WO2003089640A2

WO2003089640A2 - Biofertilizer for plants based on rhizobium bacteria having an improved nitrogen fixing capacity

Info

Publication number: WO2003089640A2
Application number: PCT/MX2003/000033
Authority: WO
Inventors: Humberto Peralta Diaz; Jaime Mora Celis
Original assignee: Universidad Nacional Autonoma De Mexico
Priority date: 2002-04-19
Filing date: 2003-04-11
Publication date: 2003-10-30
Also published as: WO2003089640A3; AU2003218828A1; MXPA02003920A; AU2003218828A8

Abstract

The invention relates to a biofertilizer which is applied to plants in order to increase the nitrogen uptake capacity thereof. Generally, soils are fertilized with nitrogen, phosphorous and potassium with nitrogen being the most scarce. The aforementioned biofertilizer can be based on Rhizobium bacteria having an improved nitrogen fixing capacity, together with the plants, specifically pulses, i.e. those with seeds that grow in pods. According to the invention, said biofertilizer improves the fixing capacity of the plants by overexpressing the nitrogenase genes in the bacteria. Nitrogenase is the enzyme responsible for the nitrogen fixing catalysis. The use of the inventive biofertilizer is intended to replace completely chemical nitrogenous fertilizers which are applied to crops on a large scale. Inoculation with genus Rhizobium bacteria is a cheap and environmentally harmless process which can be applied to any type of pulse.

Description

Biofertilizer for plants based on Rhizobium bacteria with improved nitrogen fixation capacity.

Object of the invention

The object of the present invention is the nitrogen biofertilization of plant crops, especially legumes, by improving the ability of biological nitrogen fixation by microorganisms. The improvement of this capacity is due to the overexpression of the bacterial nitrogenase enzyme complex, which is responsible for the catalysis of nitrogen fixation.

Background of the invention.

Agricultural crops should usually be fertilized with nitrogen, phosphorus and potassium, but in the soil the main shortage is nitrogen. Nitrogen fertilization implies high economic costs for the farmer, but the most serious effects of mass fertilization in the world are environmental: the deterioration of soils, environmental pollution from the manufacture of fertilizers and the contamination with mtrogenated products of surface aquatic mantles and underground. The abundance of nitrogen compounds in water produces the phenomenon called eutrophication, which is the abnormal growth of bacteria that use these rich sources. This depletes the dissolved oxygen in the water and causes the mass death of organisms such as fish. Also, the consumption of water with a high content of mtrogenated compounds such as nitrates and nitrites causes severe damage to human health. Therefore, one of the most important challenges for agriculture is to eliminate the use of nitrogen fertilizers and reduce the economic cost but without affecting crop productivity. Currently, several crops of nutritional importance for man have benefited from the use of biofertilizers, that is, the use of organisms and / or their living and / or dead products to improve the qualities of soils that allow increasing crops or The quality of products. Some of the practices include: adding dried seaweed to the soil, leaving the stubble from the previous crop for degradation, adding guano to the soil, inoculating bacteria or fungi. Among the most used biofertilizers are inoculants based on nitrogen fixing bacteria such as Rhizobium, Azospirillum and Azotobacter. As for Rhizobium and its related genera {Bradyrhizobium, Mesorhizobium, Azorhizobium, Allorhizobium), its beneficial effect as a biofertilizer occurs in leguminous plants (peas, beans, lentils, beans, alfalfa, clover, among others) and non-legumes (such as betabels) wheat, sorghum and corn). In this regard, the cultivation of legumes has very important advantages since that its seed contains a high proportion of proteins and amino acids that can constitute the greatest protein contribution as it happens in the population of Mexico and the rest of Latin America, Africa, Asia and part of Europe. Nitrogen fixation is the assimilation of nitrogen present very abundantly in the air (N ₂ , 79% by volume) transforming it into ammonium, which can now be taken by any other organism such as plants or even other bacteria. Among the fixing bacteria there are those that fix nitrogen for growth such as Klebsiella or Azotobacter (which are normally found in soil and especially near the roots of plants) and those that fix nitrogen symbiotically such as Rhizobium, Sinorhizobium and Bradyrhizobium, that is, in a closer relationship with plants such as legumes in which they inhabit a root structure called the nodule and the ammonium produced is transported to the stem and leaves and subsequently to the seed. It is important to mention that the ammonium that is supplied by the microorganism can constitute all or most of the nitrogen that the crop requires. The use of nitrogen fixing bacteria in crops is called biofertilization or inoculation and is used with some success worldwide, but the main problem is the low efficiency of the inoculants, so its use in the field has not been extensive. The production of a biofertilizer in industry represents a much lower cost than producing a fertilizer. For example, for one hectare of beans, about 500 pesos are required to fertilize and to inoculate that same surface with bacteria, without reducing the yield, approximately 10 pesos. Then, an economically and ecologically more suitable alternative to achieve the objective mentioned in the field is the improvement of the fixation of atmospheric nitrogen that occurs in the cultivation of plants, especially in legumes in association with soil bacteria such as Rhizobium. In Mexico, beans are the most important legumes in the country by quantity of consumption and nutritional quality of the seed.

Producing a biofertilizer for plants with improved nitrogen fixation efficiency has been the objective of several research groups for several years as it represents very important economic, agricultural and environmental benefits. The improvement of the nitrogen fixation process could be obtained through the use of organisms such as genetically modified Rhizobium bacteria. However, despite the varied strategies that have been carried out, researchers have reported very limited successes in their attempt to improve the symbiotic fixation of estrogen. Among these attempts are the following.

