .........A.........G...............................C.........................T........AT............ A.tumefaci .........A...................................................................T........AT............ A.rubi .........A.........G...............................C.........................T........AT............ A.vitis .........A.........G...............................C.........................T........AT............ R.etli ......................................................................................AT............ Phaseoli ......................................................................................AT............ Viceae ......................................................................................AT............ Trifolii ......................................................................................AT............ A.rhizogen ......................................................................................AT............ tropicii_D ......................................................................................AT............ Mongolense ......................................................................................AT............ Gallicum ..............................................G.......................................AT............ Hinansis ......................................................................................AT............ WSM1271 .AG................................................................................CT............... WSM1497 .AG................................................................................CT............... WSM1284 .AG................................................................................CT............... WSM1283 .AG................................................................................CT............... N18 .AG................................................................................CT............... N87 .AG................................................................................CT............... N17 .AG................................................................................CT............... M.ciceri .AG................................................................................CT............... M.loti .AG................................................................................CT............... M.tianshan .AG................................................................................CT............... M.meditere .AG................................................................................CT............... N45 .................................................................................................... M.plurifar .................................................................................................... M.huakuii .................................................................................................... M.amorphae .................................................................................................... B.elkani ..G..............C.G...............................C.A.............................C...A............ B.japonicu ..G..............C.G...............................C.A.............................C...A............ Azorhizobi .AG......TG......C...................................A......................CA.....CT..A............ 701 711 721 731 741 751 761 771 781 791 consensus GGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGT-AGTCCACGCCGTAAACGATGAATGTTAGCC-GTCGGCAAGTTTACTTGTCGGTGGCG S.fredii ...............................................................................GC........GT......... S.xinjiang ...............................................................................GC........GT......... S.saheli ............................................C..................................GC........GT......... S.teranga ...............................................................................GC...C....GT......... S.meliloti ...............................................................................GC........GT......... R.galegae ..................................................N................................................. Hautalense .................................................................................................... A.tumefaci ...............................................................................GC...A....GT......... A.rubi ...............................................................................GC....G...GT......... A.vitis ...................................................TT................................G.............. R.etli ...............................................................................GC...A....GT......... Phaseoli ..........................................T....................................GC...A....GT......... Viceae .........................................................................C.....GC...A....GT......... Trifolii ...............................................................................GC...A....GT......... A.rhizogen ...............................................................................GC...A....GT......... tropicii_D ...............................................................................GC...A....GT......... Mongolense ......................................................C............................................. Gallicum .................................................................................................... Hinansis ...............................................................................GC...A....GT......... WSM1271 ...........................................................T....GA.C................................ WSM1497 ...........................................................T....GA.C................................ WSM1284 ...........................................................T....GA.C................................ WSM1283 ...........................................................T....GA.C................................ N18 ...........................................................T....GA.C................................ N87 ...........................................................T....GA.C................................ N17 ...........................................................T....GA.C................................ M.ciceri ...........................................................T....GA.C................................ M.loti ...........................................................T....GA.C................................ M.tianshan ...............................................................G.A.C........T....................... M.meditere ...............................................................G.A.C........T....................... N45 ...............................................................G.A.C........T....................... M.plurifar ...............................................................G.A.C........T....................... M.huakuii ...............................................................G.A.C........T....................... M.amorphae ...............................................................G.A.C........T....................... B.elkani ..CA...............................................................CC.......TA.TGG.......CACTA...... B.japonicu ..CA...............................................................CC.......TA.TGG.......CACTA...... Azorhizobi ...............................................................G...C........T..GG..C..G..CT..A......


