WO2021119867A1

WO2021119867A1 - Strain of pseudomonas protegens rgm 2331 and use thereof to produce a biostimulant with antifungal properties to promote growth in plants

Info

Publication number: WO2021119867A1
Application number: PCT/CL2020/050127
Authority: WO
Inventors: Yessenia VEGA ORREGO; Ernesto MOYA ELIZONDO
Original assignee: Universidad de Concepción
Priority date: 2019-12-20
Filing date: 2020-10-13
Publication date: 2021-06-24
Also published as: MX2022007604A; CL2019003791A1

Abstract

The present technology relates to a strain of Pseudomonas protegens, RGM 2331, capable of producing the phytohormone indole acetic acid (IAA) and solubilising phosphorus (P), these features allowing same to be used to produce a biostimulant with antifungal properties, to promote growth in plants. The biostimulant and the use thereof directly on the foliar part or on the roots are also claimed.

Description

A strain of Pseudomonas protens RGM 2331 and its use in the preparation of a biostimulant with antifungal properties for the promotion of plant growth.

TECHNICAL SECTOR

This technology is related to the agricultural industry, in particular a bacterial strain is presented as a biostimulant with antifungal properties for the promotion of plant growth.

STATE OF THE ART

Sustainable agriculture must ensure global food security while promoting healthy ecosystems and supporting the sustainable management of land, water and natural resources. For this, agriculture must satisfy the needs of present and future generations for its products and services, while guaranteeing profitability, environmental health and social and economic equity (FAO). Currently the increase in the world population has required an increase in crop yields to maintain profitability and meet the demands of the markets in quality, health and production, for this they resort to the use of agrochemicals and mineral fertilizers which are being questioned due to its effect on human health (Wolansky, 2011), eutrophication problems, Zn deficiency and a decrease in the activity and diversity of the native microbiota present in soils (Babalola and Glick, 2012).

In this context, the development of new products aimed at promoting healthy ecosystems, improving profitability, take on enormous value to help solve the current agricultural problem. These products in Chile can be found as "biostimulants", the vast majority of these are based on algae, and those that are based on microorganisms, their main function is to control diseases based mainly on the use of bacteria of the Bacillus genus. Of the biostimulants based on microorganisms (Biological) marketed in Chile, at least 50% that are used are imported (Red Agrícola, 2017), the responses of these products in crops are variable since their behavior is affected by the environment, which is closely related to the environment from which they were isolated, presenting less adaptability to the soil and environmental conditions that exist in Chile (Toju et al., 2018).

At the research level, the trend is focused on growth-promoting bacteria (PGPR: "Plant Growth Promoting Rhizobacteria") applied as biological controllers, while their direct growth-promoting properties (phosphorus solubilization, IAA production, others) have It has been widely studied under in vitro or controlled conditions, but with few experiences at the field level where they rarely have significant results in the field (Backer et al., 2018). Populations of Pseudomona fluorescens have been characterized by producing antibiotics such as 2,4-diacetylfloroglucinol (2,4-DAPG), pioluteorine, pyrrolnitrine, phenazine-l-carboxylic acid 2-hydroxy phenazines and phenazine-l-carboxamide (Dilantha et al., 2005). Gaeumannomyces graminis var. tritici is highly susceptible to 2,4-DAPG, and the bacteria-plant interaction is benefited by monoculture, generating a decline in the disease through the years of monoculture without generating resistance from the fungus (Weller et al. , 2002). This decline or suppression of the disease has been described in the world (Weller et al., 2002) and in Chile (Campillo et al., 2001) on farms where wheat is continuously grown. Pseudomonas fluorescens that produce compounds such as 2,4-DAPG have also shown indirect growth promoting activity by controlling the disease in wheat, but also promoting plant growth (Campillo et al., 2001).

