WO1987006796A1

WO1987006796A1 - Alteration of plant response to microorganisms

Info

Publication number: WO1987006796A1
Application number: PCT/AU1987/000137
Authority: WO
Inventors: Barry Garth Rolfe; Hancai Chen; Steven Phillip Djordjevic; John William Redmond; Michael Batley
Original assignee: The Australian National University; Macquarie University
Priority date: 1986-05-13
Filing date: 1987-05-13
Publication date: 1987-11-19
Also published as: EP0326545A4; JPH02500002A; EP0326545A1

Abstract

Composition for use in altering the response of plants to microorganisms comprises the exopolysaccharide(s) derived from bacteria of the genus Rhizobium, or oligopolysaccharide(s) consisting of or containing one or more of the repeat-units of said exopolysaccharide(s). A method for treatment of plants with the composition either before, after, or simultaneously with exposure of the plants to microorganisms is also disclosed.

Description

ALTERATION OF PLANT RESPONSE TO MICROORGANISMS

This invention relates to a composition and ^'method for altering the response of plants to microorganisms. In one particular aspect, this invention relates to the alteration of the response of plants to microorganisms of the genus Rhizobium, however it is to be understood that in its broadest aspect the invention extends to alteration of the response to other microorganisms, including other bacteria, fungi and viruses.

It will be well understood that the traditional methods of control of the influence or effects of microorganisms have been based on the use of chemical sprays and pesticides. It is recognised, however, that many such chemical agents are ecologically undesirable due to the production of unacceptable levels of residues and so on. Accordingly, the production of bio-degradable, ecologically acceptable, means for the control of microorganisms is highly desirable.

In work leading to the present invention, it has been recognised that certain biological molecules are able to elicit responses in plants which in effect either turn "on" or turn "off" the ability of the plant to respond to microorganisms, particularly invading microorganisms. In general terms, therefore, these biological molecules provide the basis upon which can be developed, for example, a system for increasing the protection of plants from potential pathogens.

According to a first aspect of the present invention, there is provided a composition for use in altering the response of plants to microorganisms, which comprises the exopolysaccharide(s) derived from bacteria of the genus Rhizobium, or oligopoly- saccharide(s) consisting of or containing one or more of the repeat-units of said exopolysaccharide(s) .

In another aspect, this invention provides a method for altering the response of plants to microorganisms, which.^'comprises the step of treating said plants with an effective amount of a composition» as broadly described above, either before, after or simultaneously with exposure of said plants to the microorganisms.

In one particular embodiment of this invention, the composition comprises the exopoly¬ saccharide derived from Rhizobium sp. strain NGR 234 or Rhizobium trifolii or derivatives thereof, characterised by having an oligopolysaccharide repeat-unit as set out in Figure la or lb attached hereto.

In general terms, this invention is based on the use of bacterial exopolysaccharide(s) or oligosaccharide(s) derived therefrom, for example, for the regulation of the plants' defences against invading organisms to achieve higher crop yields, or to produce more efficient nitrogen fixation and hence reduce production costs.

The composition of the present invention may, for example, be formulated as a spray, dust or powder with conventional horticulturally- or agriculturally-acceptable diluents or carriers for topical administration to the plants, particularly young plants, to influence or affect their response to invading organisms.

A characteristic of many wild-type strains of Rhizobium is their ability to produce copious amounts of exopolysaccharides and form mucoid colonies. It has been proposed that these exo¬ polysaccharides have important functions in the plant- Rhi-zobium symbiosis, such as determination of host specificity. As broadly outlined above, it has now been discovered that these exopolysaccharides, or oligosaccharides consisting of or containing one or more repeat-units thereof, can be used to affect the response of plants to microorganisms.

The following detailed description relates specifically to the application of the broad concept of the invention in the development of nitrogen-fixing nodules in plants. It will be appreciated, however, that this specific description is included by way of illustration of the present invention, and is not intended to constitute a limitation thereof. Accompanying Figure 1 shows the chemical structures of oligosaccharide repeat-units used in the specific example below;

(a) is the structure of the acidic oligosaccharide repeat-unit of ANU 280 (a derivative of Rhizobium strain NGR234) ;

(b) is the structure of the acidic oligosaccharide repeat-unit of ANU 843 (a strain of R.trifolii) .

A complex multi-step interaction between the

_#soil bacterium Rhizobium and specific leguminous plants results in the induction of nitrogen-fixing nodules on legume roots (Rolfe et.al., 1981). The early steps of the interaction are characterised by the distortion or curling of the root hair cells. The cell walls of the root hairs are penetrated after 24hr by a compatible Rhizobium strain and an infection thread is synthesised by the plant after the nucleus of this cell has migrated to the infection site (Callaham and Torrey, 1981; Ridge and Rolfe 1985).

The bacteria are carried toward the root cortex inside the infection thread where they actively divide. Shortly before, or concurrent with the initiation of infection thread synthesis, cortical cell division is thought to be induced by Rhizobium strain, presumably by diffusable substances released by the bacterium (Bauer, 1981; Bauer et.al. 1985) .

Another feature of the Rhizobium-legume interaction is the host specificity displayed. Fast- growing ("temperate") Rhizobium strains, for example, usually nodulate only one plant species effectively, whilst slow-growing Bradyrhizobium strains typically have a broad host-range. In contrast, the fast-growing Rhizobium strain NGR234 (Trinick 1980) possesses an unusually extensive host-range which includes a variety of tropical and temperate legumes as well as the non-legume tropical tree Parasponia andersonii (Trinick and Galbraith 1980) .

Since the initial interaction between the symbionts occurs at the surface of the two organisms, cell-surface molecules may be important in determining the outcome of the infection. Rhizobium bacteria characteristically produce large amounts of exopoly- saccharides (EPS) on various laboratory media and the colonies formed are mucoid (Muc ) in appearance.

Rhizobium polysaccharides, particularly EPS and lipopolysaccharides (LPS) have been postulated to be involved in the early recognition steps between the plant and the bacterium including specific adhesion to the root hair surfaces (Dazzo et.al. 1978) and the dtermination of host-specificity (Dudman 1977) . Whilst results concerning the true role of EPS remain controversial, a good correlation exists between the ability of Rhizobium strains to produce normal EPS on laboratory media and the ability to produce nitrogen-fixing nodules (Fix ) on legume plants. A number of transposon (Tn5) induced Muc^" mutants of

R.trifolii (Chakravorty et.al. 1982) and NGR234 (Chen et.al. 1985) are still able to infect host legumes (although often poorly) but are unable to initiate nitrogen-fixing nodules. Examination of nodules induced by Muc mutants of R.trifolii and strain

NGR234 by electron microscopy have shown that nodules on some plant species are poorly developed, contain fewer dividing cells and intracellular bacteria and little or no indication of the presence of bacteroids (Chakravorty et.al. 1982; Chen et.al. 1985). In contrast, the majority of so-called calcoflor-dark (Muc~) mutants in R.meliloti were unable to induce detectable root hair curling or infection thread formation on alfalfa plants thus indicating that EPS is involved in nodule invasion as opposed to nodule formation (Leigh et.al. 1985) .

The first indications that EPS may be important for nodule development came from cell mixing experiments using an invasive Muc^" strain SU846 and a Muc⁺, Nod^" strain SU847. The Muc⁺, Nod^" strain SU847 has an extensive deletion in the native Sym plas id which removes many essential nodulation and nitrogen fixing genes. The Muc Nod strain inoculated alone onto clover plants formed poorly developed nodules which were unable to fix nitrogen. However, a mixed inoculum containing these two strains could induce a proportion of fully-functional nitrogen-fixing nodules on clovers. No evidence of genetic transfer was detected between the two strains (Rolfe et.al. 1980) .

The approach used to determine the role of EPS in symbiosis involved the isolation of specific non-mucoid (Muc ) mutants. Over ninety Tn5-induced EPS defective strains of NGR234 which were found to map on the chromosome of this strain (Chen et.al. 1985) have been isolated. In addition, the precise structure of the EPS of the broad host range bacterium NGR234 (Djordjevic et.al. 1986 a, b) has been d termined.

It has now been shown that several Tn5-induced Muc mutants of NGR234 produce very little (<5% of the amount produced by the parent strain ANU280) or no detective amounts of acidic type EPS or induce nitrogen fixing nodules on several host plants. In addition, it has been shown that these Muc mutants can have their symbiotic phenotypes corrected by the addition of purified oligosaccharide molecules which forms the repeat-unit of the EPS synthesised by NGR234. This result demonstrates that Rhizobium EPSs play an essential role in the development of nitrogen- fixing nodules.

MATERIALS AND METHODS

Bacterial strains used in this study are listed in

Table 1.

Media

All media used (BMM, Fahraeus) have been described earlier (Rolfe et.al.1980) . Coinoculation experiments

Muc mutant strains of ANU280 and Sym plasmid-cured derivatives of both strains ANU280 and ANU843 were grown on BMM agar plates for 2 days. Suspensions of both strains were grown to the same optical density and mixed in an approximate 1:1 ratio before inoculating onto sterile 3 day old Leucaena seedlings. Isolation and purification of Polysaccharides

Strains ANU280, ANU2811, ANU2820, and ANU2840, were grown in a glutamic-aσid-D-mannitol-salts medium as described earlier (Djordjevic et.al. 1986b) . EPS was isolated by using an Amicon DC10L hollow-fibre filtration system fitted with a O.lμ filter and further purified by precipitation as the cetyltri- m hy1ammonium (CTAB) salt as described previously (Djordjevic et.al., 1986a). Acidic oligosaccharide was similarly isolated by hollow-fibre filtration using a (H10P3-20) filter. Acidic oligosaccharide was not precipitated as the CTAB salt. Crude oligo- saccharides were fractioned on a DEAE-Sephadex A25 column and analysed for hexose and uronic-acid as described previously (Djordjevic et.al. 1986) . Sterile seedlings germinated on BMM agar were transferred to Fahraeus agar and allowed to settle to the agar surface for 16hrs. Aliquots (5 or 10μl) of stock solutions (5mg/ml) of EPS or its derived

..oligosaccharide were applied along the length of 3 day-old seedlings. These acidic saccharides were added either simultaneously or 24hr before or after inoculation with Rhizobium bacteria. Plant assays

Macroptilium atrbpurpureum (DC) Urban (siratro) and Trifolii repens (L) c.v. 5826 (white clover) , were tested for nodulation-using the plate method (Rolfe et.al., 1980; Cen et al., 1982) . Leucaena leucocephala (Lam) Wit. var peru was tested for nodulation according to the method of Chen et.al. (1985) .

Preparation of nodule sections for light microscopy. Specimens for light microscopy were prepared as described earlier (Chen et.al., 1985) .

RESULTS Exopolysaccharide Mutants

Mutants ANU2811, ANU2820 and ANU2840 are Tn5- induced mutants of strain NGR234 defective in exopoly¬ saccharide synthesis. These strains form non-mucoid (Muc ) colonies on all laboratory media and have defective symbiotic phenotypes (properties) on different legume plants (Chen et.al., 1985) . The Tn5 ^*: insertion in these strains has been shown to reside in different genomic DNA fragments (Table 1) . Little or no uronic-acid containing saccharides could be detected by colourimetric analysis of both retentates obtained by hollow-fibre filtration. Chromatographic analysis of the surface polysaccharides of these Muc strains showed that they fail to produce any detectable acidic oligosaccharide. In contrast, the parent strain ANU280 produces gram quantities of acidic EPS and related oligosaccharide when grown under the same culture conditions. Despite the different location of Tn5 in each of these Muc^" mutants, strains ANU2811, ANU2840 and ANU2820, have a common nodulation-defective phenotype on Leucaena plants but a different phenotype on siratro plants. These Muc^" mutants produce grossly disorganised callus-like structures on the roots of Leucaena plants which -do not fix atmospheric nitrogen whereas the parent strain induces indeterminate, nitrogen-fixing nodules (Chen et.al., 1985) . On siratro, Muc mutants ANU2820 and ANU2811 produce poor, non-nitrogen fixing nodules, while strain ANU2840 forms nitrogen-fixing nodules (Chen et.al., 1985) . Although Muc strain ANU2840 fails to produce any detectable level of acidic oligosaccharide, colimetric analysis of the O.lμm retentate (isolated from strain ANU2840) indicates that a small amount of acidic EPS may be produced. In view of the total absence of acidic oligosaccharide, this apparent low level of uronic-acid containing material in the O.lμm retentate may be an artifact. Coinoculation of Muc and Muc strains on Leucaena plants.

The Sym plasmid cured derivative of NGR234, (ANU265) produces Muc colonies, but is unable to initiate any detectable symbiotic response on any legume since all the essential nodulation genes as well as the nitrogenase genes have been shown to be deleted from this strain (Morrison et.al., 1983). An NMR analysis, of the isolated oligosaccharide repeat-unit from strain ANU265 showed that its

.chemical structure is very similar to the parent strain. ANU280. When strain ANU265 was mixed in equal amounts with each of the Muc^" mutants and coinoculated onto Leucaena plants, nitrogen-fixing nodules were produced (Table 2) . Sections through these nodules showed a meristematic zone with well formed vascular bundles, and an extensive "bacteroid zone" containing rod shaped non-swolleή bacteria. Both strain ANU265 and* the Muc mutants were isolated from the nitrogen-fixing nodules. The isolated bacteria retained their original colony morphology (Muc , for ANU265, and Muc^" for the mutant strains) , antibiotic resistant markers (Sp^r for ANU265 and Rf , Sm^r and m^r for the Muc mutants) and their original nodulation - phenotypes on Leucaena plants, thus indicating that no detectable genetic transfer had occurred between these strains. To determine if the ANU265 background strain was important for the correction of the Muc^" mutants a foreign Rhizobium strain was used instead of ANU265. The Sym plasmid cured R.trifolii strain ANU845 (instead of ANU265) was mixed with the Muc^" mutant ANU2840. Nitrogen-fixing nodules were not produced (Table 2) . Effect of the addition of purified EPS or related oligosaccharide and Muc^" mutants to Leucaena

The acidic EPS from the parent strain ANU280 had been isolated and chemically sequenced (Djordjevic et.al., 1986a), and the structure of the oligo¬ saccharide repeat unit is reproduced in Figure la. Purified EPS or related oligosaccharide isolated from strain ANU280 was then inoculated onto Leucaena plants together with one of the Muc mutants. This was done to determine if the helper effect of strain ANU265 could be substituted for by the addition of purified EPS from the parent strain ANU280 or from the Sym plasmid-cured strain ANU265. In all cases, the coinoculation of the EPS or the oligosaccharide repeat-unit enabled the Muc mutants to induce nitrogen-fixing nodules on Leucaena plants, although some calli were still produced (Table 3) . As expected, since the EPS from the Sym plasmid- cured strain ANU265 and from the parent strain ANU280 were shown to be identical, EPS isolated from ANU265 also permitted the Muc mutants to initiate nitrogen-fixing nodules on Leucaena plants.

No evidence of the Muc bacteria (able to nodulate Leucaena) were detecte when the contents of 30 pigmented leucaena nodules were analysed and the Muc bacteria isolated from the nodules invariably retained their defective phenotype.

To measure the effect of EPS concentration on the above results, different amounts of purified EPS from ANU280 was added to plants in addition to the Muc^" mutants ANU2811 and ANU2840. A standard volume of 10μl was added to Leucaena seedlings at concentrations ranging from O.lmg/ml to lOmg/ml prior to the bacterial inoculum. Nodules, as distinct from callus like structures could be found on the lateral roots of Leucaena seedlings when as little as 5μg of the EPS was added (Table 4) .

Effect of the addition of EPS before and after the inoculation of Muc mutants to Leucaena

Aliquots (50μg) of EPS were added to 10 Leucaena plants either (a) 24h prior to, (b) at the same time as, or (c) 24h after the Muc^" bacteria were inoculated onto Leucaena plants. In each case nitrogen-fixing

•nodules were formed indicating that this parameter was not crucial for the time periods tested.

The effect of the addition of EPS and Muc strains to siratro plants.

Parallel experiments such as those described above for Leucaena plants were also done on siratro plants where the parent strain induces nitrogen-fixing determinate nodules and the Muc mutants ANU2811 and ANU2820 alone induce normal-looking nodules which fail to fix nitrogen. The addition of EPS from ANU280 24hrs prior to inoculation with the Muc strains ANU2811 or ANU2820 enabled these bacteria to produce determinate nitrogen-fixing nodules on siratro plants (Table 5) like the parent strain.

Addition of R.trifolii EPS or related oligosaccharide and R.trifolii Muc mutant ANU437 to white clover.

The second group of plants to be tested were the temperate legumes, clovers. The structure of the EPS of the R.trifolii strain ANU843 was determined using a similar approach as used to determine the structure of the NGR234 EPS. The structure of the oligosaccharide of ANU843 used in these plant assays is reproduced in Figure lb. The Muc^" Rhizobium strain ANU437 is a Tn5-induced mutant derived from strain ANU794. Previous work has shown that strain ANU437 produces levels of EPS which are at least 1000 fold less than that produced by the parent strain and forms small, ineffective nodules on clovers which rapidly senesce (Chakravorty et.al. 1982) .

To test whether the addition of the R.trifolii strain ANU843 EPS or related oligosaccharide could correct the nodulation-defective phenotype of ANU437 Muc^" mutant, 50μg of either the R.trifolii EPS or related oligosaccharide was added to white clover seedlings 24h prior to the inoculation of strain ANU 437. After 5 weeks, 25% of the seedlings produced a proportion of nodules which resembled the pigmented nitrogen-fixing nodules produced by the parent strain. Acetylene reduction assays confirmed that nitrogen reduction was occurring (Table 6) . Bacteria isolated from these clover nodules were found to retain all the features of an inoculum, containing strain ANU437 alone.

DISCUSSION

The Sym plas id-cured strain ANU265, derived from Rhizobium sp. strain ANU280, has a Muc phenotype. It is, however, unable to nodulate test plants because it does not contain genes essential for nodulation or nitrogen-fixation. Coinoculation of this strain with different Muc^" mutants of strain ANU280 enables the formation of nitrogen-fixing nodules which were shown to contain both of the inoculated strains. This coinfection phenomenon has been reported previously for temperate legumes (Rolfe et.al. , 1980 a,b) . Purified EPS or related oligosaccharide were added to iπfectable roots to see if these saccharides could substitute for the addition of the Muc , non-invasive strain ANU265 used in the coinoculation

5 experiments. Similar phenotypic correction was achieved by the use of homologous, purified exopoly¬ saccharides or derived oligosaccharide. The homologous saccharide was effective when used at various concentrations and when added either 24hrs

10 ^*: be-fόre or up to 24hrs after inoculation of the Muc

.bacteria.. The nature of the type of saccharide on the helper strain is important because mixing experiments involving the Muc R.trifolii strain ANU845 (a Sym plasmid. cured derivative of ANU843) and Muc mutants

15 of ANU280, were unable to induce nitrogen-fixing nodules on Leucaena. This indicated that the ability to obtain correction of the Fix phenotype in mixing experiments depended on the presence of a Sym plasmid cured strain .that produces homologous EPS. Similarly,

20 defective nodulation properties of Muc R.trifolii strain ANU437 were corrected by addition of EPS and related oligosaccharide isolated from R.trifolii ANU843. Heterologous EPS and oligosaccharide from strain ANU280 was unable to restore an effective

25. nodulation phenotype to strain ANU437. The observed specificity of action of surface saccharides indicates that^*, they have more than a simple passive role of masking surface determinants on the Rhizobium surface. Recently, specific oligosaccharides derived from the

30 cleavage of host or plant pathogen cell walls have been postulated to regulate specific plant functions, such^*, as growth, differentiation and disease resistance (Keim et.al., 1985; and Albershei et.al., 1985). It is possible that the EPS or a specific derivative of

35 15 this molecule may have a function similar to that of oligosaccharins. The action of EPS and/or related oligosaccharide may also rely on the presence of plant enzymes (Dazzo et.al, 1982; Solheim et.al., 1984 and Bhagwhat et.al., 1984) which degrade the bacterial EPS polymer to active oligomers which have a specific role in effective nodule formation.

TABLE 1 Bacterial Strains.

NGR234 derivatives, relevant Phenotype of Plant genetic characteristics, and Response location of Tn5 in EcoRI Leucaena digested total DNA Siratro

ANU280 Parent Strain, Sm Laroe Nod Large Nod Rf derivative of Fix Fix NGR234 (Muc ) (Tn5 not present)

ANU265 Sym .plasmid derivative Nod Nod of NGR234 Sm^r, Sp^r (Muc ) (Tn5 not present)

ANU2811 Tn5 induced Muc Callus Nod mutant of ANU28 (Fix^") Km^r, Sm^r, Rf^r (Tn5 - 7.0kb)

ANU2840 Tn5 induced Muc Callus Large Nod mutant of ANU280 (Fix^") Fi Km^r, Sm^r, Rf^r (Tn5 - 7.5kb)

ANU2820 Tn5 induced Muc^" Callus Small Fix mutant of ANU280 (Fix^") Sm , Rf^r, Km^r (Tn5- 18.0kb)

R.trifolii strains, relevant genetic character¬ istics, and location of Tn5 on EcoRI digested total DNA Response on White Clover

ANU794 Muc⁺ Sm^r Nod Fix nodules

(Tn5 not present)

ANU437 Tn5 induced Muc Nod Fix nodules. derivative of ANU794 Sm , Km . (Tn5 - 9.4kb)

ANU843 Parent strain Muc + +^■ Nod Fix nodules. (Tn5 not present)

ANU845 Sym plasmid cured Nod derivative of ANU843 Muc Rf (Tn5 not present) TABLE 2 - Mixing Experiments on Leucaena

Inoculum Phenotype of No.Plants % Plants forming strains inoc.plants tested. nitrogen-fixing- nodules.

ANU265 Nod 14 0

ANU2811 Roots with calli 14 0

ANU2811 ^& + ANU265 Fix nodules

(C-,H₂ reducing) 14 60

ANU2820 Roots with calli 12 0

ANU2820 ^& + ANU265 Fix Nodules

(C-,H₂ reducing) 12 60

ANU2840 Roots with calli 35 0

ANU2840 ANU265 Fix nodules

(C-H- reducing) 35 90

ANU845 Nod^" 12 0

ANU2840 & ANU845 Small Fix nodules 12

Plants with Fix nodules reduce acetylene at approximately 30-40% of the efficiency of the parent strain.

18

TABLE 3 Effect of the addition of EPS or oligosaccharide repeat-unit isolated from ANU280 on Leucaena leucocephala nodulation.

Strain Plant nodulation No.Plants % Plants

Inoculated Response Tested forming

Fix nods

ANU280 (parent strain)

Large Fix nodules 100 100

& occasional small calli.

ANU2811 Small calli 100 < 2

£NU2840 Small calli 100 1

ANU2820 Small calli 20 0

ANU2811 & EPS from ANU280₊

Large Fix nodules 30 35 and small calli.

ANU2811 & oligosaccharide repeat-unit from ANU280

Large Fix nodules 10 30 and small calli.

A2840 & EPS from ANU280

Large Fix nodules 100 40 and small calli.

ANU2840 & oligosaccharide₊repeat-unit from ANU280

Large Fix nodules 20 40 and small calli.

ANU2820 & EPS from ANU280₊

Large Fix nodules 17 25 and small calli.

ANU2820 & oligosaccharide repeat-unit from ANU280

Large Fix nodules 10 30 and small calli.

Nitrogen fixing nodules induced by the addition of EPS or oligosaccharide repeat unit and Muc^" mutants on Leucaena reduce acetylene 10-30% as effectively as Fix nodules induced by the parent strain ANU280. Percentages of plants forming Fix nodules are averages of batch experiments containing 10 plant replicas. In each batch of 10 plants, the percentage 19 of plants forming Fix nodules varies from 30-80% with each Muc^" mutant. EPS and oligosaccharide repeat unit was added 24hrs prior to inoculation with Muc bacteria.

TABLE 4 Effect of adding different amounts of EPS together with Muc^" mutants ANU2811 and ANU2840 on Leucaena nodulation.

Amount of No. Plants % Plants forming added EPS (μg) Tested. Fix nodules

100 80 35

50 50 40

5 20 40

1 20 30

Percentages of plants forming Fix nodules are averages of 10 plant batch experiments. EPS was added 24hrs prior to inoculation with either Muc mutant.

TABLE 5 Effect of the addition of purified EPS or oligosaccharide repeat-unit on siratro nodulation.

Inoculation Plant No. lants % Plants Strain Nodulation Tested forming Fix Responses Nodules

ANU28 0 (parent strain)

Large Fix 250 >90

ANU2811 Small Fix^" 80 2

ANU2820 Small Fix^" 20 0

ANU2811 & EPS from ANU|80 Large Fix 40 40

ANU280 & EPS from ANU28_0 Large Fix 30 40

ANU2820 & oligosacchar.de repeat unit from ANU280 Large Fix 20 40

EPS and oligosaccharide repeat-unit were added 24hrs prior to inoculation of Muc bacteria.

TABLE 6 The effect of adding EPS or oligosaccharide repeat-unit isolated from R.trifolii strain ANU843 on nodulation of white clover.

Plant No. Plants % Plants

Strain Nodulation Tested Forming Fix

Response Nodules

ANU794 .Pigmented Fix⁺ >200 >95 ANU437 Small Fix^" >200 0

437 & EPS from ANU843

Pigmented Fix_ 20 25 and Small Fix^"

437 & oligosaccharide repeat-unit from ANU843 Pigmented Fix_ 20 30 and small Fix^"

437 & EPS from ANU280

Small Fix^" 20

437 & oligosaccharide repeat-unit from ANU280 Small Fix^" 20 0

EPS and oligosaccharide repeat-unit were added 24hrs prior to the inoculation of Muc^" mutant ANU437.

REFERENCES

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Claims

CLAIMS :

1. A composition for use in altering the response of plants to microorganisms, which comprises the exopolysaccharide(s) derived from bacteria of the genus Rhizobium, or oligopolysaccharide(s) consisting of or containing one or more of the repeat-units of said exopolysaccharide(s) .

2. A composition according to claim 1, comprising the exopolysaccharide(s) derived from Rhizobium sp. strain NGR 234 or Rhizobium trifolii, or derivatives thereof.

3. A composition according to claim 1, wherein the exopolysaccharide(s) or oligopolysaccharide(s) are characterised as consisting of or containing one or more of the repeat-units as set out in Figure la or Figure lb.

4. A composition according to claim 1, which comprises bacteria of an exopolysaccharide-producing strain of the genus Rhizobium.

5. A composition according to any one of claims 1 to 4, further comprising a conventional horticulturally- or agriculturally-acceptable diluent or carrier.

6. A composition according to claim 4, formulated as a spray, dust, or powder for topical administration to plants.

7. A method for altering the response of plants to microorganisms, which comprises the step of treating said plants with an effective amount of a composition which comprises the exopolysaccharide(s) derived from bacteria of the genus Rhizobium, or oligopolysaccharide(s) consisting of or containing one or more of the repeat-units of said exopolysaccharide(s) , either before, after, or simultaneously with exposure of said plants to the microorganisms.

8. A method according to claim 7, wherein said plants are treated with a composition according to any of claims 2 to 6.

9. A method according to claim 7, wherein said step of treating said plants is carried out either before, after, or simultaneously with exposure of said plants to potentially plant-infective or plant- pathogenic microorganisms.

10. A method according to claim 7, wherein said step of treating said plants is carried out either before, after, or simultaneously with exposure of said plants to microorganisms involved in nitrogen- fixation in said plants.