801 811 821 831 841 851 861 871 881 891 consensus CAGCTAACGCATTAAACATTCCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAAGGAATT-GACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTT S.fredii .................................................................................................... S.xinjiang ..NN................................................................................................ S.saheli .................................................................................................... S.teranga ..........................................................................-......................... S.meliloti ..........................................................................-......................... R.galegae ..........................................................................-......................... Hautalense .................................................................................................... A.tumefaci ..........................................................................-......................... A.rubi ......................................................N...................-......................... A.vitis .................................................................................................... R.etli ...............................................................T.................................... Phaseoli ...............................................................G.................................... Viceae .................................................................................................... Trifolii .................................................................................................... A.rhizogen ..........................................................................-......................... tropicii_D ..NN................................................................................................ Mongolense .................................................................................................... Gallicum ..CG................................................................................................ Hinansis ..........................................................................-......................... WSM1271 ...............G.TC................................................................................. WSM1497 ...............G.TC................................................................................. WSM1284 ...............G.TC................................................................................. WSM1283 ...............G.TC................................................................................. N18 ...............G.TC................................................................................. N87 ...............G.TC................................................................................. N17 ...............G.TC................................................................................. M.ciceri ...............G.TC................................................................................. M.loti ...............G.TC................................................................................. M.tianshan ...............G.T.C................................................................................ M.meditere ...............G.T.C................................................................................ N45 ...............G.T.C................................................................................ M.plurifar ...............G.T.C................................................................................ M.huakuii ..NN...........G.T.C................................................................................ M.amorphae ...............G.T.C................................................................................ B.elkani ..........T....G.................................................................................... B.japonicu ..........T....G..........................................................-......................... Azorhizobi ..........C....G...C......................................................-......................... 901 911 921 931 941 951 961 971 981 991 consensus AATTCGAAGCAACGCGCAGAACCTTACCAGCCCTTGACATC-CCGGTCGCGG TTCCAGAGATGGA CCTTCAGTTCGGCTGGACCG-GAGACAGGTGC S.fredii .............................................A......A.A.G......C.TAT.................T.............. S.xinjiang .............................................A......A.A.G......C.TAT.................T.............. S.saheli ....................................................A.A.G......C.TATT............................... S.teranga ....................................................A..A........TTTT................................ S.meliloti .............................................A......A.A.G......C.TAT.................T.............. R.galegae ........................................G...C.GACA.--C.A........T.GTG-...CC.....--......G..C........ Hautalense ........................................G...C.G.CA.--CCA........TGGTG-...CC.....--......G..C........ A.tumefaci ...............................T........T..G..GTTT..GCAGTG....CATTGT.........A......C..CA..-........ A.rubi ...............................T........T..G..GTTT..GCAGTG....CATTGT.........A......C..CA..-........ A.vitis ...............................T...........T.TGAC..--CCA.G....C.TGGTT-...CT.....--....ACA........... R.etli ........................................G...C-GGCGAC-C.G........C.GGG-...CC.....--......G..C........ Phaseoli ........................................G...C-GGCTAC-..G........C.AGG-...CC.....--......G..C........ Viceae ........................................G...C-GGCTAC-..G........C.AGG-...CC.....--......G..C........ Trifolii ........................................G...C-GGCTAC-..G........C.AGG-...CC.....--......G..C........ A.rhizogen ...........................................T.-.GTTAC-CCGT......ATGGGG-.C..C.....--T.G.GCA........... tropicii_D ...........................................T.-.GTTAC-C..T......A.GGGG-.C..C.....--T.G.GCA........... Mongolense ........................................G...C-GGC.AC-C.A........T.GGG-...CC.....--......G..C........ Gallicum ........................................G...CCGGCTAC-C.A........T.GGG-...CC.....--......G..C........ Hinansis ........................................G...C-GGA.AC-C.A.G......T.GTG-.C..C.....--....A.G..A........ WSM1271 ....................................................T.............TA......................T......... WSM1497 ....................................................T.............TA......................T......... WSM1284 ....................................................T.............TA......................T......... WSM1283 ....................................................T.............TA......................T......... N18 ....................................................T.............TA......................T......... N87 ....................................................T.............TA......................T......... N17 ....................................................T.............TA......................T......... M.ciceri ....................................................T.............TA......................T......... M.loti ....................................................T.............TT......................T......... M.tianshan ....................................................T.............TT......................T......... M.meditere ....................................................TC............GA......................T......... N45 ....................................................T.............AA......................T......... M.plurifar ....................................................T.A.....A....TTT......................T......... M.huakuii ....................................................T.............TT......................T......... M.amorphae ....................................................T.............TT......................T......... B.elkani .......C............................................AC........C...GTT...................GAG-........ B.japonicu .......C................................GT..A.GAC...TCG.-.......T-GA.....TC.....--A.C.T.GAGC........ Azorhizobi ................................T.......G.G.A.GA-..AC....G....C...TTT....-CAG.AA-......TGC.C........