In R. leguminosarum, pea and bean symbiote, and other rhizobium species Dr. Bascones' group (in Generation of new hydrogen-recycling Rhizobiaceae strains by introduction of a novel hup minitransposon, Applied and Environmental Microbiology 664292-9, 2000) expressed the region of hydrogen intake, one of the operons that make it up is that of hydrogenase, with this function it is intended to recover part of the energy that escapes in the fixation catalysis in the form of hydrogen. In Sinorhizobium meliloti, alfalfa symbiote, Spaink (in Symbiotic properties of rhizobia cantaining a flavonoid-independent hybrid nodD product, Journal of Bacteriology 169 1423-32, 1989) constructed and expressed a nodD hybrid gene that does not respond to flavonoids with the intention to increase the nodulation capacity and therefore the fixation. Also in S. meliloti, Bosworth (in Alfalfa yield response to inoculation with recombinant strains of Rhizobium meliloti with extra copy of dctABD and / or modified nifA expression, Applied and Environmental Microbiology 60 3815-32,

1994) increased the number of copies of the genes responsible for the transport of C4 dicarboxylic acids, called dct and the expression of the nifA binding regulatory gene with the intention of increasing the inflow and metabolism of organic acids such as succinic and thus increase fixing efficiency In R. etli, bean symbiote, Castillo (Master's Thesis in Basic Biomedical Research,

CIFN-UNAM, 1999) obtained and tested recombinant symbiotic plasmids to combine the best nodulation and fixation properties that each of them granted separately. Also in R. etli Miranda (in Rhizobium etli chromosome mutants with derepressed expression of cytochrome oxidases and enchanced symbiotic nitrogen accumulation, Applied Microbiology and Biotechnology 45 182-8, 1996) modified the expression of the genes that code for symbiotic cytochrome oxidase {cbb ) to increase the respiratory capacity of the bacteria inside the nodule. None of the previous experiments achieved a symbiotic benefit in the greenhouse (determined either by nitrogenase activity, nitrogen content in seed and plants or in yield) greater than 22% with respect to the use of parent strains. Recently, Olah (in Mutation in the ntrR gene, a member of the vap gene family, increases the symbiotic efficiency of Sinorhizobium meliloti, Molecular Plant-Microbe Interactions 14 887-94, 2001) performed the mutation of the ntrR gene in S. meliloti that caused a 24% increase in plant nitrogen in greenhouse tests. It should be noted that only one of the previous experiments

(Bosworth, 1994), was carried out in the field under controlled conditions, and the rest of the mentioned strains do not meet the biosafety requirements for the release and field testing of genetically modified microorganisms, due to containing exogenous material as markers of resistance to antibiotics, genes from other organisms or vector sequences that are not allowed for release or as biofertilizers.

The bacteria of the Rhizobium genus are associated with the family of leguminous plants, which are all those whose seeds are given in pods and is the most widespread in the planet with about 20 thousand species, among them are some such as beans, peas, beans, lentils, alfalfa, clover and others that are used as human food or as fodder for cattle. But recently, works have been published in which Rhizobium is also associated with grassy plants such as wheat, corn and barley, so that these plants receive benefits such as increased growth and production, although it is not clearly known by what mechanism. Fischer's work (in Genetic regulation of nitrogen fixation in Rhizobia, Microbiological Reviews 58 352-386, 1994) summarizes the knowledge available about the genetic regulation of symbiotic nitrogen fixation in Rhizobium. The catalytic activity of nitrogen fixation is carried out by the nitrogenase enzyme complex composed of nitrogenase reductase, nitrogenase component alpha and nitrogenase component beta, which is encoded by the nifH, nifD and nifK genes (SEQ ID NO: 2, 3 and 4), respectively, with the additional participation of more than 20 proteins. R. etli, bean symbiote, presents two reiterations of the complete nifHDK operons (copies a and b) and a third repetition called nitric or copy c, coupled to a truncated nifD gene as explained in the work of

Valderrama (in Regulatory proteins and cis-acting elements involved in the transcriptional control of Rhizobium etli reiterated nifH genes, Journal of Bacteriology 178 3119-3126, 1996). In this same work, the analysis of genetic expression showed that the operon c in the free-life and nodule assay is transcribed 4 to 10 times more than the operons a and b. The transcription of the nitrogenase enzyme complex operon, made up of the nifHDK genes, depends on a promoter recognized by the sigma 54 factor and the NifA nitrogen fixation activator. The mechanism of transcription initiation is as follows: the RNA polymerase complex is located on the promoter forming a closed transcriptional complex, the NifA protein is coupled to DNA sequences located above the promoter, an additional protein causes an acute fold of the DNA strands between the NifA coupling site and the promoter, NifA comes into contact with the RNA polymerase and hydrolyzes ATP and the energy released by the ATP hydrolysis achieves the opening of the closed complex for the start of transcription. Although the same elements participate in the regulation of the operons described a, b and c, the difference in their transcriptional activation lies in the distance that exists from the promoter to the NifA coupling sequence (called UAS).

