PLANT GROWTH PROMOTING BACTERIA OF THE BACILLUS GENUS

FIELD OF THE INVENTION

The invention refers to a process for increasing the production of biomass and spores of plant growth promoting bacteria of the Bacillus genus. The process comprises incubating the microorganism in a suitable culture medium and cultivating it under specific physicochemical conditions, which generates a significant increase in biomass production, using less time, and with high sporulation efficiency.

DESCRIPTION OF PRIOR ART

Environmental damage caused by the use of nitrogen fertilizers, as well as the demand for environmentally safe measures by control agencies, have encouraged the search for sustainable management strategies that reduce the environmental impact of agricultural activities. An option to solve this problem is the use of plant growth-promoting microorganisms, which has proven to be effective in various plant systems (1-6). Plant growth-promoting rhizobacteria (PGPR), including that of the Bacillus genus, colonize the rhizosphere of plants and have the ability to promote plant growth, either through direct mechanisms such as soil nutrient solubilization and production of phytohormones, or by indirect mechanisms such as enzyme production, nutrient competition, and generation of systemic resistance. These microorganisms can be produced rapidly in culture media and can be stored for long periods of time given they produce spores (7-10).

Various strains of some species of Bacillus (e.g., B. subtilis, B. amyloliquefacines, B. cereus, B. mycoides, B. anthracis and B. thurigiensis) have been identified as plant growth promoters and useful in agricultural activities (11-16). However, the definition of PGPR is made at the strain level, not at the species level. There are some publications that describe processes for the production of different growth promoting strains of the Bacillus genus.

WO2009031874, WO2004024865, and WO20050118011 disclose the use of various strains of Bacillus sp. to promote plant growth. US20030228679 describes compositions and methods to increase plant growth through inoculation with plant growth-promoting bacteria of the B. subtilis and B. thuringiensis species. CN101381692 describes a culture medium to produce PGPR made of complex nutrient sources such as molasses syrup and fermented corn juice.

Although several processes to produce Bacillus genus have been reported, these pose some issues due to the small amount of biomass obtained or low sporulation efficiency, which generally does not exceed 50%. Similarly, various production methods that involve improved culture media to increase the production of Bacillus sp. spores have been published, but the results are still very poor in terms of sporulation efficiency (17- 23).

BRIEF DESCRIPTION OF THE INVENTION

The present invention refers to a process that allows increasing the production of biomass and spores from microorganisms of the Bacillus genus with high sporulation efficiency (over 85%), employing a suitable culture medium (SBM medium) and specific physicochemical conditions of aeration, stirring, pH, and temperature. The process of the invention reduces manipulation during the production process and increases biomass production and sporulation efficiency, which makes it much more appropriate and affordable for large scale production of the microorganism.

BRIEF DESCRIPTION OF THE FIGURE

FIG. 1 Total dry weight of banana plants inoculated with spores and vegetative cells of Bacillus subtilis EA-CB0575 at different microorganism concentrations and inoculation times. DETAILED DESCRIPTION OF THE INVENTION

The process of the present invention involves, as an initial step, the activation of a microorganism of the Bacillus genus by cultivating it in an enriched solid culture medium (TSA), inoculating a preinoculum in a suitable culture medium, and incubating using specific physicochemical conditions in order to obtain biomass and/or spores.

In a preferred embodiment of the invention, the microorganism of the Bacillus genus is selected from the group consisting of Bacillus amyloliquefaciens EA-CB0158, Bacillus pumilus EA-CB0177, Bacillus amyloliquefaciens EA-CB0123, Bacillus subtilis EA- CB0575, Bacillus altitudinis EA-CB0686, Bacillus megaterium EA-CB0784, Bacillus pumilus 1077 and Bacillus subtilis EA-CB1121.

The culture medium suitable to produce PGPR microorganisms according to the invention (hereinafter SBM medium) consists of one or more carbon sources, one or more nitrogen sources, salts, macronutrients, micronutrients, pH buffers, and antifoaming agents. To prepare the SBM culture medium, the carbon source, the nitrogen source, and the macronutrients are mixed in distilled water, the resulting mixture is sterilized; once sterilized, previously sterilized aqueous solutions (stock) containing salts at concentrations between 0.1 and 1.0 molar are added.