1001 1011 1021 1031 1041 1051 1061 1071 1081 1091 consensus TGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGCCCTTAGTTGCCAGCATT AGTTGGGCACTCTAA S.fredii ....................................................................................T............... S.xinjiang ....................................................................................T............... S.saheli .................................................................N..................TG.............. S.teranga ....................................................................................TG.............. S.meliloti ....................................................................................C............... R.galegae ....................................................................................C............... Hautalense ....................................................................................C............... A.tumefaci ....................................................................................T............... A.rubi .................N..................................................................TG.............. A.vitis ....................................................................................C............... R.etli ....................................................................................TG.............. Phaseoli ....................................................................................C............... Viceae ....................................................................................C............... Trifolii ....................................................................................C............... A.rhizogen ....................................................................................C............... tropicii_D ....................................................................................C............... Mongolense ....................................................................................C............... Gallicum ....................................................................................T............... Hinansis ....................................................................................T............... WSM1271 ....................................................................................A............... WSM1497 ....................................................................................A............... WSM1284 ....................................................................................A............... WSM1283 ....................................................................................A............... N18 ....................................................................................A............... N87 ....................................................................................A............... N17 ....................................................................................A............... M.ciceri ....................................................................................A............... M.loti ....................................................................................A............... M.tianshan ....................................................................................A............... M.meditere ....................................................................................C............... N45 ....................................................................................C............... M.plurifar ...............................................................................T....C............... M.huakuii ....................................................................................C............... M.amorphae ....................................................................................A............... B.elkani ...............................................................C..T..........T.C....T.....A......... B.japonicu ...............................................................C..T..........T.C....T.....A......... Azorhizobi ....................................................................T..........T....C............... 1101 1111 1121 1131 1141 1151 1161 1171 1181 1191 consensus GGGGACTGCCGGTGATAAGCCGAGAGGAAGGTGGGGATGACGTCAAGTCCTCATGGCCCTTACGGGCTGGGCTACACACGTGCTACAATGGTGGTGACAG S.fredii .................................................................................................... S.xinjiang .................................................................................................... S.saheli .................................................................................................... S.teranga .................................................................................................... S.meliloti .................................................................................................... R.galegae .................................................................................................... Hautalense .................................................................................................... A.tumefaci .................................................................................................... A.rubi .................................................................................................... A.vitis .................................................................................................... R.etli .................................................................................................... Phaseoli .................................................................................................... Viceae .................................................................................................... Trifolii .................................................................................................... A.rhizogen .................................................................................................... tropicii_D .................................................................................................... Mongolense ..................C................................................................................. Gallicum .................................................................................................... Hinansis .................................................................................................... WSM1271 .................................................................................................... WSM1497 .................................................................................................... WSM1284 .................................................................................................... WSM1283 .................................................................................................... N18 .................................................................................................... N87 .................................................................................................... N17 .................................................................................................... M.ciceri .................................................................................................... M.loti .................................................................................................... M.tianshan .................................................................................................... M.meditere .................................................................................................... N45 .................................................................................................... M.plurifar .................................................................................................... M.huakuii .................................................................................................... M.amorphae .................................................................................................... B.elkani ..A...................C....................................................................C.......A B.japonicu ..A...................C....................................................................C.......A Azorhizobi A.....................C....................................................................C.......A


1201 1211 1221 1231 1241 1251 1261 1271 1281 1291 consensus TGGGCAGCGAGACCGCGAGGTCGAGCTAATCTCCAAAAGCCATCTCAGTTCGGATTGCACTCTGCAACTCGAGTGCATGAAGTTGGAATCGCTAGTAATC S.fredii .................................................................................................... S.xinjiang .................................................................................................... S.saheli .................................................................................................... S.teranga .................................................................................................... S.meliloti .................................................................................................... R.galegae .........GAGGA....TCC............................................................................... Hautalense ..........AGGA....TCC............................................................................... A.tumefaci .............A....T................................................................................. A.rubi .............A....T................................................................................. A.vitis .................................................................................................... R.etli ...........CA......TGT.............................................................................. Phaseoli ...........CA......TGT.............................................................................. Viceae ...........CA......TGT.............................................................................. Trifolii ...........CA......TGT.............................................................................. A.rhizogen ...........CA......TGT.............................................................................. tropicii_D .N.........CA......TNT....................................................N......................... Mongolense ...........CA......TGT.........................................C.................................... Gallicum ...........CA......TGT.............................................................................. Hinansis ...........CA.....CTGT..TG.G............................................................G........... WSM1271 ......................................A............................................................. WSM1497 ......................................A............................................................. WSM1284 ......................................A............................................................. WSM1283 ......................................A............................................................. N18 .................................................................................................... N87 .................................................................................................... N17 ..........A......................................................................................... M.ciceri ......................................A............................................................. M.loti .................................................................................................... M.tianshan .................................................................................................... M.meditere .................................................................................................... N45 .................................................................................................... M.plurifar .................................................................................................... M.huakuii ........................NN................................................N......................... M.amorphae .................................................................................................... B.elkani ....AT..T.AGGG....CCCTTC..A......A....T..G...............GG..............CC......................... B.japonicu ....AT..T.AGGG....CCCTTC..A......A.......G...............GG..............CC......................... Azorhizobi ....AT.....C.T......GT....A..............G.......................................................... 1301 1311 1321 1331 1341 1351 1361 1371 1381 1391 consensus GCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTTGGTTTTACCCGAAGGCGGTGCGCTAACC-GC S.fredii ..A...........T..........................................................C..........TA.............. S.xinjiang ..A...........T..........................................................C..........TA..N........... S.saheli ..A...........T..........................................................C..........TA.............. S.teranga ..A...........T..........................................................C..........TA.............. S.meliloti ..A...........T..........................................................C..........TA.............. R.galegae ...........C........................................................................TA.............. Hautalense ...........C........................................................................TA.............. A.tumefaci ..A...........T.....................................................................TA.............. A.rubi ..A...........T.......................................................................C............. A.vitis ..A...........T.....................................................................TC.............. R.etli ....................................................................................TA.............. Phaseoli ....................................................................................TA.............. Viceae ....................................................................................TA.............. Trifolii ....................................................................................TA.............. A.rhizogen ....................................................................................TA.............. tropicii_D ....................................................................................TA..N........... Mongolense ....................................................................................TA.............. Gallicum ....................................................................................TA.............. Hinansis ..A...........T.....................................................................TA.............. WSM1271 ......................................................................................C..T.......... WSM1497 ......................................................................................C..T.......... WSM1284 ......................................................................................C..T.......... WSM1283 ......................................................................................C..T.......... N18 ......................................................................................C..T.......... N87 ......................................................................................C..T.......... N17 ......................................................................................C..T.......... M.ciceri ......................................................................................C..T.......... M.loti ......................................................................................C..T.......... M.tianshan ......................................................................................C..T.......... M.meditere ......................................................................................C..T.......... N45 ......................................................................................C..T.......... M.plurifar ......................................................................................C..T.......... M.huakuii ......................................................................................C..T.......... M.amorphae ......................................................................................C..T.......... B.elkani .T.........C...A..............................................................T....A...............A B.japonicu .T.........C...A..............................................................T....A.............C.. Azorhizobi .T.............A.......................C...............................C..............T.............

