WO2021261984A1

WO2021261984A1 - Biostimulant based on bacteria for better adaptation of plants to hydric and osmotic stresses

Info

Publication number: WO2021261984A1
Application number: PCT/MA2021/000012
Authority: WO
Inventors: Mohamed Hafidi; Lamfeddal Kouisni; Ahmed NAFIS; Yedir Ouhdouch
Original assignee: Ocp Sa
Priority date: 2020-06-22
Filing date: 2021-06-18
Publication date: 2021-12-30
Also published as: MA50114A1; CN116133525A; MA50114B1; AU2021296688A1; KR20240010707A; CA3189825A1; EP4168532A1

Abstract

This invention describes the obtaining of a biostimulant based on 3 bacterial strains for boosting plant resistance to hydric and osmotic stresses. These bacteria are capable of increasing the hydric potential of soils by means of the degradation of waxes (BDC) which coat the components of soils and reduce the capacity of the latter to store sufficient rain and/or irrigation water. In addition, these bacteria help plants to save water more successfully via optimal and continual potassium nutrition (BSK). These three bacteria (BDC + BSK) are coded N1, N4 and N3 and identified (N1 = Streptomyces acrimycini N4= Streptomyces mutabilis and N3= Polyangium brachysporum). This invention also describes a new bacterial inoculum formulation based on the optimisation of certain biological properties in these bacteria (N1, N4 and N3), namely their properties in increasing the water retention capacity of soils and in solubilising and mobilising the forms of potassium for the plant from complex inorganic forms which are not accessible to the plant without the microbial partner thereof (BCD and BSK). By including together propagules of these bacteria and perlite as a matrix, the ability of the (BDC and BSK) to increase the sponge capacity of soils and to solubilise the insoluble forms of potassium and make them accessible to the plant will be increased. As a result, the role of the (BDC and BSK- Plant) partnership on the development of the host plant will be optimised by intimately combining the impact of these bacteria on the plant directly (control of absorption, conduction and transpiration) and indirectly (availability of the water in soils in a uniform manner, by modification of the components that cause water loss by leaching, evaporation).

Description

DETAILED DESCRIPTION OF THE INVENTION

Bacteria-based biostimulant for better adaptation of plants to water and osmotic stress.

Figures and graphs listing

Figure 1: Diagram of the technique for evaluating the degree of hydrophobicity of a sandy soil (a) and determination of the volume of water retained (b)

Figure 2: Elemental analysis of sand at low altitude a: BNT: untreated sand, b: BT: sand treated with butanone. (% CK in BNT is 29 against 14 in BT)

Figure 3a: Macroscopic and microscopic aspects of isolates (NI, N3 & N4) degrading commercial waxes.

Figure 3b: Phylogenetic tree of bacterial strains capable of degrading waxes (NI, N4 and N3) retained for this invention. (NI, N4 and N3) are characterized by the sequences Seq N3, Seq N4, & Seq NI (Plate 1) and submitted to the IDA service of the CCMMs with accessions: Nl = CCMMP1, N4 = CCMMP2 andN3 = CCMM P3

Figure 4: Examples of use of insoluble forms of K and P expressed by the extent of growth of strains (NI, N4 and N3) on screening media (Aleksandrov & NBRIP)

Figure 5: Protocol followed for the evaluation of root colonization of alfalfa and Aradopsis seedlings by strains (NI, N4 and N 3).

Figure 6. The supernatant obtained from the N3 culture mixed with water improves the wetting and penetration of water repellent soil. (Legend: drop of water and drop of water + 50% culture juice of the N3 strain,)

GRAPHS

Graph 1: PGP traits of NI, N4 and N4 strains: Germination index, TCP solubilization of Mica and auxin production

Graph n ° 2. Some of the physicochemical parameters of the sandy soil used

Graph 3. Effect of inoculation of sandy soil by strains (NI, N4 and N3) on water retention capacity

Graph 4: The design of the experimental protocol for agronomic tests

Graph 5. Biostimulating effect of the different parts of alfalfa seedlings by the strains (NI, N3 & N4) compared to the commercial product (C) control expansive and uninoculated vermiculite (NI).