In the operon c the distance is sufficient for the double helix of the DNA to cover from 7.8 to 8.3 complete helical turns, depending on the degree of winding, which produces a suitable activating complex between the binding regulatory protein

NifA and the transcriptional complex placed in the sigma-54 RNA-factor polymerase promoter (SEQ ID NO: 1), while in operons a and b the distance is 8.3 to 8.7 helical turns that produce very disadvantaged complexes to be transcribed Naturally, in this organism the symbiotic efficiency in association with the bean is given by the nitrogenase complex produced from the transcription of operons a and b, while the protein products of operon c have no significant contribution in catalysis, as explained in Romero's work (in Effect of naturally occurring nif reiterations on symbiotic effectiveness in Rhizobium phaseoli.

Appl. Environ. Microbiol 54: 848-850, 1988) who demonstrated that a mutation in any of the complete operons a or b decreases the fixative capacity of the strain by 50%, just as the mutation in the nifHc gene has no effect.

Description of the invention

To produce a biofertilizer for plants with improved nitrogen fixation capacity, especially the association of Rhizobium bacteria with leguminous plants that is potentially used in the field safely and without any environmental risk, the following was done. A nitrogen nitrogen operon (SEQ ID NO: 2, 3 and 4) that is transcriptionally controlled by the promoter region of nifHc (copy c, SEQ ID NO: 1) was constructed in the laboratory using conventional techniques of molecular genetics. it was replaced by double recombination by an operon resident in the Rhizobium genome and its expression caused greater activity of the nitrogenase enzymatic ecomplex that was reflected at the end of the crop in greater production and nitrogen content in the seed. Specifically, it was expressed in Rhizobium etli bacteria associated with bean plants and the results of the greenhouse and field experiments are shown below. It is very important to highlight that the strains obtained with increased nitrogen fixing capacity, unlike those described in Background, do not contain exogenous genetic material such as antibiotic resistance genes or foreign plasmid vector genes, for this reason they were authorized for controlled environmental release for field experiments by the National Biosafety Commission since they do not present any environmental risk and can be used massively in the field.

Procedure for carrying out the invention.

The technical procedure is explained below. The genetic construction mentioned in this application pr. c nifHDK (SEQ ID NO: 1, 2, 3 and 4) does not exist as such in nature although the material with which it was made is present in the genome of the bacterium Rhizobium etli. and was obtained using conventional genetic engineering techniques in the laboratory. One of the R. etli nitrogenase operons is cloned into plasmid pCQ12 which was digested with the enzymes EcoRI and BglII and the Corresponding DNA segment was separated and purified. The nifHc promoter region was obtained from plasmid pCQ23 with Bgiπ digestion and the corresponding segment was separated and purified. Both fragments were ligated and plasmid pHP40 was obtained. Subsequently, the DNA segment containing the nifHc promoter region and the nifHDK nitrogenase operon were separated from the previous plasmid by EcoRI digestion, cloned into the vector pWS233 and the plasmid pHP789 was obtained (Figure 1). With the construction in this vector it was possible to obtain recombinant double strains by bacterial conjugation that have incorporated this construction into their genome since the vector plasmid used in the construction of the chimeric operon, called pr. c nifHDK, presents the sacRB genes that favor the obtaining of double recombinants by means of a 10% sucrose growth that smooths the cells that contain the suicide plasmid still incorporated as a simple recombinant as mentioned by Gay and others (in Positive selection procedure for entrapment of insertion sequence elements in gram-negative bacteria, Journal of Bacteriology 164 918-921, 1988). Thus, only bacteria that have suffered double recombination grow after sucrose treatment.

After obtaining the double recombinants at an approximate frequency of lxl O ^{"7, they} were isolated and grown in PY medium with nalidixic acid. To confirm the exchange, southern hybridization and PCR were used. The construction effect was determined in greenhouse tests. and field.

The expression of the construction in the Rhizobium strains analyzed is characterized by increasing the expression levels of the nitrogenase enzyme complex in bacteria of the Rhizobium genus by presenting a promoter region with high transcription coupled to the complete nifHDK operon (SEQ ID NO: 2, 3 and 4). It is important to mention that there is currently no report or strain, except those addressed in this application, that are capable of increasing Rhizobium's fixing capacity. Therefore, the main characteristic of Rhizobium strains that contain this genetic construction in association with a legume such as beans is that they reach increased levels of seed production and nutritive content of the same since by fixing more nitrogen, it is incorporated to proteins and amino acids in the seed in greater quantity.

The construction effect was determined in greenhouse and field tests. In summary, the expression of the genetic construction produced on average, with respect to inoculation with the wild strain in greenhouse, an increased nitrogenase activity of 40% (determined as ethylene micromoles / h mi g dry nodule weight), a higher content of nitrogen in the plant of 20%, a higher yield of up to 49% (in mg of seed / plant) and higher content of nitrogen in the bean seed of 35% to 65% (in mg N / plant), which had an impact in a protein content 50% higher in the seed. The expression of genetic construction pr. c nijHDK & other strains of Rhizobium bean symbiotes such as CIAT652 and AM652 produced an average of 20% more nitrogen in the plant, 40% more yield and 50% higher nitrogen content in seed compared to inoculation of wild strains. The increase in nitrogen in seed has a direct impact on its nutritional quality and therefore, the seed produced using this bacterium as a crop biofertilizer is up to 50% more nutritious. In the field, several trials were carried out in experimental plots as shown in the following tables, with modalities of rolled irrigation, drip irrigation, rhizorriego and rain. It was inoculated as is normally done with other types of crops such as alfalfa and soy, for which it was used as a vermiculite support (inert synthetic material).