1401 1411 1421 1431 1441 1451 1461 1471 1481 1491 consensus AAGG-AGGCAGCCGACCACGGTAGGGTCAGCGACTGGGGTGAAGTCGTAACAAGGTAGCCGTAGGGGAACC----------------------------- S.fredii ............TA...................................................................................... S.xinjiang ............TA...................................................................................... S.saheli ............TA...................................................................................... S.teranga ............TA...................................................................................... S.meliloti ............TA...................................................................................... R.galegae ............TA...................................................................................... Hautalense ............TA.........................................................TGCGGCTGGATCACCTCC........... A.tumefaci ............TA...................................................................................... A.rubi .....G.....G........................................................................................ A.vitis ............GA...................................................................................... R.etli ............TA.........................................................TGCGGCTGGATCACCTCCTTTCT...... Phaseoli ............TA.........................................................TGCGGCTGGATCACCTCCTTTCT...... Viceae ............TA...........................................--------------............................. Trifolii ............TA........................................-..C..-----------............................. A.rhizogen ............TA.......................................................NN............................. tropicii_D ............TA...................................................................................... Mongolense ............TA...........................................--------------............................. Gallicum ............TA...................................................A.....TGCGGCTGGATCACCTCCTT......... Hinansis ............TA.................................................--------............................. WSM1271 ...........G.............................................--------------............................. WSM1497 ...........G.............................................--------------............................. WSM1284 ...........G.............................................--------------............................. WSM1283 ...........G.............................................--------------............................. N18 ...........G.............................................--------------............................. N87 ...........G.............................................--------------............................. N17 ...........G.............................................--------------............................. M.ciceri ...........G.............................................--------------............................. M.loti ...........G........................................................................................ M.tianshan ...........G...........................................................TGCGGCTGGATCACCTCCTT......... M.meditere ...........G.............................................--------------............................. N45 ...........G.............................................--------------............................. M.plurifar ...........G........................................................................................ M.huakuii ...........G.......................N....N...................N....................................... M.amorphae ...........G.............-.............................................TGCGGCTGGATCACCTCC........... B.elkani ....GG..-.....G........................................................TGCGGCTGGATCACCTCCTTTCTAAGGAT B.japonicu ....G.........G..................................................................................... Azorhizobi ...........G..................T.....................................................................

Appendix 4 – Distance matrix of 16S rRNA gene sequence of some RNB in α-proteobacteria showing Kimura distance values 197

R.mongolense 0.0

R.gallicum 0.0064 0.0

R.l.phaseoli 0.0252 0.0261 0.0

R.etli 0.0186 0.0196 0.0118 0.0

R.l trifolii 0.0245 0.0278 0.0028 0.0121 0.0

R.viciae 0.0252 0.0259 0.0028 0.0121 0.0021 0.0

R.hainanensis 0.029 0.0277 0.0311 0.0251 0.0329 0.0311 0.0

R.tropicii 0.0365 0.0354 0.0207 0.0221 0.0187 0.0209 0.0341 0.0

A.rhizogenes 0.0335 0.0339 0.0178 0.0207 0.0165 0.0179 0.0319 0.0042 0.0

R.huautlense 0.0319 0.0324 0.0408 0.0422 0.0399 0.0417 0.0473 0.0482 0.0445 0.0

R.galegae 0.0349 0.036 0.0444 0.0451 0.0433 0.0448 0.0492 0.0527 0.0497 0.0063 0.0

