WO1997026363A2

WO1997026363A2 - Method of inducing nitrogen fixation in plants

Info

Publication number: WO1997026363A2
Application number: PCT/GB1997/000120
Authority: WO
Inventors: Edward Charles Daniel Cocking
Original assignee: Edward Charles Daniel Cocking
Priority date: 1996-01-19
Filing date: 1997-01-17
Publication date: 1997-07-24
Also published as: AU1394697A; GB9601110D0; WO1997026363A3

Abstract

The invention relates to a method of inducing nitrogen fixation in non-leguminous plants by inoculating the plant with a nitrogen-fixing bacterium, wherein the bacterium is exposed to a nod-factor inducing agent. The invention also relates to a composition comprising a non-leguminuous plant seed mixed with a plant growth medium and an inoculum of a nitrogen-fixing bacterium exposed to an agent capable of inducing nod-factor production in the bacterium. In another aspect the invention relates to a plant growth medium for a non-leguminous plant comprising a bacterium capable of fixing nitrogen wherein the bacterium has previously been exposed to a nod-factor inducing agent.

Description

METHOD OF INDUCING NITROGEN FIXATION IN PLANTS

The invention relates to plant biotechnology and particularly to me stimulation of nitrogen-fixation in non-leguminous plants by interaction with nitrogen-fixing bacteria.

Legumes, such as clover, have nodules containing nitrogen-fixing bacteria on their roots which fix atmospheric nitrogen so that it is available to the plant as a nutrient. Apart from the commercially unimportant Parasponia genus of plants (tropical shrubs in the elm family), non-legumes do not naturally form such nodules. It would be desirable to enable commercially important non-leguminous cereal crops such as wheat, rice, maize, barley, millet, sorghum and rice; tomatoes and other horticulturally important crops, and especially oilseed rape (Brassica napus and other Brassica species) to fix nitrogen as this would offer the possibility of reducing the amount of nitrogenous fertilizers used at present. Indeed Cocking et al ((1994) Agro-Food Industry Hi-Tech Jan/Feb 21-24) remarked that "The introduction of symbiotic biological nitrogen fixation into the major non- legume crops of the world would be one of the most significant contributions biotechnology could make to agriculture.".

Nodulation of legumes by the nitrogen-fixing bacteria rhizobia is generally considered to be controlled by rhizobia nodulation (nod) genes. These nod genes encode enzymes involved in the syndiesis of Nod factors that induce morphological changes in legume roots. The four nod genes A,B,C and D are u e minimum number of genes required for the formation of such extracellular metabolites which induce the root hair curling response in legumes. It has been demonstrated that the expression of some of the rhizobia symbiotic genes wim the associated production of rhizobia Nod factors, can induce changes in the growth of rice root hairs comparable to the curling and distortion observed in root hair of legumes during interaction wiύi rhizobia.

Some naturally occurring rhizobia induce nitrogen-fixing root nodules on a wide range of legumes. For instance, strain NGR234 nodulates many legumes, including species in the genera Arachis, Glycine, Lablab and Vigna. Additionally, this wide host range strain NGR234 induces effective root nodules on the non-legume Parasponia. This unique association has prompted a genetic analysis of the nitrogen fixing symbiosis between Parasponia and rhizobia. The infection of Parasponia by rhizobia requires the nod regulatory gene, together with host-specific nodulation (hsri) genes. Thus, nodulation of Parasponia requires the co¬ ordinated expression of nodA,B and C genes together with rhizobia nsn genes under the regulation of the nodD product. This knowledge enabled uie construction of a new rhizobium strain (ANU536) which possessed multiple copies of the nodD gene on a high copy number vector. This strain was able to induce nodule-like strucmres on the roots of rice seedlings at a low frequency of 0.1-0.25%. These nodule-like strucmres were small, white and contained membrane encapsulated bacteria; one of these strucmres was observed to have an internal strucmre resembling that found within legume root nodules. However, nitrogen fixation activity has not been detected.

A wide range of nod genes have so far been identified in rhizobia (see Werner D, Symbiosis of Plants and Microbes (1992), published Chapman & Hall, especially pages 68 and 69 and Table 3.7 below). However, many of the nod factors have not been characterised. From Wemer (1992): Table 3.7 Nodulation (nod) genes in Rhizobium

(R), Bradyrhizobium (B), Sinorhizobium (S) and Azorhizobium (A).

Gene Functions of gene products nodABC Essential for root hair curling and cortical cell division. NodABC likely synthesize a phytohormone-like substance. R. meliloti: Rmnod-1 identified as a Nacyl-tri-N-acetyl-/°-l,4-D- glucosamine tetrasaccharide bearing a sulphate group on carbon 6 of the reducing sugar moiety. nodbl, D2,D3 Transcriptional regulator required for the expression of the other nod genes. nodE Host range, reported sequence similarity to the^άMJ gene of f. coli, condensing enzyme of fatty acid synthase. Membrane protein. Mutants lacking ΝodE are affected in capsular polysaccharide biosynthesis. nodF Host range, reported sequence similarity to acyl carrier protein. NodF carries 4'-phosphopantetheine as a prosthetic group. At least rwo different forms of ΝodF can be isolated from a ΝodFe- overproducing strain of R. leguminosarum bv . viciae. nodG Host range, reported sequence similarity to dehydrogenases. nodti Host range, many modify factor produced by NodABC. may function as a sulphate transferase for the synthesis of ΝodRm-I. nodi Reported sequence similarity to ATP binding transport proteins. R. leguminosarum bv. trifolii mutants defective in Nodi have a decrease in the 3-hydroxybutyrate substitution of the capsular polysaccharide. nodi Membrane protein, may act in conjunction with nod . nodK Reported only in B. sp. (Parasponia). Function unknown. nodL Host range, reported sequence similarity to acetyltransferase. nodM Host range, reported sequence similarity to amidotransferase. nodN Reported. Involved in biosynthesis of hair deformation factor. nodO Function unknown, encoded protein is exported. Reported to bind calcium. nod? Function unknown, gene hybridizes to E. coli genomic DΝA. nodQ Host range, reported sequence similarity to GTP binding proteins, EF-tu and EF-1. May modify factor produced by NodAB . nodR Reported only in R. leguminosarum bv. trifolii. Host range. nodS Function unknown. MW 23 kDa. nodT Host range. Function unknown. nodV Function unknown. MW 62 kDa. nodV Reported only in B. japonicum. Important for host range, sequence similarity to membrane sensor family. nodW Reported only in B. japonicum. Important for host range, sequence similarity to family of transcriptional regulatory proteins. nodX Reported only in R. leguminosarum strains capable of nodulating Afghanistan pea. nodY Reported only in B. japonicum, but found in all rhizobia by hybridization. Function unknown. nodZ Host range required for growth under microaerobic conditions. nodlA Host range. Essential for nodulation of selected soybean cultivars. Reported only from B. japonicum. Shows sequence similarity to MerR, a transcriptional regulatory protein. nolD, nolE, Host range in R. meliloti. nolF, nolG

Expression of nod genes may be regulated in legumes by inducers such as the flavones or flavonones exuded by legume roots.