- Irrigation regime of 25%; - 50% irrigation regime; - 75% irrigation regime Climate change has been a hot topic for several years. Numerous studies deal with the subject and make observations and perspectives that may worry us about the future. Agriculture faces a major challenge in many parts of the world (Ainsworth and Orth, 2010). The climate change we are experiencing is illustrated in particular by increasingly frequent and longer periods of drought. The consequences on crop yields can be dramatic. Water and osmotic stresses are and always will be the most important abiotic stresses at the global level. Arable land suffers from a lack of water which causes frequent water stress. This phenomenon is not only amplified by climate change but also by the presence of non-wettable organic matter in the form of waxes which coat the components of the soils and reduce the latter's capacity to store sufficient rainwater and / or soil. irrigation. Potassium (K) is an essential element involved in water management at the plant level. Indeed, For plants, potassium (K) plays an important role in water and osmotic status: (i) by regulating the opening and closing of stomata, (ii) by the osmotic adjustment of the plant (turgor) , (iii) and by root growth. In addition to these 3 functions, the University of Halle has demonstrated a significant impact of potassium on the useful water reserve of the soil (Damm 2012).

There are solutions today that increase the water holding capacity of soils. However, these solutions are either expensive or difficult to apply. For example, adding clay to the soil requires 300t of clay / hectare (Carter and Hetherington 2006).

The search for other cheaper, ecological and efficient additional routes has become a requirement. The development of a new biostimulant product based on microorganisms to improve the wettability of soils, boost the water retention capacity of the soils and the good management of perspiration to strengthen the water economy by plants is an urgent need. This invention provides an alternative based on an innovative concept inspired by nature.

This invention describes obtaining a plant biostimulant based on bacteria isolated from the root system of desert plants (rhizospheric soils). The choice of this habitat is not trivial. Our goal was to understand how the plants in this stressed habitat manage to resist and cope with the lack of water. These bacteria which help desert plants are able to reduce water and osmotic stress thanks to their following essential properties:

Degradation of waxes (BDC) that coat soil particles and reduce the soil's ability to store sufficient water, atmospheric or dew, rain and / or irrigation.

Solubilization and mobilization of insoluble forms of potassium into forms that can be assimilated by plants (B SK) to control water savings.

It also describes a new formulation of bacterial inoculum based on the optimization of certain biological properties in these bacteria (BDC + BSK) namely: their ability to degrade waxes, to solubilize and to mobilize potassium in forms that can be assimilated by the plant. from complex inorganic potassium forms which are not accessible to the plant without its microbial partner (BDC + B SK). By including together propagules of these bacteria and a source of mineral potassium soaked in soybean flour juice, the ability of (BDC + BSK) to break down several waxes and to solubilize potassium, phosphorus and to produce hormone type auxin and make them accessible to plants. Therefore, the role of the partnership (BDC + BSK- Plant) on the development of the host plant will be optimized by intimately combining the impact of these (BCD + BSK) on the plant directly (water, mineral, hormonal effect, etc. ) and indirectly (alteration of potassium and inorganic phosphate).

These bacteria (BCD + BSK) are cultivated on liquid nutrient medium obtained from soybean flour juice after filtration (3g / L of water). After a period of culture depending on the physiological characteristics of (BCD + BSK), the propagules are recovered by filtration, washed with sterile water to remove all chemical traces from the nutrient medium used then ground in a Waring Blender and stored at room temperature and at 4 ° C. Granules of potassium (Mica) (50 µm diameter) or commercial perlite are immersed in a sterile solution composed (i) of soybean flour juice (3 g / L), previously sterilized (ii) of propagules (1 g dry weight per liter of mixture). After 2 hours of immersion with stirring, the Mica and / or perlite granules impregnated with the bacteria (BCD + BSK) are incubated at 28 ° C. for 2 days. The perlite and Mica granules thus colonized by (BCD + BSK) forming propagules on the surface are then dried in air and placed in flasks to store them at room temperature and / or at 4 ° C.

Another advantage of this invention is the fact that the seeds or seeds can be inoculated directly with the propagules of (BCD + BSK) by the method of seed bacterization.

For the definition of the invention, it is:

A new method of isolating bacteria, a new biotechnology and a new biostimulant.

For the technical problem (s) that the invention would solve?