In a preferred embodiment of the invention, the SMB medium includes one or more components selected from the group consisting of glucose, yeast extract or meat extract, MgS0 ₄ , MnCl _2. KH ₂ P0 ₄ , peptone, CaCl _2. ZnS0 ₄ , NaCl, and FeS0 ₄ in a solid, semisolid or liquid matrix.

In an even more preferred embodiment of the invention, the SBM culture medium contains 1.04 g/L glucose, 0.6 g/L magnesium sulfate heptahydrate, 5.0 g/L yeast extract or meat extract, 6.0 g/L K ₂ HP0 ₄ , 3.0 g/L peptone, 0.01 g/L NaCl, and a stock of salts consisting of 1.14 mL/L FeS0 ₄ *7H ₂ 0, 0.1M, 300 μί/L ZnS0 ₄ *7H ₂ 0 0.1M, 9.9 mL/L CaCl ₂ 0.1M, and 30.0 mL/L MnCl ₂ 0.1M. The process of the invention can be performed either in a flask or in a bioreactor. Physicochemical conditions necessary to carry out the process of the invention include temperature, pH, aeration, fermentation and stirring time. Temperature must be maintain between 25°C and 37°C, pH must be between 5.0 and 7.5, aeration must be between 8 and 16 L/min, fermentation time must be between 48 and 72 hours, and continuous stirring must be between 300 and 600 rpm. pH can be adjusted by adding strong acids or strong bases such as H2SO4 and NaOH, while antifoaming agents, preferably of the silicone type, can be added to control foaming.

In a preferred embodiment of the invention, the process is carried out using the SBM culture medium in a 14 L bioreactor, maintaining a temperature of 30 °C for 60 hours with a pH of at least 5.5, aeration of 1.5 vvm, and continuous stirring at 430 rpm. The culture medium and the above conditions allow maximizing production of biomass and spores from microorganism Bacillus genus, yielding an amount of up to lxlO ¹⁰ CFU/mL, with sporulation efficiency greater than 92%. Centrifugation, microfiltration, decantation or thermal shock may be used to recover spores and/or biomass.

Once the microorganism has been obtained, either in the form of spores or vegetative cells, solid formulations or liquid compositions can be prepared together with one or more adjuvants and/or acceptable carriers, corresponding to another embodiment of the present invention. To prepare pesta, nor talc -based solid formulations, previously sterilized solid components are mixed with a suspension containing the microorganism and then dried at a temperature not exceeding 60°C, whereas for liquid formulations, the vegetative cells or spores of the microorganism are suspended in sterile water or other solvent and subsequently homogenized.

Preferred embodiments of the invention include talc-based formulations comprising between 5.0% and 25.0% (w/v) bacterial suspension of B. subtillis, between 70.0% and 99.0% (w/v) industrial talc, between 0.05% and 2.0% (w/v) carboxymethyl cellulose (CMC), and between 1.0 % and 30.0% (w/v) CaC0 ₃ . Similarly, other embodiments involve pesta-based formulations comprising between 5.0% and 25.0% (w/v) bacterial suspension of B. subtillis, between 50.0% and 75.0% (w/v) flour or semolina flour, between 1.0% and 15.0% (w/v) xanthan gum, and between 2.0% and 20.0% (w/v) industrial kaolin.

Formulations of the invention can be applied either around the stem of the plant or directly into the soil. The formulation to be applied in greenhouse can be obtained by dissolving the solid or liquid formulation in water at a ratio between 1 : 1 and 1 : 100000 based on the microorganism, yielding suspensions between lxlO ⁵ and lxlO ¹⁰ CFU/mL, which may be applied in amounts between 0.001 and 5.0 L/ha. If the formulation is to be applied in field, its volume and/or concentration must be adjusted so that a greater colonization of the microorganism is achieved as plants will be full grown and there may be more competition with microorganisms in the soil.

