RU2846734C1 - Bacillus atrophaeus bacteria strain ad22 as soya plant productivity increasing agent and their protection against phytopathogenic fungi - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention is a strain of endophytic bacteria Bacillus atrophaeus AD22, deposited in Network bioresource collection in field of genetic technologies for agriculture on 13.06.2023 under number RCAM 06418, as a means to increase productivity of soya plants and protection thereof from phytopathogenic fungi.

EFFECT: invention makes it possible to increase productivity of soya plants and to protect them against phytopathogenic fungi.

1 cl, 4 tbl

Description

The invention relates to biotechnology and agriculture and concerns a new strain of endophytic bacteria Bacillus atrophaeus AD22 as a means for increasing the productivity of soybean plants and protecting them from phytopathogenic microorganisms.

In modern crop production, crop losses due to pests and diseases can be quite significant. According to the Food and Agriculture Organization of the United Nations (FAO), when taking into account the combined impact of diseases, pests, and weeds, damage to agricultural produce before harvest can range from 25 to 40%, with fungal phytopathogens causing the most significant damage. Today, protecting plants from fungal diseases using chemical and biological pesticides is a priority in agriculture.

However, the use of chemical pesticides is declining due to growing negative consumer attitudes towards them due to potential adverse effects on people and negative impacts on the soil.

Currently, many microbiological preparations for agriculture are known for various purposes: growth-promoting, as well as suppressing the development of phytopathogenic bacteria and fungi.

Known is Russian Federation patent No. 2495119 for the invention “ Bacillus subtilis 8a bacterial strain as a means of increasing plant productivity and protecting them from phytopathogenic microorganisms” with priority from 11/29/2012, IPC C12N 1/20, C12R 1/125, A01N 63/00, published on 10/10/2013.

The invention relates to biotechnology, specifically to the Bacillus subtilis 8A strain, which is deposited under accession number RCAM 00876 in the collection of the All-Russian Research Institute of Agricultural Microbiology of the Russian Academy of Agricultural Sciences. Bacillus subtilis 8A exhibits high fungicidal activity against phytopathogenic fungi and bactericidal activity against phytopathogenic bacteria, as well as growth-promoting activity against various agricultural crops. The strain can be used in agriculture as an effective means of increasing plant productivity and protecting them from phytopathogenic microorganisms.

Known is Russian Federation Patent No. 2551968 for the invention “ Bacillus pumilus A 1.5 bacterial strain as a means of increasing plant productivity and protecting them from diseases caused by phytopathogenic microorganisms” with priority dated 01.08.2013, IPC C12N 1/20, A01N 63/00, C12R 1/07, published 10.02.2015.

The strain of endophytic bacteria Bacillus pumilus A1.5 has fungicidal and bactericidal activity against phytopathogenic bacteria, is deposited in the collection of the All-Russian Research Institute of Agricultural Microbiology of the Russian Academy of Agricultural Sciences under the number RCAM02057 and can be used as a means of increasing plant productivity and protecting them from diseases caused by phytopathogenic microorganisms, and also expands the range of products that protect plants from phytopathogenic microorganisms.

Known patent of the Russian Federation No. 2626543 for the invention "Bacterial strain Paenibacillus mucilaginosus , a method for stimulating growth and protecting plants from diseases and the use of the bacterial strain Paenibacillus mucilaginosus as a fertilizer and biological control agent (antipathogenic agent) in the prevention and/or treatment of plant diseases" with priority of 09.11.2015, IPC C12N 1/20, C05F 11/08, published 16.05.2017.

A group of inventions based on the Paenibacillus mucilaginosus strain is described. It stimulates plant growth and has an increased capacity to synthesize bactericins with a broad spectrum of suppression of phytopathogenic microflora. The strain is deposited in the All-Russian Collection of Industrial Microorganisms under registration number VKPM B-12259. The use of the Paenibacillus mucilaginosus strain VKPM B-12259 as a fertilizer for agricultural crops and for the prevention and treatment of fungal plant diseases. A method for stimulating growth and protecting plants from fungal diseases is proposed, which involves pre-sowing treatment of soil, plant seeds, and crops with a suspension of Paenibacillus mucilaginosus strain VKPM B-12259 cell culture at a concentration of at least 120 million spores/ml. The inventions ensure an increase in the yield of agricultural crops and their resistance to fungal diseases.

Known patent of the Russian Federation No. 2701500 for the invention "Strain of spore-forming bacteria Bacillus amyloliquefaciens , having a fungicidal effect against phytopathogenic fungi that cause diseases of vegetable plants, a biological preparation based on it" with priority of September 28, 2018, IPC C12N 1/20, A01N 63/02, C12R 1/07, published on September 26, 2019.

The group of inventions includes the spore-forming bacterial strain Bacillus amyloliquefaciens VKPM B-11443 and a biological product based on it. This strain exhibits fungicidal activity against phytopathogenic fungi that cause vegetable plant diseases. The product based on this strain comprises a culture medium consisting of a complex of spores, vegetative cells, and metabolic products, and a carrier in the form of microkaolin or microdiatomaceous earth granules in a volume ratio of 1:3 to 1:5. This strain and the biological product based on it exhibit high fungicidal activity across a broad spectrum.