Investigations carried out in Chile determined the presence of Pseudomonas protens bacteria with antimicrobial genes associated with the production of 2,4-DAPG, pioluteorine and pyrrolnitrin and with inhibitory activity on G. graminis var. tritici and other fungi that affect wheat (Moya-Elizondo et al., 2013; Cattan, 2014), while in works carried out by Moya-Elizondo and Tejeda-Tribiño (2014) bacteria have been isolated from Chilean native potatoes with chitinolytic, glucanolytic activity and surfactant production, which shows the potential of the microflora present in soils in southern Chile for disease control, but it is not known if these Chilean strains have any growth-promoting effect. BRIEF DESCRIPTION OF THE FIGURES

Figure 1: It is observed the phosphate solubilization halos formed by some Chilean strains of Pseudomonas protens and Pantoea spp. on Pikovskaya medium Figure 2: Grouping by quantitative phosphate solubilization capacity for different strains of Pseudomonas protens and Pantoea spp. in Pikovskaya liquid medium which was determined by Ward's cluster analysis. It is observed that the Ca6 strain obtained the highest values for phosphate solubilization.

Figure 3: Grouping by indole-3-acetic acid (IAA) production capacity for different strains of Pseudomonas protens and Pantoea spp. determined by Ward's cluster analysis.

Figure 4: Average results of IAA production of the bacterial strain Ca6 in different culture media, where the -T media are media with tryptophan and the -S media have the salts of the KB culture medium. Figure 5: Average of three measurements of the percentage of blight in the potato crop.

Figure 6: Average of the yield of the three central rows, for each of the treatments in the potato crop.

Figure 7: Average of the sum of the number of fruits per plant, of three measurements, for each of the treatments in a commercial strawberry orchard cv. Saint Andrea.

Figure 8: Average number of fruits per plant from three measurements made on different dates for each of the treatments in a commercial strawberry orchard.

DESCRIPTION OF THE INVENTION

The present technology corresponds to the use of a bacterial strain as a biostimulant with antifungal properties for the promotion of plant growth.

The invention is based on the use of the bacterial strain for growth promotion, which also produce antimicrobial compounds. This technology corresponds to the Pseudomonas protens Ca6 strain, deposited in the Chilean Collection of Microbial Genetic Resources with registration number RGM 2331, which was deposited on September 30, 2016.

This bacterium was isolated from Andisol soils, which constitute only 1% of the world's soils (FAO), this explains that the characteristics that this bacterium presents is rare and allows us to assume that it is not widely distributed worldwide and that it has greater adaptability to the soils and climatic conditions that exist in Chile.

The biostimulant comprises a liquid bacterial suspension> 10 ⁸ CFU mL ^~ ¹ obtained from the fermentation of the bacterial strain in King B liquid medium (KB) for 48 hours at 24 ° C and 150 rpm of agitation. KB medium is the best culture medium for formulation because in this medium the bacteria increase their IAA production capacity.

The biostimulant based on this bacterial strain isolated in Chile increases crop yield through mechanisms such as the production of the phytohormone indole acetic acid (IAA) and the solubilization of phosphorus (P) (Figure 1, 2 and 3 ). The production of a phytohormone is of great importance since they are molecules that in low concentrations are capable of generating stimuli on plants, IAA being one of the phytohormones that causes the greatest stimulus on plants, mainly by promoting root growth. On the other hand, the solubilization of phosphate (P) is of great importance since the data obtained in the previous study of this bacterium values were high compared to other bacteria, so they have comparative advantages, even more within the country where the soils They have a large amount of immobilized P which forces producers to apply large amounts of P increasing production costs, therefore the use of these bacteria would imply a decrease since they use the phosphorus that is immobile in the soil. All these data indicate that this strain is capable of promoting plant growth through two mechanisms, the production of IAA (Figure 3) and mainly by the solubilization of phosphorus (Figure 2) and indirectly they are capable of controlling diseases by improving the phytosanitary status in plants, due to the production of antimicrobial compounds. These results are contrasted with the biostimulants present on the market, which are mainly phosphorus solubilizers, but are not IAA producers.

The biostimulant has a high adaptability to Chilean soil, promoting plant growth, ensuring availability and absorption of macronutrients such as phosphorus, the production of phytohormone (IAA), and also protects crops against various pathogens.

The application recommendation of this product, due to its protection characteristics, should be in accordance with the following model: a) Potatoes: a. Tuber treatment: Application to direct sowing to the tuber by applying to the planting furrow using a manual sprayer or by using potato planting machinery with an integrated nozzle, the application of the product can be in conjunction with fungicides or individually. This application allows to achieve the initial endophytic colonization, to achieve a homogeneous emergence of the crop together with the initial protection of the seedling. b. Foliar treatments: Foliar applications are by manual or mechanical sprayers, and it is recommended to carry out at least three applications in the season to achieve an increase in yields, which can be alternated with fungicide applications to improve the effect of disease control. b) Strawberries a. Root treatment: to achieve an initial endophytic colonization of the bacteria in the plants, the roots and / or crowns must be suspended in the liquid solution for 10 minutes prior to transplantation. b. Foliar treatment: Foliar applications are by manual or mechanical sprayers, and it is recommended to carry out at least three applications in the season to achieve an increase in yields, which can be alternated with applications of fungicides to improve the effect of disease control.