In summary, the strain derived from R. etli presenting the genetic construction pr. c nifHDK called strain HP310 showed significantly different increases at p <0.05 in nitrogen yield up to 25% with respect to the strain from which it originates (CFN42) and from 10 to 70% compared to non-inoculated plots. The strain HP789 derived from the wild strain CIAT652 and presents the construction pr. c nifHDK showed increases of up to 145% with respect to the wild strain CIAT652 and up to 185% with respect to non-inoculated control plots. Strain HP652, which is derived from strain AM652 and has the construction pr. c nifHDK showed increases of up to 20% compared to CIAT652 strain and 34 to 78% compared to non-inoculated plots. In several of the experiments no significant increases in production were achieved by biofertilization, nor were they observed with nitrogen chemical fertilization. This is because it has been observed that in very productive bean genotypes it is difficult to increase yield. However, a fundamental parameter is that of the nitrogen content in seed, since it represents the nutritive quality of the seed.

With biofertilization with the strains that present the genetic construction pr. c nifHDK very important increases were achieved. With this, it is proven that there is practical application of genetic construction to make more efficient the use of these strains as crop biofertilizers. The Rhizobium etli strain was deposited with the pr. c nifHDK with registration NRRL B-30606 on July 12, 2002 in the Culture Collection,

Agriculture Research Service, United States Department of Agriculture (Peoría, DI.).

Practical examples.

This invention relates directly to the use of bacteria as a biofertilizer for plant cultivation. It can be used in the cultivation of legumes, due to its ability to interact commonly with this type of plants, but there are reports that its use is very favorable for other types of crops such as cereals (wheat) and tubers (beet). In the case of legumes, the bacteria have an improved capacity nitrogen fixation which can perfectly supply nitrogen fertilizers without diminishing yield. For the use of the invention, present in strains of the Rhizobium bacteria, in the application as a biofertilizer, the bean culture was specifically tested by the following procedure. Greenhouse tests were performed for which Rhizobium bacteria with the genetic construction of nitrogenase overexpression was grown in liquid and the cells collected and washed with sterile water. They were used to inoculate bean seedlings at an average density of lxlO ⁷ bacteria per plant and the nitrogenase activity was analyzed in an acetylene reduction test and detection by gas chromatography on the dates 18, 25 and 32 days after inoculation ( dpi) At the end of the test, the seed was collected, the yield was quantified; The nitrogen contained in the seed was analyzed by pyrolysis spectrophotometry (Figures 2 to 10).

For field experiments, the bacterium was also grown and prepared for biofertilization, mixing in this case with sterile vermiculite as a solid support. The bean seed was inoculated at an approximate density of 10 x ⁶ cells per seed and was sown manually in plots of 6 meters with five rows each, in four to five random replicas in experimental fields of the central region of the country. At the end of the test the seed was collected, the yield was quantified and the nitrogen present in them was analyzed as indicated above. The following tables show the results obtained in the field.

Table 1. Performance data, nitrogen content and nitrogen yield in seeds of field plants. Zacatepec, December 1999. Rolled irrigation. May flower variety M38.

N in seed

Production Incr Performance N Incr.

Strain (mgN / g weight (Ton / Ha) (%) • (Kg N / Ha)% dry)

CFN42 ^• 0.544 29 33.9 (2.9) 184 * 72

HP310 0.456 8 38.7 (7.2) 176 * 64

CIAT652 0.435 3 34.6 (4.7) 150 * 40

HP789 0.649 54 47.1 (8.6) 306 * 185

AM652 0.469 11 34.8 (8.7) 163 * 52

HP652 0.602 43 41.7 (4.2) 251 * 134

S / I 0.420. 25.6 (5.5) 107. + N 0.465 10 25.5 (8.3) 118 10

Production of four plots in random arrangement. %, regarding plots without inoculation. In parentheses, standard deviation. * significantly different with p <0.05 compared to treatment without inoculation (S / I). CFN42 wild strain, HP310 derived from CFN42 with nitrogenase overexpression, CIAT652 wild, HP789 derived from CIAT652 with nitrogenase overexpression, AM652 phb mutant, HP652 derived from AM652 with nitrogenase overexpression.

In this experiment, the inoculation with the wild and modified strains has a variable impact on the yield, obtaining the best productions with the modified strains with overexpression of nitrogenase HP789 and HP652 of 54 and 43% respectively on the uninoculated treatment. Additionally and as very important characteristics, there is the increase in nitrogen content and nitrogen yield in seed. In the treatment without inoculating the nitrogen content in seed is 26 mgN / g dry weight and the yield of N is 107 Kg N / Ha total and very similar to the fertilized treatment (25.6 and 118 respectively). But the seed obtained with inoculation of the modified strains HP789 and HP652 is 47.1 and 41.7 mgN / g p.s. and in nitrogen yield it is 306 and 251 Kg N / Ha respectively, increasing this parameter by 185 and 134% respectively.

Table 2. Performance, nitrogen content and nitrogen yield data in field plant seeds. Celaya, March 2000. Drip irrigation and rhizorriego. May flower variety M38.

Production Incr N in seed Yield N Incr.