1) Effect Optimization (BDC) to increase the water potential of soils by removing waxes that prevent water absorption by soil particles and boosting the soil's ability to hold more water. Among the advantages:

^■ Reduce the impact of climate change on crops

^■ Increase the capacity of soils to retain water

^■ Optimizing the use of water

^■ Reduce water evaporation from sandy soils

^■ Increase the area of arable land and improve agricultural yields

^■ Improve the conditions for plants to adapt to water stress

^■ Homogenize seed germination in water-repellent soils

^■ Reduce soil erosion

2) Optimization of the effect (BSK) for better management of water saving by the host plant through optimal and permanent potassium nutrition. The latter will allow good absorption, conduction and control of perspiration in stressful conditions. hydrous and osmotic. The optimal and permanent presence of potassium will also have a positive impact on the useful water reserve of the soil.

To better describe the invention, this example:

As a first step, obtaining (BDC + BSK) a- Isolation of (BDC + BSK) from sand samples collected from rhizospheric soils of certain desert plants adapted to water and osmotic stress. With this in mind, the samples were taken in the region of Merzouga and others (Morocco). Likewise, plant samples were collected from the sites studied. b- Study of the characteristics of the collected sandy soils:

The study of the water retention capacity and hydrophobicity of sandy soils was carried out by two techniques, namely the "MED: Molarity Ethanol Drop" technique (figure la). This technique is based on the concentration of ethanol necessary to penetrate the soil in less than 10 seconds (MED). The second technique consists of measuring the retained volume (figure lb).

> Determination and assessment of the hydrophobicity of the sand (standard standard: MED)

Time taken for the drop of water to disappear on the surface of the non-wetting soil:

✓ <1 second Not significant

✓ 1-10 seconds Very low water repellency

✓ 10-50 seconds Low water repellency

✓ 50-260 seconds Moderate water repellency

✓> 260 seconds Moderate to severe water repellency Rhizospheric soils collected near the roots of desert plants have very low water repellency (with a time of less than 10 seconds). However, bare sands far from the root system of plants show pronounced hydrophobicity with a time that is between 250-260 seconds.

To determine the volume of water retained in the sand, we followed the following protocol: 1- Install a cloth (as filters) in the two funnels.

2- Place each funnel on a different graduated test tube.

3- Take the two sand samples (50 grams, 1 treated with butanone to remove the waxes surrounding the grains of sand and the other untreated) and place each in the funnel.

4- Put 100 ml of distilled water in a graduated beaker.

5- Start the stopwatch and slowly pour in the 100 ml of water.

6- Using the graduated cylinder, measure the quantity of water collected.

NB: The treatment of the water-repellent sand with butanone made it possible to dissolve the thin layers of waxes which surround the grains of sand, thus increasing the contact surface of the grains of the latter with water.

After carrying out the test, we noticed that:

- The time taken for water to pass through the treated sand (removal of waxes with butanone) is long compared to untreated sand.

- The volume of water collected in treated sand is lower compared to untreated sand, so treated sand retains more water.

- The flow velocity of untreated sand, vis-à-vis the water, is large compared to that of the treated sample. c- Determination of the elemental composition of the desert sand explored

The elemental composition of the collected and treated (BT) and untreated (BNT) sand with butanone was determined by scanning electron microscope analysis (Figures 2a and 2b).

The results show that the grains of sand contain a significant amount of organic matter in the form of wax. This was confirmed during the removal of this thin wax skin by butanone. Indeed, the carbon percentages found are lower compared to the case where the sand is untreated (figure 2). For the choice of butanone as a solvent for removing waxed organic matter from sand, butanone is volatile and has no pronounced antimicrobial effect. This choice gave us access to bacteria trapped in the wax and which cannot be isolated by the standard soil microbiology technique. d- Isolation and identification of bacteria capable of degrading wax: In order to isolate the bacteria having the ability to degrade wax, two methods were adopted. Method 1: The rhizospheric or non-rhizospheric sand grains are washed with sterile distilled water and dried were seeded (deposited) directly in a minimum culture medium (MM) without any source of organic carbon. The composition of MM consists of the following ingredients (for 1 liter): (KNO ₃ 2g, K ₂ HPO ₄ lg, MgS0 ₄ 0.5 g, NaCl 0.5g, CaC0 3g, FeS0 ₄ 0.01g and noble agar or agarose 14g As can be seen, the only source of organic carbon is that which surrounds the grains of sand.