APPLICATION EXAMPLES

Example 1: isolation and characterization of the strains.

The Ca6 bacterium (RGM 2331), which corresponds to the Pseudomonas protens species, was obtained from the Cajón sector, a place located in the Araucanía region, and is characterized by producing different antimicrobial compounds such as 2,4-diacetylfloroglucinol (2 , 4-DAPG), pyoluteorine and pyrrolnitrine. The characterization of the species was done by analysis of the 16S rRNA gene, and amplification with specific primers of the genes ph / D, pltB, and prnC associated with the production of 2,4-DAPG, pyoluteorine and pyrrolnitrin, respectively. It should be noted that the bacteria that are being worked on in this proposal are genetically sequenced in the 16S ribosomal RNA gene, and correspond to Pseudomonas protens species that have mutations in their genomic sequence that make them different from other species described in the Bank of sequence of genes existing worldwide. On the other hand, these strains have demonstrated the ability to solubilize immobilized phosphorus, produce indole acetic acid and other biosurfactant properties, which are not common characteristics of isolates of the same species, such as P. protens strain Pf5 isolated in the United States (Paulsen et al. ., 2005; Ramette et al., 2011).

Example 2: phosphorus solubilization tests and IAA production.

The tests were carried out on different strains, including Strain Ca6. To quantify the phosphate solubilization capacity of the bacterial strains, Pikovskaya's liquid medium was used. The bacteria were inoculated into the medium and the quantitative solubilization of phosphate was determined. The Ca6 strains were able to solubilize phosphorus at 123.3 mg L ¹ (Figures 1 and 2). These results agree with those reported by Kumar et al. (2014) where they used strains of the genus Pseudomonas which They solubilized phosphate reaching a maximum of 15.19 mg of P ₂ O ₄ L ^_1 , values that were eight times lower than that observed in the Ca6 strain, which reached a maximum of 123.3 mg of P ₂ O ₄ L ^_1 . In order to measure the production of IAA by the bacteria, they were multiplied on trypticase soy agar and incubated for 48 h. The AIA concentration was evaluated using the methodology described by Bric et al., 1991. The Ca6 strain obtained IAA production levels of 0.79 mg L ^ (Figure 3).

Example 3: Formulation.

The formulation comprises a liquid bacterial suspension> 10 ⁸ CFU mL ¹ obtained from the fermentation of the bacterial strain in King B (KB) liquid medium for 48 hours at 24 ° C and 150 rpm of stirring. For its preparation, an aliquot of 10 pL of the original strain of the bacterium is taken, which is kept in 20% glycerol at -80 ° C, and is placed to grow in 10 mL of KB medium for 48 hours and then inoculated with 2 mL of this suspension (~ 10 ⁷ CFU mL ¹ ) per 100 mL. KB medium is the best culture medium for the formulation because in this medium the bacteria increase their IAA production capacity (Figure 4).

This formulation has proven effectiveness in promoting growth when applied in concentrations of 5% diluted in water. It must be prepared fresh for each application, but can be kept refrigerated at 4 ° C for 30 days, in which the bacteria survive.

Example 4: tests on crops.

Field trials were carried out in different crops and geographical areas. The strains arranged in the formulation were applied to the roots in the case of strawberries prior to transplantation, while in the potato crop the bacterial strains were applied directly to the seeds. After the initial application, between 3 to 4 foliar applications were made, formulated in KB culture broth, obtaining various results in the promotion of growth associated with an increase in yields, the results vary according to the culture where the bacteria were applied:

1. Potatoes: The trial was established on October 3, 2019 in the experimental field "El Nogal" in the town of Chillán. Potatoes from the PUKARA variety, which is the most planted in the region, the trial consisted of twelve treatments and four repetitions applied in plots of 20 m ² with 5 rows of 4 m long, the tubers were planted approximately every 33 cm (3 tubers per subway). The treatments were applied at the time of planting on the tubers with a spray. To prepare the formulations, the bacteria were allowed to grow in KB broth for 48 hours in an orbital shaker at 150 rpm with a temperature of 25 ° C, in addition to applying the treatments, 11 plots were inoculated with the Rhizoctonia sotaní fungus, leaving one as healthy control, to ensure that the disease is present and to be able to measure disease control parameters on the part of the bacteria. During the development of the crop, two applications of the treatments were made, one when the plants reached full emergence, and the next when they were in 50% of flowering. In addition, two applications of fungicides and one application of nitrogen fertilizers were made in the first aporca of the crop. To determine the growth promotion capacity of part of the applied bacteria, in the establishment of the culture a 50% fertilization (Nitrogen, phosphorus and potassium) was carried out in all the treatments with bacteria, in the TI and T2 treatments, which are the controls, an amount of fertilizers equal to that of a commercial establishment was applied for the normal development of the crop. In the potato crop, the Ca6 strain increased yields with a 50% fertilization compared to the control by 17.6%, on the other hand, it was able to reduce the blight of the plants by 56% compared to the control without bacteria Positive Ctrl (Figure 5 and 6).

2. Strawberries: Strawberries of cv. San Andrea on November 17, 2018, in the town of San Nicolás in a commercial orchard of a Prodesal user. For the establishment of the trial, 6 treatments were applied plus a water control and a positive control based on a commercial formulation of Trichoderma, which is what producers in the area currently use to reduce diseases and increase plant growth. Each of the treatments with four repetitions, a plot corresponded to 24 strawberry plants. For the formulation of the treatments, the bacteria were allowed to grow in KB broth for 48 hours in an orbital shaker at 150 rpm with a temperature of 25 ° C. For each treatment the formulations were applied to the root of the plant, the root was soaked with the treatments before being planted. To carry out the measurements, only the 16 central plants were measured, leaving 8 border plants for each of the plots. After planting, applications were made every 15 days of those formulated during the December-March period by spraying. All treatments were fertilized in January via irrigation. During the flowering and fruiting period of the crop, three evaluations were carried out, in February the temperatures and the lack of water drastically reduced the production, so no measurements were made until March, the period in which the plant. In the commercial strawberry orchard, the Ca6 bacteria managed to increase the number of fruits by 39.1% with respect to the control in which they use a commercial product based on Trichoderma. (Figure 7 and 8).

Claims

1. A bacterial strain for the promotion of plant growth CHARACTERIZED because it corresponds to the strain of Pseudomonas protens Ca6, deposited in the Chilean Collection of Microbial Genetic Resources with registration number RGM 2331.

2. The bacterial strain, according to claim 1, CHARACTERIZED because it has the ability to produce the phytohormone indole acetic acid (IAA) and solubilize phosphorus (P).

3. Use of the bacterial strain, according to claim 1, CHARACTERIZED because it is used for the elaboration of a biostimulant with antifungal properties for the promotion of plant growth.

4. Use of the bacterial strain, according to claim 1, CHARACTERIZED because its use allows the growth of roots stimulated by the production of IAA.

5. Use of the bacterial strain, according to claim 1, CHARACTERIZED because its use allows the solubilization of immobilized phosphate in soils.

6. Use of the bacterial strain, according to claim 1, CHARACTERIZED in that its use allows, indirectly, to control diseases by improving the phytosanitary status in plants, by the production of antimicrobial compounds.

7. The biostimulant for the promotion of plant growth CHARACTERIZED because it comprises a suspension of the RGM 2331 strain in King B (KB) liquid medium at a concentration of at least> 10 ⁸ CFU mL ^_1 .

8. Use of the biostimulant, according to claim 7, CHARACTERIZED in that it is used by applying it directly to the sowing furrow using a manual sprayer or by using sowing machinery, the application of the product can be in conjunction with fungicides or individually.

9. Use of the biostimulant, according to claim 7, CHARACTERIZED because it is used by applying it to the leaf segments, using manual or mechanical sprayers, and it is recommended to carry out at least three applications in the season to achieve an increase in yields.

10. Use of the biostimulant, according to claim 7, CHARACTERIZED because it is used by applying it directly to the roots to achieve initial endophytic colonization, through immersion in the liquid solution for 10 minutes prior to transplantation.