WO2023167579A1

WO2023167579A1 - Endophytic bacterial strains, probiotic mixtures, formulation and method, for stimulating the filling of plant fruits

Info

Publication number: WO2023167579A1
Application number: PCT/MX2022/050117
Authority: WO
Inventors: Cristóbal FONSECA SEPÚLVEDA; Gloria Margarita MACEDO RAYGOZA
Original assignee: Fonseca Sepulveda Cristobal
Priority date: 2022-03-04
Filing date: 2022-11-29
Publication date: 2023-09-07
Also published as: WO2023167579A9; MX2022002758A

Abstract

The invention relates to the Caulobacter vibrioides strain with deposit number CM-CNRG TB186, the Providencia rettgeri strain with deposit number CM-CNRG TB187, and the Bacillus pumilus strain with deposit number CM-CNRG TB188. These strains are endophytes of the plant Vaccinium corymbosum from which they have been isolated and have agronomic potential due to the properties they exhibit. The invention also relates to mixtures of at least two of the bacteria, formulations containing one of them with at least one prebiotic substance and at least one diluent and a method for stimulating plant growth by applying the formulation.

Description

ENDOPHYTIC BACTERIAL STRAINS, PROBIOTIC MIXTURES, FORMULATION AND METHOD, TO STIMULATE THE FILLING OF VEGETABLE FRUITS

TECHNICAL FIELD OF THE INVENTION

The present invention is related to the technical field of Biotechnology and Agriculture, since it provides endophytic bacterial strains isolated from Vaccinium corymbosum (blueberry), probiotic mixtures that comprise the bacterial strains, formulation and method, to stimulate the filling of vegetable fruits. , through the use of the formulation, which contains the bacterial strains.

BACKGROUND OF THE INVENTION

Blueberry cultivation is currently widespread in countries such as China, Japan, Chile, New Zealand, Argentina and Mexico. According to the FAO, the global area of the crop has increased by approximately 15,000 ha between 2010 and 2014. Mexico is among the countries that have promoted the cultivation of this shrub. In 2019, SIAP reported the amount of 3786.45 ha planted with blueberries in Mexico, mainly in the states of Jalisco, Michoacán, Sinaloa and Puebla. The indiscriminate use of chemical products for the harvest of this crop has led producers to opt for an organic production system, using organic sources to supply the nutritional requirements of the plant. This decision of an organic treatment is reflected in the added value of the product, however, it presents two restrictions: the use of herbicides and the few sources of nitrogen contribution for the crop.

The use of microorganisms as biofertilizers has emerged as an alternative since it is not only friendly to the environment, but also as an alternative to increase crop yield, in addition to providing the different nutrients, including Nitrogen, for a correct filling of vegetable fruits.

The patent document WO2015118516 (A1) refers to soil bacteria to inoculate stressed soils. Among the strains that the invention provides is an exopolysaccharide-producing AP strain, preferably selected from the group consisting of Bacillus simplex S 28 NCAIM (P) B 001432, Kocuria rosea S 225 NCAIM (P) B, 001426, Leuconostoc mesenteroides K2009 25/ 4 NCAIM (P) B 001372, Pseudomonas chlororaphis K2009 13/4 B NCAIM (P) B 001370, and Pseudomonas chlororaphis K2009 9/4-B NCAIM (P) B 001371. This document WO2015118516 (A1) already proposes the use of strains bacteria for stressed soils, where strains of the genera Bacillus spp. and Kocuria spp., but no strain corresponds to the species proposed by the present invention. Furthermore, this document makes no reference to the isolation of any strain of the genus Caulobacter spp. The purpose of the strains in this document is very different from the use proposed by the present invention. Furthermore, these strains were isolated from soils outside of Mexico, so their growth and invasion characteristics of microorganisms are determined by their genetic constitution and the environment (geographical region) where they developed. Therefore, the bacterial strains of the document WO2015118516 (A1) will not have the same effectiveness of growth, invasion and promotion of the filling of vegetable fruits, if they were used in Mexican soils. That is why there is great interest in isolating, validating the possible antagonistic characteristics and promoting the filling of vegetable fruits, of endophytic bacterial strains in Mexico, to guarantee a high effectiveness of said isolated strains.