Strain (Ton / Ha) (%) (mgN / g dry weight (Kg N / Ha)%

CFN42 4.24 0 46.7 (8.0) 198 * 30

HP310 4.29 0 38.6 (2.4) 166 * 10

CIAT652 4.22 0 56.5 (3.8) 238 * 57

HP789 4.08 0 50.9 (1.4) 208 * 37

HP652 4.34 0 53.6 (3.5) 233 * 53

S / I 4.38 - 34.6 (2.0) 153

+ N 4.48 2 71.3 (6.0) 319 * 110 Production of five plots in random arrangement. %, regarding plots without inoculation. In parentheses, standard deviation. * significantly different with p <0.05 compared to treatment without inoculation (Sil). CFN42 wild strain, HP310 derived from CFN42 with nitrogenase overexpression, CIAT652 wild, HP789 derived from CIAT652 with nitrogenase overexpression, HP652 derived from the AM652 phb mutant with nitrogenase overexpression.

In the experiment in Table 2, inoculation with wild and modified strains no increase in production was obtained as well as the fertilized treatment was only 10% greater than the non-inoculated. As in the previous one, the two parameters that indicate the nutritional quality of the seed (nitrogen content and nitrogen yield in seed) were evaluated. In the treatment without inoculating the nitrogen content in seed is 34 mgN / g dry weight and the yield of N is 153 Kg N / Ha total, while in the fertilized it was 71.3 and 319, respectively. On this occasion, the wild strain CIAT652 produced notable increases (56.5 and 238, respectively). The HP652 strain obtained a very similar performance (53.6 and 233, respectively). It can also be noted the large increase in production in general for the entire experiment due mainly to the drip irrigation regime. Rizorriego refers to the incorporation of the inoculant in the irrigation system.

Table 3. Performance data, nitrogen content and estrogen yield in field plant seeds. Celaya, June 2000. Rolled irrigation. May flower variety M38.

Incr Production N in seed Yield I> Incr

Strain

(Ton / Ha) (%) (mgN / g dry weight (Kg N / Ha)%

CFN42 2.09 0 45.7 (2) 95 0

HP310 2.15 0 55.6 (2) 119 23 *

CIAT652 2.10 0 62.5 (2) 131 35 *

HP789 2.03 0 61.7 (2) 125 29 *

HP652 2.23 0 66.3 (2) 148 52 *

S / I 2.23 - 43.4 (2) 97 -

+ N 2.57 15 72.6 (1) 187 93 *

Production of five plots in random arrangement. %, regarding plots without inoculation. In parentheses, standard deviation. * significantly different with p <0.05 compared to treatment without inoculation (SΛ). CFN42 wild strain, HP310 derived from CFN42 with nitrogenase overexpression, CIAT652 wild, HP789 derived from CIAT652 with nitrogenase overexpression, HP652 derived from the AM652 phb mutant with nitrogenase overexpression.

In the experiment in Table 3, the inoculation of wild and modified strains did not impact seed production. Only the fertilized treatment was 15% higher than the uninoculated. Regarding the two parameters that indicate the nutritional quality of the seed (nitrogen content and nitrogen yield in seed) in the treatment without inoculating the nitrogen content in seed was 43.4 mgN / g dry weight and the yield of N 97 Kg Total N / Ha, while the fertilized was 72.6 and 187, respectively. The modified strain HP310 obtained a 23% increase in N yield and the modified strains HP789 and HP652 29 and 52% compared to the treatment without inoculation. The irrigation regime was rolled or gravity irrigation.

Table 4. Performance data, estrogen content and nitrogen yield in field plant seeds. Texcoco, June 2000. Rolled irrigation. Black variety M8025.

N in seed

• Incr reduction. Performance N Incr.

Strain (mgN / g weight (Ton / Ha) (%) (Kg N / Ha)% dry)

CFN42 3.43 8 46.1 (3) 158 65 *

HP310 3.40 7 47.5 (1) 163 70 *

CIAT652 3.45 8 57.1 (2) 197 107 *

HP789 3.33 5 49.3 (3) 164 72 *

HP652 3.13 0 54.3 (1) 170 78 *

S / I 3.18 - 29.9 (2) 95.42 -

+ N 2.83 0 66.3 (1) 188.0 97 *

Production of five plots in random arrangement. %, regarding plots without inoculation. In parentheses, standard deviation. * significantly different with p <0.05 compared to treatment without inoculation (S / I). CFN42 wild strain, HP310 derived from CFN42 with nitrogenase overexpression, CIAT652 wild, HP789 derived from CIAT652 with nitrogenase overexpression, HP652 derived from the AM652 phb mutant with nitrogenase overexpression. In this field the irrigation was by rolled irrigation and in general a very favorable response in production is noted. Only inoculation with wild and modified strains produced increases in seed yields between 6 and 8% (except HP652). In the determination of nitrogen content and nitrogen yield in seed, the highest increases were with chemical fertilization (66.3 mgN / g dry weight and 188

Kg N Ha), followed by inoculation with HP652, 54.3 and 170, respectively) and the wild strain CIAT652 (57.1 and 197, respectively). The HP310 strain obtained a 5% increase in nitrogen yield compared to the strain from which it comes, CFN42.

Table 5. Performance data, nitrogen content and nitrogen yield in seeds of field plants. Juchitepec, June 2000. Rain. Black variety M8025.

„Incr Production . - _τ , Performance P Incr.