Method 2: From 200g of rhizospheric or non-rhizospheric rice, dried, treated with butanone to extract waxes and propagules from microorganisms. After evaporation of the butanone, the suspension of propagules was seeded on the MM + 5 g of commercial wax (for 1 liter MM + agar-agar not usable by bacteria). Three waxes were used.

The colonies developed were purified and tested for their ability to degrade a wide range of commercial waxes, namely, Carnauba wax, Candelilla wax and Mimosa wax. The controls were used: MM + Agar-agar and wax + agar-agar

In total, 13 isolates showed the ability to degrade a wide range of waxes and their morphological and molecular diversities were investigated. Morphological diversity was studied by determining the shape, the type of Gram, the macroscopic appearance of the colonies, the microscopic aspects of the cells, the diffusible pigments produced. In the case of actinobacteria, the color of the aerial and substrate mycelium (Figure 3a) was noted. The molecular diversity of the isolates was carried out via the isolation, purification, amplification and sequencing of the gene which codes for 16S RNA by the “Macrogen-South Korea” analysis platform. The results showed that the isolates are not pathogenic and show taxonomic diversity (Figure 3b). Three strains (NI, N4 and N3) were selected. Taxonomically, the N3 isolate is identified as Voyl anyium bmchyspomm, the N 4 isolate identified as Streptomyces mutabilis and NI is identified as Streptomyces acumycini. In the second step, study of the properties or traits of plant growth promotion (PGP) of the strains (NI = CCMMP1, N4 = CCMMP2 and N3 = CCMMP 3) capable of degrading the selected waxes a) Qualitative study of the solubilization of insoluble forms of K, P

To study the PGP traits of bacteria (NI, N4 and N3) degrading the selected waxes, the K and P solubilization tests were carried out on solid culture media. The solubilization of P was carried out in NBRIP medium (National Botanical Research Institute of Phosphate) supplemented with tricalcium phosphate (TCP) (Ca (P03) 2 as an inorganic source of P. The solubilization of K was carried out on Aleksandrov medium with Mica as the only source of insoluble K. The results obtained show the capacity of these three strains (NI, N4 and N3) to use the insoluble forms of K and P. The extent of the use of the insoluble forms of K and P is evaluated by the intensity of growth. Aleksandrov and NBRIP media without any soluble source of K and P represent negative controls. Aleksandrov and NBRIP media with soluble sources of K and P represent positive controls. Aleksandrov medium whose source of P is (TCP) and source of K is Mica used for the solubilization of K and P in the same culture medium. An example of the growth obtained is given in figure 4. b) Quantitative study of the solubli K, P, Indolo Acetic Acid (IAA) production and the effect on seed germination of Arabidopsis taliana as a model plant.

The solubilization performance of the insoluble forms of K and P of these isolates was measured via the analysis of the culture juices by ISP (Inductively Coupled Plasma). The AIA assay was performed in the juice of cultures as well. Germination tests were carried out on Arabidopsis taliana seeds as a model plant. Before germinating the seeds, the latter were disinfected and bacterized by stem cells (NI, N4 and N 3) cultured for three days in Bennett's medium in order to produce sufficient biomass. Subsequently, the seeds of the model plant were cultivated under optimal growth conditions and the results are noted after 5 days. The results of these traits promoting plant growth of the strains (NI, N4 and N 3) are shown in graph 1 c) Study of the colonization of the roots of Aradopsis taliania and alfalfa plants by stem cells (NI, N4 and N 3).

This study of the colonization of plant roots by stem cells (NI, N4 and N 3) was carried out according to the diagram in FIG. 5. The results obtained showed that the three strains did indeed colonize the roots of the tested plantlets. . No negative effect on the health of the seedlings was observed during the test period.

In the third step, study of the performance of strains (NI, N4 and N 3) in increasing the water retention capacity of sandy soil.

The sandy soil used is characterized by the physicochemical parameters recorded in graph n ° 2.

The strains (NI, N4 and N3) are cultivated on liquid nutrient medium obtained from soybean flour juice after filtration (3 g / L of water). After 3 days, the propagules are recovered by filtration, washed with sterile water to remove all chemical traces from the nutrient medium used, then ground in a Waring Blender then dried. 100 g of the water-repellent sand of composition indicated in Table 2 are inoculated with 10 ⁶ CFU (colony-forming unit) of each strain. The dried propagules were mixed well with the sand.

