WO2016009397A1

WO2016009397A1 - Biofertilizer, method for its preparation and uses thereof

Info

Publication number: WO2016009397A1
Application number: PCT/IB2015/055424
Authority: WO
Inventors: Daniela Maria Spera; Marisa TERRERI; Emanuela SERVO; Matteo DI MUZIO
Original assignee: Consorzio Di Ricerche Applicate Alla Biotecnologia
Priority date: 2014-07-18
Filing date: 2015-07-17
Publication date: 2016-01-21

Abstract

The present invention relates to a dry biofertilizer in the form of granules based on alginate and a PGPM(Plant Growth Promoting Microorganisms)-conditioned medium. In some embodiments, in addition to the alginate and the PGPM-conditioned medium, the biofertilizer also comprises PGPMs entrapped/encapsulated within the granule. The plant growth promoting microorganism ( PGPM) is selected from among: Azospirillum brasilense, Gluconacetobacter diazotrophicus, Burkholderia cepacia and Herbaspirillum seropedicae; preferably, it is Azospirillum brasiliense. In particular, the biofertilizer of the present invention is obtained from an alginate mixture dissolved in a PGPM-conditioned medium and the mixture is then allowed to drip into a solution of CaCl₂ in such a way as to obtain granules. Optionally, PGPMs are also added to the mixture and are thus retained within the granule. Furthermore, the present invention relates to the use of said biofertilizer in agriculture, in particular to increase the vegetable biomass and/or productivity of plants.

Description

"BIOFERTILIZER, METHOD FOR ITS PREPARATION AND USES

THEREOF"

DESCRIPTION

The present invention relates to an alginate-based biofertilizer in the form of granules, a PGPM-conditioned medium and optionally also PGPMs. Furthermore, the present invention relates to a method for producing said biofertilizer granules and the use thereof in agriculture to increase the vegetable biomass and/or plant productivity.

Plant growth and crop productivity is largely influenced by the microflora residing in the soil surrounding the plants, in particular, the microflora of the rhizosphere.

The microflora comprises bacteria, fungi, algae and yeast. Some bacteria are harmful because they negatively impact plant growth. In contrast, other bacteria, called PGPMs (Plant Growth Promoting Microorganisms), are capable of colonizing, in particular, the root apparatus and promoting plant growth, for example by positively influencing nitrogen fixation or the mobilization of recalcitrant soil nutrients such as sulphur or iron. PGPMs have aroused the interest of the scientific community because of the possibility of using them as natural biofertilizers in replacement of or alongside classic chemical fertilizers that are very dangerous for the environment.

In this context, in recent years, most efforts have been made with the aim of identifying new PGPM-based biofertilizers and/or evaluating their compatibility with the commercially relevant crops presently available. Biofertilizers contain live microorganisms and the majority of them are liquids, so that in order to prevent the death of the microorganisms, they must be immediately and often repeatedly applied to assure good colonization of the soil by the PGPMs.

To remedy these problems, dehydrated biofertilizers have been developed by encapsulating the microorganisms within polymers.

In this regard, the object of the present invention is to provide a dry PGPM-based biofertilizer or derivatives which impart a longer half-life to the PGPMs, serves to increase the PGPM concentration at the root level, disperses the least possible amount of PGPMs in the soil and reduces the competition of PGPMs with the other soil microorganisms.

The biofertilizer of the present invention is a dry biofertilizer in the form of granules based on alginate and a PGPM-conditioned medium. In some embodiments, in addition to the alginate and the PGPM-conditioned medium, PGPMs entrapped/encapsulated within the granule are also present.

In particular, the biofertilizer of the present invention is obtained from an alginate mixture dissolved in a PGPM-conditioned medium and the mixture is then allowed to drip into a solution of CaCI₂ in such a way as to obtain granules. Optionally, PGPMs are also added to the mixture and are thus retained within the granule.