^Strain (Ton / Ha) (%) ^ ^ ⁹ "* (Kg N / Ha)%

CFN42 1.44 0 47.2 (2) 68 34 *

HP310 1.38 0 52.5 (2) 72 44 *

CIAT652 1.29 0 62.9 (2) 82 62 *

HP789 1.41 0 57.0 (2) 81 60 *

HP652 1.47 0 58.9 (1) 86 71 *

S / I 1.56 - 32.1 (1) 50 -

+ N 1.44 0 70.2 (1) 101 101

Production of five plots in random arrangement. %, regarding plots without inoculation. In parentheses, standard deviation. * significantly different with p <0.05 compared to treatment without inoculation (S / I).

CFN42 wild strain, HP310 derived from CFN42 with nitrogenase overexpression, wild CIAT652, HP789 derived from CIAT652 with nitrogenase overexpression, HP652 derived from the AM652 phb mutant with nitrogenase overexpression.

In the experiment in Table 5, neither the inoculated strains nor the fertilization produced increases in production. The above may indicate the abundance of nitrogen in that type of soil. However, the total nitrogen yield was favorably impacted by the inoculation of the wild strains (34 and 62%, CFN42 and CIAT652, respectively), the modified strains (44, 60 and 71%, strains HP310, HP789 and HP652, respectively ) and fertilization (101%) over that obtained in the plots without inoculation. The irrigation regime was due to rain in the summer season and production was reduced overall.

Table 6. Summary of field experiments with genetically modified strains of Rhizobium that overexpress in nitrogenase enzyme complex. It is compared with respect to nitrogen fertilization.

% increase in N yield (Kg N / Ha) compared to fertilized control

State Morelos Guanajuato State of Mexico

Location Zacatepec Celaya Celaya Texcoco Juchitepec irrigation

Type of irrigation irrigation and irrigation rain drip irrigation <rhizorriegc

Strain

CFN42 72 62 49 84 66

HP310 64 52 64 86 11

CIAT652 40 74 70 105 80

HP789 185 65 66 87 79

HP652 134 73 79 90 85

Controls

Not inoculated 91 47 52 51 49

(YES)

Fertilized

100 100 100 100 100

(+ N)

Underlined, data in which the genetically modified strain gave better results than its wild strain.

Table 6 summarizes the field experiments carried out with the application of nitrogenase overexpression in Rhizobium etli microorganisms in beans. The irrigation regime is indicated. In underlining it can be noted that almost for all Experiments the modified strains achieved the best nitrogen yields in the inoculation. 100% is indicated by chemically fertilized treatment. Figure 1 shows the diagram of the constructed plasmid. As can be seen in Figures 2 to 10, the expression of the genetic construct called pr. c nifHDK (SEQ BD NO: 1, 2, 3 and 4) produced on average, with respect to inoculation with the wild strain in greenhouse, an increased nitrogenase activity of 40% (determined as ethylene micromoles / h mi g dry weight of nodule), a higher nitrogen content in the plant of 20%, a higher yield of up to 49% (in mg of seed / plant) and a higher nitrogen content in the bean seed of 35% to 65% (in mg N / plant), which resulted in a 50% higher protein content in the seed. The expression of the genetic construct in Rhizobium CIAT652 and AM652 strains (strain derived from CIAT652 that presents a mutation for phb), used as a biofertilizer in common bean plants {Phaseolus vulgaris L.) produced an average of 20% more nitrogen in the plant, 40% higher yield and 50% higher seed nitrogen content, compared with the use of wild strains. The increase in nitrogen in seed has a direct impact on its nutritional quality and therefore, the seed produced using this bacterium as a crop biofertilizer is up to 50% more nutritious. In the field, several trials were carried out in experimental plots with modalities of rolled irrigation, drip irrigation, rhizorriego and rain. It was inoculated as is normally done with other types of crops such as alfalfa and soy, for which it was used as a vermiculite support (inert synthetic material). As could be seen in the previous tables in general, strain HP310 showed significantly different increases ap <0.05 in nitrogen yield up to 25% with respect to the strain from which it comes (CFN42) and from 10 to 70% compared to the plots not inoculated. The HP789 strain showed increases of up to 145% compared to the wild strain CIAT652 and up to

185% compared to non-inoculated control plots. The HP652 strain showed increases of up to 20% compared to the CIAT652 strain and from 34 to 78% compared to the non-inoculated plots. In several of the experiments, no significant increases in production were achieved due to biofertilization, nor were they observed for nitrogen fertilization. This is because it has been observed that in very productive bean genotypes it is difficult to increase yield. However, a fundamental parameter is that of the seed nitrogen content, since it represents the nutritional quality in the form of proteins and free amino acids that it contains. In that regard, with biofertilization in general and in particular with strains with genetic construction to overexpress nitrogenase, very significant increases were achieved.

With the above, it is verified that there is practical application of genetic construction pr. c nifHDK (SEQ JD NO: 1, 2, 3 and 4), reason for this request. The potential use of Rhizobium organisms as biofertilizers for containing the genetic construct with which nitrogenase is overexpressed (pr. C nifHDK) is supported by field experiments. Although the tests were performed on the bean crop, this is only an example of how to carry out the application of the invention. Additionally it can be applied in other legumes such as peanuts, soybeans, alfalfa, clover, lentil, beans, peas, etc. and even in other crops such as beets, wheat, corn and sorghum. In the construction of the strains, their possible use in the Mexican field was always considered and therefore they comply with all the requirements for the authorization of the release of genetically modified organisms established by the National Biosafety Commission, the competent authority in the field. The use of this biofertilizer in the field is completely harmless based on decades of research and application as biofertilizers and does not harm the environment as do the nitrogen fertilizers that are usually applied. The preparation of the bacteria for use as a biofertilizer and the application in planting is simple and very low cost, so the replacement of the nitrogen chemical fertilizer represents an important economic benefit for the agricultural producer. Additionally, the seed produced has better nutritional characteristics for the consumer in general.