The growth promoting activity of the microorganism can also be assessed in vitro or in vivo, either in greenhouse or in field, in various types of crops such as bananas, corn, tomato, and chrysanthemum, among others. In order to establish the in vitro activity, different biochemical tests can be performed to quantify the production of metabolites (hormones, antibiotics, or siderophores) and determine the ability of phosphate solubilization and nitrogen fixation. Various inhibition assays against phytopathogenic microorganisms such as Fusarium oxysporum, Fusarium solani, Ralstonia solanacearum and Mycosphaerella fijiensis, among others, can also be carried out using enriched culture media. These assays can be performed in Petri dishes or microplates of PGRP co-cultures with phytopathogenic agents, aiming to identify growth inhibition of the harmful microorganism when exposed to PGPR. The following examples further illustrate the invention, but it is understood that the invented concept is not limited thereto.

EXAMPLES

Example 1. Obtaining and identifying strains of plant growth promoting microorganisms of the Bacillus genus Strains of Bacillus sp. were isolated from the rhizosphere of plantain plants (Musa AAA) originating from Urabd (Antioquia), Colombia. Rhizosphere soil and plant roots from production fields were used for this isolation process. A suspension of the samples was prepared in a phosphate buffer and subsequent serial dilutions were plated on TSA (trypticase soy agar) culture medium.

To select the Bacillus microorganisms, the samples were subjected to thermal shock at 80°C for 20 minutes and resistant organisms were purified and stored in TSB medium with 20% glycerol at -80°C (24). Table 1 shows the strains of PGPR microorganisms and their isolation source. The microorganisms were identified using 16s rDNA gene sequencing (24).

Table 1. PGPR bacteria. Isolation and Identification

Example 2. Designing and optimizing the culture medium

After a review of relevant literature (20, 21, 25-30), the most important sources of nutrients and macronutrients for the growth of microorganisms of the Bacillus genus were selected, and Placket and Burman (PBD) experiments were designed in order to identify the factors that have significant effects. Assessed factors include the sources of carbon (CeHuOe), magnesium (MgS04*7H20), manganese (MnCl ₂ *4H ₂ 0), phosphorus-potassium (KH2PO4), and nitrogen (yeast extract, meat extract, peptone, and (NtL^SCv), while on the other hand, variables include viable biomass concentration (CFU/mL), spores, and sporulation rate, defined as the ratio of spores to biomass. An analysis of variance (ANOVA) with a significance level of 5% was carried out. Table 2 shows concentration ranges for each analyzed factor.

Table 2. Plackett and Burman design for the culture medium of the invention

Treatment No. 11 showed the best result in terms of the culture medium, using B. subtilis EA-CB0575 as PGPR for this specific case. It was determined that MgS04*7H ₂ 0 and meat extract are significant factors for the variable total biomass response, while glucose is a significant factor for spore production. The highest spore production reached at this stage of medium design was 1.1 x 10 ⁹ CFU/mL, with a sporulation rate of 93%.

In order to establish the most key components, individual optimization of each response variable and multivariable optimization was performed using first a full factorial design and then a central composite design. The first design significantly improved production, yielding 1.6 x 10 CFU/mL with a sporulation rate of 95%, whereas the second design yielded 1.4 x 10 ⁹ CFU/mL with a sporulation rate of 94%.

In this case, the joint maximization of variables determined that a culture medium with concentrations between 1.0 and 3.0 g/L glucose; between 0.3 and 0.6 g/L MgS04*7H20; between 5.0 and 10.0 g/L yeast extract or meat extract; between 4.0 and 6.0 g/L K2HPO4; between 2.0 and 5.0 g/L peptone; between 0.01 g/L NaCl, and a stock of salts composed of 1.14 mL/L FeS0 ₄ *7H ₂ 0, 0.1M, 300 μί/L ZnS0 ₄ *7H ₂ 0 0.1M, 9.9 mL/L CaCl ₂ 0.1M and 30.0 mL/L MnCl ₂ 0.1M maximizes spore production and sporulation rate.