It is known uiat the flavonoid naringenin increases root nodulation and nitrogen fixation when added to the rhizosphere during inoculation of alfalfa cultivar Rijka T9 with Rhizobium meliloti (Jain et al (1990) World J Microbiol 6:434-436). However, attempts to extend these observations to other alfalfa cultivars did not increase nodulation (Phillips (1992) Phenolic Metabolism in Plants, Chapter 7, page 219, Ed. Stafford and Ibraheim, Plenum Press, New York) which strongly suggests that the observations could not be extended to other legumes.

The present invention aims to provide a method for inducing nitrogen fixation in non-legume plants.

Various aspects of the present invention are defined in the accompanying claims.

According to the invention there is provided a method of inducing nitrogen fixation in a non-leguminous plant by inoculating the plant with a nitrogen- fixing bacterium; wherein the bacterium is exposed to a nod-factor inducing agent.

The term "inoculating" is intended to embrace any means by which the plant and bacterium are brought into nitrogen-fixing interaction.

Unexpectedly, the inventor has found that exposure of die bacterium to a nod-factor inducing agent in the meuiod of the invention induces the bacterium to produce Nod factors which stimulate the entry of the bacterium into the non-leguminous plant root system and nitrogen fixation.

The results presented herein demonstrate for the first time that nod-factor inducing agents such as flavonoids can stimulate the interaction of nitrogen fixing bacteria with a non-legume crop, such as wheat, resulting in nitrogen fixation.

In a further aspect the invention provides a non-leguminous plant in a nitrogen fixing interaction with a bacterium obtainable by the method of the invention.

The phrase "nitrogen-fixing interaction" is intended to include any plant/bacterial interaction in which atmospheric nitrogen is made available to the plant as a nutrient. The interaction may, for example, result in uie formation of root or stem nodules containing the bacterium, or, by bacterial invasion of lateral roots, in the formation of short, thickened lateral roots (STLRs) which are very similar in appearance to the normal short lateral roots of the plant.

Preferably, t e bacterium for use in the method of the invention is oxygen tolerant. By "oxygen tolerant" we include any bacterium which is able to fix atmospheric nitrogen in the presence of at least 0.1% oxygen. Examples of suitably tolerant rhizobia bacteria comprise stem-nodulating rhizobia including Azorhizobium species such as A caulinodans (eg. strain ORS571 ATCC deposit No. 43989) and Bradyrhizobium species such as B. ORS310, which normally nodulates stems of Aeschynomene indicia (Van Rhiju and Vanderleyden (1995) J. Microbiol Rev. 59: 124-142).

The nitrogenase of A caulinodans is tolerant of up to approximately 3% oxygen and the nitrogenase of B. ORS310 is tolerant of up to approximately 0.5% oxygen.

Oxygen tolerant bacteria are preferred because, unlike leguminous plants, non-leguminous plants do not normally contain molecules such as leghaemoglobin which can take up free oxygen to minimize inhibition of nitrogen fixation activity due to inactivation of nitrogenase.

Other bacteria suitable for use in the method of the invention include the Rhizobia which namrally infect legumes (especially temperate legumes) via an infection thread in uie root hairs such as Rhizobium leguminosarum, R. meliloti, R. loti, R. phaseoli, R. japonicum, R. lupini and R. tήfolii.

Preferably, Rhizobia used in the meuiods of he invention are hose which disturb the epidermis themselves or which can infect the plant by "crack- entry", that is, take advantage of a dismrbance of the epidermis (for example resulting from physical injury or the emergence of an organ such as a lateral root) and in either case thereby form a nodule. Such Rhizobia include those which namrally form nodules on uie roots of Parasponia species and those which infect the roots (including lateral roots) or other tropical plants (especially legumes), such as Aeschynomenes spp. (for example A. afraspera), Arachis Lypogea, Neptunia oleracea, Stylosanthes spp. and Sesbania rostrata. The Rhizobia also include those which induce nodules on the stems of Sesbania, Aeschynomenes, and Neptunia species. Rhizobia which infect Parasponia and cause nodulation include:

Normal Host Examples of strains

P. rugosa P. andersonii CP283

Cajanus cajan NGR 70

Centrosema pubescens

Crotalaήa anagyroides

Flemingia congesta Inocarpus fragiferum

Macroptilium lathyroides NGR 86, NGR 169

Phaseolus calcaratus

Stizolobium deeringianum NGR 179

Stylosanthes gracilis Lablab purpuresu NGR 234

Acacia farnesiana NGR 71

Leucaena leucocephala NGR 7, 8, 44, 63, 75, 89, 14/1 , 19,

43, 95, 98, 99, 107, 111 , 112

Mimosa invisa } NGR 181, 189, 83, 135 M. pudica } 114, 190 (from Trinick & Galbraith (1980) New Phytol. 86, 17-26).

The Parasponia-wfecting Rhizobium strains CP283 and 501 are particular examples of effective Rhizobia.

The nod-factor inducing agents useful in the present invention comprise any agent which can induce nod-factors in the bacterium used to inoculate the non-leguminous plant. Preferably, the inducing agents are obtained from plant exudates.

Particularly preferred inducing agents are flavonoids. Dakora, F.D. (1995) Aust. J. Plant Physiol 22, 87-99 provides a review of plant flavonoids.

Flavonoids are naturally occurring plant products which originate from the phenylpropanoid pathway (Table A). Each molecule has a C₁₅ skeleton formed from condensation of three malonate units with a phenylalanine- derived -C₃ precursor. Their distribution is widespread in higher plants.

Isoflavone Dihydroflavonol

Flavan-3,4-diol

8a Table A. Proposed pathways of phenylpropanoid metabolism (a-c) and flavonoid biosynthesis (1-7). Enzymes: a, phenylalanine ammonia-lyase (PAL); b, cinnamate 4-hydroxylase: c. 4-coumarate: CoA ligase (4CL): 1, chalcone synthase (CHS); 2, chalcone isomerase (CHI); 3, flavone synthase; 4, 'isoflavone synthase' (possibly two enzymes); 5, flavanone 3-hydroxylate; 6, flavonol synthase; and 7, dihydroflavonol 4-reductase (DFR).