Description of the figures. Fig. 1. It is a scheme of plasmid pHP789 with the genetic construct pr. c nifHDK

(SEQ ID NO: 1, 2, 3 and 4), which shows the site called EcoRI for cutting with the restriction enzyme EcoRI and the BglII site for cutting with the restriction enzyme BglII. Both were used to obtain genetic construction.

Fig. 2. It is a graph about the activity of nifrogenase produced by the genetically modified strain HP310 (with pr. C nifHDK SEQ ID NO: 1, 2, 3 and 4) and the wild strain CFN42, in black bean greenhouse plants jamapa at 18, 25 and 32 days post inoculation (dpi), where * denotes significant differences at the 95% confidence level (p <0.05).

Fig. 3 It is a graph about the seed yield that produces the inoculation of jamapa black bean plants with the genetically modified strain HP310, when compared with the strain from which CFN42 comes, in jamapa black bean greenhouse plants.

Fig. 4 It is a graph about the nitrogen content of seed that produces the inoculation of jamapa black bean plants with the genetically modified strain HP310, when compared to the strain from which CFN42 comes, in jamapa black bean greenhouse plants.

Fig. 5. It is a graph about the nitrogenase activity produced by the genetically modified strain HP789 (with pr. C nifHDK SEQ ID NO: 1, 2, 3 and 4) and the wild strain

CIAT652, in jamapa black bean greenhouse plants at 18, 25 and 32 dpi.

Fig. 6 It is a graph about the seed yield that produces the inoculation of jamapa black bean plants with the genetically modified strain HP789, when compared with the strain from which CIAT652 comes, in jamapa black bean greenhouse plants.

Fig. 7 It is a graph about the seed nitrogen content produced by the inoculation of jamapa black bean plants with the genetically modified strain HP789, when compared with the strain from which CIAT652 comes, in jamapa black bean greenhouse plants .

Fig. 8. It is a graph about the nitrogenase activity produced by the genetically modified strain HP652 (with pr. C nifHDK SEQ ID NO: 1, 2, 3 and 4) and the mutant strain phb AM652, in bean greenhouse plants Jamapa black at 18, 25 and 32 dpi.

Fig. 9 It is a graph on seed yield that produces the inoculation of jamapa black bean plants with the genetically modified strain HP652, when compared with the strain from which AM652 (phb mutant) comes from, in black bean greenhouse plants jamapa

Fig. 10 It is a graph about the seed nitrogen content produced by the inoculation of jamapa black bean plants with the genetically modified strain HP652, when compared with the strain from which AM652 (phb mutant) comes from, in greenhouse plants of black jamapa beans.

Methods used

Cultivation of strains. Escherichia coli was grown in LB medium (5% peptone, 2% casein and 5% NaCl) at 37 degrees. R. etli was grown in PY medium (2% peptone, 0.5% casein) at 30 degrees. Antibiotics were added as required (tettacycline 10 mg / ml, nalidixic acid 20 mg / ml.

Isolation and digestion of total and plasmid DNA. Total DNA was extracted from cultures of R. etli grown in PY. It was centrifuged and the collected cells were resuspended in 470 μl of 50 mM Tris-HCl buffer solution / 20 mM EDTA at pH 7.0. They were used with 20 μl of proteinase K 10 mg / ml and 10 SDS 10% for 1 hour at 37 degrees. It was treated with phenol / chloroform / isoamyl alcohol 24: 24: 1 and chloroform / isoamyl alcohol 24: 1. 2/3 volume of isopropanol was added, the liquid was centrifuged and discarded. The pellet was washed with 1 ml of 70% ethanol and dried in a Savant vacuum dryer. It was resuspended in 50 μl of 10 mM Tris-HC1 / 1 mM EDTA pH 8.0. Plasmid DNA was obtained from E. coli grown in LB. It was centrifuged and the collected cells were resuspended in 200 µl of 50 mM Tris-HCl buffer 20 mM EDTA / 5 mg / ml RNAse at pH 8.0. 200μl of 0.4N NaOH / 1% SDS was added. 170 µl of 5M ammonium acetate was added to pH4.5 and stirred and centrifuged. To the lysate was added 1 ml of frozen ethanol. The supernatant was centrifuged and discarded. The DNA pellet was washed with 1 ml of 70% ethyl alcohol and dried in a Savant vacuum dryer. It was resuspended in 20 μl of 10 mM Tris-HC1 / 1 mM EDTA pH 8.0. DNA digestion was done with 5 μl of total or plasmid DNA, 2 μl of REact buffer solution, 1 μml of the enzyme and 12 of ultrapure water. It was digested 4 or 16 hours at 37 ° C depending on whether it is plasmid or total DNA. The digested DNA was run on 0.8% agarose gels stained with ethidium bromide (0.5 mg / ml). The ligaments were made with 5 μl of digested insert DNA and 2 μl of digested vector, 2 μl of ligation buffer solution, 1 μl of T4 DNA ligase and 10 of ultrapure water. They were incubated at 12 ° C for 16 hours.