A.tumefaciens 0.0556 0.055 0.0544 0.0543 0.0541 0.0549 0.0546 0.0474 0.0475 0.0376 0.0421 0.0

A.rubi 0.0642 0.0628 0.0652 0.0637 0.0643 0.0659 0.0648 0.0574 0.0575 0.0405 0.045 0.0148 0.0

A.vitis 0.057 0.0586 0.065 0.0657 0.064 0.0656 0.0639 0.0657 0.0611 0.039 0.0383 0.0376 0.0443 0.0

S.fredii 0.0418 0.0421 0.0422 0.0429 0.0411 0.0426 0.0409 0.0368 0.04 0.0473 0.0527 0.0448 0.0531 0.0541 0.0

S.xinjiangensis 0.0419 0.0407 0.0423 0.0438 0.0411 0.0427 0.041 0.0369 0.04 0.0474 0.0528 0.0449 0.0533 0.0543 0.0 0.0

S.saheli 0.048 0.0482 0.0484 0.0476 0.0473 0.0488 0.0456 0.0422 0.0446 0.0528 0.0552 0.051 0.0563 0.0566 0.0084 0.0084 0.0

S.terangae 0.0472 0.0474 0.0483 0.046 0.0472 0.0487 0.0439 0.0474 0.0467 0.0496 0.0512 0.0494 0.0555 0.0542 0.0163 0.0163 0.0113 0.0

S.meliloti 0.048 0.0498 0.0407 0.043 0.0396 0.0411 0.0425 0.0368 0.0385 0.0505 0.0559 0.0495 0.0578 0.0588 0.0098 0.0099 0.017 0.0249 0.0

WSM1271 0.0643 0.0642 0.0605 0.0666 0.0597 0.0604 0.0667 0.0611 0.062 0.0535 0.0579 0.0694 0.0662 0.0694 0.0484 0.0484 0.0546 0.0507 0.0446 0.0

WSM1497 0.0643 0.0641 0.0605 0.0666 0.0597 0.0604 0.0666 0.0611 0.0619 0.0535 0.0579 0.0694 0.0662 0.0693 0.0483 0.0484 0.0546 0.0507 0.0446 0.0 0.0

WSM1284 0.0643 0.0641 0.0605 0.0666 0.0597 0.0604 0.0666 0.0611 0.0619 0.0535 0.0579 0.0694 0.0662 0.0693 0.0483 0.0484 0.0546 0.0507 0.0446 0.0 0.0 0.0

WSM1283 0.065 0.0649 0.0613 0.0673 0.0605 0.0611 0.0674 0.0618 0.0627 0.0528 0.0571 0.0702 0.067 0.0701 0.0491 0.0492 0.0553 0.0515 0.0453 0.0007 0.0007 0.0007 0.0

N18 0.0658 0.0656 0.062 0.0666 0.0613 0.0619 0.0682 0.0626 0.0634 0.055 0.0594 0.0709 0.0677 0.0708 0.0498 0.0499 0.0561 0.0491 0.0461 0.0042 0.0042 0.0042 0.0049 0.0

N87 0.0658 0.0656 0.062 0.0666 0.0613 0.0619 0.0682 0.0626 0.0634 0.055 0.0594 0.0709 0.0677 0.0708 0.0498 0.0499 0.0561 0.0491 0.0461 0.0042 0.0042 0.0042 0.0049 0.0 0.0

N17 0.0666 0.0664 0.0628 0.0673 0.062 0.0627 0.0689 0.0634 0.0642 0.0543 0.0586 0.0717 0.0685 0.0716 0.0506 0.0507 0.0568 0.0499 0.0468 0.0049 0.0049 0.0049 0.0042 0.0007 0.0007 0.0

M.ciceri 0.0643 0.0651 0.0606 0.066 0.0583 0.0605 0.0676 0.0612 0.062 0.054 0.0579 0.0695 0.0663 0.0694 0.0484 0.0485 0.0546 0.0508 0.0447 0.0 0.0 0.0 0.0007 0.0042 0.0042 0.0049 0.0

M.loti 0.0651 0.0659 0.0607 0.066 0.0597 0.0613 0.0656 0.0612 0.0614 0.0533 0.0542 0.0686 0.0655 0.0655 0.0478 0.0479 0.051 0.0472 0.0441 0.0028 0.0028 0.0028 0.0035 0.0056 0.0056 0.0064 0.0028 0.0

M.tianshanense 0.061 0.0602 0.0606 0.0665 0.0627 0.062 0.0662 0.0621 0.063 0.0485 0.0533 0.0647 0.0615 0.0632 0.0411 0.0411 0.0471 0.0434 0.0479 0.0119 0.0119 0.0119 0.0126 0.0147 0.0147 0.0154 0.0121 0.0119 0.0

M.mediterraneum 0.0579 0.0594 0.0613 0.0651 0.059 0.0613 0.0659 0.0603 0.0604 0.0477 0.0516 0.0638 0.0606 0.0615 0.0422 0.0423 0.0483 0.0453 0.0484 0.0142 0.0142 0.0142 0.0149 0.0157 0.0157 0.0164 0.0135 0.0157 0.0049 0.0

N45 0.0595 0.0601 0.059 0.0658 0.0583 0.0589 0.0658 0.0611 0.0612 0.0489 0.0531 0.0653 0.0622 0.0623 0.0407 0.0408 0.0469 0.0431 0.0461 0.0183 0.0183 0.0183 0.019 0.0154 0.0154 0.0161 0.0186 0.02 0.0098 0.0085 0.0

M.plurifarium 0.0611 0.0627 0.0608 0.0669 0.0598 0.0614 0.0688 0.059 0.0607 0.0488 0.0542 0.0631 0.06 0.0618 0.0395 0.0396 0.0456 0.0433 0.0449 0.02 0.02 0.02 0.0207 0.02 0.02 0.0207 0.02 0.0198 0.0098 0.0121 0.0071 0.0

M.huakuii 0.0582 0.0583 0.058 0.0649 0.0569 0.0585 0.0627 0.0593 0.0595 0.0467 0.0506 0.0627 0.0596 0.0597 0.039 0.039 0.0451 0.0413 0.0444 0.0172 0.0172 0.0172 0.0179 0.0157 0.0157 0.0164 0.0172 0.017 0.007 0.0078 0.0028 0.0042 0.0

M.amorphae 0.0596 0.0588 0.0585 0.0644 0.0605 0.0597 0.064 0.0598 0.0608 0.047 0.0519 0.0632 0.0601 0.0609 0.0396 0.0397 0.0457 0.0419 0.0457 0.0155 0.0154 0.0154 0.0161 0.0155 0.0155 0.0162 0.0157 0.0155 0.0054 0.0092 0.0042 0.0042 0.0014 0.0

B.elkanii 0.1188 0.1149 0.1242 0.1156 0.1258 0.1275 0.1217 0.125 0.1265 0.1074 0.1141 0.129 0.1265 0.1311 0.1127 0.1129 0.1127 0.111 0.1187 0.1144 0.1144 0.1144 0.1135 0.1151 0.1151 0.1142 0.1161 0.1155 0.1059 0.1084 0.1084 0.113 0.1093 0.1077 0.0

B.japonicum 0.1116 0.1112 0.1209 0.1116 0.1195 0.1221 0.1191 0.1197 0.1204 0.1056 0.1097 0.1284 0.1276 0.1319 0.1164 0.1167 0.1172 0.1155 0.1232 0.1234 0.1233 0.1233 0.1224 0.1231 0.1231 0.1223 0.1235 0.1237 0.1162 0.114 0.1155 0.1185 0.1167 0.1179 0.0206 0.0

Azorhizobium 0.0978 0.0991 0.0977 0.0959 0.0962 0.0987 0.1023 0.1031 0.1027 0.1009 0.1034 0.1065 0.1032 0.1072 0.0982 0.0984 0.0999 0.1021 0.0997 0.099 0.0989 0.0989 0.0997 0.0973 0.0973 0.0981 0.0991 0.0978 0.0908 0.0926 0.0943 0.0925 0.0905 0.0917 0.1008 0.1 0.0

R.m

ongo

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R.h

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R.tr

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R.h

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S.te

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S.m

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1271

W

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W

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1284

W

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1283

N

18

N87

N

17

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M.ti

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M.m

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Appendix 5 – Statistical analysis for N2 fixation effectiveness 198

MANOVA MAIN EFFECT: TREATMENT

{1} {2} {3} {4} {5} {6} {7} {8} {9} {10} {11} {12} {13} {14} {15} {16} {17} {18}

TREATMEN 0.259 0.1565 0.01775 0.06125 0.04625 0.054 0.004 0.05425 0.047 0.11025 0.11725 0.0915 0.121 0.14225 0.12 0.1335 0.132 0.00375

N {1} 0.0000001 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

WSN1271 {2} 0.0000001 0 0.0000003 0 0.0000001 0 0.0000001 0 0.0063126 0.019298 0.0001978 0.0335213 0.3850927 0.0290276 0.1632922 0.1380172 0

N15 {3} 0 0 0.0099193 0.0855687 0.030098 0.4018745 0.0290276 0.077863 0.0000005 0.0000001 0.0000328 0 0 0.0000001 0 0 0.393428

N17 {4} 0 0.0000003 0.0099193 0.3607597 0.6577309 0.0008905 0.6687988 0.3850927 0.0039541 0.001125 0.0685 0.0005537 0.0000069 0.0006704 0.0000449 0.0000613 0.0008496

N18 {5} 0 0 0.0855687 0.3607597 0.6358269 0.0121152 0.624995 0.9634106 0.0002414 0.0000582 0.0074541 0.0000265 0.0000002 0.0000328 0.0000018 0.0000025 0.0116435

N39 {6} 0 0.0000001 0.030098 0.6577309 0.6358269 0.003323 0.9877996 0.6687988 0.0010739 0.00028 0.0250754 0.0001323 0.0000014 0.0001618 0.0000096 0.0000133 0.0031806

N45 {7} 0 0 0.4018745 0.0008905 0.0121152 0.003323 0.0031806 0.0107498 0 0 0.0000017 0 0 0 0 0 0.9877996

N46 {8} 0 0.0000001 0.0290276 0.6687988 0.624995 0.9877996 0.0031806 0.6577309 0.001125 0.0002942 0.0260158 0.0001391 0.0000015 0.0001702 0.0000101 0.000014 0.003044

N87 {9} 0 0 0.077863 0.3850927 0.9634106 0.6687988 0.0107498 0.6577309 0.00028 0.000068 0.0084319 0.0000311 0.0000003 0.0000384 0.0000021 0.0000029 0.0103269

N1 {10} 0 0.0063126 0.0000005 0.0039541 0.0002414 0.0010739 0 0.001125 0.00028 0.6687988 0.2543201 0.5116858 0.0544016 0.5515872 0.1588466 0.1869787 0

N5 {11} 0 0.019298 0.0000001 0.001125 0.0000582 0.00028 0 0.0002942 0.000068 0.6687988 0.1194131 0.8186228 0.1303167 0.8664381 0.3224611 0.3687583 0

N19 {12} 0 0.0001978 0.0000328 0.0685 0.0074541 0.0250754 0.0000017 0.0260158 0.0084319 0.2543201 0.1194131 0.0754271 0.0029129 0.0855687 0.0126043 0.0159353 0.0000016

N36 {13} 0 0.0335213 0 0.0005537 0.0000265 0.0001323 0 0.0001391 0.0000311 0.5116858 0.8186228 0.0754271 0.1971526 0.9512278 0.4457597 0.5019578 0

N48 {14} 0 0.3850927 0 0.0000069 0.0000002 0.0000014 0 0.0000015 0.0000003 0.0544016 0.1303167 0.0029129 0.1971526 0.1772082 0.5930024 0.5314413 0

N59 {15} 0 0.0290276 0.0000001 0.0006704 0.0000328 0.0001618 0 0.0001702 0.0000384 0.5515872 0.8664381 0.0855687 0.9512278 0.1772082 0.4104321 0.464072 0

N64 {16} 0 0.1632922 0 0.0000449 0.0000018 0.0000096 0 0.0000101 0.0000021 0.1588466 0.3224611 0.0126043 0.4457597 0.5930024 0.4104321 0.9269002 0

N84 {17} 0 0.1380172 0 0.0000613 0.0000025 0.0000133 0 0.000014 0.0000029 0.1869787 0.3687583 0.0159353 0.5019578 0.5314413 0.464072 0.9269002 0

M {18} 0 0 0.393428 0.0008496 0.0116435 0.0031806 0.9877996 0.003044 0.0103269 0 0 0.0000016 0 0 0 0 0

LSD tests for differences between treatments, p is 0.01

STATIST summary of all effects; design: ANOVA significance level is 0.01because of unequal variancesGENERA 1-TREATMENMANOVA

df MS df MS Effect Effect Effect Error Error F p-level

1 17 0.016 54 0.00053 30.856 0

Appendix 6 – Plate photos showing the intrinsic antibiotic resistance of re-isolates and WSM1271 199

Gentamycin

Kanamycin

Ampicillin Ampicillin

Chloramphenicol Chloramphenicol

Gentamycin

Kanamycin

N5

N5

N5

N5

N19

N19

N19

N19

N36

N36

N36

N36

N48

N48

N48

N48

N59

N59

N59

N59

N64

N64

N64

N64

N84

N84

N84

N84

1271

1271

1271

1271

Gentamycin

Kanamycin


Chloramphenicol Chloramphenicol

Gentamycin

Kanamycin

N5

N5

N5

N5

N19

N19

N19

N19

N36

N36

N36

N36

N48

N48

N48

N48

N59

N59

N59

N59

N64

N64

N64

N64

N84

N84

N84

N84

1271

1271

1271

1271

Nalidixic acid Nalidixic acid

Spectinomycin Spectinomycin

Streptomycin Streptomycin

Tetracycline Tetracycline

N5

N5

N5

N5

N19

N19

N19

N19

N36

N36

N36

N36

N48

N48

N48

N48

N59

N59

N59

N59

N64

N64

N64

N64

N84

N84

N84

N84

1271

1271

1271

1271



Streptomycin Streptomycin


N5

N5

N5

N5

N19

N19

N19

N19

N36

N36

N36

N36

N48

N48

N48

N48

N59

N59

N59

N59

N64

N64

N64

N64

N84

N84

N84

N84

1271

1271

1271

1271

Appendix 7 – Plate photos showing the intrinsic antibiotic resistance of novel isolates, WSM1271 and control species 200

NZ - Mesorhizobium loti (NZP2213), MEL - Sinorhizobium meliloti (USDA1002), LEG - Rhizobium leguminosarum (USDA2370)


Chloramphenicol

Gentamycin

Kanamycin

N87 1271

1271

Chloramphenicol

Gentamycin

Kanamycin

N46

N46

N46

N46

N87

N87

N87

1271

1271

1271

1271

1271

1271

1271

Chloramphenicol

Gentamycin

Kanamycin

N17

N17

N17

N17

N18

N18

N18

N18

N39

N39

N39

N39

N45

N45

N45

N45


Chloramphenicol

Gentamycin

Kanamycin

N87 1271

1271

Chloramphenicol

Gentamycin

Kanamycin

N46

N46

N46

N46

N87

N87

N87

1271

1271

1271

1271

1271

1271

1271

Chloramphenicol

Gentamycin

Kanamycin

N17

N17

N17

N17

N18

N18

N18

N18

N39

N39

N39

N39

N45

N45

N45

N45



Streptomycin


N59

N87 1271

1271N46

N46

N46

N46

N87

N87

N87

1271

1271

1271

1271

1271

1271

1271

N17

N17

N17

N17

N18

N18

N18

N18

N39

N39

N39

N39

N45

N45

N45

N45

Streptomycin



Streptomycin


N59

N87 1271

1271N46

N46

N46

N46

N87

N87

N87

1271

1271

1271

1271

1271

1271

1271

N17

N17

N17

N17

N18

N18

N18

N18

N39

N39

N39

N39

N45

N45

N45

N45

Streptomycin

Ampicillin

Chloramphenicol

Gentamycin

Kanamycin

NZ

NZ

NZ

NZ

MEL

MEL

MEL

MEL

LEG

LEG

LEG

LEG

Nalidixic acid

Spectinomycin

Streptomycin

Tetracycline

NZ

NZ

NZ

NZ

MEL

MEL

MEL

MEL

LEG

LEG

LEG

LEG

Ampicillin

Chloramphenicol

Gentamycin

Kanamycin

NZ

NZ

NZ

NZ

MEL

MEL

MEL

MEL

LEG

LEG

LEG

LEG

Nalidixic acid

Spectinomycin

Streptomycin

Tetracycline

NZ

NZ

NZ

NZ

MEL

MEL

MEL

MEL

LEG

LEG

LEG

LEG

Appendix 8 – Plate photos showing the pH tolerance of re-isolates, novel isolates and WSM1271 201

pH 5.0

pH 5.0

pH 4.5 pH 5.0

pH 4.5 pH 5.0

pH 4.5

pH 4.5

N5

N59

N17

N46

N19

N64

N18

N87

N36

N84

N39

1271

N48

N1271

N45

1271

N5

N59

N17

N46

N19

N64

N18

N87

N36

N84

N39

1271

N48

1271

N45

1271

pH 5.0

pH 5.0

pH 4.5 pH 5.0

pH 4.5 pH 5.0

pH 4.5

pH 4.5

N5

N59

N17

N46

N19

N64

N18

N87

N36

N84

N39

1271

N48

N1271

N45

1271

N5

N59

N17

N46

N19

N64

N18

N87

N36

N84

N39

1271

N48

1271

N45

1271

N5

N59

N17

N46

N19

N64

N18

N87

N36

N84

N39

1271

N48

N1271

N45

1271

N5

N59

N17

N46

N19

N64

N18

N87

N36

N84

N39

1271

N48

1271

N45

1271

pH 9.0

pH 9.0

pH 7.0 pH 9.0

pH 7.0 pH 9.0

pH 7.0

pH 7.0

N5

N59

N17

N46

N19

N64

N18

N87

N36

N84

N39

1271

N48

N1271

N45

1271

N5

N59

N17

N46

N19

N64

N18

N87

N36

N84

N39

1271

N48

1271

N45

1271

pH 9.0

pH 9.0

pH 7.0 pH 9.0

pH 7.0 pH 9.0

pH 7.0

pH 7.0

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