In a main embodiment, the biofertilizer of the present invention advantageously serves to enhance the development of the vegetable biomass, improving the majority of the critical parameters as a result (for example, the length of the stem, of the roots and of the plant, the number of leaves, the weight of the plant etc.), without using live microorganisms, such as PGPMs, and thus avoiding the problems related to maintaining the number and viability of the PGPMs. In fact, the Applicant has found that the substances contained in the PGPM-conditioned medium (essentially phytohormones) enable plant growth and productivity to be stimulated without any need for there to be any microorganisms present. Furthermore, the Applicant has surprisingly found that, in the embodiment in which the biofertilizer granule also comprises PGPMs, it advantageously entraps up to 10⁹ PGPMs, i.e. each biofertilizer granule is capable of containing a number of PGPMs that is more than sufficient for the demands of each seed. Advantageously, the half-life of the PGPMs within the granule of the present invention is longer than in the presently available dry biofertilizers because, even when, during storage, the granule becomes hydrated, the substances contained in the conditioned medium preserve the viability and functionality of the microorganisms. This means an increase in the concentration of viable PGPMs at the level of the roots. Furthermore, the PGPMs are well entrapped inside the granule and thus the bacteria are not dispersed in the soil; this reduces PGPM competition with other soil microorganisms.

Finally, a further non-negligible advantageous aspect of the biofertilizer of the present invention is that the PGPMs entrapped/encapsulated in the granules are tolerant to biotic and abiotic stresses.

The invention will be described below in a detailed manner and will be illustrated, for non-limiting demonstrative purposes, also with the aid of the following figures, in which:

- Figure 1 shows the length of the plants (A) and the length of the stem (B, i.e. the length from the longest leaf of the collar) of plants of Lactuca sativa germinated from: seeds alone (Ctrl); seeds + alginate-based granules + Azospirillum brasilense-conditioned medium and Azospirillum brasilense (Trial 1 ); seeds + alginate- based granules + Azospirillum brasilense -conditioned medium (Trial 2); and seeds + alginate-based granules and Azospirillum

brasilense (Trial 3).

- Figure 2 shows the length of the collar (A) and the length of the main root (B) of plants of Lactuca sativa germinated from: seeds alone (Ctrl); seeds + alginate-based granules + Azospirillum brasilense -conditioned medium and Azospirillum brasilense (Trial 1 ); seeds + alginate-based granules + Azospirillum brasilense- conditioned medium (Trial 2); and seeds + alginate-based granules and Azospirillum brasilense (Trial 3).

- Figure 3 shows the weight of whole plants (A) and of plants without roots (B) of Lactuca sativa germinated from: seeds alone (Ctrl); seeds + alginate-based granules + Azospirillum brasilense- conditioned medium and Azospirillum brasilense (Trial 1 ); seeds + alginate-based granules + Azospirillum brasilense -conditioned medium (Trial 2); and seeds + alginate-based granules and Azospirillum brasilense (Trial 3).

- Figure 4 shows the weight of the roots and the number of leaves of plants of Lactuca sativa germinated from: seeds alone (Ctrl); seeds

+ alginate-based granules + Azospirillum brasilense -conditioned medium and Azospirillum brasilense (Trial 1 ); seeds + alginate- based granules + Azospirillum brasilense -conditioned medium (Trial 2); and seeds + alginate-based granules and Azospirillum brasilense (Trial 3).

The present invention relates to a biofertilizer composition/formulation comprising an alginate or a salt thereof, preferably sodium alginate, and a PGPM-conditioned medium. The composition/formulation is preferably dry (dehydrated), more preferably in the form of granules. Optionally, the biofertilizer composition/formulation further comprises one or more PGPMs.

Therefore, the biofertilizer of the present invention is definable as a natural fertilizer which in some embodiments preferably consists in granules of alginate and PGPM-conditioned medium, and in other embodiments consists in granules of alginate and PGPM-conditioned medium in which PGPMs are contained/entrapped.

In the context of the present invention, granule means a capsule, a pellet, a pill or a dry (dehydrated) bead of alginate and PGPM-conditioned medium, preferably of alginate dissolved in a PGPM-conditioned medium. In some embodiments, said granule further comprises PGPMs entrapped inside the granule.

PGPM-conditioned medium means the supernatant of a PGPM culture. That is, the PGPMs are cultured, using the normal techniques known to every person skilled in the art, in a suitable culture medium, for example buffered Luria-Bertani (LB) broth, for a predetermined time (for example about 24-48 hours) at a suitable temperature typical of each PGPM (for example about 30°C for Azospirillum brasilense).

At the end of culturing, the PGPMs are allowed to settle, preferably by centrifugation, so as to obtain a bacterial phase and a culture broth phase without bacteria. The culture broth without bacteria is also known as a supernatant and the supernatant, collected after a certain period of culturing the bacteria, is called a bacteria-conditioned medium (agar or broth; in this case PGPM-conditioned) because it contains the metabolites secreted by or in any case derived from the microorganisms during the culturing thereof.