The patent document CN201710185079 (A) describes biological fungicides to prevent and control diseases and pests in fruit trees. Biological fungicides may comprise a mixture composed of a mixture of strains of the genus Bacillus spp., a culture medium, and a carrier. The mixture of bacilli comprises: Bacillus licheniformis, Bacillus pumilus and Bacillus thuringiensis. As you can see this patent document only proposes the use of strains of the genus Bacillus spp., and they are to control diseases and pests in fruit trees, so they are not specifically intended to promote the filling of vegetable fruits. Furthermore, if these bacterial strains originate from the People's Republic of China, they will hardly have the same effect in promoting the filling of vegetable fruits.

The patent document KR2007137133 (A) discloses a method of cultivating fruits with a high content of vitamin and calcium by means of a foliar spray of a fermented liquid using seaweed, sugar, powdered sugar derived from cornstarch, garlic, fishmeal and similar to improve the added value of the fruit at low cost. The method comprises preparing fermented seaweed powder, with sugar, powdered sugar derived from cornstarch is dissolved in water and fermented with a culture of Bacillus subtilis ATCC 9372 to obtain a fermented seaweed extract. The crushed garlic and water are heated in a double boiler, filtered, mixed with sugar, powdered sugar derived from cornstarch and a culture of Lactobacillus acidophilus ATCC 9224 and fermented to give fermented garlic extract. The fermented seaweed extract is mixed with the fermented garlic extract in a 1:51 weight ratio and foliar sprayed 3 times at a time interval of 6 weeks prior to harvest. Blueback fish meal is mixed with a culture of Lactobacillus acidophilus ATCC 9224, sugar and yeast extract and fermented to blueback fish extract. The fish powder is dissolved in water, enzymatically degraded with a protease and filtered to give an enzymatic fish hydrolyzate. Blueback Fish Powder is mixed with Enzymatic Fish Hydrolyzate at a 1 to 51 weight ratio and foliar sprayed before and after the rainy season and 5 to 4 weeks before harvest. Seaweed powder, sugar, and cornstarch-derived powdered sugar are dissolved in water, fermented with a culture of Bacillus pumilus ATCC 7061, foliar sprayed after flowering, 2 weeks after the first spray, and after the fruit set. Document KR2007137133 (A) departs from the spirit of the present invention, since the bacterial strains that are used are to generate a fermented extract, the which is mixed with other substances, so the strains are not applied live to the plants.

Therefore, with the purpose of contributing to the solution of the aforementioned drawbacks, endophytic bacterial strains of Vaccinium corymbosum (blueberry) plants were isolated, which showed stimulation in the filling of vegetable fruits; Therefore, probiotic mixtures with said endophytic strains, formulations and a method were also developed to stimulate the filling of vegetable fruits.

The characteristic details of the present invention are clearly shown in the following description and examples, which are attached merely for the purpose of illustrating some embodiments of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The object of the present invention is an isolated strain of Caulobacter vibrioides identified as B18, characterized in that it comprises the characteristics of the strain deposited under accession number CM-CNRG TB186.

The object of the present invention is also an isolated strain of Providencia rettgeri identified as B105, characterized in that it comprises the characteristics of the strain deposited under accession number CM-CNRG TB187.

Said invention also has as its object an isolated strain of Bacillus pumilus identified as B124, characterized in that it comprises the characteristics of the strain deposited under accession number CM-CNRG TB188.

The bacterial strains, isolated and deposited: CM-CNRG TB186 (Caulobacter vibrioides), CM-CNRG TB187 (Providencia rettgeri), and CM-CNRG TB188 (Bacillus pumilus) where said bacterial strains are endophytes of plants of Vaccinium corymbosum (blueberry); and it was found that the bacterial strains exhibit stimulating activity in the filling of vegetable fruits, for example, in vegetable fruits they belong to plants of the Ericaceae family, specifically in vegetable fruits that are from plants that belong to the genus Vaccinium, and more specifically in vegetable fruits of plants belonging to the species Vaccinium corymbosum.

One more object of the present invention is a probiotic mixture, which can comprise at least two isolated bacterial strains, in accordance with the bacterial strains described in the present invention.

The mixture of the previous claim, wherein said bacterial strains are endophytes of Vaccinium corymbosum (blueberry) plants.

Yet another object of this invention is a formulation to stimulate the filling of vegetable grains, which may comprise: at least one isolated bacterial strain, which is selected from the following group of deposited strains: CM-CNRG TB186 (Caulobacter vibrioides), CM-CNRG TB187 (Providencia rettgeri), and CM-CNRG TB188 (Bacillus pumilus), and their mixtures between them; at least one prebiotic substance; and at least one diluent.