Transformation. Plasmids were introduced by heat shock. The DNA was added to 100 µl of E. coli cells and incubated for 5 minutes on ice, 2.5 minutes at 42 ° C and 5 minutes on ice. 1 ml of LB was added and incubated at 37 ° C for 1 hour. It was plated in LB boxes with antibiotics for selection of the incoming vector. It was incubated for a day at 37 ° C.

Recombinant crosses. E. coli strain HB101 containing plasmid pHP789 is grown for 16 hours at 37 ° C in LB. R. etli is grown at 30 ° C in PY. They are mixed and centrifuged at 6,000 x g for two minutes. They are resuspended and placed in a PY box without antibiotics incubating for a day at 30 ° C. Roasts were taken to stretch PY with antibiotic. It was incubated at 30 ° C for two days.

Southern hybridization. The problem DNA was digested and ran in gel. It was treated with 1M NaOH 0.5M NaCl for 30 minutes and with 0.5M Tris base pH7.5 1.5M NaCl for 45 minutes. The DNA was transferred to a nylon membrane by capillarity. The buffer and transfer solution is SSC6x (SSC 20x: sodium citrate 88.2g, sodium chloride 175.3 g per liter). The DNA was fixed with ultraviolet light. He hybridized for 16 hours at

65 degrees with radioactively labeled DNA tester with 32P. Washed with SSC O.lx 1% SDS 30 minutes, SSC O.lx SDS 1% 30 minutes at 37 ° C and SSC O.lx SDS 0.1% at 65. It was placed in a cassette for exposure of radiographic plaque.

Plant trials The jamapa black bean seeds were treated with 1% sodium hypochlorite for 5 minutes and washed with sterile water. They were germinated and planted in pots with sterilized vermiculite. ⁶ R. etli cells washed with lxlO were inoculated with

0.85% NaCl. The pots were watered with water and salt solution without nitrogen. At 18, 25 and 32 days post inoculation (dpi) they were collected for acetylene reduction assay. 20 plants of each strain were allowed to mature to obtain seed at 80 dpi. For the acetylene reduction test, the roots were taken and placed in hermetically sealable bottles. They were injected with acetylene and a sample of 0.4 ml was analyzed in gas chromatograph Varies 3700 to determine the conversion into ethylene. The calculation of the activity implies a molecular extinction factor for ethylene, bottle capacity, elapsed time, injected volume and dry nodule weight. Inoculations were prepared with lxlO ⁹ cells per gram of vermiculite, added to bean seed and planted in 5 replicate plots of 5 rows of 6 meters each. Plots were planted without inoculation and plots for chemical nitrogen fertilization with 75 kg N / Ha.

Nitrogen analysis in seed and plant. The samples were milled and the nitrogen content was determined by flame spectrophotometry with an Antek 7000 apparatus. Albumin was included to perform calibration curves.

Statistic analysis. The t-test was applied to the nitrogenase activity, nitrogen content in seed and plants and greenhouse and field yield data to determine significant differences. The Rhizobium etli strain that presents the genetic construction mentioned in the

Claims 1 to 16 was deposited in the Culture Collection, Agriculture Research

Service, United States Department of Agriculture (Peoría, 111.) on July 12, 2002 with registration number NRRL B-30606.

Having described the background on nitrogen fixation and the strategies carried out to improve it, taking into account that the genetic construction that is the reason for this request does not exist as such in nature, we claim as novel what is contained in the following:

Claims

1. Genetic construction that contains the sequence identified with SEQ ID NO: 1,

2. 3 and 4. 2. Vector containing the genetic construction of Claim 1.

3. Vector according to Claim 2 wherein the vector can be a cosmid.

4. Vector according to Claim 2 wherein the vector may be a plasmid.

5. Escherichia coli strain named HB101 / pHP789 containing the vector plasmid with the genetic construct mentioned in Claim 1.

6. Rhizobium strain containing the genetic construct mentioned in the

Claim 1.

7. Vector construction process mentioned in Claim 2 which consists of obtaining the Rhizobium nifHDK nitrogenase operon by cutting with EcoRI and BglU, linking it to the promoter region of high transcriptional rate of nifHc, cloning this fragment to a vector plasmid and with the plasmid obtained perform double recombinants in bacteria.

8. Process for the preparation of an expression vector based on Claim 7, consisting of: expressing a complete nifHDK nitrogenase operon under the regulatory control of the nifHc promoter region.

9. Process according to claims 7 and 8 wherein a complete nitrogenase operon is expressed under the regulatory control of a promoter region of high transcriptional capacity {nifHc) in bacteria.

10. Process according to claims 7, 8 and 9 wherein the genetic construction is preferably expressed in microorganisms of the Rhizobium genus as

Rhizobium leguminosarum (pea, bean and lentil symbiote), Rhizobium trifolii (clover symbiont), Sinorhizobium meliloti (alfalfa symbiont), Bradyrhizobium japonicum (soybean and peanut symbiont) and the other species of genus and best animals The expression of this genetic construction.

11. Process according to Claim 7 wherein the foreign genetic material from the vector is not incorporated into the genome of the Rhizobium bacteria allowing its use as biofertilizers.

12. The use of the genetic construct of Claim 1 present in bacteria for use as biofertilizers.

13. The use of the vectors of Claims 2 to 4 in bacteria as biofertilizers.

14. The use of the strain of Claim 6 as a biofertilizer.

15. The use of the bacteria obtained based on Claims 9 to 12 in legumes.

16. The use according to Claim 15 wherein the legume can be bean.