More specifically, the PGPM-conditioned medium is rich in phytohormones (phytostimulating agent), preferably hormones such as auxins, most preferably indoleacetic acid. The quantity of phytohormones contained in the PGPM-conditioned medium preferably ranges from 15 to 60 ppm, more preferably it ranges from 20 to 40 ppm, even more preferably from 28 to 36 ppm.

The concentration of phytohormones such as indoleacetic acid is normally calculated using the method of Loper and Scroth (1986). In particular, an aliquot of the culture broth is removed and centrifuged. The recovered supernatant is mixed with orthophosphoric acid (usually two drops of about 40 μΙ of orthophosphoric acid) and with a Salkowski-reactive material. The appearance of a pink colour indicates the presence of indoleacetic acid in the supernatant. At this point the absorbance of the sample is read by using a spectrophotometer set to a wavelength of 530 nm.

As said previously, PGPMs are microorganisms that promote plant growth, and are generally present in the soil rhizosphere. For the purposes of the present invention, the PGPMs preferably used are selected from among: Azospihilum brasilense, Gluconacetobacter diazotrophicus, Burkholdeha cepacia and Herbaspirillum seropedicae.

The PGPMs that are preferred for the purposes of the present invention are of the genus Azospirillum, preferably of the species Azospirillum brasilense. The granule has an average size preferably in the range of 1 mm to 2 mm in diameter.

Preferably, the quantity of PGPMs (expressed in CFU, i.e. "colony-forming units") per capsule/granule ranges from 0.5-3x10⁹, more preferably 0.5- 1 .5x10⁹, even more preferably 0.75-1 .5 x10⁹, even more preferably 1 - 3x10⁹ CFU per millilitre.

A further aspect of the present invention relates to a method for obtaining a biofertilizer in granules, said method comprising the steps of:

i. Preparing a culture of PGPMs;

ii. Obtaining the PGPM-conditioned medium;

iii. Preparing a matrix comprising an alginate or a salt thereof and said

PGPM-conditioned medium;

iv. Allowing the matrix obtained in step (iii) to drip into a solution of

CaCl₂, preferably under stirring, in order to obtain granules; and v. Drying the granules obtained in step (iv).

Alternatively, in step (iii) a matrix comprising an alginate or a salt thereof, the PGPM-conditioned medium and the PGPMs is prepared.

Preferably, the matrix can comprise a nutrient for the bacteria, for example a milk-based nutrient such as skim milk, or starch, dextrose, yeast extract, peptone bacteriological, or nutrient broth.

The step (i) of culturing the PGPMs is carried out using the normal techniques known to every person skilled in the art. In particular, the PGPMs are cultured in a suitable culture medium, for example buffered Luria-Bertani (LB) broth, for a predetermined time, preferably around 24- 48 hours), at a suitable temperature typical of each PGPM; for example, for Azospirillum brasilense the culture is preferably grown at about 30°C. Before the culture is prepared, usually in a large volume, it is preferable to pre-cultivate the PGPMs on a small scale and then add the pre-culture in a larger volume for the actual culture.

Step (ii) is carried out at the end of culturing of the PGPMs, which are allowed to settle, preferably by centrifugation, so as to obtain a bacterial phase (i.e. a bacterial pellet) and a culture broth phase without bacteria. Preferably, the bacterial pellet is re-suspended in a culture medium, preferably peptone-based, so that the bacterial suspension comprises 0.5- 3x10⁹, more preferably 0.5-1 .5x10⁹, even more preferably 0.75-1 .5 x10⁹, even more preferably 1 -3x10⁹ CFU per millilitre.

The culture broth without bacteria is also known as a supernatant and the supernatant, collected after a certain period of culturing the bacteria, is called a bacteria-conditioned medium (agar or broth; in this case PGPM- conditioned) because it contains the metabolites secreted by or in any case derived from the microorganisms during the culturing thereof.

More specifically, the PGPM-conditioned medium is rich in phytohormones (phytostimulating agent), preferably hormones such as auxins, most preferably indoleacetic acid. The quantity of phytohormones contained in the PGPM-conditioned medium contained in the PGPM-conditioned medium preferably ranges from 20-50 ppm, more preferably it ranges from 25-40 ppm, even more preferably from 30 to 35 ppm.

Step (iii) comprises preparing a matrix comprising an alginate or a salt thereof and the PGPM-conditioned medium as described above.