Flavonoid structures, namely flavones, flavanones, isoflavones and chalcones (Table A), induce transcription of nodulation (nod) genes in Rhizobium cells as the first step towards root nodule formation and symbiotic N₂ fixation. Anthocyanidins such as delphinidin, petunidin and malvidin are also able to transcribe nod genes in Rhizobium legumino¬ sarum biovar phaseoli. The molecules identified are released from seed coats during imbibition and from root exudates of sterile young seedlings. This pool of active molecules is increased in exudate by the release of additional nod-gene inducers when rhizobial cells are present on infectible roots.

Table B. Variation in the nodulating signal in different legume- rhizobial symbioses. Harborne (1991): Chapter 11 of "Herbivores: their interactions with secondary plant metabolites" 2 Ed. Vol 1: The Chemical Participants Academic Press. Inc.

Symbiosis Inducing flavonoids from roots

Rhizobium trifolii 7,4' -dihydroxy flavone on clover 7-methoxy-4 ' -hydroxy flavone 7,4'-dihydroxy-3 '-methoxy flavone

Rhizobium meliloti 5 ,7 ,3 ' ,4 '-tetrahydroxyflavone (luteolin) on alfalfa Rhizobium leguminosarum 5,7,3',4'-tetrahydroxyflavone (eriodictyol) on pea 5,7,4'-trihydroxyflavone 7-glucoside

Useful flavonoids can be provided as exudate from the seedlings of plants, particularly legumes which normally have nodules on their roots containing the nitrogen fixing bacterium used to inoculate the non- leguminous plant.

Flavonoids can be purified from the exudate of seedlings using conventional methods such as those described by Messens et al (1991 , Mol. Plant-Microbe Interactions, vol.4, No.3, pp.262-267). They extracted 7,4'-Dihydroxyflavanone (liquiritigenin) as the major nod-factor inducing component of exudate obtained from seedlings of the tropical legume Sesbania rostrata which normally includes nitrogen-fixing nodules on its roots and stems containing the bacterium Azorhizobium caulinodans strain ORS571. O her flavonoids which were shown by Messens et al to imitate the inducing effect of liquiritigenin were 5,7,4'- trihydroxyflavanone (naringenin) and 4,2',4'-trihydroxychalcone (isoliquiritigenin).

Thus, in a preferred embodiment the method of the invention comprises inoculating a non-leguminous plant with Azorhizobium wherein uie bacterium is exposed to one or more nod-factor inducing flavonoids selected from naringenin, liquiritigenin and isoliquiritigenin. Preferably, the bacterium is Azorhizobium caulinodans.

Non-flavonoid nod-factor inducing agents for use in the invention may include betaines, preferably trigonelline and stachydrine. The latter two betaines were identified by Phillips et al (1992) Plant Physiol 99: 1526-

10 1531 as major compounds inducing nod-factor synthesis in seed exudates from the legume Alfalfa.

The inventors have also identified di-iodo 4-hydroxybenzoic acid (Pfaltz Bauer Inc) as a nod-factor inducer in Azorhizobia inoculated wheat seedlings and the related 4-hydroxybenzoic acid as a nod-factor inducer in Rhizobium NGR234.

It will be appreciated by a person skilled in the art that the preferred amount of the agent for use in uie meύiod of the invention to induce nod- factor production in a particular bacterium and allow it to form a nitrogen- fixing interaction with a particular non-leguminous plant can be determined using routine tests involving nothing more than trial and error.

Preferably, with a sufficient amount of inducing agent the bacterium is exposed to an inducing agent solution having a molarity of from 1 x IO^"6 to 5 x 10^"4 and especially from 2 x IO^"5 to 1 x 10

Preferably, seedlings of the non-leguminous plants are inoculated with uie nitrogen-fixing bacterium in the presence of the nod factor inducing agent shortly after germination. For example, wheat seedlings may be inoculated approximately 3 to 6 days after germination.

Alternatively, the bacterium can be pretreated with the inducing agent prior to inoculation of the plant seedlings.

Preferably, the seeds are sterilised prior to germination and grown under sterile conditions prior to inoculation.

In an alternative preferred embodiment the non-leguminous plant

11 protoplast is exposed for interaction with the bacterium in the root hairs of the plant wiuiout release of the protoplasts from the plant.

Preferably, such exposure is achieved by degrading the plant cell wall at the apices of the root hairs enzymatically.

The bacterium is preferably exposed to the nod-factor inducing agent during or prior to this step.

A suitable experimental protocol for exposing plant protoplasts in accordance with the above preferred embodiment of the invention is described in UK Patent No. GB 2175919B, the disclosure of which is incorporated herein by reference. Particular reference is made to examples 3 and 4 on pages 10 to 12 and example 6 on pages 14 to 16.

It will be appreciated that the procedure described in UK patent GB 2175919B is modified in the present invention in that the bacterium is exposed to a nod-factor inducing agent.

In a further aspect the invention provides a composition comprising a non- leguminous plant seed in combination with a nitrogen-fixing bacterium which is exposed to a nod-factor inducing agent such as a flavonoid.

In the embodiment in which non-leguminous plant seeds are mixed with a plant growth medium such as peat in combination whh a bacterial inoculum and a nod-factor inducer, it is preferred that the growth medium is treated with a solution of the inducer. Alternatively, uie bacterium can be pre-treated by incubation with the inducer.

Preferably, the composition comprises sterile plant growth medium, such

12 as peat, mixed with sterilised seeds and the nod-factor inducer together with a bacterial inoculum.

In use, when the plant seed germinates the bacterium invades the emerging roots of the seedlings in the presence of the nod-factor inducing agent to establish a nitrogen-fixing interaction.

Preferably, the composition comprises a non-legume seed or a somatic non-legume plant embryo enclosed within a synthetic coating comprising a growth medium. Such coated seeds are well known, wiύi alginate beads being the most widely studied coating material.

Preferably, the coating comprises a pharmaceutical type capsule as described by Dupuis et al (1994) Bio/Technology Vol.12 April pg.385-389 the disclosure of which is incorporated herein by reference. In uiis example, the capsule body acted as a strong water soluble hull, covered on its inner surface by a watertight film composed of polyvinyl chloride (PVC), polyvinyl acetate (PVA) and bentone as a thickener, to control the nutrient supply and the subsequent development of the somatic embryo. A germination medium and the somatic embryo were then placed in die capsule.

In the present invention a bacterial inoculum is added to the growth medium. The nod-factor inducing agent can also be included in die growth medium, but it is preferably used to induce nod-factor expression in the bacterium before the bacterium is added to uie growth medium.

In a further aspect, the invention provides a growth medium for a non- leguminous plant comprising a bacterium capable of fixing nitrogen; wherein the bacterium is exposed to a nod-factor inducing agent. In this

13 aspect, the medium can be prepared simply by inoculating a known medium with a bacterium which has been pretreated with an inducing agent.