After incubation for 3 weeks, we were able to confirm the ability of the strains (NI, N4 and N3) to improve the sponge effect of particles in the sandy soil tested. Sand inoculated with CCMMP3 strain N3 (Polyangium brachysporum) showed water retention capacity with an increase of 10% (graph 3).

In the penultimate step of this invention, agronomic trials under controlled conditions in a greenhouse after inoculation of alfalfa with the strains (NI, N3 and N4)

To validate the ability to improve water retention by inoculated sandy soil and its effect on growth promotion, we carried out agronomic trials in a greenhouse on a model plant "alfalfa". Three strains (NI, N4 and N3) were chosen in order to study their effects on the promotion of the growth of the chosen model plant (Medicago sativa) subjected to increasing irrigation regimes and under conditions of temperature and humidity. controlled. Strain N3 is identified as Poyl angium bmchyspomm, Isolate N 4 identified as Streptomyces mutabilis and NI is identified as Streptomyces acrimycini. Jars of 200 grams of water-repellent soil of composition indicated in graph 2 were used. For inoculation, four seeds of alfalfa bacterialized by each strain per pot. The 4 alfalfa seedlings are bacterized and germinated on Petri dishes and then expose to a water regime of 25, 50 and 75% compared to the field capacity of the water-repellent soil used. Two types of soil were used, one with 0.4 g of insoluble K (Mica) and the other normal without any source of K. A soil mixed with 25% expanding vermiculite was used as a commercial control. The culture was carried out for 1 month and according to the protocol recorded in graph 4.

The results obtained are very interesting. In fact, the N3 strain identified as Poyl angium bmchyspomm showed a biostimulating capacity of alfalfa seedlings with a yield of more than 50% compared to the commercial expansive vermiculite control product (Graph 5).

After harvest, the results of alfalfa plants were found to be more interesting in terms of the biomass yield of the obtained plant material. The biostimulatory effect of the N3 strain (Polyangium brachysporum) is the most important. In fact, this strain has made it possible to increase the yield in terms of plant biomass by more than 50% and it is the same strain that has made it possible to obtain the increase in water retention of about 10% of the soil. used. For the K contents of the different parts of alfalfa seedlings, compared to the uninoculated controls, the potassium contents of the different parts of the alfalfa seedlings were improved for the seedlings inoculated with the N3 strain.

In the last step of this invention, it was shown that the process of degradation of waxes by strains NI, N4 and N3 is linked to the excretion of one or more molecules of the bio-surfactant type. The dissolving process of Mica probably involves the destruction of the strong ionic bonds existing between the constituent elements of this potassium rock. The strain N3 related to Polyangium brachysporum) showed the best ability to degrade waxes. The bio-surfactant present in the fermentation juice of this strain reduced the surface tension of a water-repellent soil (Figure 6)

References Ainsworth, EA, Ort, DR (2010). How do we improve crop production in a warming worl d? Plant Physiology 154, 526D530.

Damm S., Hofmann B., Gransee A. & Christen O. (2012): Zum Einfluss langjâhrig differen zierter Kaliumdüngung auf Parameter des Bodenwasserhaushaltes, Archives of Agrono my and Soil Science, DOI: l 0.1080 / 03650340.2011.606217

Damm S., Hofmann B., Gransee A. & Christen O. (2012): Wirkung differenzierter KD Düngung auf bodenphysikalische Eigenschaften, Bodenwassergehalt und Ertrag von Zu ckerriiben und Sommergerste auf einem Tschemosem im Mitteldeutschen Trockenge and Archives Science, Agronomy and Soil of Mitteldeutschen Trockengebi DOI: 10.1080 / 03650340.2012.6639

Carter D and Hetherington R. (2006) Claying water repelient soils. Department of Agriculture and Food, Western Australia Agdex 514.

NI Sequence (16S rDNA)

ACGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAACGATGAACCACCTTCGGGTG

GGGATTAGTGGCGAACGGGTGAGTAACACGTGGGCAATCTGCCCTGCACTCTGGGACA

AGCCCTGGAAACGGGGTCTAATACCGGATACTGACCCGCTTGGGCATCCAAGCGGTTCG

AAAGCTCCGGCGGTGCAGGATGAGCCCGCGGCCTATCAGCTTGTTGGTGAGGTAATGG

CTCACCAAGGCGACGACGGGTAGCCGGCCTGAGAGGGCGACCGGCCACACTGGGACTG

AGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGAA

AGCCTGATGCAGCGACGCCGCGTGAGGGATGACGGCCTTCGGGTTGTAAACCTCTTTCA

GCAGGGAAGAAGCGAAAGTGACGGTACCTGCAGAAGAAGCGCCGGCTAACTACGTGCC

AGCAGCCGCGGTAATACGTAGGGCGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGAG

CTCGTAGGCGGCTTGTCACGTCGGTTGTGAAAGCCCGGGGCTTAACCCCGGGTCTGCA

GTCGATACGGGCAGGCTAGAGTTCGGTAGGGGAGATCGGAATTCCTGGTGTAGCGGT

GAAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGATCTCTGGGCCGATAC

TGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCAC

GCCGTAAACGGTGGGCACTAGGTGTGGGCAACATTCCACGTTGTCCGTGCCGCAGCTA

ACGCATTAAGTGCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTG

ACGGGGGCCCGCACAAGCGGCGGAGCATGTGGCTTAATTCGACGCAACGCGAAGAACC

TTACCAAGGCTTGACATACACCGGAAAACCCTGGAGACAGGGTCCCCCTTGTGGTCGGT

GTACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCG

CAACGAGCGCAACCCTTGTCCCGTGTTGCCAGCAGGCCCTTGTGGTGCTGGGGACTCAC

GGGAGACCGCCGGGGTCAACTCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGCC

CCTTATGTCTTGGGCTGCACACGTGCTACAATGGCCGGTACAATGAGCTGCGATACCGC

GAGGTGGAGCGAATCTCAAAAAGCCGGTCTCAGTTCGGATTGGGGTCTGCAACTCGAC

CCCATGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCATTGCTGCGGTGAATACGTTCC

CGGGCCTTGTACACACCGCCCGTCACGTCACGAAAGTCGGTAACACCCGAAGCCGGTGG

CCCAACCCCTTGTGGGAGGGAGCTGTCGAAGGTGGGACTGGCGATTGGGACGAAGT

> N3 Sequence (16S rDNA)

GCTCAGATTGAACGCTGGCGGCATGCCTTACACATGCAAGTCGAACGGCAGCACGGGA

GCAATCCTGGTGGCGAGTGGCGAACGGGTGAGTAATACATCGGAACGTGCCCAGTAGT

GGGGGATAGCCCGGCGAAAGCCGGATTAATACCGCATACGACCTGAGGGTGAAAGCG

GGGGACCGCAAGGCCTCGCGCTATTGGAGCGGCCGATGTCAGATTAGCTAGTTGGTGG

GGTAAAGGCCTACCAAGGCGACGATCTGTAGCTGGTCTGAGAGGACGACCAGCCACAC

TGGGCGCAAGCCTGATCCAGCCATGCCGCGTGCGGGAAGAAGGCCTTCGGGTTGTAAA

AAGAATAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCAAGC

GTTAATCGGAATTACTGGGCGTAAAGCGTGCGCAGGCGGTTGTGCAAGACAGATGTGA

AATCCCCGGGCTCAACCTGGGAACTGCATTTGTGACTGCACGGCTAGAGTGCGGCAGA

GGGAGATGGAATTCCGCGTGTAGCAGTGAAATGCGTAGATATGCGGAGGAACACCGAT

GGCGAAGGCAATCTCCTGGGCCTGCACTGACGCTCATGCACGAAAGCGTGGGGAGCAA

ACAGGATTAGATACCCTGGTAGTCCACGCCCTAAACGATGTCAACTAGTTGTTGGACGG

CTTGCTGTTCAGTAACGTAGCTAACGCGTGAAGTTGACCGCCTGGGGAGTACGGCCGC

AAGGTTGAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGATGATGTGGTTT

AATTCGATGCAACGCGAAAAACCTTACCTACCCTTGACATGCCAGGAATCCTGCAGAGAT GTGGGAGTGCTCGAAAGAGAACCTGGACACAGGTGCTGCATGGCCGTCGTCAGCTCGT

GTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCATTAGTTGCTACG

AAAGGGCACTCTAATGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTC

AGGTCATCATGGCCCTTATGGGTAGGGCTACACACGTCATACAATGGCCGGTACAGAG

GGCTGCCAACCCGCGAGGGGGAGCTAATCCCAGAAAACCGGTCGTAGTCCGGATCGCA

GTCTGCAACTCGACTGCGTGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGTCGCG

GTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGGGAGCGGGTTCTGC

CAGAAGTGGGTAGCCTAACCGCAAGGAGGGCGCTTACCACGGCAGGGTTCGTGACTGG

GGTGAAGTCGT

> N4 Sequence (16S rDNA)