As an alternative to the alginate or a salt thereof and the PGPM- conditioned medium, it is also possible to add the PGPMs, i.e. the bacterial suspension obtained by re-suspending the bacterial pellet obtained from the PGPM culture as described above. In other words, a suspension comprising 0.5-3x10⁹, more preferably 0.5-1 .5x10⁹, even more preferably 0.75-1 .5 x10⁹, even more preferably 1 -3x10⁹ CFU per millilitre is added to the matrix of alginate (or a salt thereof) and PGPM-conditioned medium.

These two alternatives make it possible to obtain biofertilizer granules based on alginate and PGPM-conditioned medium and biofertilizer granules based on alginate, PGPM-conditioned medium and PGPMs. During step (iv), the matrix obtained according to step (iii) is allowed to drip, preferably using a syringe or a pipette, into a solution of CaC^, preferably under stirring, to obtain granules. The solution of CaCI₂ preferably has a concentration of 1 -2M.

Once the drops of the matrix have fallen into the solution of CaC^, the alginate will cross-link in a time of more or less 30-40 minutes.

Step (v) comprises drying the granules, preferably at about 25°C, most preferably for about 12-24 hours.

Therefore, a further aspect of the present invention is a biofertilizer in the form of granules obtained with the method described above.

The biofertilizer of the present invention is particularly useful in the agricultural sector, in particular for stimulating plant growth and plant productivity.

For this purpose the granules of the present invention can be distributed in the soil where the vegetable varieties/plants of interest are planted/grown, or else it is possible to bind the seeds to the granules, preferably in a 1 :1 ratio, for example using a vegetable oil.

Therefore, a further aspect of the present invention is a method per stimulating/improving plant growth and/or increasing the productivity thereof which comprises a step of associating, preferably binding, the biofertilizer granules of the present invention to the seeds of the vegetable varieties/plants of interest. Binding is preferably achieved by using a vegetable oil. Preferably, the ratio between granules and seeds is 1 :1 . Preparation of the bacterial culture

The plant growth-promoting microorganism (PGMP) used in the present invention is, by way of example, Azospirillum brasilense.

The bacteria were pre-cultured in 20 ml of buffered Luria-Bertani (LB) broth. The pre-culture was incubated at 30°C for 24 hours. The pre-culture of PGPMs was transferred into 200 ml of buffered LB broth and incubated at 30 °C, under stirring at 1 10 rpm, for 48 hours.

After 48 hours, the bacterial cells were collected by centrifugation at 5000 rpm (4192 g) for 20 minutes at 25 °C, washed twice with a saline solution containing 0.8% NaCI and for each wash they were collected by centrifugation at 5000 rpm (4192 g) for 20 minutes at 25 °C.

The bacterial pellet was re-suspended in 8 ml of a 1 % peptone solution. The concentration of bacteria per millilitre was about 1 .5 x 10⁹ CFU/ml. The supernatant, i.e. the medium conditioned by the PGPM, in this case Azospirillum brasilense, was set aside (at 4 °C) so as to be used in the subsequent encapsulation step.

Both the saline solution and the peptone solution were sterilized prior to use at 121 °C for 20 minutes.

Encapsulation of the PGPMs

The process for producing capsules (granules, beads, pills, pellets) of alginate was carried out with two methods.

The encapsulation matrix used with both methods is made up of a solution comprising 2% sodium alginate and 0.75% skim milk (any other bacterial nutrient can be used).

The matrix is sterilized at 121 °C for 20 minutes before being used.

According to one method, the sodium alginate and skim milk are dissolved in the supernatant of the bacterial culture, i.e. in the PGPM-conditioned medium obtained in the previous step.

In the specific case in which Azospirillum brasilense is used as the PGPM, the supernatant contains the auxin indol-3-acetic acid (IAA) produced by the PGMP, which is capable of stimulating plant growth and, in particular, the development of the root apparatus. The quantity of IAA present in the conditioned medium obtained under the growth conditions indicate is about 32ppm).

The alternative method comprises also adding the suspension of bacterial cells (i.e. the PGPMs, in this case Azospirillum brasilense) to the matrix of sodium alginate and skim milk dissolved in the PGPM-conditioned medium, under aseptic conditions.

The matrix prepared with and without bacterial cells is loaded into a syringe and allowed to drip into a solution of CaC 1 M, placed under stirring by means of a magnetic stirrer (60ml of matrix in 200ml of CaC 1 M).

The entire process is carried out under aseptic conditions.

After 30 minutes, i.e. the time necessary for the cross-linking of the alginate, the capsules/beads/granules of alginate formed are washed twice with sterile distilled water.

Encapsulation was also achieved by dissolving the alginate in distilled water, applying the protocol proposed by Ivanova E. et al (2005).

Drying of the beads

The alginate beads were placed in a Petri dish and allowed to dry overnight in a temperature-controlled oven at 25 °C.

Evaluation of the efficacy of the alginate beads

The alginate beads obtained were then bound to lettuce seeds (Lactuca sativa), in such a way that an individual bead was bound to each seed, by using condensed cedarwood oil, which is a mineral oil. In particular, the seeds were immersed in the mineral oil and subsequently placed on the alginate beads.

The concentration of bacteria in the alginate beads was 1 .5 x 10⁹ CFU/ml and was determined on the basis of a viable cell count using serial dilutions and seeding on plated LB.

The following trials were set up to test efficacy:

Trial Sample 1 : seeds of Lactuca sativa + alginate bead prepared with the PGPM-conditioned medium specifically containing IAA (32 ppm) + PGPM in this case Azospirillum brasilense; e

- Trial Sample 2: seeds of Lactuca sativa + alginate bead prepared with the PGPM-conditioned medium specifically containing IAA (32 ppm); and

Trial Sample 3: seeds of Lactuca sativa + alginate bead containing Azospirillum brasilense obtained with Ivanova's method. - Control (CTRL): seeds of Lactuca sativa.

21 seedlings were evaluated per series. The parameters measured in order to evaluate the efficiency of the microbial inoculants encapsulated in the alginate beads were:

The length of the whole plant (in centimetres);

The length of the stem from the collar to the longest leaf (in centimetres);

The length of the collar (in centimetres);

The length of the main root (in centimetres);

The weight of the plant (in grams);

The weight of the plant without roots (in grams);

- The weight of the roots (in grams); and

The number of leaves.

The concentration of phytohormones such as indoleacetic acid is normally calculated using the method of Loper and Scroth (1986). In particular, an aliquot of the culture broth is removed and centrifuged at about 5000 rpm (4192 g) for about 30 minutes. The recovered supernatant is mixed with orthophosphonc acid (usually two drops of about 40 μΙ of orthophosphonc acid) and with a Salkowski-reactive material. The appearance of a pink colour indicates the presence of indoleacetic acid in the supernatant. At this point the absorbance of the sample is read by using a spectrophotometer set to a wavelength of 530 nm. To determine the concentration of indoleacetic acid, a calibration curve is also constructed. The calibration curve was prepared according to the method of Vasanthakumar and McManus (2004). 5.7 mg of IAA 5.7 mg of IAA powder was then prepared and brought to a final volume of 50 ml with distilled H2O, after the addition of 2 drops of NaOH 2 M to facilitate the solubilization of the IAA. Successive serial dilutions were made from the mother solution, containing 1 14 ppm (ppm stands for "parts per milions" and can indicate, for example, the milligrams per litre) of IAA; furthermore, for each dilution, a reading was taken of the absorbance at 530 nm versus distilled H2O, after the addition of the Salkowski-reactive material and orthophosforic acid, in glass cuvettes. Results

The results associated with each of the parameters measured for the controls and the trial sample are shown in Table I and in Figs. 1 -4.

Statistical analysis of the results

The averages of the different groups were compared to groups of two variables, using the T-test.

The hypothesis (HO) that the sample of Trial 1 was equal to the control was tested, with a 95% confidence level. The hypothesis (HO) that the sample of Trial 2 was equal to the control was tested, with a 95% confidence level. The hypothesis (HO) that the sample of Trial 3 was equal to the control was tested, with a 95% confidence level.

The results of the test of statistical significance for each of the measured parameters are shown in Tables II, III and IV.

Table II

The microorganisms indicated as PGPMs are promoters of plant growth. Numerous field studies have positively evaluated the use of PGPMs in agriculture in the role of biofertilizers, which provide plants not only with micro and macro nutrients, but also with natural substances acting as phytohormones.

A great deal of research has been conducted on the effects exerted by the inoculation of microbial consortia containing PGPM on plants and the studies have highlighted the ability of such microbial consortia to enhance the development of the vegetable biomass.

The results shown above demonstrate that the treatment with granules (capsules) based on alginate, PGPM-conditioned medium and PGPMs (Trial 1 ), and the one carried out with granules (capsules) based on alginate and PGPM-conditioned medium (Trial 2) are both capable of improving all of the parameters measured, in a statistically significant manner compared to the controls, with the exception of collar length (in this case the controls are significantly better than the treated plants).

The treatment with granules (capsule) based on alginate, PGPM- conditioned medium and PGPMs (Trial 1 ) is capable of improving all of the parameters measured to a significant degree compared to the treatment carried out with alginate-based granules and PGPMs (Trial 3, the granules prepared with Ivanova's method, in which the alginate is dissolved in distilled water).

The treatment with granules (capsules) based on alginate and PGPM- conditioned medium (Trial 2) is slightly less efficacious than the treatment carried out with alginate-based granules and PGPMs (Trial 3, i.e. the granules prepared with Ivanova's method, in which the alginate is dissolved in distilled water). However, these granules have the advantage of not containing living materials (i.e. PGPMs) and can therefore be used in situations in which the viable component of PGPMs can create problems, for example in some applications which require that the presence of a viable microorganism be excluded, for example because the conditions of transport of the composition/formulation are not conducive to the viability of microorganisms, or because use of a composition/formulation containing live microorganisms is expressly precluded.

It is interesting to note that the parameters that are most increased through both treatment methods, compared to the controls, are: length of the main root and weight of the whole root apparatus; this greater development of the root apparatus of treated plants was already evident upon visual analysis, which made it possible to observe a greater ramification, as well as a greater robustness and thickness of the roots. The advantages of this type of technology are:

•Greater concentration of PGPMs at the level of the plant roots;

"Less dispersion of PGPMs in the ground;

•Reduction in competition with other soil microorganisms; and

•A longer half-life of the PGPMs.

Claims

1. A biofertilizer composition/formulation comprising at least an alginate or a salt thereof and at least a PGPM-conditioned medium, said composition being formulated as granules, beads, capsules, pills or pellets.

2. The composition according to claim 1 , further comprising at least a PGPM and/or at least a nutrient for microorganisms, preferably milk.

3. The composition according to claim 1 or 2, wherein said PGPM is selected from among: Azospirillum brasilense, Gluconacetobacter diazotrophicus, Burkholderia cepacia and Herbaspirillum seropedicae; preferably, it is Azospirillum brasiliense.

4. The composition according to any one of claims 1 -3, wherein the concentration of said PGPM ranges from 0.5-3x10⁹, preferably 0.5- 1 .5x10⁹, more preferably 0.75-1 .5 x10⁹, still more preferably 1 - 3x10⁹ CFU per millilitre.

5.

The composition according to any one of claims 1 -4, wherein said conditioned medium comprises at least a phytostimulating agent, preferably indoleacetic acid.

6.

The composition according to claim 5, wherein the concentration of said phytostimulating agent in said conditioned medium ranges from 15 to 60 ppm; preferably, it ranges from 20 to 40 ppm, more preferably from 28 to 36 ppm.

7.

The composition according to any one of claims 1 -6, wherein the dimensions of said granules, beads, capsules, pills or pellets range from 1 mm to 2mm.

8. A process for obtaining the composition according to any one of claims 1 -7 comprising the steps of:

(i) Preparing a culture of PGPM;

(ii) Obtaining the PGPM-conditioned medium;

(iii) Preparing a matrix comprising an alginate or a salt thereof and said PGPM-conditioned medium;

(iv) Allowing the matrix obtained in step (iii) to drip into a solution of CaCl₂, preferably under stirring, in order to obtain granules; and

(v) Drying the granules obtained in step (iv).

9. A method according to claim 8 wherein the matrix of step (iii) comprises an alginate or a salt thereof, the PGPM-conditioned medium and at least one PGPM.

10. The method according to claim 8, wherein the step (II) comprises further adding to said conditioned medium and to said matrix, a suspension of PGPM at a concentration that preferably ranges from 0.5-3x10⁹, preferably 0.5-1 ,5x10⁹, more preferably 0.75-1 .5 x10⁹, still more preferably 1 -3x10⁹ CFU per millilitre.

1 1 . The method according to any one of claims 8-10, wherein the matrix according to step (iii) further comprises a nutrient for microorganisms, preferably milk.

12. A use of the composition according to any one of claims 1 -7 in agriculture, preferably to increase the vegetable biomass and/or productivity of plants.