An embodiment of the formulation of the present invention is where the bacterial strain, either individual or mixed, is in an amount of 0.01%, the prebiotic substance in 4.99%, and the diluent in 95%, with respect to the total volume of the formulation.

Said invention also has as its object a method for stimulating the filling of vegetable fruits, which comprises, applying to vegetable fruits, a sufficient quantity of a formulation to stimulate the filling of vegetable fruits, in accordance with the present invention. A variant of the method to stimulate growth in vegetable plants is when the sufficient quantity of the formulation is 1 L for every 4,166 vegetable plants of Vaccinium corymbosum.

One more embodiment of the method in question, is when the application of the formulation is in the stage called "fruit mooring" which is when the plants have already flowered and pollination has taken place and the fruits begin to form.

A further embodiment of said method for stimulating growth in vegetable plants, is when the vegetable plants are from the Ericaceae family, preferably from the Vaccinium genus, and more preferably from the Vaccinium corymbosum species.

EXAMPLES

The following examples are included for the sole purpose of demonstrating the conception of the present invention and some preferred embodiments, so they should not be considered as a limitation for the scope of protection of the present invention.

Example 1 . Isolations of endophytic bactrian strains of Vaccinium corymbosum (blueberry) plants.

A symbiosis study was established in different regions of the state of Jalisco, between the months of September 2020 and July 2021, with the purpose of evaluating the potential of bacterial strains in the production of auxins and production of the inhibition hormone of acetylene, ACC deaminase. The strains were isolated from Vaccinium corymbosum (blueberry), using the endophyte extraction method, the tissues from which the strains were isolated were leaves, stem, root, flower, fruit and petiole because in the literature it has been found that these strains are concentrated there. A total of 3 strains of the genera Bacillus (1 strain), Caulobacter (1 strain) and Providencia (1 strain) were isolated from a total of 145 isolated strains, because these strains showed greater production of ACC deaminase, an enzyme that inhibits the production of ethylene, the fruit ripening molecule, more obtaining micronutrients such as Fe, in addition to the greater production of plant growth hormones such as auxins.

Specifically, the 3 bacterial strains were selected because they showed the following activities:

- Caulobacter vibrioides B18 (Proteobacteria), identified as B18, was selected because it fixes nitrogen, produces siderophores, produces oxidases, hydrocyanic acid, ammonium and chitinases, as well as ACC deaminase and is the strain that produces the most polyhydroxybutyrate.

- Providencia rettgeri B105 (Proteobacteria), identified as B105, was selected because it fixes nitrogen, produces siderophores, hydrocyanic acid, ammonium, chitinases, ACC deaminase, and catalases.

- Bacillus pumilus B124 (Firmicutes), identified as B124, was selected because it fixes nitrogen, solubilizes phosphates, produces siderophores, produces catalases, chitinases, ammonium, ACC deaminase, polyhydroxybutyrate, glucanases, and is the one that produces the highest amount of proteases.

Example 2. Agronomic tests of the 3 isolated bacterial strains.

The following agronomic tests were performed on the selected blueberry isolates: nitrogen fixation test (NFB), phosphate solubilization test (NBRIP Ca, and NBRIP Fe), iron chelation test (siderophores), production test phytohormones (auxins) and ACC deaminase (ACC) enzyme production test. In this case, as our main objective was to select microorganisms that induce plant growth, we emphasized the production of auxins, since this is a phytohormone related to growth, as well as nitrogen fixation, an essential element for the plant. and especially the production of the enzyme ACC deaminase, an enzyme whose function is to inhibit the synthesis of ethylene, a molecule related to fruit ripening. However, we also consider the results of the other tests to generate a product that not only achieves adequate growth, but also provides other properties that can be beneficial to the plant. By analyzing all the isolates, we selected the following 3 microorganisms, the results of which are shown in Table 1.

Table 1 shows that the 3 strains are positive for the nitrogen fixation test, due to the color change that was generated in the culture medium. For the solubilization of phosphates, the halo generated around the colony inoculated in the medium is measured. The strain corresponding to B124 was the only one that generated a halo in the solubilization of calcium phosphates, being the strain with the highest phosphate solubilization of the three. selected generating a halo of 0.4 cm, the rest of the strains were negative for this test. With iron phosphates, no isolate including the three selected strains managed to solubilize said phosphates.

Table 1. Results of the agronomic tests of the 3 strains selected for the formulation of filling of vegetable fruits.

Where, lig + is a minimal production. + is little production. ++ is moderate production and +++ is abundant production.