Example 3. Production of biomass and spores from Bacillus genus in a bioreactor

The preinoculum was prepared by transferring three colonies of a solid culture of B. subtilis to 250 mL of SBM culture media. Stirring at 150 rpm was carried out for 24 hours at 30°C. The ΌΟβοο of the preinoculum was adjusted to 2.0 with SBM sterile medium. The preinoculum was added at a ratio of 1 to 10% v/v.

An inoculum containing between 1 x 10 ⁵ and 1 x 10 ⁹ CFU/mL of the Bacillus sp. microorganism was added to 7 liters of SMB culture medium held in a BioFlo ^® 110 bioreactor (New Brunswick Scientific Co.) with a ring diffuser, two turbine impellers, and devices for measuring temperature, pH, and dissolved oxygen. To ensure the required physicochemical conditions, pH was adjusted to 5.5 and temperature was maintained at 30°C for 60 hours, with 1.5 vvm aeration, and stirring at

430 rpm. Biomass production was between 1.0 x 10 8° CFU/mL and 1.0 x 101 ¹ 0 ^U CFU/mL, with sporulation percentages greater than 92%. Table 3 shows each of the produced strains and the corresponding sporulation efficiency.

Table 3. Sporulation percentages of Bacillus sp. using SBM medium

Example 4. Assessing the effect of pH

The effect of pH on growth parameters of the B. subtilis strains cultured in a bioreactor using SMB culture medium was assessed using an univariate design. Results (Table 4) indicate that pH in a range between 6.5 and 7.0 even without control, does not affect the production of total biomass and/or spores.

Table 4. Assessment of the effect of pH on the production of Bacillus subtilis EA-

CB0575

Example 5. Assessing the effect of stirring and aeration

The effect of stirring and aeration when using a 14 L bioreactor in the process of the present invention was assessed for B. subtilis EA-CB0575 through a central composite design (CCD) with star points, aiming to optimize the production of biomass and spores. Biomass and spores were quantified in CFU/mL through surface plaiting and dry weight in g/L, reducing sugars at the end of fermentation were also quantified.

According to the results, stirring between 400 and 450 rpm and aeration between 10 to 12 L/min maximizes the production of spores and the sporulation percentage, obtaining values between 8.0 x 10 ⁹ and 1.0 x 10 ¹⁰ spores/mL and a sporulation efficiency of about 94%.

Example 6. PGPR microorganism formulations

Microorganisms of the Bacillus genus obtained according to Example 3 can be formulated either to obtain aqueous suspensions of concentrations between 1 x 10 ⁶ and 1 x 10 ¹¹ CFU/mL of spores or vegetative cells, or to obtain pesta or talc-based solid formulations by incorporating adjuvants such as CMC, kaolin, xanthan gum, and calcium carbonate at concentrations between 0.5% and 25.0 % (w/w).

To prepare said formulations, the adjuvants must be initially sterilized and then mixed with the microbial active principle and water in a ratio of 1 : 10 to 1 :100 (v/v). Once mixed, they are left to dry at 60°C for 30 minutes and, lastly, they are hermetically packaged and stored in suitable conditions.

Example 7. Applying a PGPR microorganism to bananas

A PGPR microorganism of Bacillus sp., particularly B. subtilis EA-CB0575, was applied to banana (Musa AAA) at different stages of plant growth. The plants were immersed for more than 60 minutes in aqueous suspensions of the microorganism at

6 7 8

concentrations of 1.0 x 10 , 1.0 x 10 , and 1.0 x 10 CFU/ mL, using spores or vegetative cells. Subsequently, the plants were cultured in peat for the corresponding hardening and rooting stages in a greenhouse at temperatures above 30°C and relative humidity greater than 80%.

Fig. 1 shows the dry weight of plants four months after the application of spores and vegetative cells of Bacillus subtilis EA-CB0575 in a greenhouse in banana plants using various inoculation times and concentrations of the microorganism.

Results show that there is a significant increase (from 11% to 120%) in the total dry weight when banana plant roots are inoculated with spores or vegetative cells of Bacillus. Best inoculation times are those over half an hour and at concentrations greater than 1 x 10 ⁶ CFU/mL of spores or vegetative cells.