In a further aspect, uie invention provides a method of inducing nitrogen fixation in a non-leguminous plant comprising providing a non-leguminous plant or reproduction material of said plant including heterologous genetic material capable of expression to produce a nod-factor inducing agent and inoculating the plant with a nitrogen-fixing bacterium.

Methods have been developed for the production of transgenic wheat, rice, maize and oilseed rape and for most of the other major non-legume crops (S.L. Kothari, et al. Transgenic Rice, in Transgenic Plants Vol 2., Edit. Shain-Dow Kung and Ray Wu, p3-20, 1993, Academic Press). The procedures available for the transfer of genes into these major non-legume crops include the use of protoplasts, with direct delivery of the gene by electroporation or chemically induced uptake of the gene followed by regeneration of fertile transgenic plants expressing the introduced foreign gene. Another somewhat similar procedure is to utilise biolistics, the so- called particle gun bombardment method, involving the high velocity microprojectile delivery of me gene(s) into plant cells (without having to remove their cell walls to produce protoplasts) which are capable of regenerating shoots and, ultimately, fertile plants. The bacterium Agrobacterium tumefaciens can also be used to deliver genes into cells of the major non-legume crops from which transgenic plants can be regenerated using standard tissue culture procedures. The gene or genes to be inserted for the required flavonoid syndiesis and secretion in die root system or target crops will be introduced into the non-legume crops using a plasmid construct containing a suitable promoter, terminator and uie gene of interest. The pathway of flavonoid synthesis is well established

14 in plants (CJ. Beggs, et al (1986) Photo control of flavonoid biosynthesis. In "Photomorphogenesis in Plants", Eds. R.E. Kenerick and G.H.M. Kronenberg, pp 467-502 (Martin Nijhoff, Dordrecht) and the failure of any particular non-legume crop to synthesise a specific flavonoid required for activation of interacting nitrogen-fixing bacterium such as rhizobia will be linked to the absence of the required enzyme under the control of a specific gene. This specific "flavonoid synthesis" gene can be produced from legumes by standard molecular procedures involving isolation of messenger RNA and reverse transcription of cDNA followed by introduction into the target non-legume crop plant by delivery into isolated protoplasts, or by incorporating into totipotent cells through biolistics or the use of Agrobacterium tumefaciens as the gene vector. The cDNA can be tailored with a suitable promoter (preferably a root-specific promoter) and termination sequence. Putative transgenic non-legume crops would be evaluated at the molecular level for the presence of the "flavonoid synthesis" transgene. Their required flavonoid synthesis capability can be demonstrated using the common acetylene reduction assay for nitrogen fixation (Witty J.F. [1979] Soil. Biol. Biochem. 11 : 209-210).

Alternatively, expression of a nod-factor inducing agent by a non- leguminous plant can be provided by blocking a step in a biosynuietic pathway of the plant whereby an intermediate capable of inducing nod- factors accumulates so that the bacterium can be exposed thereto. For example, the conversion of the intermediate naringenin (a nod-factor inducing agent) to Dihydrokaempferol by the enzyme Flavanone-3'- hydroxylate can be blocked by a variety of techniques in which expression of the gene encoding the enzyme is suppressed. Suitable suppression methods are described by Courtney-Gutterson et al (1994) Bio/Technology vol.12, pg.268-271 and the disclosure of that document is incorporated herein by reference.

15 Courtney-Gutterson et al (1994) suppressed the expression of the gene for the biosynthetic enzyme chalcone synthase using chimeric anti-sense gene constructs or the introduction of an additional copy of the enzyme- encoding endogenous gene (see Experimental Protocol Section on page 271).

Suitable methods for making transgenic plants capable of expressing flavonoid biosynthetic pathway genes are described in WO90/11682 (DNA Plant Technology Corporation) die disclosure of which is incorporated herein by reference. Also described are methods for suppressing the expression of flavonoid biosynthetic genes.

Methods suitable for introducing exogenous DNA into a plant using Agrobacterium as a vector, whereby the transformed plant is capable of expressing die exogenous DNA, are described in WO92/03041 (Florigene BV) and the disclosure of that document is incorporated herein by reference.

Preferred embodiments of the invention will now be described by way of example with reference to the following figures in which:

Figure 1 shows: a) Formation of short thickened lateral roots (STLRs; arrowed) on a 2 Id-old wheat seedling inoculated mt Az. caulinodans in die presence of 10^M naringenin. b) Root of a seedling inoculated wititi Az. caulinodans, without naringenin, which lacks STLRs. c) Longitudinal section of a STLR from (a) showing crack entry invasion (arrowed), widi azorhizobia penetrating between cells at the base of the merging lateral root. d) The region arrowed in (c) showing azorhizobia between

16 cells. e) "Pockets" of azorhizobia, surrounded by fibrillar material, in die intercellular space between root cortical cells. f) Azorhizobia within a wheat root cortical cell. AR, azorhizobia; cw, cell wall; cy, cytoplasm.

Figure 2 shows die gene sequence of die chalcone synthase of Petunia hybrida (Koes et al, 1989, Gene 81, 245-257);

Example 1(a)

Induction of mtrogen fixation in a non-leguminous plant by inoculation with a nitrogen fixing bacterium exposed to a nod-factor inducing agent.

Az caulinodans IRBG314, nod- V44 Nod A::Tn5 and ORS 571 (ATCC deposit No. 43989) ntf^" 57004 (nif04-Tn5) were maintained on YEM medium (Vincent J M. Int. Biological Programme Handbook 15, Oxford. Blackwell Scientific Publications (1970). After 3 d ays (d) of culture in TGYE liquid medium (Ladha J K, et al. Appl. & Environ. Microbiol. 55:454-460 (1989)) die rhizobia were used for inoculation. Seeds of wheat (Triticum aestivum var Canon) were surface sterilized [70% v/v ethanol (1 min) followed by 30% v/v Domestos bleach (lh)], washed with sterile water and germinated on water-agar (0.8% w/v; Sigma), 25 °C, 16th photoperiod, 250 μmo s 'm^"2 daylight fluorescent illumination.

After 3d, germinating seedlings were transferred to autoclaved tubes (24 x 200 mm) each containing 25 ml agar-solidified (0.8% w/v; Sigma) N- free Fahraeus medium (Fahraeus G. J. Gen. Microbiol. 16:374-381 (1957)), one seedling/tube, and grown under sterile conditions. The flavonoid nod-factor inducing agent naringenin (Sigma), from a stock

17 solution (10 mg ml^"1 in ethanol), was added to the autoclaved Fahraeus medium, following cooling of the latter to 80 °C, to give the required naringenin concentration. Az. caulinodans Nod factors P2 fraction (Mergaert P, et al. Proc. Nad. Acad. Sci. USA, 90: 1551-1555 (1993) were similarly added from an ethanol stock solution (lμg μl^'x) to a final concentration of IO^"6. After 3 d in tubes, each seedling was inoculated widi 0.2ml of the appropriate rhizobial culture ( ~ 10⁹ bacteria ml^"1). Nitrogenase activity was deteπnined by the acetylene reduction assay using 10% acetylene with 90% air in tubes closed with subaseals (Witty J F. Soil Biol. Biochem. 11:209-210 (1979). The number of short diickened lateral roots (STLRs) was determined by direct measurement of the number of lateral roots which were more than 0.5 mm wide at the base and up to 2 mm in length.

18 Table 1: Naringenin stimulation of nitrogenase activity and STLR formation in wheat inoculated with Azorhizobium caulinodans

14d 21d 28d

Naringenin

Nano moles STLRs Nano moles STLRs Nano moles STLRs Molarity ethylene/ ethylene/ ethylene/ pla.it/24h plant/24h plant/24h

0 0 0 0 0 0 0

0* 729.4+561.9 3.3 ± 1.2 N.D N.D N.D N.D

2x10^{" 5} 143.2± 117.8 5.8±2.4 93.9±71.7 4.0± 1.4 106 8

10 lxlO^"4 1802.9±921.6 9.3±4.5 880.2±587.5 14.8±9.5 732.2±371,5 15.6±9.0

N.D. Not Determined ± Standard Deviation

10⁶M Nod factor (P2 fraction) from Az caulinodans added.

Table 1:

Nitrogenase activity was determined using the acetylene reduction assay at various times post inoculation. Uninoculated seedlings, with and without naringenin, and seedlings inoculated with lz. caulinodans nif^~ and Az caulinodans nod , widi and without naringenin, failed to produce ethylene or STLRs either at 14d, 2 Id or 28d. Thirty replicates were assayed per treatment. All seedlings inoculated with Az. caulinodans in the presence of naringenin showed some positive ethylene production and die formation of STLRs.

Example lb

Roots were fixed in 2.0% (v/v) glutaraldehyde for 24h at 4°C, followed by 1.0% (w/v) osmium tetroxide (2h, 4°C). Fixatives were prepared in 0.1M sodium phosphate buffer, pH 7.0. Specimens were dehydrated uirough 10% (v/v) ethanol to absolute ethanol (30 min each) and embedded in LR White medium grade resin (The London Resin Co. , Basingstoke, UK) (Davey M R et al. J. Exp. Bot. 44:863-867 (1993). For light microscopy (Fig. 1 c,d) sections were cut to 2 μm on glass knives, collected on glass slides and stained with 0.5 % (w/v) toluidine blue in 0.1 % (w/v) sodium tetraborate (2 min, 60°C), prior to mounting in styromount (Raymond Lamb, Lond, UK). Ultra-thin sections were collected on Pioloform-coated (Agar Scientific, Stansted, UK) copper grids, stained with lead citrate and examined at 80 kV in a Jeol 100-S transmission electron microscope (Davey M R et al. J. Exp. Bot. 44:863- 867 (1993).

Results

A highly stimulatory effect of the flavonoid naringenin on nitrogen

20 fixation and on the formation of short, thickened lateral roots (STLRs) was observed (Table 1) in wheat inoculated with Az. caulinodans, compared with inoculated seedlings wiuiout naringenin (compare Fig la and b). The finding that a mixmre of Nod factors, isolated from Az caulinodans, also stimulates nitrogenase activity of inoculated wheat seedlings and STLR formation (Table 1), suggests that the stimulation by naringenin results from the flavonoid inducing nodulation genes for Nod factor synthesis comparable to the situation in the Azorhizobium-Sesbania symbiosis. This is further supported by the finding that inoculation widi a nod^" strain of Az. caulinodans, which fails to produce Nod factors, did not result in nitrogen fixing wheat seedlings, or die formation of STLRs invaded by rhizobia, in seedlings at eidier 14, 21 or 28 days post inoculation (Table 1). Viewed overall, these results indicate that die formation of lateral roots invaded by crack entry (Fig lc-f), and the onset of nitrogen fixation in wheat inoculated with azorhizobia, are controlled by the same types of signals that are known to operate during the symbiotic interaction between rhizobia and legumes, allowing symbiotic nitrogen fixation to be established in non-legume crops, such as wheat.

Intercellular infection by azorhizobia in wheat resulting from crack entry (Fig lc,d) is followed by multiplication of die bacteria forming intercellular infection pockets filled with azorhizobia (Fig le). The presence of azorhizobia in wheat cells (Fig lf) probably results from localised cell wall degradation. In wheat seedlings inoculated with azorhizobia in the presence of naringenin, invaded lateral roots can be readily identified after inoculation by the marked swelling at their bases resulting from this crack invasion (Fig lc,d). A similar swelling at the base of laterals, resulting in the formation of STLRs, also occurs following inoculation wiui azorhizobia in the presence of Nod factors from Az. caulinodans (Table 1). No STLRs were produced following

21 inoculation with azorhizobia in the absence of naringenin, or by naringenin without inoculation (Table 1). These observations, coupled widi die fact that Nod factors from Az. caulinodans are known to induce swellings at the base of lateral roots of die legume Sesbania rostrata, suggest that Nod factors, induced in Az. caulinodans by naringenin in die above experiment, are controlling the invasion of wheat roots by azorhizobia and the development of nitrogen fixation in the resulting STLRs.

The inventor also observed diat narmgenin stimulates nitrogen fixation in wheat when otiier rhizobia, such as Bradyrhizobium ORS310 which normally nodulates stems of Aeschynomene indica are used for inoculation (data not shown). This suggests that narmgenin may be a generally active nod gene inducing signal for various rhizobia interacting with non- legumes, leading to nitrogen fixation.

Nitrogenase activity in S. rostrata seedlings inoculated widi Az. caulinodans under the tube growth conditions mentioned previously, and assayed as in Table 1, was increased from 161 nmoles ethylene/plant/24 h to 689 mnoles ethylene/plant/24 h by lO^M naringenin. The significant stimulation of nitrogen fixation in inoculated wheat by naringenin (Table 1), coupled with the formation of STLRs invaded by azorhizobia, indicates that the nitrogenase activity is arising from rhizobia within die root system. The fact that wheat seedlings inoculated with the nod- strain of Az. caulinodans, which is capable of nitrogen fixation in the free-living state in the presence of up to 3% oxygen, failed to show nitrogenase activity with and without naringenin, as did the wild-type Az. caulinodans used for inoculation wiuiout naringenin (Table 1), also indicates diat nitrogenase activity is arising from rhizobia within die root system of the wheat seedlings. Moreover, the nitrogen fixation activity of surface azorhizobia would be completely inhibited by die concentration of oxygen

22 used in the acetylene reduction assay system. The faUure of a nif^~ mutant of Az. caulinodans (Table i) to produce ethylene in die assay for nitrogenase activity demonstrated the absence of azorhizobia stress-induced plant etirylene production.