In the case of siderophores, the three strains showed to be effective for the production of these chelators, with the B124 strain being the one with the highest production of siderophores. In the production of auxins, the strain that produced the greatest amount of this phytohormone is the B105 strain, obtaining 3 out of 3 crosses, while the B18 and B124 strains were not able to produce this molecule. Finally, the production of the enzyme ACC deaminase, as We observe in the table, the three strains produce a good quantity of this enzyme when obtaining 3 crosses of 3

DEPOSIT OF ISOLATED BACTERIAL STRAINS

Seeing that the isolated bacterial strains Caulobacter vibrioides identified as B18, Providencia rettgeri identified as B105, and Bacillus pumilus identified as B124, have agronomic potential, they were deposited on January 21, 2022, in the Microorganism Collection of the National Center for Genetic resources belonging to the National Institute of Forestry, Agriculture and Livestock Research, with registration number 1006 before the World Federation of Culture Collection (CM-CNRG) and International Depositary Authority with notification 308 for purposes of patent procedure in accordance with the Budapest Treaty ; and who has his address at Boulevard de la Biodiversidad 400, C. P. 47600. Tepatitlán de Morelos, Jalisco, Mexico. The bacterial strain Caulobacter vibrioides identified as B18 corresponded to the deposit number CM-CNRG TB186, the Providencia rettgeri strain identified as B105 was deposited with the number CM-CNRG TB187 and Bacillus pumilus identified as B124 deposited with the accession CM-CNRG TB188.

Claims

CLAIMS An isolated strain of Caulobacter vibrioides identified as B18, characterized in that it comprises the characteristics of the strain deposited under accession number CM-CNRG TB186. An isolated strain of Providencia rettgeri identified as B105, characterized in that it comprises the characteristics of the strain deposited under accession number CM-CNRG TB187. An isolated strain of Bacillus pumilus identified as B124, characterized in that it comprises the characteristics of the strain deposited under accession number CM-CNRG TB188. The strains of the preceding claims, wherein said bacterial strains are endophytes of Vaccinium corymbosum (blueberry) plants. The strains according to the previous claims, where the bacterial strains exhibit stimulating activity in the filling of vegetable fruits. The strains of the previous claim, where the vegetable fruits belong to plants of the Ericaceae family. The strains according to the preceding claim, where the vegetable fruits are from plants belonging to the genus Vaccinium. The strains according to the previous claim, where the vegetable fruits belong to vegetable plants belonging to the species Vaccinium corymbosum. A probiotic mixture, characterized in that it comprises at least two isolated bacterial strains, in accordance with the preceding claims. The mixture of the previous claim, wherein said bacterial strains are endophytes of Vaccinium corymbosum (blueberry) plants. The mixture of claim 9, wherein the bacterial strains exhibit stimulating activity on the growth of vegetable plants. The mixture of the previous claim, where the vegetable plants belong to the Ericaceae family. The mixture according to the preceding claim, wherein the vegetable plants belong to the genus Vaccinium. The mixture according to the previous claim, where the vegetable plants belong to the species Vaccinium corymbosum. A formulation to stimulate the filling of vegetable fruits, characterized in that it comprises: i) at least one isolated bacterial strain, which is selected from the following group of deposited strains: CM-CNRG TB186, CM-CNRG TB187, CM-CNRG TB188, and their mixtures between them;

ii) at least one prebiotic substance; and iii) at least one diluent. The formulation of the preceding claim, where the bacterial strain, whether individual or mixed, is in an amount of 0.01%, the prebiotic substance 4.99%, and the excipient 95%, with respect to the total volume of the formulation. The formulation of claim 15, where the fruits are from vegetable plants of the Ericaceae family. The formulation according to the preceding claim, where the fruits are from vegetable plants of the genus Vaccinium. The formulation of the previous claim, where the fruits are from vegetable plants that belong to the Vaccinium corymbosum species. A method to stimulate the growth of vegetable plants, characterized in that it comprises, applying to vegetable fruits, a sufficient amount of a formulation to stimulate the growth of vegetable plants, in accordance with claims 15 to 19. The method of the previous claim, where the sufficient amount of the formulation is 1 L for every 4,166 Vaccinium corymbosum plant plants. The method according to claim 20, where the application of the formulation is in the stage called "fruit mooring" of the vegetable plants. The method of claim 20, where the fruits are from vegetable plants belonging to the Ericaceae family. The method according to the preceding claim, where the fruits are from vegetable plants that are of the genus Vaccinium. The method of the previous claim, where the fruits are from vegetable plants of the Vaccinium corymbosum species.