CTCAGGACGAACGCTGTCGGCGTGCTTAACACATGCAAGTCGAACGATGAACCACTTCG

GTGGGGATTAGTGGCGAACGGGTGAGTAACACGTGGGCAATCTGCCCTGCACTCTGGG

ACAAGCCCTGGAAACGGGGTCTAATACCGGATACTGAGGTCCGCAGGCATCTGTGGTTC

TCGAAAGCTCCGGCGGTGCAGGATGAGCCCGCGGCCTATCAGCTTGTTGGTGAGGTAA

CGGCTCACCAAGGCGACGACGGGTAGCCGGCCTGAGAGGGCGACCGGCCACACTGGG

ACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGG

CGAAAGCCTGATGCAGCGACGCCGCGTGAGGGATGACGGCCTTCGGGTTGTAAACCTC

TTTCAGCAGGGAAGAAGCGAAAGTGACGGTACCTGCAGAAGAAGCGCCGGCTAACTAC

GTGCCAGCAGCCGCGGTAATACGTAGGGCGCGAGCGTTGTCCGGAATTATTGGGCGTA

AAGAGCTCGTAGGCGGCTTGTCGCGTCGGTTGTGAAAGCCCGGGGCTTAACCCCGGGT

CTGCAGTCGATACGGGCAGGCTAGAGTTCGGTAGGGGAGATCGGAATTCCTGGTGTAG

CGGTGAAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGATCTCTGGGCC

GATACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTA

GTCCACGCCGTAAACGGTGGGCACTAGGTGTGGGCGACATTCCACGTCGTCCGTGCCG

CAGCTAACGCATTAAGTGCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAG

GAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGTGGCTTAATTCGACGCAACGCGA

AGAACCTTACCAAGGCTTGACATACACCGGAAACGTCTGGAGACAGGCGCCCCCTTGTG

GTCGGTGTACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAA

GTCCCGCAACGAGCGCAACCCTTGTCCCGTGTTGCCAGCAAGCCCTTCGGGGTGTTGGG

GACTCACGGGAGACCGCCGGGGTCAACTCGGAGGAAGGTGGGGACGACGTCAAGTCA

TCATGCCCCTTATGTCTTGGGCTGCACACGTGCTACAATGGCCGGTACAATGAGCTGCG

ATACCGCGAGGTGGAGCGAATCTCAAAAAGCCGGTCTCAGTTCGGATTGGGGTCTGCA

ACTCGACCCCATGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCATTGCTGCGGTGAAT

ACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCACGAAAGTCGGTAACACCCGAAG

CCGGTGGCCCAACCCCTTGTGGGAGGGAGCTGTCGAAGGTGGGACTGGCGATTGGGA

CGAAGTC

Claims

1. Method for the isolation of bacterial strains deposited at the CCMM under No. CCMM PI, CCMM P2 and CCMMP 3 characterized by the sequences Seq NI, SeqN4, & SeqN3

2. Use of the strains according to claim 1 or mutant strains, for inoculating perlite particles or potassium rocks or seeds and obtaining biostimulants.

3. Use of the strains of claim 1 or of potassium rock particles, perlite particle or inoculated seeds obtained according to claim 2, for the degradation of waxes and the solubilization of potassium to boost the resistance of plants to water and osmotic stress. .

4. Use of the strains of claim 1 or mutant strains, for obtaining active compounds by fermentation in a liquid medium of said strains after centrifugation, filtration and purification.

5. Use of the strains of claim 1 or mutant strains, or of a filtrate of cultures obtained according to claim 4 or of an active compound obtained according to claim 4.

6. Use of the strains of claim 1, or of a filtrate of cultures or active compounds obtained according to claim 4 or of an active compound obtained according to claim 5

7. Use of the strains of claim 1 or mutant strains, or of a filtrate of cultures obtained according to claim 4 or of an active compound obtained according to claim 6, for stimulating the growth of plants.