These results demonstrate mat the flavonoid naringenin activates Az. caulinodans to produce Nod factors which stimulate the entry of azorhizobia into the wheat root system, inducing the formation of (STLRs). The signalling system operating in wheat interacting with azorhizobia, in t e presence of naringenin, may be comparable to that in the legume Sesbania rostrata during symbiotic nitrogen fixation with Az. caulinodans.

Similar results were obtained widi otiier non-legumes including rice, maize, oil-seed rape and Arabidopsis thaliana, which suggests that such naringenin induced stimulation of nitrogen fixation is likely to occur in all non-legumes..

Example 2

Protocol for Induction of Rhizobium Uptake into Non-Legume Root Hairs

Rice seeds (dehusked) are surface sterilised in 30% (v/v) Domestos solution and germinated on nitrogen-free agar at 28 °C in die dark. This medium (Fahraeus) has the following composition:- [CaCl₂ (0.1 g), MgSO₄.7H₂0 (0.12 g), KH₂PO₄ (0.1 g), Na₂HPO₄.2H₂O (0.15 g), Fe citrate (0.005 g), traces of: Mn,Cu,Zn,B,Mo] per litre widi 0.8% agar, pH 6.5-7.0.

40-48 hour old seedlings are incubated for 5 minutes in isotonic enzyme

23 mixmre of die following composition: [Worthington CEL (1 g), Novozyme 2,3,4 (0.5 g), Pectolyase (0.1 g), mannitol (8.0 g)] per 100 ml, pH 5.6-5.8, then transferred to the Rhizobium uptake treatment.

Exponential phase Rhizobia are cultivated by centrifugation and resuspension in fresh yeast extract-mannitol solution (0.5 ml) having the composition [K₂HPO₄ (0.5 g), MgSO₄.7H₂0 (0.2 g), NaCl (0.1 g), mannitol (10.0 g), yeast extract (Difco)(0.4 g)] per litre, pH 6.8-7.0. Immediately prior to the root immersion this preparation is mixed with 1 ml of polyethylene glycol solution of composition [Polyethylene glycol M.W. 6000 (20.0 g), CaCl₂.2H₂0 (0.15 g)] per 100 ml [Difco is a Registered TM].

The enzyme-treated roots are immersed in die polyetiiylene glycol widi Rhizobia for 5 minutes followed by two washings widi mannitol solution [Mannitol (9.0 g) per 100 ml, pH 6.5-7.0].

The seedlings are transferred to nitrogen-free agar in square Petri dishes which can be stacked vertically for optimum seedling growth. All seedlings survive the enzyme treatment altiiough die root growtii maybe impaired in some cases. Root samples are removed for light and electron microscopic examination.

Fusion of Protoplasts Extruding from Root Hair Tips or Isolated from Root Hairs with Protoplasts Isolated from other Plant Species

Gene transfer by protoplast fusion is a well established procedure for the transfer of clusters of nuclear or cytoplasmic genes, and die use of protoplasts being released from root hairs now enables the basic strategy of somatic hybridisation to be applied to the intact plant. Such protoplast

24 fusion using eidier chemical or electrical procedures (Davey, M.R. and Kumar, A., Int. Rev. Cytol. Suppl. 16, 219-299 (1983)) may be used to transfer nuclear genes (controlling for instance symbiotic associations) or cytoplasmic genes (conveying for instance male sterility) without impairment of the functional integrity of the plant. Exposed plasma membranes readily regenerate a new cell wall and root hairs with exposed plasma membranes, after fusion, may be stabilised in this way. If required, protoplasts can be isolated from root hairs and used for somatic hybridisation by fusion with otiier isolated protoplasts.

This technique is illustrated in Example 3 below.

Example 3

Protocols for Uptake of Rhizobium into Non-Legume Root Hairs

These procedures are generally applicable to all non-legume crop species in which it is possible to achieve enzymatic degradation of the apices of root hairs. This first procedure involves fusion of protoplasts containing Rhizobia (diese protoplasts are isolated enzymatically from nodules of the legume) with the exposed plasma membrane of die non-legume root hair whereby the root hair of the non-legume will contain Rhizobia in its cytoplasm. The second procedure involves fusion of protoplasts (subprotoplasts) released from the tips of root hairs of enzymatically- treated root hairs of legumes with die exposed plasma membrane of the non-legume root hair; die hybrid root hair on the non-legume then behaves like a legume root hair and interacts with Rhizobia in the usual way that legumes do during their normal infection with Rhizobia.

25 Procedure (1)

a) Rice seeds (or seeds of any other non-legume) are surface sterdised in 30% (v/v) Domestos and germinated on nitrogen-free agar (see Example 2) at 28° in the dark, and 2-day-old seedlings are incubated in isotonic enzyme mixmre for 5 minutes to expose the plasma membranes at the surface of their root hairs.

b) Protoplasts are isolated from young nodules of the legume using the procedure described by Davey and Cocking, 1973, Namre, 224,

460, which involves incubating die sliced nodule in a cell wall degrading enzyme mixmre in a suitable plasmolyticum.

c) Seedlings of the non-legume following the treatment as detaded in (a) are mixed witii nodule protoplasts (which contain Rhizobium) such that die root hairs are mixed widi these nodule protoplasts widi a ratio of approximately four nodule protoplasts to every root hair. The root hair system of the non-legume with associated isolated root nodule protoplasts is then incubated in autoclaved 30% w/v polyediylene glycol (PEG) M.W. 6000 containing 0.01

CaCl₂.2H₂0 and left for 10 minutes at room temperamre. The PEG solution is then diluated at 5 minute intervals by the addition of 50% of its volume by nitrogen-free medium (see Example 2, but widiout agar), and tiien by the addition of a further 50% of its volume and tiien by a further 50% of its volume until die PEG has been replaced by diis medium. Fusion of die nodule protoplasts with the exposed protoplast of the root hair takes place and wdl result in die non-legume seedlings possessing root hairs contøining Rhizobia.

26 Procedure (2)

a) The procedure is as in (l)(a).

b) Subprotoplasts are isolated from root hairs of the legume by treating root hairs of seedlings with the enzyme mixmre under conditions which cause extrusion of the protoplast from the root hairs. The subprotoplasts are collected by flotation.

c) Using PEG as the fusion agent as described in (l)(c) fusion of these root hair subprotoplasts from legumes with the exposed protoplast of the root hair of the non-legume takes place and results in the non-legume seedlings possessing root hairs which resemble physiologically (as far as Rhizobium infection is concemed) root hairs of me legume. Such treated seedlings are then incubated with

Rhizobia of die required legume specificity for infection of the non- legume to take place.