Additionally, an assay in greenhouse and in field was conducted to assess other growth promoting microorganisms of the Bacillus genus (B. megaterium and B. cereus) in banana plants (Musa AAA). Table 5 shows formulation applied, strained used, and fruit production time, which decreased up to 1.5 months in relation the absolute control.

Table 5. Formulations assessed in greenhouse and field in banana (Musa AAA)

• Different letters denote significant differences (p <0.05) Example 8. Assessing growth promoting activity of PGPR in other crops

Growth promoting activity of PGPR microorganisms of the Bacillus genus was assessed in corn, coriander, and tomato plants using vegetative cells. This assessment was performed by inoculating the microorganism into seeds, and then into the substrate of the seedlings that germinated after the first inoculation, at a concentration of 1 x 10 ⁸ CFU/mL. Table 6 shows the results of the strains assessed in the aforementioned crops.

Table 6. Growth promotion of various Bacillus genus PGPR

· Different letters denote significant differences (p <0.05)

According to the results, total dry plant weight significantly increased in corn between 10.5% and 65.5%; in cilantro between 7.6% and 25.7%; and in tomato between 50.0% 100% in relation to the control without inoculation after growing for 3 weeks in a greenhouse.

To determine the in vitro growth promoting potential of various Bacillus sp., production of siderophores, auxins, phosphate solubilization, nitrogen fixation, and phytopathogenic antagonism capacity were assessed in vitro. Biochemical tests carried out in vitro determined the presence or absence of PGPR characteristics in the assessed isolates. The Salkowsky colorimetric method (31) was used to assess auxin production; the CAS colorimetric method (32) was used to assess siderophore production; the protocol reported by Parson y Strickland 1972 (33) was used to determine phosphate solubilization; and the studied strain was cultured in nitrogen-free Nfb medium(34) to assess nitrogen fixation.

Co-cultures of PGPR and phytopathogenic agents Fusarium oxysporum EAP004, Fusarium solani EAP-005, Botritys cinerea EAP-001, Mycosphaerella fijiensis, and Ralstonia solanacearum EAP-009 were performed to assess phytopathogenic antagonism using PDA culture medium; except for Ralstonia solanacearum, where BGTA culture medium was used. Table 7 illustrates the antagonism results and PGPR biochemical traits of the Bacillus sp. strains, showing that B. subtillis EA-CB0575 and EA-CB0158 have the highest potential.

Example 9. Assessing antagonism and PGPR biochemical traits for Bacillus genus.

To determine the in vitro growth promoting potential of various Bacillus genus, production of siderophores, auxins, phosphate solubilization, nitrogen fixation, and phytopathogenic antagonism capacity were assessed in vitro. Biochemical tests carried out in vitro determined the presence or absence of PGPR characteristics in the assessed isolates. The Salkowsky colorimetric method (31) was used to assess auxin production; the CAS colorimetric method (32) was used to assess siderophore production; the protocol reported by Parson y Strickland 1972 (33) was used to determine phosphate solubilization; and the studied strain was cultured in nitrogen-free Nfb medium (34) to assess nitrogen fixation.

Co-cultures of PGPR and phytopathogenic agents Fusarium oxysporum EAP004, Fusarium solani EAP-005, Botritys cinerea EAP-001, Mycosphaerella fijiensis, and Ralstonia solanacearum EAP-009 were performed to assess phytopathogenic antagonism using PDA culture medium; except for Ralstonia solanacearum, where BGTA culture medium was used. Table 7 illustrates the antagonism results and PGPR biochemical traits of the Bacillus sp. strains, showing that B. subtillis EA-CB0575 and EA-CB0158 have the highest potential. Table 7. PGPR traits and phytopathogenic antagonism by Bacillus genus.

(+): Low antagonism; (+ +): Average antagonism; (+ + +): High antagonism.

(-): Antagonism or compound production not detected

n/d: Not determined.

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It should be understood that the present invention is not limited to the embodiments described and illustrated herein. As it will be apparent to one skilled in the art, there are potential variations and modifications that do not depart from the spirit of the invention, which is only defined by the following claims: