DD263074A5 - Process for binding nitrogen into non-legumes - Google Patents

Abstract

The invention relates to a rhizobium transformant which binds symbiotically stock substance in non-leguminous plants and additionally comprises a transconjugant. For example, the novel rhizobium transformant can symbiotically bind nitrogen to non-legumes belonging to the Poaeceae family, such as wheat, barley, sorghum, rice, rapeseed. The invention further relates to a material for treating seeds of a non-legume plant containing a rhizobium transformant which symbiotically binds nitrogen in the non-legume plant. Furthermore, the invention relates to a process for the preparation of rhizobia F2 transconjugants which bind symbiotically nitrogen in Nichtleguminosen.

Description

Method for binding nitrogen in dense legumes Field of application of the invention

The invention relates to rhizobia transformants which infect turf leguminous plants whose seeds have been coated with a material specific for the rhizobia transformant, cause root nodule formation there and bind nitrogen in them. The non-legumes with formed root nodules (nodulated) can be grown without the use of nitrogen fertilizers. The plants and the straw remaining after harvest have a higher protein, dry matter and nitrogen content than their counterparts without formed root nodules (non-nodulated).

The invention will be further illustrated by way of specific examples in which rhizobia transconjugants are used for wort ingrowth formation in grasses (family Poaceas) including wheat, barley, sorghum, rice and Brassicaceae (family Cruceforae) such as rape. Rhizobia transconjugants that bind nitrogen in Eucalyptus (family Liyrtaceae) are also treated in the examples.

Characteristic of the known state of the art

An essential aspect of plant metabolism is the use of nitrates and other inorganic nitrogen compounds in the synthesis of organic compounds such as amino acids, proteins, chlorophylls, vitamins, hormones, and alkaloids necessary for growth and development

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important to the plant. Although plants absorb nitrates and other nitrogenous compounds from the earth, the source of nitrogen is the free dinitrogen (N_) from the atmosphere; however, the free dinitrogen "must be bound as such and converted into a form that can be utilized by the plant.

2.1. Biological nitrogen bond

Biological dinitrogen bonding (more commonly referred to as biological nitrogen bonding) is a complicated process involving the stepwise reduction of free nitrogen to ammonia through a series of intermediates. It is caused by nitrogen-binding microorganisms, of which some genera live in symbiosis with certain vascular plants. The most important genus of symbiotic nitrogen-binding bacteria is rhizobium, of which there are numerous species, each of which lives in symbiosis with one or more closely related legume species (eg, peas, beans, clover, etc.). Consequently, legumes, unlike other plants that are unable to bind nitrogen, do not require nitrogen fertilizers to grow; rather, legumes can enrich the nitrogen content of the soil. Given the economic importance of many legumes, bacterial cultures are available for inoculation of legume seeds; In addition, legume seed traits containing viable rhizobia have been disclosed (U.S. Patent 3,499,748 and U.S. Patent 4,149,869).

The symbiotic nitrogen binding looks like this: A rhizobium bacterium that is specific to a particular legume host infects the root of the legume host. Thereafter, at the root, a nodule develops in which the rhizobia live in an endosymbiotic state known as bacteroids. They perform unique functions, such as nitrogen fixation, in close cooperation with the legume host. The bacteroid behaves almost like an organelle. Interestingly, rhizobia generally does not bind explanta nitrogen. For a review of various aspects of biological nitrogen fixation in legumes, see Chapters 3, 4, and 5 in "Plant Genes Research - Genes Involved in Microbe-Plant Interactions", DPS Verma, TH Hohn , Springer-Verlag, New York, 1984.

Infection, root nodule formation (nodulation) and nitrogen fixation each require a complicated interaction between the Rhizobium and its specific Leguminosenwirt · In order for the initial infection occurs, Rhizobium and Legurainose must each "detect ¹ * · It is believed that this highly specific recognition of an interaction between a Including lectin of the legume host (a glycoprotein found on the root hairs) and polysaccharides on the bacterial surface, this interaction may be considered as a very specific "lock-and-key" mechanism, the specificity of which with an antibody-antigen interaction, without the After specific detection and attachment of rhizobium to the root of a particular legume host, infection of the host occurs in a non-pathogenic manner due to host responses to the invading host Rhizobia is controlled. In general, the rhizobia penetrate into the legume through the root hairs, and the penetration of the bacteria is mediated by a tubular structure formed by the host plant and called an infection thread, which penetrates into the inner cells of the root cortex. The rhizobia are released from the infective thread but remain in a host-derived membrane envelope called the peribacteroid membrane; In this way, the bacteroids are restricted to extracellular chambers. Disruption of this complicated sequence of events could lead to infection that would be pathogenic to the host.

After rhizobium infection of the host legume, subsequent root nodule formation occurs only in response to a complex interaction of events, where legume affects the expression of rhizobium genes, and rhizobium in turn affects the legume gene activity required for root nodule formation. The cells in the nodular tissue are highly organized in zones with plant-specific arrangements; the bacteria are usually found in the peribacteroidal membranes,

which exclude the bacteria from the epidermal and meristematic zones. The organization and morphology of the root nodule is significantly biochemical, as diffusion problems must be resolved to ensure oxygen and nutrient ingress for the bacteroids and ammonia leakage for the host plant. "A characteristic feature of effective root nodules, i. H. Nodules that actually bind nitrogen are the presence of leghemoglobin, an oxygen binding protein that appears to be involved in nodule respiration (ie, the high concentration of this protein facilitates diffusion of oxygen into the bacteroid areas) and protection of nitrogenase (a major enzyme in the nitrogen binding pathway) Leghemoglobin is obviously a true symbiotic product - the globin proteins are encoded by plant genes, and the bacteria are involved in heme synthesis.

After all, the symbiotic nitrogen fixation - which takes place in the nodule - is a joint venture. The bacteroids contain the mechanisms for nitrogen binding, and the legume partner converts the generated ammonia into an organic form, which is then used to nourish the entire plant and the bacteroids; the legume partner creates a suitable environment and energy source that enables the bacteroids to bind nitrogen. Much can go wrong with the whole process. If the VV mammalian bacteria are "mismatched", the infection will not be successful. Consequently, the whole process of root nodule formation and symbiotic nitrogen binding will not occur. In addition, successful root nodule formation does not mean that nitrogen fixation is inevitable because the resulting nodule may be defective.

2.1.1. Lequminosenqene involved in the symbiosis

Two major groups of host gene products, Leghemoglobin and Noduline, are specifically induced during symbiotic nitrogen binding. While the structure and function of Legend

Hemoglobin in the Wurzelknöllchen are relatively clear, is little known about the Noduline. Interestingly, a recent article reported that leghemoglobin plant DNA sequences are detectable in non-legumes, suggesting that the genes for these oxygen-binding proteins may be more widespread than previously thought (Hattori & Johnson, 1985, Plant. Mol Biol. 4: 285-292).

2.1.2. Rhizobium genes involved in the symbiosis

Several years ago, it was discovered that rhizobia genes ( nod ) needed for root nodule formation (nodulation) are located on a large plasmid (called symbiosis plasmid) that also carries the nitrogen-binding genes (nif) itself. It has since been found that many of the rhizobiabacteria could be induced to recognize a new legume partner by transferring to that rhizobium a symbiosis plasmid from another rhizobium species of a different host specificity. There is no suggestion that transfer of rhizobium symbiotic plasmids from one rhizobium to another could extend the host range to non-legumes. Interestingly, the incorporation of a rhizobium symbiosis plasmid into Aqrobacterium tumefaciens (a bacteriura causing tumor growth in plants called gonads ) gives Aqrobacterium the ability to produce root nodules in legumes; however, the nodules are out of order in that they do not bind nitrogen (Hooykaas, in "Molecular Genetics of the Bacteria-Plant Interaction", A. Puhler, ed., Springer-Verlag, New York, 1983, pp. 229-239).

Over the years, many evidences have shown that nodule activity is increased in legumes by the action of certain plant exudates. This action may stimulate the expression of the nod gene. All legume root exudates cause this effect, however

the exudates of a variety of non-legumes do not.

2.1.3. Biological nitrogen fixation and non legume

A non-Rhizobium bacterial genus, called Frankia , lives in symbiosis with some non-leguminous vascular plants such as Alnus (alder), Casuarina (mace tree) / Ceanothus, Eleagnus, Myrica (evergreen) and Psychotria (a tropical wood plant). A report based on data derived from in vitro co-existence of a tobacco cell culture system and rhizobia indicates that some non-legumes can produce factors that can be exploited by rhizobia (Gibson et al., 1976, Planta 128: 233- 239); however, no specific recognition, symbiotic ratio or specific interactions were suggested or observed. In fact, the only known example of a stable symbiosis between a non-legume seed plant and Rhizobium, outside the legumes, is that of Parasponia, a large tropical Malayan family (elm). In Australia, naturally occurring native Rhizobium starch was found to induce root nodule formation and nitrogen binding in Parasponia (Trinick, 1980, New Phytol., 85: 37-45 and 86: 17-26; Trinick, 1981, Current Perspectives in Nitrogen Fixation (Current ... perspectives in nitrogen bond) Gibson et al, eds Elsevier Press p 480; Trinick et al _f 1976 Arch Microbiol 108: 159-156; see also Bender et al .; 1985, Plant Science 38:... 138 -140).

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Object of the invention

The aim of the invention is to easily trigger Wurzelknöllchenbildung in non-leguminous and bind nitrogen in them.

Explanation of the essence of the invention

The invention has for its object to find a suitable Rhizobium transformants that binds symbiotically nitrogen in Nichtlegurainosen.

3. Summary of the invention

Rhizobia transformants are described that can symbiotically bind nitrogen to non-legumes. More specifically, the rhizobium transformants of the invention may a) infect the roots of non-leguminous plants derived from seeds coated with a Rhizobium transformant-specific protein mixture, b) induce nodule formation on the roots of these non-leguminous plants, and c) nitrogen in the root nodules thereby reducing the need for nitrogen fertilizers to promote plant growth and development in these legumes

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is turned off. A nutrient medium is described which is useful in the identification of various rhizobia species and the transformants of the invention.

Coated seeds of non-legumes are also described, some of which contribute to the realization of syrabotic nitrogen binding in non-legumes by the rhizobia transformants of the present invention. These seed coatings contain legume extracts, chromatographic fractions of legume extracts, crystals or purified proteins specific for the Rhizobium transformant. The seed coatings may also contain the Rhizobium transformant itself as an ingredient. Methods are described for producing rhizobiura transformants, coating the non-legumes, and effecting the symbiosis between the rhizobium transformants and the non-legumes.

3.1. definitions

The term "rhizobium transformant" as used herein means a rhizobium containing incorporated DNA that can be made by any of a number of methods including, but not limited to: transformation (ie, plasmid DNA infection), transfection (ie Infection with free DNA, phage DNA or virus DNA) or conjugation (ie, transfer of a replica plasmid from one bacterium to the other). The transformants derived from the conjugation are a subgroup of transformants of the invention and may be referred to specifically as transconjugants.

The term "parent rhizobia" as used herein means the parent species conjugated to produce rhizobium transconjugants of the present invention, as well as the rhizobia species from which plasmids or DNA sequences can be isolated and used to transform rhizobia To produce Rhizobium transformants.

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4. Brief description of the figures

Fig. 1 is a graph showing the dry matter content (A) and the nitrogen content (B) of wheat plants harvested at various times after planting. Three wheat groups are shown: Wheat in which root nodule formation was induced by Rhizobium transconjugants (+ R-N); Wheat fertilized with a nitrogenous fertilizer but not treated with Rhizobium transconjugants (-R + N) j and Wheat which did not cause root nodule formation by Rhizobium transconjugants and which was not treated with nitrogen fertilizer (-R-N).

Fig. 2 is a graph showing the dry matter content (A) and the nitrogen content (B) of barley plants harvested at various times after planting. Three barley groups are shown: barley in which root nodule formation was induced by Rhizobium transconjugants (+ R-N) j barley fertilized with a nitrogen fertilizer but not treated with Rhizobium transconjugants (-R + N); and barley in which root nodule formation was not produced by Rhizobium transconjugants and which would not be treated with nitrogen fertilizer (-R-N).

5. Detailed description of the invention

The invention relates to rhizobia transformants which induce root nodule formation in non-legumes and bind nitrogen. The non-leguminous plants with formed root nodules (= nodulated non-leguminous plants) can be grown without nitrogen fertilizers and have the same or higher protein content, dry matter content and nitrogen content than their counterparts without formed root nodules (non-nodulated counterparts) fertilized by nitrogen fertilizer. The straw remaining after the harvest of the nodulated non-legumes has a high protein content.

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More specifically, the rhizobia transformants of the present invention are capable of forming nodules in non-leguminous plants grown from seeds coated with a material specific to the particular transformant The invention relates to the Rhizobia transformants Production of the Rhizobia transformants, the material used to coat the non-legume larvae, the coated seeds themselves, methods of inducing V-root nodule formation in the non-legume plants with the Rhizobia transformants, and the resulting nodulated non-legumes having a high protein content.

For clarity of description, the invention is treated in the following subsections in the following order: a) the Rhizobium transformants; b) The coatings for the non-legume seeds; c) The realization of symbiotic nitrogen binding in non-legumesϊ and d) The nodulated non-legumes.

5.1. The rhizobia transformants

The production and identification of the rhizobia transformants of the present invention was based on the original discovery that each rhizobia species, when grown on nutrient medium outside its legume host, produces a colony of a particular color, provided that the nutrient medium is in addition to those for supporting the rhizobia Bacterial growth necessary nutrients contains a non-denatured extract derived from the leguminous host, which is the specific partner of the Rhizobiumspecies. The colony color can be exploited as a means of identifying the particular Rhizobium species. Indeed, various rhizobia species can be grown on a nutrient medium containing a mixture of the undenatured extracts of each individual species-specific legume host; each species forms colonies with a characteristic color.

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The invention is also based on the further discovery that the rhizobia transformants which infect the non-legumes, causing them to nucleate and bind nitrogen in them, form snow white colonies on nutrient medium containing extracts of the legume host partners of the parent rhizobia of the transformant, and that the. Transformant bacteroids, which are isolated from the non-leguminous root nodule and cultured on the medium defined above, which additionally contains an extract of the non-legume host, forms colonies whose color is greyish. Thus, the nutrient media provide a relatively straightforward means of identifying the transformants of the present invention. The nutrient media are described in the subsection below.

5.1.1. Nutrient media for the identification of Rhizobia transformants

The nutrient medium should contain the nutrients necessary for rhizobium growth, i. H. including, but not limited to, any of the known sources of carbon, nitrogen and salts as well as B-group vitamins and the essential amino acids such as L-alanine, L-serine and L-tryptophan which may be in the form of single amino acids, tripeptides or oligopeptides, etc . may be present. The legume extract ingredient is useful for the identification of rhizobia species, but is not required for the diet of rhizobia, i. that is, when a rhizobium is grown on a nutrient medium that either does not contain its legume host extract or contains denatured legume host extract (e.g., autoclaving the media after denaturing legume extract denatures the extract), colonies will form but become the colony of each individual Rhizobium species have a unique color. Table I below lists the characteristic colony colors produced by the various rhizobia species grown on nutrient medium containing the extract of the leguminous host of each species plated on the plate. It should be noted that incubation of the in the embodiments

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of the invention used at a temperature above 32 c caused by all on the medium grown Rhizobiakolonien the formation of red colonies; this is a reversible color change in that the characteristic color of each colony reverts when the temperature is lowered, for example to a value between 18 ^° C. and 30 ^° C.

In addition, the nutrient medium can be modified by the addition of cysteine and phenylalanine so that colonies with different shades of color characteristic of this species arise.

Table I

Colony color of various rhizobia species cultured on nutrient medium containing extract of the host-specific legume partner χ

Rhizobium R. cowpea

R. japonicum R. lequminosarum R. lupini R. meliloti R, phaseoli R. trifoli

Leguminose Host partner Vigna (peanut, Mimosa acacia, Leucaena) Glycine (soybean) Lathyrus (pea) Lupinus (lupine) Melilotus (clover) Phaseolus (bean) Trifolium (clover)

Colony-color greyish brown

reddish orange golden yellow light yellowish brown dark brown light brown

κ ο

Colonies are kept at a temperature below 32 C (e.g.

between 18 ⁰ C and 30 ⁰ C), as described in section 6.1. Incubated in more detail described nutrient medium.

The leguminous extracts used in the nutrient medium can be obtained from any part of the leguminous host plant, including but not limited to shoots, stems, roots or seeds. The extracts of young shoots

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give the fastest color reaction. The legume extracts may be prepared by dividing the plant into fine particles, homogenizing the macerated material in ethanol with a buffer at a pH of about 7.2, and pelleting the insoluble material by centrifugation. The supernatant liquid can be dialyzed using distilled water until clear, after which it can be recentrifuged. The whole process is carried out at 4 ^° C. in order to minimize the decomposition of the vegetable substances.

The legume extract may be fractionated chromatographically by a method which is a modification of the method of Allen et al., 1973, Biochem. J, 131: 155-162; Allen et al., February 1975, FEBS Letters 50 (3): 362-364; Gordon et al., August 1972 FEBS Letters 24 (2): 193-196; Peomans et al., 1982, Planta 156: 568-572; and Trowbridge, 1974, 3rd Biol. Chem. 249: 6004-6012. Briefly, this involves chromatographic separation of the extract using a column of galactose-derived CH-Sepharose 4B (Pharmacia, Sweden) and DEAE-52 (Whatman) as follows: a) The extract is first applied to the column of galactose Unlatted substances are removed and recovered by washing the column with buffer and collecting 5 ml fractions, which are spectrophotometrically analyzed for absorbance at a wavelength of 280 nm. Washing is continued until runoff The fractions showing absorbance at 280 nm are collected and used for chromatography on DEAE-52 The substances bound to the galactose-derived Sepharose 4B layer are added by addition of 4 % Glucose is eluted to the column, the eluent is collected in 5 ml fractions, which are also analyzed for extinction at 280 nm, b) Di fractions containing the substances that were not bound to the galactose-derived Sepharose 4B are placed on a DEAE-52 column which is eluted first at pH 7.2 and then at pH 9.2; 5 ml fractions are collected and

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analyzed for absorbance at 280 nm. The fractions having the maximum absorbance at pH 7.2 are pooled and the fractions with a maximum absorbance at pH 9.2 are taken together. These three fractions of a particular legume host (ie, the fraction eluted using glucose and the fractions eluted at pH 7.2 and pH 9.2) may be taken together and substituted for the whole extract in the nutrient medium described above be used. In fact, the active component of the three fractions can be crystallized and stored. These crystals derived from the legume host may be substituted for the legume host extract as part of the nutrient media used to identify the rhizobia transformants of the invention.

While not bound by any theory or explanation of the invention, it can be noted that the chromatographic fractions are likely to contain protein, and possibly glycoproteins. The affinity of the derived Sepharose 4B suggests that probably at least one of the proteins is a lectin. This could be significant because it is believed that leguminous lectins are important in the initial recognition of rhizobium partners. Once the amino acid sequences of these proteins are determined, these proteins can be made by chemical synthetic methods or by recombinant DNA techniques using prokaryotic or eukaryotic host vector expression systems to express the proteins. Expression of the protein in a host-vector eukaryotic expression system may be preferred because the eukaryotes can process the protein in a manner more similar to naturally-occurring proteins. This could be especially important if the protein is a lectin. In addition, the identified proteins can be isolated from sources other than the legume host. This theory does not exclude the possibility that other constituents present in the extract, such as carbohydrates, alkaloids, hormones, etc., are a significant factor in the

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Although the nutrient media described above are suitable for identifying the transformants of the invention, an initial characterization of the transformant DNA was performed. The rhizobia transformants according to the invention, which bind symbiotically nitrogen in non-leguminous plants, possess plasmids which are not contained in the parent rhizobia of the transconjugant. While not wishing to be bound by any theory or explanation of the invention, it is interesting to note that these plasmids may contain the DNA sequences responsible for the new host range of the Rhizobium transformants.

Methods for producing the rhizobia transformants of the invention are discussed in more detail below.

5.1.2. The change-line culture method for the production of Rhizobia transconjugants

The turnover line culture method for the production of rhizobia transconjugants of the invention involves the following: two different rhizobia species (parent generation) are alternately streaked onto a solid nutrient medium containing, in addition to the nutrients important for bacterial growth, either: a) a non-denatured mixture Extracts of the leguminous hostners of each rhizobia species; b) the three chromatographic fractions obtained from the extract of each legume host as described in section 5.1.1. obtained above: c) the crystals obtained from the chromatographic fractions of each legume host; or d) related proteins (including glycoproteins). In the following, these components, called legume extract, chromatographic fractions, crystals or proteins, of each parent rhizobium species will form colonies of a characteristic color along the line. Rizobia transconjugants (here called rhizobia F transconjugants) are inserted between the alternating rows of the parental

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lonien formed. The Rhizobia F transconjugants, unlike their parents, form milky-white colonies and can not induce root nodule formation on plants.

The rhizobia F ^ transconjugants are isolated from the milky white colonies and cultured in alternate rows with a third rhizobium parent species on solid nutrient medium, in addition to the legume extracts, chromatographic fractions, crystals or proteins used to produce the F transconjugant used a third non-denatured. Legume extract, chromatographic fractions, crystals or proteins derived from the restricted iminosome host partner of the third rhizobium parent species. Rhizobium F transconjugants are generated between the alternating rows of milky white colonies formed by the Rhizobium F transconjugant and the colored colonies formed by the third parent Rhizobium. The Rhizobium F transconjugant colonies are identified by their snow white color and are capable of infecting non-legumes seeded, treated and seeded as described herein, root nodules formed thereon and nitrogen in to bind them.

The identification and selection of the rhizobium F and F transconjugants produced above will be appreciated due to the use of -12

When the individual crosses are carried out, two types of colonies develop between the parents: a) Colonies with a color representing a mixture of the colors of the parent colonies * and b) the milky white F transconjugant colonies or the snow-white Fo transconjugant colonies. The mixed color colonies contain rhizobia, which are not stable; these rhizobia can cause root nodule formation in the legume host partners of both parent rhizobia, but only in one generation. In other words, the bacteroids recovered from the root nodules of each legume host can only be restored to this particular leguminous

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we can trigger nodule formation. The F ^ transconjugants of the milky white colonies are stable but unable to induce nodule formation in any plant. Surprisingly, the F transconjugants of the snow-white colonies are stable rhizobia, which in non-leguminous plants form root nodules and can symbiotically bind nitrogen in them.

The parent rhizobia used to generate the F transconjugants may contain a third rhizobia species, another F transconjugant, and optionally another F transconjugant. If the third parent comprises another F transconjugant, then the nutrient media will contain at least four legume extracts, chromatographic Fractions, crystals or proteins; d. H. those of the leguminous host partner for each of the two parent Rhizobia used to produce each of the two F transconjugants If the third parent comprises an F transconjugant, then the nutrient media will contain at least five host plantain extracts, chromatographic fractions, crystals or proteins. ie those of the legume host partner for each of the two parent rhizobia used to produce the F trans -conjugant and the legume host partner for each of the three parent rhizobia.

Rhizobia, which are used to produce the F transconjugant.

The alternate series of parent colonies method is a convenient solution for generating the transconjugants; however, any pattern or method of inoculation can be used to grow parent colonies so close together that conjugation can occur. So circles, ellipses, wave or spiral patterns can be used. In fact, a number of different parents may be streaked onto the plates to produce a number of different transconjugants on the same plate.

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5.1.3. Other methods for producing Rhizobia transformants

Although the exchange line culture method is a suitable method for the production of rhizobiurn F transformants. however, the invention is not limited to this method. In addition to the conjugation, transformation with the appropriate plasmid responsible for the increased host range specificity of the rhizobium is considered to be within the scope of the invention. In addition, recombinant DNA techniques including the use of phage and other vectors for the infection of Rhizobia with the appropriate sequences are considered to be within the scope of the invention.

For example, the Tn5 transposon that can be used in a rhizobial transformation scheme is a particularly useful system for rhizobia. Symbioseplasmide C Sym plasmids) can be identified by hybridization with a labeled DNA probe containing nif genes. Transfer of this Sym plasmid can be done using Tn5 to incorporate a) a labeling gene into the Sym plasmid; for example, a gene for antibiotic resistance can be incorporated into the Sym plasmid using transposon Tn5 by a conjugating donor and recipient bacteria and hybrid selection based on the acquired resistance; b) by cloning the Sym plasmid or parts thereof into E. coli and transforming Rhizobia with the plasmid itself; c) by recombining the plasmid with transport ^genes together with the marker ^gene (eg antibiotic resistance) such as ^pvw5ai _or Tn5-Mob; and d) by hybridizing the bacteria containing the plasmid with its incorporated marker gene to the recipient bacterium simultaneously with a helper bacterium for transfer promotion.

5.2. Seed coatings for non-legumes and coated seeds

To effect the symbiosis between the rhizobia transformants of the invention, the seeds of the non-legumes should

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treated or coated with a material specific for the rhizobia transformant. Such overcoats may include or exclude the rhizobium transformant itself. The presence or absence of the Rhizobia transformant in the top pull only affects the method used to cause symbiosis in the growing plant later on. "

5.2.1. Seed cover for non-glucose

The seed coat includes, but is not limited to, the following, which may include or exclude the rhizobium transformant: a) a mixture of extracts derived from each of the legume host plants that is a partner of each parent of the Rhizobia transformant. chromatographic fractions derived from each legume host plant that is a partner for each parent Rhizobium of the transformant; c) crystals derived from the legume extract and / or its chromatographic fractions or d) related proteins. As explained above, the proteins can be recovered by chemical synthetic methods, recombinant DNA techniques, or isolated from other sources.

While not wishing to be bound by any theory or explanation of the invention, these described seed coatings may include any of a number of factors, including, but not limited to, lectins, flovones, etc., which are important in achieving the symbiosis between the non-legumes Rhizobia transformants can be. Recently, it has been reported that the expression of rhizobium nodule genes involved in normal root curl and therefore symbiont / host recognition and nodule formation (nodulation) has been induced by legume root exudate (Rossen et al., 1985, EMBO 4 (13A): 3369-3373). Recently, it has been reported that a group of compounds called flavones secreted by legume roots express expression of Rhodobium nodulation genes

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(see Redmond et al., 1986, Nature 323: 632-635). Flavones are normally produced in flowers and leaves of various plants, only legumes are known to secrete flavones or to contain roots. According to the report by Redmond et al. induced certain flavones in the root exudate of pea, bean, soybean, alfalfa and clover expression of the no dA gene in R. trifoli, which was not the case with root exudate from the non-legumes corn, rice and parasponia. Stimulation of rhizobium was not symbiotically host specific from a symbiotic point of view; that is, the expression of nodA was induced in rhizobium in response to a number of different legume exudates . Thus, the flavones may be one of the early signals between a rhizobium and a legume, followed by a second, more host-specific recognition (eg, the lectin-polysaccharide recognition system) to produce this particular host-symbiont partnership. It is possible that the flavone induction hysteresis is an early event which allows legume extract treated non-legume seeds to become susceptible to infection with the F -Rhizobium transformants of the present invention. Perhaps the flavones present in the legume extracts are taken up by the non-legume seeds during imbibition or pre-germination, transported to the root of the young seedling and, when exuded, attract the rhizobium transformant.

Other factors from the legume strains contained in the seed coatings may be important in achieving F transconjugant infection of the non-legume host. For example, the seed coatings may contain lectins that are believed to be involved in the specific recognition interaction between rhizobia and its leguminous host partners. A recent report describes a particulate form of the lectin, trifolin A, which can be isolated from clover root exudates and specifically bound to R. trifoli (Truchet et al., 1986, Physiol. Plant 66: 575-582). Interestingly, lectin-containing legume exudates containing lectins provided the ability

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rhizobial mutants recognize their legume host and induce root nodule formation (see Halverson et al., 1984, Plant Physiol. 74: 84-89 and 1985, Plant Physiol. 77: 621-625). It is possible that one or more lectins are included in the seed coatings of the invention which allow the specific recognition to take place between the rhizobia transformant and its non-legume partner.

5.2.2. Methods for topping the non-legume seeds

The non-legume seeds can be coated by a variety of methods including, but not limited to, immersion and air drying, spraying, embedding (for example, in polymers), immersion and tumble drying, etc. The method chosen depends on the topcoat to be used. For example, if the rhizobia transformant is included in the top coat mixture, methods or ingredients that guarantee the viability of the bacteria should be used. These include, but are not limited to: blends containing a gel material to keep the level of moisture high; optionally, the seeds may be coated with powdered seed pods containing fungicides. In any case, the method should be chosen so that the active ingredients are not denatured or destroyed in the top train.

In the examples according to the invention, the seeds of the non-legumes were coated by immersion in the leguminous extracts and air drying; preferably three times. Two immersions gave poor penetration of the rhizobia transformants, while four immersions did not appear to enhance penetration.

5.3. Realization of syrabotic nitrogen binding in non-legumes

A number of methods can be used to ensure the infection of non-legumes by the Rhizobia transformants; the selected method depends in part on the

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Texture of the used topcoat for the non-legume seeds.

For example, non-legume seeds that have been coated as described above that does not contain the rhizobium transformant may be wetted with a suspension of the rhizobium transformant. Thus, the seed, seedling or plant can be watered with an appropriate volume of the bacterial suspension. Where the seed coat contains Rhizobium transformants, only plants and watering are required.

It may be desirable to add non-nitrogenous fertilizer depending on the nature of the plant, condition of the soil or terrain, etc. In fact, it has been found in the laboratory experiments that the addition of 2 mM nitrogen at the very beginning, sufficient to increase the initial growth of the plant at sowing the seeds, without harming the rhizobia transconjugants or "paralyze ¹ *" It simulates more natural Condition, because most soils (even unfertilized) contain a certain amount of nitrogen (not more than 2 mM), in the field the administration of nitrogen is not required.

5.4. The nodulated non-leucuminoses

The non-legumes, which are stimulated by the Rhizobia transformants according to the invention for root nodule formation, have a nitrogen and dry matter content equal to or greater than that of their nitrogen-fertilized non-nodulated counterparts. The nodulated plants have a higher nitrogen and dry matter content than their unfertilized non-nodulated counterparts. Analyzes of the amino acid compositions of the nodulated plants demonstrated that in most cases the proportion of each amino acid remains the same, but the total concentration per plant is increased. In some cases, however, higher tryptophan and leucine levels were observed.

- 25 -

Interestingly, it has now been found that the straw remaining after the harvest of some of the nodulated non-leguminous species consistently had a protein content of about 6% to about 9 % ; that is a gross difference to the protein content of 1.5% to 2 % normally found in the straw of the non-nodulated counterparts. This straw with the high protein content can be advantageously used as a source of protein, for example, in livestock feed mixtures for both livestock and domestic animals.

The rhizobia transformants of the invention, which induce root nodule formation in non-legumes and bind nitrogen in them, can reduce the use of expensive nitrogenous fertilizers and ultimately improve the soil.

5.4.1. VVurzelknöllchenmerkmale

The morphology of the root nodules formed by the rhizobia transformants of the invention on the non-legumes is apparently quite normal in appearance, but a larger percentage of smaller nodules are formed. The rhizobia apparently get into the root via infection threads, and the bacteroids are contained in chambers. In fact, a reddish color is found in the root nodule which may be due to leghemoglobin or a closely related protein. Electron microscopy of the nodules shows that the vascular bundles found in the cortex are peripheral

- 25a -

lie while the vascular bundles of the lateral roots are central. Most cells in the middle part of the root nodules are filled with bacteroids.

embodiment

The invention is explained in more detail below with reference to some examples.

Example 6: Material and Methods for the Production of Rhizobia Transconjugants and the Root Knot Formation in Non-legumes

The following examples describe root nodule formation by rhizobium transformants according to the invention in the following non-leguminous plants belonging to the grass family (Poaceae): wheat, barley, sorghum and rice. In addition, at

Ä WWV / f

- 26 -

a member of the Brassicaceae, a genus outside the grass family (d.) h. Family Cruciferae) also caused by a rhizobium transformant root nodule formation according to the invention.

Interestingly, in another plant outside the grass family, Eucalyptus (a member of the Myrtaceae family), a positive effect was noted. In each example, the turnover line culture method was used to generate Rhizobium transconjugants. The seeds of the non-legumes were coated by immersing the seeds in leguminous extracts specific for the specific Rhizobium transconjugants used for nodule formation in the non-legumes. The coated non-legume seeds were infected with the Rhizobia transconjugants by seeding the coated seeds, germinating the seeds, and casting the seedlings with a suspension of the Rhizobium transconjugant. Within 8 to 12 weeks, nitrogen-binding root nodules developed.

In the examples, laboratory experiments were performed in which the non-legumes were divided into three groups which were treated as follows: a) the first group was treated with the Rhizobium transconjugant without nitrogen fertilizer (+ R-N); b) the second group was treated with nitrogen fertilizer without the Rhizobium transconjugant (-R + N); and c) the third group was not treated with either the rhizobium transconjugant or nitrogen fertilizer (-R-N). At certain times during growth, plants were sampled and the total organic nitrogen content per plant, the amount of dry matter per plant and in some experiments the protein content per plant and its amino acid composition were determined. In some examples, field trials were conducted. The results showed that in each case the non-leguminous plants brought by the Rhizobia transconjugants for root nodule formation were able to bind nitrogen and did not require nitrogen fertilizer for growth. Rather, the

- 27 -

Non-leguminous nodules (+ R-N) in many cases have a higher nitrogen and dry matter content than the fertilized non-nodulated group (-R + N) or the untreated group (-R-N).

Unless otherwise indicated, the materials and methods described below were used in each of the following examples.

6.1. The change line culture method

The change-line culture method used in each of the following examples involves the following: two different rhizobia species (the parental generation) were streaked in alternate rows (3 mm apart) on an agar medium supplemented with nutrients required for growth, an undenatured extract of each legume host specific for each parent Rhizobium. After incubation at a growth temperature below 32 ⁰ G, z. B. between 18 ⁰ C and 30 ⁰ C, each parent Rhizobium formed colonies having a characteristic for this species Farbej between the alternating rows of the colored parent colonies were Rhizobia transconjugants (F. transconjugants) is generated. Unlike their parents, the Rhizobia-F. transconjugans formed ". milky-white colonies and failed to cause root nodule formation on plants. It should be noted that incubation at a temperature above 32 C on the agar medium used here results in the formation of red colonies by all rhizobia species coated on the plate; however, if the temperature is lowered below 32 C (eg, preferably between 18 C and 30 C), the characteristic color of each colony is restored.

The Rhizobia F transconjugants were isolated from the milky white colonies and cultured as described in alternate rows (3 mm apart) with a third rhizobium parent species on agar nutritive medium supplemented with the undenatured legume extracts used in the generation

• & ν w *

- 28 -

of the milky white colonies, contained a third non-denatured legurainose extract derived from the legume host specific for the third parental rhizobium species. Rhizobia F transconjugants were generated between the alternating rows of milky white colonies formed by the Rhizobium F transconjugant and the colored colonies formed by the third parent Rhizobium. The colonies of the Rhizobium F transcanonant were identified by their snow white color; these F transconjugants were capable of infecting non-legumes, as described and planted, inducing root nodule formation and binding nitrogen in them. The materials and methods used to produce the rhizobium transconjugants are described in more detail below.

6.1.1. Isolation of Parent Rhizobia

The rhizobia species used as parent generations were isolated from either soil samples or from leguminous root nodules that were in an advanced stage of development.

The isolation of the. Parent rhizobia from the soil was made by placing seeds of the host legume plant in which Rhizobia triggers root nodule formation into the soil samples. The plants were harvested after 3 to 4 weeks (normally) or 10 to 12 weeks, when they were slow growing plants.

The isolation of the parent rhizobia from the leguminous root nodules was as follows: The nodules were separated from the roots along with about 1 cm of the root tissue surrounding the nodule. The nodule was sterilized by immersion in 3% HgCl and rinsed in 80% ethanol. The nodules were then excised from the root tissue and crushed under aseptic conditions in a mortar. The macerated material was analyzed on the below

- 29 -

distributed agar nutritive medium. The colonies were cleaned by pickling on a medium of the same composition until single colonies were formed twice. The rhizobia isolated from the purified single colonies were streaked in alternating rows on the agar medium described to grow the alternate sets of parent Rhizobia colonies.

6.1.2. Aqar-broth

The agar medium used to grow the rhizobia in alternate lines was prepared by adding the following nutrients in the indicated amounts to 100 ml of distilled water:

KH ₂ PO ₄ 0.15 g K ₂ HPO ₄ 0.15 g KNO ₃ 2.50 g (NH) SO 0.135 g MgSO _4. 7H ₂ O 0.25 g mannitol 10.00 g Aga r 12.00 g Solution No. 1 1,00 ml yeast extract 5.00 ml

Solution No. 1 had the following composition:

MnCl ₂ . 4H ₂ O ml. 500 mg ^H 3 ^B0 3 300 mg ZnSO ^. 4 2H ₂ O 200 mg NaMoO ,, , 2H ₂ O 20 mg CuSO. , 4 5H ₂ O 2 mg CoCl ₂ . 6HO 2 mg Distilled water to fill up to a final volume from 100

The agar medium described above was then added for 20 minutes

2 bar autoclaved and at a temperature between

- 30 -

55 c and 60 C, followed by the addition of 1.0 ml each of solution no. 2, solution no. 3 and solution no. 4; 20 ml each of non-denatured legume plant extracts derived from the parent rhizobia species Vtfirt legume plant specific for each medium to be cultured on the medium, and 15 mg each of the following amino acids: L-alanine, L-serine and L-tryptophan To avoid proteins in the legume extract, the final media composition should not be autoclaved.

Solution No. 2, Solution No. 3, and Solution No. 4 had the compositions given below. Each was prepared using sterile distilled water.

Solution No. 2: 50 mg nicotinic acid 50 mg Thiamine Hcl 50 mg Pyridoxine HCl 50 mg myo-inositol 50 mg biotin

Sterile distilled water to a final volume of 100 ml

Solution No. 3: water 75 mg Potassium 100 ml Sterile distilled Solution No. 4: V / ater 15 gm CaCl ₂ . H ₂ O 100 ml Sterile distilled 6.1.3. Lequminosenpflanzenextrakt

The legume plant extracts used in the agar medium and to cover the non-leguminous macaws were prepared as described below.

The entire plant or selected parts of plants, eg. B.

<O J U / # - 31 -

clean washed roots, sterilized seeds or upper parts or, preferably, young shoots were cut into fine particles, ground in a mortar with 80% ethanol to a thin, homogeneous paste into which an equal volume of potassium sodium salt buffer, pH 7 , 2, was given the following composition:

K ₂ HPO ₄ 0.430 g

NaH ₂ PO ₄ 1.469 g

NaCl 7,200 g

Sterile distilled water to a final volume of 1000 ml.

The mixture of homogenized plant extract and buffer was allowed to stand at 4 c for 48 hours and pelleted by centrifugation at about 5000 g for 30 minutes. Then, the supernatant was dialyzed at 4 ⁰ C using sterile water for about 48 hours, during which time the water was replaced 5 to 6 times, until a clear, the color, transparent liquid, that is, the extract was recovered. The extracts were stored at 4 ⁰ C.

6.1.4. Isolation of rhizobia transconjugants

When the two parent rhizobia were cultured at a temperature below 32 C in alternate lines (3 mm apart) on the agar medium containing the appropriate leguminous extracts as described above, the parent rhizobia formed colonies of a characteristic color. However, two types of colonies developed between the rows of colored parent colonies: a) colonies with a color representing a mixture of the colors of the parent colonies and b) the milky white colonies of the rhizobium F ^ transconjugant. The ratio between the colored colonies and the milky white F transconjugant colonies varied somewhat, but on average it was about 100: 3 (mixed color colonies: milky white colonies). The mixed color colonies formed root nodules in both host legume species, but only in one generation; d. H. the rhizobia of the mixed

- 32 -

In color colonies, the legume host partners of both parental rhizobia nodded, but the rhizobia obtained from the root nodules of each legume host could only produce root nodule formation in this particular leguminous host. In contrast, the milky white F transconjugant colonies did not induce root nodule formation in any plant.

Since it was difficult to transfer a single hybrid colony to a new medium without contaminating it with one or more colored colonies, one or more biological purifications were performed by switching the milky white colonies to another agar mediura (of the same composition). were transferred until, generally after 10 to 12 weeks, a pure culture of the F -transconjugant was obtained with a consistent and uniform milky white color.

The pure F -Transkonjugantenkultur, which formed the milky white colonies was then (3 mm apart) cultured with another Rhizobiumspecies on an agar medium of the same, used for the F transconjugants composition at a temperature below 32 ⁰ C in alternating lines, which additionally contained a third legume extract derived from the legume host specific for the third parental rhizobium host. The F -transconjugant produced its characteristic milky white colonies while the parent rhizobium produced colonies with its specific color. Two colonies developed between the lines of milky-white colonies and the colored colonies: colored colonies and snow-white colonies. The snow white colonies contain the rhizobium Fp transconjugants, which in non-legumes can form nitrogen-binding V-root nodules. The snow white F transconjugant colonies were purified and isolated as described for the milky white colonies and used for root nodule formation in non-legumes.

- 33 -

After root nodule formation in the non-legume plant, the Rhizobia F transconjugant bacteroids can be isolated from the nodule and grown in an agar medium. When the agar medium has the same composition as the agar medium used to produce the transconjugant (ie, the agar medium contains the three leguminous extracts derived from the legume host specific for each parent rhizobium used to produce the F trans -conjugant) and thereto an extract of its non-plain urine host plant, then the F transconjugants isolated from the non-leguminous root nodule form colonies of a grayish color.

6.2. Preparation of non-legume seeds

Seeds of the non-legume plant were coated by dipping them three times at 20 ^° C. for 3 hours each in an aqueous solution containing 3 % calcium sulfate and up to 10 % of a mixture of the three, as described in Section 6.1.3. contained leguminous extracts used to produce the Rhizobium F transconjugant. After each immersion, the seeds were air-dried at 40 ^° C. for 12 hours.

6.3. Infection of non-legumes with Rhizobia F ₂ transconjugants

The infection of non-legumes with Rhizobia F transconjugants was carried out by sowing the coated non-legume seeds and watering the germinated seedlings with a suspension of the Rhizobium Fp transconjugant. In the following examples, laboratory and field experiments were performed in which the germinated seedlings were treated either a) with the rhizobium F ₂ transconjugant without inorganic nitrogen fertilizer (+ RN); b) with the inorganic nitrogen fertilizer without the Rhizobium F transconjugant (-R + N) or c) with either the Rhizobium F transconjugant or with the inorganic nitrogen fertilizer (-RN). The nitrogen content per plant, the "dry matter and in some cases the

- 34 -

Amino acid composition of the resulting plants were determined.

The materials and methods used are described in more detail below.

6.3.1. laboratory tests

In the laboratory experiments, the coated non-legume seeds were sown in 1-liter containers (ydydsk Papir Vaerk, Arhus) filled with 3 mm "Fibo" (R) clinker material; 4 to 5 seeds were sown in each container. The "Fibo" clinker material consists of air-filled, burned clay balls (pebbles) of about 3 mm in diameter, which are normally used as insulating material.

When the germinated seeds had grown a few centimeters above the "Fibo" clinker material surface, the seedlings in each container were poured as follows: a) The + R-Nr group was poured with a 50 ml suspension of the Rhizobium F_ transconjugant ; then the plants were watered once a week with 50 to 80 ml of a non-nitrogenous fertilizer per container; b) the -R + N group was not treated with the rhizobium F ₂ transconjugant, but instead was cast with an inorganic nitrogen fertilizer; then the plants were poured once a week with 50 to 80 ml of the same inorganic nitrogen fertilizer per container; and c) the -RN group was poured with 50 to 80 ml of the non-nitrogenous fertilizer per container. The inorganic nitrogen fertilizer and the non-nitrogenous fertilizer are defined below.

- 35 -

Vorratslösunqen

NH ₄ NO ₃ 2H ₂ O , 7H 4H ₂ O (1 , 0 M) P 110.7 mg / 1) CaSO ₄ . Na ₂ EDTA (O , 012 M) 9.0 mg / 1) KH ₂ PO ₄ 7H ₂ O final volume 7H ₂ O (O , 10 M) (11.1 mg / l) MgSO _4. : MnCl ₃ . 5H ₂ O (O , 20 M) Fe (EDTA) ^H 3 ^B0 3 , 2H FeSO ₄ ZnSO ₄ . 2 ° (2.490 g) CuSO ₄ . (3.716 g) Na ₂ MoO 1 liter ( 106.1 mg / 1) (142.2 mg / l) ( (

Fertilizer ml stock solution / liter final volume

nitrogen content non-nitrogen content 20 0 65 65 20 20 15 15

10

10 10 10 10 10

10

10 10 10 10 10

The suspension of rhizobia F ^ transconjugants used to water the seedlings were prepared by cultivating the Rhizobium F transconjugant in 300 ml flasks containing 200 ml of the following nutrient medium:

1.0 g

1.0 g

0.36 g

0.17 g

0.005 g

0.7 g

1.0 g

3.0 g

Distilled water to a final volume of 1000 ml.

KH ₂ PO ₄ 7H 2 ° K ₂ HPO ₄ 2H 2 ° MgSO _4. 6H 2 CaSO ₄ . FeCl ₃ . yeast extract KNO ₃ mannitol

- 36 -

The bacteria were cultured at 28 ° C for two to three days until the bacterial density measured spectrophotometrically at OD was 0.8, indicating that the bacteria were in the logarithmic growth phase and the culture had not yet entered the stationary phase. Then, the cells were pelleted by centrifuging at about 5000 g for 30 minutes and washed by resuspension in sterile water. This wash was repeated once or twice to remove all nitrogen compounds from the bacterial cells. The resulting cell pellet was resuspended in 1.8 liters of sterile water. This final bacterial suspension was used to cast the non-legume seedlings.

6.3.2. field trials

In the field trials, the coated non-legume seeds were sown in soil that had never been cultivated. The land was cleaned and divided into strips 30 feet wide by 90 feet long. The seeds were only seeded alternately into the strips (i.e., strips without seeds remained between the strips into which seeds had been placed) to prevent chemicals leached from one strip from entering another strip and contaminating it. Within a strip, the seeds were sown in six rows; the individual rows were 60 inches apart, and the seeds within each row were sown 8 inches apart.

The strip contained one of the following groups: a) the + RN group treated with the Rhizobium F -transconjugant and cast with the above-defined non-nitrogenous fertilizer; b) the -R + N group which does not interfere with the Treated with Rhizobium F ₂ transconjugants, but instead was cast with the above-defined inorganic nitrogen fertilizer; and c) the -RN group, which was treated with neither the Rhizobium F transconjugant nor the inorganic fertilizer, but instead was cast with the non-nitrogenous fertilizer.

- 37 -

The following procedure was used to infect the + RN group with the Rhizobium F transconjugant: a culture of the Rhizobium F transconjugant was inoculated 4 inches below each seeded seed. Soil seeding and inoculation were performed with a machine having a 300 gallon tank containing a suspension of the Rhizobia F transconjugant prepared as described above but on a much larger scale (ie, Rhizobia -F_ transconjugant was grown to log phase in a 10,000 gallon tank and diluted to a 0D _fi value of 0.8). Approximately 1.5 ml Rhizobiura F transconjugant culture per seed was inoculated into the soil below. Thereafter, seeds and seedlings were cast as described.

6.4. Protocol for the analysis of dry matter, nitrogen and protein content of nodulated non- leguminous mines

Periodically, a number of containers of each plant group from the laboratory experiments were separated into 3 plants per container and sampled for Kjeldahl analysis, which determined the total organic nitrogen content and the dry matter amount per container (ie per 3 plants). For this purpose, the aerial parts of the plant were removed, dried for 48 hours at 80 ⁰ C, weighed, crushed and ground in a mortar. Samples of the macerated material were taken for the Kjeldahl analysis performed according to the standard method. Each, Kjeldahl analysis was repeated 4 to 6 times. The results were recorded as amount of nitrogen and dry matter per plant. In all cases, the rhizobium seeded plants had a higher dry matter content and nitrogen content than the nitrogen fertilized or untreated groups.

In the field studies a number of plants were harvested periodically and the protein content per plant was determined by first of all the nitrogen content per plant

- 38 -

Using the Kjeldahl analysis, the percent nitrogen was then multiplied by a factor of 6.25. In some experiments, the amino acid composition of the protein was determined. In all cases, the rhizobium-nodulated non-legumes had a higher protein content than the plants treated with the inorganic nitrogen fertilizer and the plants treated with the non-nitrogenous fertilizer.

Example 7: Rhizobium tritici, which causes root nodule formation in wheat

Rhizobium tritici, which causes nodularity (induces nodule formation), was generated by the method described in Section 6, by first crossing R. phaseoli with R. cowpea leucaena to produce the F transconjugant, which was then crossed with R. trifoli . The R. tritici obtained in this way was used to develop root nodules in four varieties of vivae: Anja, Kraka, Vuka and Williams.

7.1. Extraction of Rhizobium tritici

For the production of R. tritici , the following parent rhizobia were used to produce the milky white F transconjugant colonies: a) R. phaseoli, isolated from the French bean cultivar, Prospector, located in a near Aarhus, MS And (b) R. cowpea leucaena, isolated from the tropical tree Leucaena leucocephala (belonging to the Fabaceae family) from Papua New Guinea.

The parental rhizobia were grown in alternate lines on the agar medium described above; the leguminous extracts used in the medium were obtained from the aerial parts of the French bean and from the leaves of Leucaena leucocephala, respectively. The colonies formed by R. phaseoli had a characteristic dark brown color while that of Γ ?. cowpea leucaena a characteristic

- 39 had a subtle greyish-brown color.

The Rhizobium F transconjugant derived from the milky white colonies was purified and cultured as described in alternating lines with a R. trifoli strain isolated from red clover grown in a soil sample found at Randers. The legume extracts used in the medium were from the aerial parts of the French bean, the leaves of Leucaena leucocephala and the aerial parts of red clover. The colonies formed by the F transconjugant were milky white while the colonies formed by R. trifoli had a characteristic light brown color.

The Rhizobium F -transconjugant, here called Rhizobium tritici , derived from the snow-white intercolon colonies, was purified and isolated as described. That took about 20 weeks. Thereafter, the F -transconjugant was used to induce root nodule formation in wheat.

7.2. Root nodule formation in wheat with the help of Rhizobium tritici

The Rhizobium tritici was used to induce root nodule formation in four wheat varieties, namely Anja, Kraka, Vuka and Williams. The wheat seeds were treated by dipping them three times in the aqueous solution described above containing 3 % calcium sulfate and up to about 10 % legume extracts used to produce R. tritici , ie the three leguminous extracts used to cover the seeds were obtained from the aerial parts of the French bean, the leaves of Leucaena leucocephala and the aerial parts of red clover. After each immersion, the seeds were air-dried and sown as described below.

7.2.1. Laboratory experiment The experimental procedure described above was used for three

- 40 - *

wheat varieties: Anja, Kraka and Vuka; ie _f 4 to 5 seeds were sown and germinated per one liter container filled with ¹ * Fibo ^H clinker material. Each of the three wheat varieties was divided into the following three groups: a) + RN, the seedlings of which were poured with a 50 ml suspension of R. tritici prepared as described above, followed by weekly pouring with the non-nitrogenous fertilizer; b) -R + N whose seedlings did not receive R. tritici but were fertilized with inorganic nitrogen fertilizer; and c) RN whose seedlings received neither R. tritici nor inorganic nitrogen fertilizers and instead were poured with the non-nitrogenous fertilizer described above. The plants treated with R. tritici developed within 8 to 10 weeks • nodules. The plants in each container were singulated on 3 plants and 10 containers were used in each group for the analysis of dry matter and nitrogen content.

7.2.2. field test

For the field trial, the coated wheat seeds were grown as described in Section 6.3.2. described, sown. The seeds of item a) + RN ₁ were treated as described with a suspension of R. tritici and poured with non-nitrogenous fertilizer; those from item b) -R + N, were not treated with R. tritici but were cast with the inorganic nitrogen fertilizer, and those from item c) -RN, were treated with neither R. tritici nor with inorganic nitrogen fertilizer, but were simply poured with the non-nitrogenous fertilizer.

7.3. Analysis of dry matter, nitrogen content and protein content of nodulated wheat

For the characterization of the nodulated wheat plants, the methods described in Section 6.4. applied analysis methods.

- 41 -

7.3.1. Results of the laboratory experiment

The wheat plants were harvested from the containers 56, 70, 87, 100 and 118 days after sowing. Dry matter per plant and nitrogen content per plant (Kjeldahl analysis) were analyzed as described above.

The results are shown in Figure 1 where the dry weight per plant (A) and the nitrogen content per plant (B) were plotted over the number of days after sowing.

For ease of understanding of the figure, the following data on the average dry matter mass and the average nitrogen content are useful for the three wheat varieties;

dry matter per 100 seeds - nitrogen   56 days 70 to the seed 118 Wheat grams 4,403 mq per seed 87 100 Anja 3,611 1,103 33 30 25 kraka 4,052 to the 0.896 24 30 21 23 20 Vuka plants 0.819 24 26 20 21 35 The number of harvested 22 26 amounted to: 11 10 8th 8th 10 7 8th 7 8th 9 7 7 12 Pflanzenqruppe 7 9 Trial 1: different time points + R-N -R + N -R-N Number of harvested plants Trial 2: + R-N -R + N -R-N

The results shown in FIG. 1 show that the rhizobium-treated IV wheat plants grow during the growth period.

- 42 -

have a higher content of nitrogen and dry matter than those fertilized with nitrogen; At the end of the experiment, the dry matter content of the + RN plants was nearly 40 % higher than that of the -R + N plants, and these two categories had a higher mass than the untreated RN wheat plants.

7.3.2. Results of the field trial

The wheat plants were harvested in the field trial after one growing period, and the protein content per plant was analyzed as described above. The results presented in Table II clearly show that those with R ^

tritici (+ RN) treated plants had higher protein content than both the inorganic nitrogen-dentin treated group (-R + N) and the non-nitrogenous fertilizer treated group (-RN),

Table protein content II. Voh wheat Per plant O / / O P / / O Pflanzenqruppe Proteinqehalt 28 14 χ + R-N-R + N-R-N 22 - 12 - ND

ND = No data (No data): these untreated plants died after 8 weeks.

8. Example: Rhizobium hordei, which causes root nodules in barley

Rhizobium hordei, which causes root nodule formation in barley, was produced by the method described in Section 6, by first crossing R. phaseoli with R. lequminosarum to recover the F transconjugant, which was then crossed with R. cowpea leucaena . The result of this

- 43 -

nene R. hordei was used to induce root nodule formation in four barley varieties: Hasso, Cerise, Harry and Igri.

8.1. Extraction of Rhizobium hordei

For the production of R. hordei , the following parent rhizobia were used to recover the milky white F transconjugant colonies: a) R. phaseoli, that of the French bean cultivar. Prospector , which had been grown in a soil sample taken near Aarhus, and b) R. lequminosarum, which had been isolated from the garden pea.

The parental rhizobia were grown in alternate lines on the agar medium described above. The legume extracts used in the medium were from the overground parts of the French bean or the garden pea. The colonies formed by R. phaseoli had a characteristic dark brown color, while the colonies formed by R. lequminosarum had a characteristic golden yellow.

The rhizobium * F transconjugant derived from the milky white colonies was purified and grown as described in alternate lines with a strain of R. cowpea leucaena that had been isolated from the tropical tree Leucaena leucocephala . The legume extracts used in the medium were obtained from the aerial parts of the French bean, the garden pea and the leaves of Leucaena leucocephala . The colonies formed by the F transconjugant were milky white, while the colonies formed by R. cowpea leucaena had a characteristic grayish brown color.

The rhizobium-F transconjugant originating from the snow-white colonies obtained between the strokes, here rhizol: called hordei , was purified and isolated as described.

- 44 -

has been written. The F -transconjugant was used to induce root nodule formation in barley.

8.1.1. Characterization of Rhizobiuro hordei

Plasmid DNA from the three parental strains, the F ^ transconjugant and the F transconjugant ( R. hordei ) were prepared using a modification of the method of Hirsch et al. (1980, 0 Gen. Microbiol. 120: 403-412). isolated, lysing the Rhizobia by incubation overnight at 4 C in 40% SDS (sodium dodecyl sulfate) and isolating the plasmid DNA separated by electrophoresis in 0.7% agarose gels. The results of such analysis indicated that the R. hordei had additional low molecular weight plasmids found in neither the three parent rhizobia nor the F transconjugant.

8.2. Root nodule formation in barley with the help of Rhizpbium hordei

Rhizobium hordei was used to induce root nodule formation in four varieties of barley; namely Hasso, Cerice, Igri and Harry. The barley seeds were treated by dipping them three times in the above-described aqueous solution containing 3 % calcium sulfate and up to about 10 % legume extracts used to produce R. hordei ; ie the leguminous extracts used to cover the seeds were obtained from the aerial parts of the French bean, the garden pea and the leaves of Leucaena leucocephala . After each immersion, the seeds were air-dried and seeded as described below.

8.2.1. laboratory test

The experimental procedure described above was carried out for each type of barley used; d. That is, 4 to 5 coated seeds were seeded and germinated per 1 liter container filled with "Fibo" clinker material. Each barley species was divided into the following three groups: a) + R-N, -

- 45 -

their seedlings were poured with a 50 ml suspension of R, hordei, prepared as described above, followed by weekly pouring with the non-nitrogenous fertilizer; b) -R + N, whose seedlings did not receive R. hordei , but were cast with the inorganic nitrogen fertilizer, and c) -RN ₁ whose seedlings received neither R. hordei nor the inorganic nitrogenous fertilizer, but instead were cast with the non-nitrogenous fertilizer were. The R. hordei treated plants developed root nodules within 8 to 10 weeks. The plants in each container were singulated on three plants each and ten containers were used by each group for the analysis of dry matter and nitrogen content.

8.3. Analysis of dry matter, nitrogen content and protein content of nodulated barley

The in section 6.4. The analytical methods described were used to characterize the nodulated barley plants.

8.3.1. Results of the laboratory experiment

The plants from the containers were harvested 59, 71, 85, 108 and 128 days after sowing. Dry matter per plant and nitrogen content per plant (Kjeldahl analysis) were analyzed as described above.

The results are shown in Fig. 2, in which the dry weight per plant (A) and the nitrogen content per plant (B) are plotted over the number of days after sowing.

To facilitate understanding of the figure, the following information about the average dry weight and the average nitrogen content of each barley variety are useful:

- 46 -

barley

Hasso Cerise igpi Ha ry

The number of harvested plants at different times was:

Number of harvested plants

dry matter nitrogen Grams per 100 seeds mq per seed 3,922 0.732 4,217 0,792 3,932. 0.775 5,068 0.826

group of plants 59 Days after 71 the 85th Seed 108 128 Trial 1: + R-N 23 23 27 31 37 -R + N 17 14 30 23 40 -R-N 14 18 28 20 32 Trial 2: + R-N 8th 8th 9 10 12 -R + N 6 5 10 8th 13 -R-N 5 6 9 7 11

The results shown in Figure 2 show that the Rhizobium-treated plants had a higher nitrogen and dry matter content during the growing period than the nitrogen fertilized plants. At the end of the experiment, the dry matter content of the + RN plants was almost 18 to 22 % higher than that of the -R + N plants, and both categories had a higher mass than the -RN barley plants treated with non-nitrogen containing fertilizer.

15 8.4. N enrichment in nodulated barley

A barley plant was in the presence of atmospheric

N, after which parts of the plant were analyzed for N content as an indication of nitrogen binding. More specifically, a barley plant (age 95 days) with small microtubers was placed in a chamber, so that the roots in

Presence of atmospheric

- 47 15

Incubated for 23 days

were. The atmosphere in the root chamber contained 80% N ¹⁵

(in which

¹⁵

After the incubation

N was 12.85 at%) and 20 % O ₂ time, the samples were analyzed for N content and the total nitrogen content of the plant was determined by the Kjeldahl method. The results are listed in Table ΙΪΙ below.

table III nodulierter Nitrogen content barley plants of parts Total N (mcj) part of plant Dry matter (Q) N (mq / g) 3.99 root 0,448 8.9 7.63 shoots 1,017 7.5 11.62 Entire plant 1.465 7.93

Roots shoots

Plant N content of parts of nodulated barley

Number of ^ c

rehearse

At%

4 3

0.450 + 0.011 0.392 + 0.003

κ 15

The N content in atmospheric air is about

0.370 at%.

The data in Table III demonstrate that both the roots and the shoots of the nodulated barley plants

15

containing more N than the atmosphere. These results show that nitrogen binding took place in the nodulated barley plant.

8.5. Morphology of root nodules in barley

Electron microscopy was performed on some of the large root nodules. This concluded the investigation of ultrathin sections that stained with uranylacatate and lead citrate

- 48 -

had been one. The cell arrangement of the barley root nodules was the same as that reported by Newcomb (1976, Can., 3: 54: 2163-2186) for pea root nodules; d. H. the vascular bundles were found in the peripheral cortex, while the middle part of the nodules was taken by bacteroid-filled plant cells.

Including light microscopy and electron microscopy (both scanned and exposed), further examination of the surface of small barley root nodules and the surface and sections of large barley root nodules was made. The observed morphology was compared with that of pea, white clover and soybean. Small root nodules on the barley plants were visible after a growth time of about 50 days and formed on about 75 % of the plants (the observation was also made on wheat plants). Large root nodules were found after about 89 to 110 days of growth. The large Wurzelknöllchen occurred rarely, but unmistakably on barley. They were about 2 to 4 mm in length and 1 to 2 ram in diameter and appeared on the main root at a location about 2 to 6 cm below the position of the old seed. Eleven of thirteen large nodules cut had a reddish brown color on the inside, ie the same color as leghemoglobin in leguminous root nodules of about 20 to 35 days of age. One of the remaining two large tubers was white and looked like a young unripe leguminous nodule, and the other was green, just like an old-fashioned leguminous nodule. In the case of barley, the infection may occur in two stages: in the initial stage, R. hordei penetrates the root, but does not cause any reaction in the form of nodule formation; in the next stage, the formation of root nodules begins after a delay period.

- 49 -

3.6. Antibiotic resistance of reisolated barley bacteria

Bacteria were reisolated from the large barley root nodules and tested for antibiotic resistance. The reisolated bacteria were resistant to spectinomycin dihydrochloride at a concentration of up to 400 μg / ml. The R. hordei -F -Transkon.juqant (ie, the vaccine) originally used to infect the barley plants has the same resistance. Both the vaccine and the reisolated material formed white opaque colonies when grown on common yeast mannitol agar. A parent of the R. hordei -F transconjugant is also resistant to spectinomycin dihydrochloride at the same concentration. This parent is the R. lequminosarum strain MAI, which forms bright yellow opaque colonies when grown on ordinary yeast mannitol agar. The spectinomycin resistance gene is not present on the Sym plasmid, so the R. hordei F transconjugant is likely to be a hybrid containing a non-symbiotic plasmid or the major chromosome of the parent R. leguminosarum MA. However, this does not exclude the possibility that the R. hordei F transconjugant also harbors the Sym plasmid of Mal.

Example 9: Rhizobium sorghi, which causes root nodules in sorghum

Rhizobium sorghi, which induces root nodule formation in sorghum, was produced by the method described in Section 6, by first crossing R. lupini with R. cowpea leucaena to produce the F transconjugant, which was then crossed with R. meliloti . The R. sorghi produced in this way was used to induce root nodule formation in three sorghum varieties, namely Safra, Dabar and Feterita.

9.1. Extraction of Rhizobium sorghi

For the production of R. sorghi , the following parent rhizobia were used to obtain the milky white F transconjugant

- 50 -

Colonies to be produced: a) R. lupini isolated from lupine found in central Jutland, and b) R. cowpea leucaena isolated from Papua New Guinea by the tropical border Leucaena leucocephala (belonging to the family Fabaceae).

The parental rhizobia were cultured in alternate lines on the previously described agar medium; the legume extract used in the medium was obtained from lupine and from the leaves of Leucaena leucocephala, respectively. The colonies formed by R. lupini had a characteristic pale yellow color, while the colonies formed by R. cowpea leucaena had a characteristic greyish-brown color.

The Rhizobium F transconjugant derived from the milky white colonies was purified and grown as described in alternating lines with a R. meliloti strain isolated from alfalfa found in Stahr. The legume extracts used in the medium were obtained from the lupine, the leaves of Leucaena leucocephala and the alfalfa. The colonies formed by the F -transconjugant were milky-white, while the colonies formed by R. meliloti had a yellowish brown color.

The Rhizobium F transconjugant, here called Rhizobium sorqhi , derived from the snow white colonies harvested between the strokes, was purified and isolated as described. The F transconjugant was used to induce root nodule formation in sorghum.

9.2. Root nodule formation in sorghum with the help of Rhizobium sorqhi

Rhizobium sorqhi was used to induce root nodule formation in three sorghum varieties, namely safra, dabar and feterita. The sorghum seeds were treated by dipping them three times in the above-described aqueous solution containing 3 % calcium sulfate and up to 10 %.

" 9 * J

- 51 -

Contained leguminous extracts used to produce R. sorghi ; ie the leguminous extracts used to cover the seeds were obtained from the lupine, the leaves of Leucaena leucocephala and from the alfalfa. After each immersion, the seeds were air-dried and sown as described below.

9; 2.1. laboratory test

The experimental procedure described above was carried out for the sorghum varieties Safra, Dabar and Feterita, ie 4-5 coated seeds were seeded and germinated in 1-liter containers filled with "Fibo" -linker material. Each sorghum variety was divided into the following three groups: a) + RN, the seedlings of which were poured with a 50 ml suspension of R. sorghi prepared as described above, followed by weekly pouring with the non-nitrogenous fertilizer; b) -R + N whose seedlings did not receive R. sorghi but were cast with the inorganic nitrogen fertilizer; and c) RN, whose seedlings received neither R. sorghi nor the inorganic nitrogen fertilizer, but instead were poured with the non-nitrogenous fertilizer. The plants treated with R. sorghi developed root nodules within 10 weeks. The plants in each container were singulated on three plants and 10 containers were used in each group for analysis.

9.2.2. field test

For the field trial, the coated sorghum seeds (Feterita variety) were grown as described in Section 6.3.2. described, sown. The seeds of item a) + RN were treated as described with a suspension of R. sorghi and poured with non-nitrogenous fertilizer; the seeds of items b) -R + N were not treated with R. sorghi but were poured with the inorganic nitrogen fertilizer; and the seeds of item ό) -RN were treated neither with R. sorqhi nor with inorganic nitrogen fertilizer, but simply with the

- poured 52 non-nitrogen fertilizer.

9.3. Analysis of dry matter, nitrogen content and protein content of nodular sorghum

The analysis methods described in Section 6.4 were used to characterize the nodulated sorghum plants.

9.3.1. Results of the laboratory experiment

The plants were harvested 76 to 152 days after sowing. Dry matter per plant and nitrogen content per plant (Kjeldahl analysis) were analyzed as described above.

The following data on the average dry matter mass and the average nitrogen content are useful:

sorghum

Safar Dabar Feterita

The number of harvested plants at the different times was:

Total number of plants harvested 76 to 152 days after plant group of the seed

+ R-N 121

-R + N 62

-R-N 70

9.3.2. Results of the field trial

In the field trial, the sorghum plants were harvested after one growing period, and the protein content per plant and the amino acid composition became as above

dry matter % Nitrogen Grams per 100 seeds per seed 3,724 2.32 2,944 2.17 3.840. 1.42

- 53 -

described, analyzed. The results listed in Table IV clearly show that the plants treated with R, sorqhi (+ RN) had a higher protein content than the group treated with nitrogen fertilizer (-R + N) or the untreated group (-RN).

Analysis of the amino acid composition showed that the relative amino acid composition of the rhizobium-treated plants is about the same as that of the nitrogen-fertilized plants; however, tryptophan and leucine appear to be elevated in Rhizobium-treated plants.

Table IV Protein content of sorghum group of plants Protein content of seeds per plant + R-N 34 to 52 % -R + N 16 % -R-N 4 % κ

* Since the plants of the -RN group were received after 14 days, no seed could be obtained. The nitrogen and protein content of the plants was determined as described.

10. Example: Rhizobium oryzae, which causes root nodule formation in rice

Rhizobium oryzae, which induces root nodule growth in rice, was produced by the method described in Section 6, by first crossing R. meliloti with R. cowpea leucaena to recover the F transconjugant, which was then crossed with R. trifoli . The R. oryzae thus produced was used to induce Wurzelknöllchenbxldung in rice.

-54-

10.1. Extraction of Rhizobium oryzae

For the production of R. oryzae , the following parent rhizobia were used to produce the milky white F. transconjugant colonies: a) R. meliloti isolated from alfalfa found near Victoria, and b) R. cowpea leucaena, isolated from the tropical tree Leucaena leucocephala (belonging to the family Fabaceae) from Papua New Guinea.

The parental rhizobia were grown in alternate lines on the agar medium described above; the leguminous extracts used in the medium were obtained from the alfalfa or from the leaves of Leucaena leucocephala . The colonies formed by R. meliloti had a characteristic yellowish-brown color, while the colonies formed by R. cowpea leucaena had a characteristic greyish-brown color.

The Rhizobium F -tender conjugate derived from the milky white colonies was purified and cultured as described in alternating lines with a R. trifoli strain isolated from Randers-derived red clover. The legume extracts used in the medium were obtained from the alfalfa, the leaves of Leucaena leucocephala and the red clover. The colonies formed by the F -transconjugant were milky-white, while the colonies formed by R. trifoli had a characteristic light brown color.

The Rhizobium F. transconjugant, here called Rhizobium oryzae , derived from the snow white colonies recovered between the strokes, was purified and isolated as described; the F ^ transconjugant was used to induce root nodule formation in rice.

10.2. V-rooting in rice with the help of Rhizobium oryzae

The rice seeds were treated as described by

- 55 -

3 times immersed in the above-described aqueous solution containing 3 % calcium sulfate uhd up to about 10 % legume extracts used to produce R. oryzae ; ie the leguminous extracts used to cover the seeds were obtained from alfalfa, the leaves of Leucaena leucocephala and the red clover. After each immersion, the seeds were air dried and seeded as described below.

10.2.1. field test

For the field trial, the coated rice seeds were grown as described in Section 6.3.2. described, sown. The seeds of items a) + RN would, as described, is treated with a suspension of R. oryzae and .nichtstickstoffhaltigem fertilizer poured% seeds of item b) -R + N were not treated with R. oryzae but with the inorganic Nitrogen fertilizer poured; and the seeds of item c) -RN were not treated with either R. oryzae or the inorganic nitrogen fertilizer but simply poured with the non-nitrogen fertilizer.

10.3. Analysis of protein content and nitrogen content of nodulated rice

The conditions described in Section 6.4. described methods for the characterization of the nodulated rice plants.

10.3.1. Results of the field trial

The plants were harvested every 4 1/2 months over a period of 2 1/2 years (2 cultures per ounce), and the protein content per plant was analyzed as described above.

The results shown in Table V clearly show that the R. oryzae (+ RN) treated rice plants had a higher protein content than the nitrogen fertilizer treated group (-R + N) and also the untreated group (-RN).

- 56 -

table V rice protein content from Protein content per plant Pflanzeng roup 12.0 to 18 % + R-N 1.5 to 3 % -R + N 0.5 to 1 % .-R-N

Example 11: Rhizobium as nitrogen fertilizer for Eucalyptus

A positive effect of Rhizobium as nitrogen fertilizer has been observed in Eucalyptus (Myrtaceae family), a plant species outside the grass family. Of the two species tested, E. qlobulus reacted very clearly with increased biomass. Root nodules, however, were not detected.

11.1. Recovery of Rhizobium 081324

Rhizobium 081324, which has a favorable effect on Eucalyptus, was produced by the method of Section 6, by first crossing R. cowpea leucaena with R. leguminosarum to recover the F. transconjugant, which was then crossed with R. trifoli , The F -transconjugant thus obtained, strain 081324 *, was used to treat two Eucalyptus species. This rhizobium strain does not infect any of the other plant hosts described in the examples.

11.2. Treatment of Eucalyptus with Rhizobium 081324

Eucalyptus seeds were pre-germinated in a leguminous plant extract of pea, bean, lupine, clover, alfalfa and leucaena as described for cereals. The treated seeds were sown in coarse sand and untreated seeds were sown as a control.

After germination, the pre-treated seedlings were

- 57 -

nitrogenous fertilizer and a suspension of one of six different Rhizobium F transconjugants (+ RN). After germination, one group of untreated seedlings was cast with a nitrogen fertilizer (-R + N) and another group received non-nitrogenous fertilizer (-RN). Four holes later, all of the seedlings treated with five of the six rhizobium strains had entered; but those treated with Rhizobium strain 081324 survived. The surviving plants were individually planted in six liter pots with soil and a topcoat of "Fibo ^H " clinker material to retain moisture in the pot and prevent fungal growth. The bottom of the pots were perforated so that excess water could flow out. but the amount of non-nitrogenous fertilizer applied was adjusted so that no stagnant wetness or bacterial contamination was possible There were a total of 28 pots, 14 of them + RN and 14 -R + N. The 14 + RN pots were re-vaccinated with Rhizobium 081324. After a growth time of 4 months, the + RN eucalyptus plants had two to three times as much biomass as the + RN plants.The examination of a -R + N plant did not yield root nodules, but the detection of small nodules is very difficult when the Roots grow in soil because the soil particles adhere to the roots and discolour the fine roots, despite the fact that in this er + RN eucalyptus plant no root nodules were found, the results show that the increased biomass is due to the nitrogen binding by rhizobia.

12. Example: Rhizobium Rl, the Wurzelknöll chenbildung triggers in Brassicaceae

A member of the Brassicaceae, another plant outside the grass family, also responded positively to Rhizobium treatment. The treatment of rape ( Brassica napus ) with Rhizobium Rl (010824) caused plant growth and the appearance of root nodules.

- 58 -

12.1. Recovery of Rhizobium Rl 010824

Rhizobium R1 (010824), which induces root nodule formation in oilseed rape, was produced by the method described in Section 6, by first crossing R.phaseoli with R. cowpea leucaena to produce the F -transconjugant, which was then crossed with R. trifoli has been

 / DR. NIELSEN: ARE THESE CROSSINGS CORRECT? 7

12.2. Treatment of rape with Rhizobium Rl 010824

The rapeseeds were pregerminated in a legume plant extract of bean, leucaena and clover as previously described for cereals. The treated seeds were sown in two liter containers, and after germination, one group (-R + N) was fertilized normally, a second group (-RN) was poured only with non-nitrogenous fertilizer, and a third group (+ RN ), which was also cast with non-nitrogenous fertilizer, was treated with six different rhizobia strains. After 3 1/2 months, the -R-N plants still had only the first three juvenile leaves, and the + R-N plants treated with three rhizobia strains, R4, R5 and raw materials, were also poorly developed. However, one of the rhizobia transconjugants, strain Rl or 010824, was advantageous. The plant treated with R110824 was almost the same size and small size as the largest -R + N plant, and small root nodules were visible.

13. Example: Total nitrogen analysis of nodulated non-leucuminoses.

To demonstrate that the root nodules produced by the rhizobia transformants of the invention in non-legumes were active in nitrogen binding, Kjeldahl analyzes of the content of the plants were carried out on organic nitrogen.

- 59 -

15.1. Materials and methods

All non-gelatinoses tested were divided into three groups? divided: a) one was treated with the rhizobium transconjugant and a non-nitrogenous fertilizer (+ R-N); fr) the second group was treated with nitrogen fertilizer without the rhizobiura transconjugant (-R + N); and c) the third group was not treated with either the rhizobium transconjugant or the nitrogen fertilizer (-R-N). This group was grown in the greenhouse under otherwise identical conditions and harvested several times during the growing period until the seeds were ripe. Three to five plants were grown in each container and at harvest their shoots were taken together. From this material, a sample was taken and analyzed for the total content of organic nitrogen. The results given in the subsections below show that the nodulated non-leguminous plants were able to bind nitrogen.

13.2. Wheat and barley plants

Two varieties of wheat, namely Cornette and Ralle, were nodulated (as shown in Section 7) with the help of Rhizobium tritici (brought to root nodule formation). The nitrogen content, expressed in mg of nitrogen per plant top, and the dry matter of the plant top were analyzed. The results are given in Table VII below.

- 60 -

• and Table VII 4) + R-N 4) 36, 5 Ve rsuch 1 2 39, -R-N = 2) 3 N-content 23 (η = 85 + O, 5 -R + N O, (n ί ⁶ ' O, 59 + 83 8th (n = 2) 2) 72.3 82 69 + O 6 days plant 27 88 + 1, 3 90, 89 + 26.3 0.8 O, 51 + 13, 3 56 O, 28 + 120 8th 2, 67 + 0.6 37.0 72 61 + O, 7 30 TM 1, Dry matter of nodulated wheat 21 37 + 1. 3 257, 56 + 62.3 1.9 O, 66 + 8, 1 N 120 O, 61 + 25 5 3, 99 + 1.4 205.5 117 21 + O, 6 50 TM 2, 112 53 + O, 3 331 99 + 5.8 2.4 O, 44 + 44, 2 N 244 + R-N 1, 81 + 5 5, Ol + 0.4 166.4 117 53 + O, 2 64 TM 3, (η β 68 7 400 64 + 83.2 1.39 O ₁ 69 + 54, 2 N 314 05 + O, 6 6 56 + 2.1 82 O + O, 2 76 TM 4, 14 + 38 2 666, 71 + 60.3 O, 6 + 3, 1 N 431, 49 + 1, 2 7, 52 + 1.4 113 96 + O, 3 97 TM 5, 26 + 23 4 668, 55 + 113.4 O, 42 + 12, N 523 78 + O, 9 94 + 3.5 94 + O 115 TM 5, 04 + 700 77 + 79.4 67 N 565, 38 + 3 11 43 + 1.9 90, ? -N 4 124 TM 8th, 28 + 5 Ve rsuch O, * 2) + 12, 1 N 47 + 1 -R + N 39, η = 30 + O, O 19 + 2 304 (Π ⁼ 91 + O, 51 + 23, 2 158 49 + 7 7, 46 + 117 54 + O, 6 54 TM 3, 98 + 1 705 64 + O, 54 + 2, 1 N 447, 11 + 6 12 48 + 131 04 + O, 3 71 TM 6 15 + 871, 57 + O, 71 + 11, 1 N 513 12 15 + 18 + O, 80 TM 7, 1128 !, 85 + 72 N 903 45 U ^ 56 + 92 TM 11 N

κ TM. = dry matter

The plants in experiment 1 were grown under suboptimal lighting conditions.

- 61 -

The results shown in Table VII show that the dry weight of the + RN plants is about 82 % of that of the -R + N plants and that the nitrogen content of the + RN plants is about 80 % of the -R + N plants.

Five varieties of barley, namely Oenny, Taarn, Lina, Grith and Triumph were brought to root nodule formation using Rhizobium hordei as described in Section 8 above. The nitrogen content, expressed as mg of nitrogen per plant top, and the dry matter of the plant top were analyzed. The results are listed in Table VIII below.

The rhizobium nodulated barley plants reach the same size and dry mass as the plants treated with normal nitrogen fertilizer. The nitrogen content of the + RN barley is about 83 % that of the -R + N plants and is 20 times as high as the nitrogen content of the -RN plants. The nitrogen found in the -RN plants stems from the seed and does not increase during the growth period.

The effect of rhizobium transconjugants as a replacement for nitrogen fertilizers is mainly due to the action of small root nodules present on 75 % of the plants. Since 3 to 5 plants per container were drawn, it was not possible to separate the roots and to analyze only the plants with Wurzelknöllchen. It is expected that root nodule formation on 100 % of the plants and the development of large root nodules would be even more effective.

To exclude the possibility that the nodulated + RN non-leguminous plants use the Rhizobium inoculum or the bacteria which may later spread in the containers as nitrogen source, the following experiment was performed: The wheat variety Cornette and the barley variety Taarn were each labeled with R lequminosarum strain Ml using the same procedures, quantities and conditions

- 62 -

Table VIII N content and dry matter of nodulated barley

plant + R-N 10) Ve Search 1 5) [ 3) 19 -R-N 5) 5.2 days (n = + 9 -R + N + 21.7 + 38.4 0 (n = 0.1 TM 43.85 + 0 (n = + 0.9 + 0.7 35 , 15 + 4.2 31 N 0.99 + 28 2 71.84 + 44.5 +120.8 0 , 27 + 0.1 TM 121.53 + 0 2 2.15 + 1.3 + 0.8 41 , 40 + 7.1 50 N 2.56 + 50 , 7 224.95 + 36.8 + 94.3 0 , 41 + 0.1 TM 198.41 + 0 , 5 4.03 + 1.1 + 2.4 49 , 87 + 4.8 64 N 2.61 + 56 , 7 269.92 + 76.9 +196.4 0 , 35 + 0.3 TM 301.04 + 0 , 5 4.05 + 2.9 + 1.6 55 , 58 + 15.7 76 N 4.09 +106 ,1 296.76 + 106.5 0 , 61 + 0.1 TM 499.23 + 1 .6 5.22 + 1,2 64 , 19 + 11.5 96 N 5.34 +123 , 9 506.33 + 124.3 0 , 35 + 0.1 DW 535.47 + 1 , 4 9.90 + 3.7 , 10 + 123 5.55 , 7 556.38 , 44 + ,1 10.34 3) + R-N 6) Ve Search 2 49 -R-N 11.8 (n = + 20 -R + N 0 (n = 0.1 TM 118.89 + 0 (n = 53 , 76 + 9.9 54 N 3.30 + 46 .6 264.84 0 , 45 + 0.1 TM 313.06 + 0 , 4 7.78 68 , 49 + 2.6 71 N 5.81 +279 ,1 405.53 0 , 41 + 0.1 TM 711.29 + 1 , 6 7.78 57 , 42 + 19.5 80 N 7.85 + 147 , 6 617.75 0 , 53 + 0.2 TM 802.30 + 1 2 10.26 , 07 + 92 N 10.08 3 837.08 , 46 + , 6 11,99

TM = dry matter

- 63 -

inoculated for inoculating these non-leguminous plants with the Rhizobium transconjugant, R. tritici and R. hordei, respectively. The plants inoculated with R. lequminosarum were poured with non-nitrogenous fertilizer and the nitrogen storage was compared with that of the -RN plants. Neither the plants inoculated with R. lequminosarum nor the -RN plants accumulated nitrogen. The conclusion is that a nonspecific bacterial vaccine as such does not deliver nitrogenous fertilizer to the plant. On the contrary, in Tables VII and

VIII nitrogen storage shown that in response to nodulation (root nodules formation) is carried out with R. tritici b ^zw · R. hordei must be due to nitrogen fixation.

13.3. Sorghum and rice plants

Three varieties of sorghum, namely Dabar, Safra and Feterita, were prepared for root nodule formation as described in Section 9, using Rhizobium sorqhi . The nitrogen content, expressed in mg of nitrogen per plant top, and the dry matter of the plant top were analyzed. The results are in the table below

IX listed.

- 64 -

Table IX N content and dry matter of nodular sorghum

+ R-N-R + N-R-N

days mg / χ plant 30 TM N 59 TM N 80 TM N 101 TM N 125 TM N

(n = 6) (n "- 3) (n = 3)

33.83 + 10.1 49.60 + 22.5 25,50 + 4,2

0.40 + 0.1 1.42 + 0.7 0.26 + O

102.93 + 15.2 329.51 + 87.7 42.84 + 8.0

1.00 + 0.2 5.93 + 2.3 0.31 + O

179.84 + 62.2 602.34 + 44.9 43.77 +21.5

2.00 + 0.8 9.86 + 2.7 0.31 + 0

439.83 + -95.8 576.18 + 75.4 53.71 +12.4

4.40 + 0.9 11.02 + 0.9 0.31 + O

1242.94 + 391.4 1198.99 +596.0 56.76 + 7.8

11.65 + 2.4 12.44 + 6.8 0.27 + O

TM = dry matter

A rice variety M-20! Was made into root nodule formation using Rhizobium oryzae as described in Section 10. The nitrogen content, expressed as mg of nitrogen per plant top, and the dry matter of the plant top were analyzed. The results are shown in Table X below.

- 65 -

days TM table + R-N X naduliertem rice -R-N 62 N : and dry matter of 32.28 -R + N 23.44 N-content TM IS 0.32 48.87 0.16 74 N 48.04 1.10 21.63 TM 1.15 94.73 0.16 98 N 107.47 2.65 29.9 TM 2.91 115.40 0.36 128 N 174.89 3.45 22.16 5.16 130.83 0.38 * TM = 4.33 dry matter

The results show that the dry matter of the + R-N plants initially lagged behind that of the -R + N plants; however, at the end of the experimental period, the dry matter accumulated by the + R-N plants will be similar to and may exceed that of the -R + N plants. The nitrogen storage in the + R-N plants and the -R + N plants seems to be about the same.

13.4. rape

Two rapeseed varieties, Hanna and Topas, were made root nodule-forming as described in Section 12 using Rhizobium R110824. The nitrogen content, expressed as mg of nitrogen per plant top and the dry matter of the plant top were analyzed. The results are listed in Table XI below.

iö4 y

- 66 -

Table XI N content and dry matter of nodulated oilseed rape

-R + N -R-N

47 TM · 67.25 + 17.4344.91 + 31.6 9.71 + 0.2

6.71 + 0.1 0.22 + 0.2

59 TM 99.40 + 14.3477.07 + 62.8 13.90 + 4.5

7.03 + 0.2 0.11 +0.1

73 'TM 248.56 + 24.3 766.47 + 72.7 14.92 + 9.9

10.25 + 1.9 0.10 + 0.04

Prlanze + R-N + 17.4 TM 67.25 + 0.3 N 0.89 + 14.3 TM 99.40 + 0.4 N 1.64 + 24.3 TM 248.56 + 0.7 N 2.00 * 72.5 TM 424.80 + 1.1 N 3.42 + 22.2 TM 547.37 + 1,9 N 5.73 +241.8 TM 1,013.59 + 0.9 N 10.24 TM 2,231.55 N 16,66

98 TM 424.80 * 72.5 704.73 + 12.11 + 7.9

17.66 + 5.1 0.10 + 0.05

110 TM 547.37 + 22.2 1049.77 +388.6 17.86 +12.1

24.33 + 7.9 0.26 + 0.2

129 TM 1013.59 +241.8 1955.60 + 330.0 15.39 + 6.0

33.21 + 8.6 0.16 + 0.09

147 TM 2231.55, ND ND

ND ND

TM = dry matter

η = 4 plants per sample ND = No data (No data)

The results show that the + RN plants stored about 50 % of the dry matter accumulated by the -R + N plants, and the nitrogen content of the + RN plants was about 30 % of that of the -R + N plants. Although the storage was lower than that of the nitrogen fertilized plants, the small root nodules observed in the rape plants explain the survival of the nodulated rape plants grown in the absence of nitrogen fertilizer (compare the results for + RN with those for -RN where neither dry matter nor nitrogen accumulates -

- 67 -

were melted).

14, microorganism bank

The following rhizobium strains were obtained from the American Type Culture Collection (ATCC), Rockville, MD _; deposited and received the following registration numbers:

Rhizobium Registration no.

R. tritici 53407 - R. hordei 53404 R. sorqhi 53405 R. oryzae 53406 R. Rl (010824)

The scope of the invention is not limited by the deposited microorganisms, as the deposited sample is to be understood as an illustration of an aspect of the invention and all microorganisms which have an equivalent function are within the scope of the invention. Indeed, various modifications of the invention in addition to those illustrated and described will become apparent to those skilled in the art from the description and the accompanying drawings. It is intended that such modifications fall within the scope of the appended claims.

Claims (8)

Berlin, July 18, 1988 68 124/13 Claims . , 1. Method for binding nitrogen. in.llichtleguminosen, characterized in that the seeds of a Hichtleguminose (a) be treated with a material which enables the infection of said seeds when germinated with a Rhiaobia transformant; b) are sown, c) be brought into contact with a rhizobia transformant capable of infecting said treated germinating seeds, and d) grown without or almost without the addition of nitrogen fertilizer. A method according to claim 1, characterized in that steps a) and c) are combined by combining said 'material and' said rhizobia transformant and treatment of said seeds before sowing with said combination. A method according to claim 1 or 2, characterized in that said seeds are of intact legumes to the pea ^; the Poaceae, like wheat, preferably include the Anja, Kraka, or Vuka tribes; Barley, preferably the strains Hasso, Cerise, Harry or Igri; Sorghum, preferably 'the Tribe Safra, Dabar or Peterita; or rice. 4. The method according to claim 1 or 2, characterized in that '. the mentioned seeds of Hichtleguininosen belong to the family of Cruciferae, such as cabbage or oilseed rape, or to the family of IJyrtaceae, such as Eucalyptus. 5. A process for the production of Rhizobia Fp transconjugates for use in a. Method according to one of claims 4 to 4, characterized by a) Spreading a first Rhizobium parent and a second Rhizobium parent alternately on a solid broth containing, in addition to the nutrients required for growth: (i) an undenatured extract of a legume / male partner of each parent Rhizobium or (ii) chromatographic fractions from an undenatured extract of a legume host partner of each parent rhizobium or (iii) crystals from the chromatographic fractions of a non-denatured legume extract. , \ 7irtspartners of each parent rhiaobium, or (iv) proteins related to the extract, chromatographic primes or ie-a crystals; b) cultivating the Rliizobia parents at a temperature of about 13 ⁰ G to about 30 ° G, so that the Rhizobia Eltem form colonies of a particular color; c) Selecting a milky-white Rhizobium F ^ -Trahsconjugant colony, .between the Rhizobia parent colonies grows; ^ " d) spreading the rhizobium F. transkon in alternating rows with a third Rhizobium parent on the IJährmedium of step a), which also contains a Leguminos extract, chromatographic fractions, crystals or proteins of the third Rhizobia parent, and ' e) culturing the Rhizobium transconjugants-I ^ and the third Rhizobium parent at 'a temperature of about 18 ⁰ C to about 30 ⁰ C so that the Rhizobia parents form colonies; f) Selecting a rhizobium P ₂ transconjugant of a snow-white colony that grows between the RhXZObIa-F ₁ transconjugant and the third rhizobia parent. 6. The method according to claim 5, characterized in that the third Rhizobia parent a third Rhizobia species., A second Rhizobium P. transconjugant or a second Rhizobium-j? ₂ transconjugant. Material for the treatment of seeds of soft legume for use in a method according to one of claims 1, 3 or 4, characterized in that it consists of (1) a nondenatured legume row, (ii) chromatographic prions of such extract, (iii ) a material made of crystals from such an extract or (iv) proteins related to such extract from each legume vyoster partner for each rhizobium parent of a rhizobium transformant which is the symbiotiseh. Nitrogen in the soft legume binds. 8. Material according to claim 7, for use in one of the arrival. .a 1 to 4, characterized in that it also contains the rhizobia transformants, which, symbiotically binds nitrogen in Hichtleguniinose.