Example 4

Production of compositions of non-legume seeds coated with a growth medium containing a bacterium exposed to a nod-factor inducing agent.

Somatic wheat embryos can be coated widi a pharmaceutical type capsule as described by Dupuis et al (1994) Bio/Technology, Vol.12 April 385- 389.

Pharmaceutical type capsule preparation. The capsules (Elanco

Qualicaps^®, Lilly, France) consisting of gelatine containing glycerol as a plasticizer, were used without heads. The capsule body was 2cm long

27 widi a 8mm external diameter. It was coated on its inner surface with a layer of an organic solvent solution containing various polymers by pouring this solution inside die capsule and rotating by hand. Before air drying, the excess of mixmre was then removed by turning the capsule on end under sterile conditions. The film components used as polymers were PVC (Sigma, France). PVA (Mowilith M70^®, Hoechst. Germany) or Poly-DL-Lactid^® (Boehringer Ingelheim KG, Germany). Natrosol HHR 250^® (Aqualon, France), Tylose^® (Hoechst, Germany), Methocel^® (SEPPIC) and Bentone SDI^® (Eurindis, France) were used as tiiickeners. After drying, the capsule was filled with 0.5ml of germination medium containing 6g/l of Phytagel^® mixed with an inoculum (approx. 0.2ml of a IO⁹ ml^"1 culture medium) of Az. caulinodans strain ORS571 which had been pretreated with a 10^ molar solution of the nod-factor inducing agent naringenin.. Finally, a 0.5 to 1.5mm long torpedo shaped embryo was placed on the intemal medium without special orientation. As the embryoes converted witiiout arrest into plants, the capsules were placed directly in germination conditions.

Capsule cap. Different metiiods for closing the capsule can be used. A fdm cap was obtained by placing in die capsule opening 0.5ml of a liquid polymeric mixmre or oil, then subjecting it to air drying (polymers) or lower temperamres (oil). Suitable ods include jojoba oil (Sigma), rape seed oil (Robbe). Where cotton or rockwoll is used as a cap, fibers are then sprayed with silicon before being inserted by hand into the opening.

28 Example 5

Production of transgenic non-legume capable of producing flavonoids for forming a nitrogen-fixing interaction with a bacterium.

Flavonoid biosyntiiesis in plants is based on a series of common steps; the key enzyme involved is chalcone svntiiase which catalyses die syndiesis of tetrahydroxy chalcone from coumaryl-CoA and malonyl-CoA. Tetrahydroxy chalcone is readily converted to naringenin by chalcone isomerase of general occurrence in plants. A preferred example of commercially important transgenic non-legume crop is rice, which does not naturally syntiiesise the nod factor inducer naringenin. To make it suitable for use according to die invention, die rice is preferably manipulated genetically to produce naringenin by inserting die gene for chalcone synthase; the apparent inability of rice to produce this flavonoid namrally is probably due to die absence in rice of the chalcone syndiase, ChsA gene.

(2) Isolation of chalcone synthase cDNA

A cDNA library was prepared in λgtlO using poly A+RNA isolated from flowers of the petunia (Ausubel et al., 1990 Current Protocols in Molecular Biology. Greene Publishing Associates, New York). Approximately 96,000 plaques of the library were screened for hybridisation to a full length CHS DNA clone of petunia. Hybridisation was carried out under low stringency conditions (2xSSC, 45 °C) resulting in die production of high background activity.

Backgrounds were reduced by screening the library with a 33 base pair oligonucleotide (5'-CCTCCAGCAAAGCAACCCTGTTG

29 GTACATCATG-3' 2xSSC, 55 °C). This sequence is located at positions 448-520 in the petunia cDNA CHS clone (Reif et al, 1985 Mol. Gen. Genet. 199: 208-215). EcoRI fragments of positively hybridising lambda clones were subcloned into pBluescripfKS for sequence analysis. Partial sequence analysis of the 5' and 5' ends was performed using primers witiiin die plasmid multiple cloning region. Full sequence analysis was performed by preparing nested deletions (Ausubel et al., 1990).

(3) Insertion of the chalcone synthase gene into Agrobacterium tumefaciens

Oligonucleotide mutagenesis was performed to introduce an Ncol site at the start codon and a BamHIsite just after the stop codon for the predicted amino acid sequence so that a perfect fusion could be obtained. The coding sequence was cloned into an expression cassette based on die 35S promoter, [The 35S promoter (from cauliflower mosaic virus) has been fully characterised and is standard in transgenic plant production (Ausubel et al., 1990)] and a 5' untranslated region of a chlorophylla/b binding protein gene of petunia (Cab22L) and die nopaline syntiiase termination region. This expression construct was introduced into the binary vector pAGS802, which contains a 35S-nptII-ocs construct to confer kanamycin resistance on plant tissue, a lacZα region for ease of cloning and replication origins of ColEI and pVSP (Courtney-Gutterson et al., 1994 Bio/Technology 12: 268-271). The binary vectors were introduced into Agrobacterium tumefaciens strain LBA4404 by electroporation.

30 (4) Production of transgenic rice expressing and secreting naringenin

This procedure is designed to produce rice plant transgenic for chalcone synthase. No specific root promoter is required since a general expression of the gene in all parts of die plant is desirable to ensure production of naringenin and secretion from the plant (including the roots). A supervirulent strain of A. tumefaciens LBA4404 (plG121Hm), containing the chalcone synthase gene; utilising die procedure of Yukoh Hiei et al., 1994 The Plant Journal 6(2): 271-282, was employed. Rice tissues (shoot apices, roots and calli derived from roots) were immersed in die bacterial suspension for several minutes and tiien transferred without rinsing on to 2N6-AS medium (all tissues except for shoot apices) or N6S3-AS medium (shoot apices), and incubated at 25 °C in darkness for 3 days. After the co-cultivation, the materials were rinsed thoroughly with 250 mg l^"1 cefotaxime in sterile water and placed on 2N6-CH medium. Colonies of cells that had proliferated were plated on a regeneration medium, N6S3- CH, and incubated at 25 °C under continuous illumination (about 2000 lux). Regenerated plants (R₀ generation) were eventually transferred to soil in pots and grown to mamrity in a greenhouse as described by Yukoh Hiei et al., 1994. The regenerated rice transgenic plants (tested by Soutiiern and Northern analysis) containing and expressing die chalcone syntiiase gene were screened for secretion of naringenin using the stimulation of nitrogen fixation assay described in Example 1(a).

Detatis of the gene sequence of the chalcone synthase of P. hybrida given in Figure 2 are as follows:

ID PHCHSA standard; DNA; PLN; 4966 BP. XX

AC X14591 ;

31 XX

DT 13-NOV-1991 (Rel. 30; Created)

DT 14-NOV-1991 (Rel. 30, Last updated, Version 8)

XX DE P.hybrida chsA gene for chalcone synthase

XX

KW chalcone synthase; chsA gene.

XX

OS Petunia hybrida OC Eukaryota;Plantae;Embryobionta; Magnoliophyta; Magnoliopsida;

OC Asteridae; Solanales; Solanaceae. XX

RN [1] RP 1-4966

RA van Tunen A.J.;

RT ;

RL Submitted (07-MAR- 1989) to the EMBL/GenBank/DDBJ databases .

RL Van Tunen A.J. , Vrije Universiteit, Department of Genetics, de

RL Boelelaan 1087, 1081 HV Amsterdam , The Netherlands.

XX

RN [2]

RP 1109-3973 RX MEDLINE; 90034197.

RA Koes R.E., Spelt C.E., van Den Elzen P.J.M. , Mol J.N.M. ;

RT "Cloning and molecular characterization of the chalcone syntiiase

RT multigene family of Petunia hybrida"; RL Gene 81 :245-257(1989).

XX

DR EPD; 35055; Ph chalcone synth. A.

DR SWISS-PROT; P08894; CHSA PETHY.

XX CC ChsA is die major expressed member of the genefamdy in various

CC floral tissues and in seedlings treated with UV light. It is

CC relatively low expressed in tissue culture material XX FH Key Location/Qualifiers

FH

FT source 1..4966

FT /organism = "Petunia hybrida"

FT /strain= "Violet 30"

32 /tissue_type= "leaf"

/clone ib— "genomic"

/clone= "VIP 17, VIP 71 "

FT /chromosome = " V"

FT CAAT signal 1060..1064

FT TATA^" signal 1117..1123

FT CDS join(1226..1403,2751..3742)

FT /gene= "chsA"

FT /EC_number= "2.3.1.74"

FT /product = "chalcone syntiiase"

FT /note= "pid:g20525"

FT mRNA join(l 147..1403,2751..one-of(3884"3911))

FT /gene= "chsA"

FT exon 1147..1403

FT /number = 1

FT intron 1404..2750

FT /number = 1

FT exon 2751..one-of(3884^A3911)

FT /number =2

FT polyA_ signal 3853..3858

FT polyA_ signal 3859..3864

FT polyA signal 3882..3888

Example 6

Isolation of nod-inducing agents from plants.

Exudate from seedlings or roots of plants such as the legume Sesbania rostrata can be subjected to standard purification procedures and die nod- factor inducing components separated and identified to prepare nod-factor inducing agents useful in die mediod of the invention.

Suitable experimental protocols are described by Messens et al (1991) Mol. Plant-Microbe Interactions, Vol.4, No.3, pp.262-267 where standard techniques involving crude exudate fractionation by reversed-phase chromatogrpahy and spectroscopic analysis to identify components

33 displaying nod-inducing activity in an Azorhizobium reporter strain harbouring a nod A::lacZ reporter fusion. The disclosure of Messens et al is incorporated herein by reference.

34

Claims

1. A method of inducing nitrogen fixation in a non-leguminous plant by inoculating the plant widi a nitrogen-fixing bacterium, wherein the bacterium is exposed to a nod-factor inducing agent.

2. A method as claimed in Claim 1 wherein the nod-factor inducing agent is provided as an exudate from a plant which normally forms a symbiotic nitrogen-fixing interaction with the bacterium.

3. A mefhod as claimed in Claim 1 or 2 wherein the nod-factor inducing agent can induce nod-factor expression in the bacterium used to inoculate the non-leguminous plant.

4. A method as claimed in Claim 2 or 3 wherein the nod-factor inducing agent is a flavonoid.

5. A mediod as claimed in Claim 4 wherein die flavonoid is selected from naringenin, liquiritigenin, and isoliquiritigenin.

6. A method as claimed in one of Claims 1 to 5 wherein the bacterium is a species selected from one or more of the genera Rhizobium, Azorhizobium and Brady rhizobium.

7. A mediod as claimed in Claim 6 wherein the bacterium is of the kind which namrally cause nodulation otiier than solely by infecting root hairs of legumes via an infection thread.

8. A mediod as claimed in Claim 7 wherein the bacterium is capable of nodulating the stem of a plant.

35

9. A mediod as claimed in any one of the preceding claims wherein the bacterium is tolerant of oxygen levels of more than 0.01 % .

10. A metiiod as claimed in any one of die preceding claims wherein the bacterium is an Azorhizobium species or a Bradyrhizobium species.

11. A method as claimed in Claim 10 wherein the bacterium is Azorhizobium caulinodans.

12. A mediod as claimed in any one of Claims 1 to 11 wherein the bacterium is exposed to a solution of tiie nod-factor inducing agent.

13. A metiiod as claimed in Claim 12 wherein die inducing agent solution has a molarity of from lO^{^6} to 10

14. A method of inducing nitrogen fixation in a non-leguminous plant comprising inoculating the plant widi a nitrogen fixing bacterium in the presence of a flavonoid.

15. A metiiod as claimed in Claim 14 wherein the flavonoid is naringenin.

16. A method as claimed in Claim 14 or Claim 15 wherein the bacterium is Azorhizobium caulinodans.

17. A method as claimed in any one of Claims 1 to 16 wherein the nod- factor inducing agent is produced by expression of a heterologous genetic material in the plant.

18. Use of a flavonoid in a method comprising inoculating a non-

36 leguminous plant with a nitrogen-fixing bacterium whereby the bacterium and plant form a mtrogen fixing interaction.

19. Use of a flavonoid as claimed in Claim 18 wherein the flavonoid is naringenin.

20. Use of a flavonoid as claimed in Claim 18 or 19 wherein the bacterium is Azorhizobium caulinodans.

21. A non-leguminous plant having a nitrogen-fixing interaction with a bacterium obtainable by a method as claimed in any one of Claims 1 to 17, or a use as claimed in any one of Claims 18 to 20.

22. A nitrogen fixing bacterium which has been made competent to form a nitrogen fixing interaction with a non-leguminous plant, other tiian a plant of the Parasponia genus.

23. A composition comprising a non-leguminous plant seed mixed widi a plant growth medium and an inoculum of a nitrogen-fixing bacterium exposed to an agent capable of inducing nod-factor production in die bacterium.

24. A composition as claimed in Claim 23 wherein die agent is a flavonoid.

25. A plant growth medium for a non-leguminous plant comprising a bacterium capable of fixing nitrogen; wherein the bacterium has previously been exposed to a nod-factor inducing agent.

37