Known patent of the Russian Federation No. 2625977 for the invention " Bacillus amyloliquefaciens OPS-32 bacterial strain for obtaining a complex-action biopreparation for protecting agricultural plants from phytopathogenic fungi, stimulating their growth and increasing yields" with priority of 10.06.2016, IPC C12N 1/20, A01N 63/02, C12R 1/07, published 20.07.2017.

The Bacillus amyloliquefaciens OPS-32 bacterial strain, which exhibits antagonistic activity against phytopathogenic microorganisms, has been deposited in the All-Russian Collection of Industrial Microorganisms under registration number VKPM B-12464. This strain can be used to create a complex biopreparation for use in plant growing, including organic farming, and can increase crop yields.

Known patent of the Russian Federation No. 2099947 for the invention "Biopreparation Fitosporin for plant protection against diseases" with priority from 15.11.1996 in the name of the Institute of Microbiology and Virology of the National Academy of Sciences of Ukraine (UA) and the Scientific and Production Association "Bashkir" (RU), IPC A01N 63/00; C12N 1/20; C12R 1:125, published. 27.12.1997 (prototype).

The basis of the biological product "Fitosporin" is the bacterial strain Bacillus subtilis 26D with a cell concentration ^{of 10-10} cells per 1 ml of saline solution in an amount of 92-98% by volume and a filler in an amount of 2-8% by volume. Moreover, the strain is characterized by high antagonistic activity against phytopathogenic bacteria and fungi, allowing its use to protect various types of agricultural, ornamental, and woody plants.

The objective of the invention is to identify a strain suitable for use in agriculture as a means of increasing the productivity of soybean plants and protecting them from phytopathogenic fungi.

This problem is solved by using the endophytic bacterial strain Bacillus atrophaeus AD22, isolated from the rhizosphere soil of lentils in Omsk and deposited on June 13, 2023, in the Network Bioresource Collection in the Field of Genetic Technologies for Agriculture under accession number RCAM 06418, as a means of increasing the productivity of soybean plants and protecting them from phytopathogenic fungi in agriculture. A deposition certificate is attached.

The bacterial strain Bacillus atrophaeus AD22 is characterized by the following morphological-cultural and physiological-biochemical characteristics.

Bacterial cells have the shape of regular straight rods measuring 3-10 µm in length and 0.25-1.0 µm in diameter with rounded ends.

The strain forms ellipsoid spores measuring (1-2) 1 µm, located in the center of the cell.

The strain stains positively for Gram.

The strain grows in liquid and semi-liquid nutrient medium, is microaerophilic, and has respiratory metabolism.

Culture medium: mannitol yeast agar.

Cultivation for 48 hours. On nutrient agar, the strain forms dry, cream-colored colonies that darken over time, with a pasty consistency and jagged edges. Colony diameter is approximately 10 mm. The optimal growth temperature is 28°C. At temperatures above 45°C and below 15°C, growth is slow. The optimal pH of the medium is 7. Growth also occurs at pH values between 4.5 and 8.0.

The strain uses maltose, lactose, glucose, sucrose, starch, sorbitol and mannitol as its sole carbon source.

The strain is capable of growing at 45°C and 5% NaCl solution.

It was identified and experimentally established that the strain of endophytic bacteria Bacillus atrophaeus AD22 has fungicidal activity against phytopathogenic fungi Fusarium graminearum , Alternaria alternata , Cladosporium cladosporioides in bacterized plants.

To evaluate the antagonistic activity of the bacterial strain Bacillus atrophaeus AD22, a series of studies were conducted against phytopathogenic fungi using the following methodology.

On separate Petri dishes with agarized mannitol-yeast medium (Yap SF and ST Lim. Response of Rhizobium Sp UMKL-20 to sodium-chloride stress // Arch. Microbiol. - 1983. - №135. - 224-228 p.) 0.1 ml of the culture of the studied phytopathogenic fungus was sown in a lawn, namely: Fusarium graminearum , Alternaria alternata , Cladosporium cladosporioides . One hour after the phytopathogen was sown, a hole was made in the surface of each Petri dish on the agar using a cork drill, into which 0.1 ml of the culture liquid of the bacterial strain Bacillus atrophaeus AD22 with a titer of ≈10 ⁷ CFU/ml was placed.

For a comparative assessment of the fungicidal activity of the studied strain, using the method described above, 0.1 ml of the culture fluid of the prototype strain Bacillus subtilis 26D, the producer of the biopreparation Fitosporin, was placed in other Petri dishes.

All Petri dishes were then incubated at 28°C for 7 days, after which the sizes of the inhibited zones of the phytopathogenic fungus were measured. The results of the experiment are presented in Table 1.

Table 1. The effect of Bacillus atrophaeus AD22 and Bacillus subtilis 26D strains on the degree of suppression of the growth of phytopathogenic fungi

Phytopathogenic fungi Dimensions of suppression zones, mm when exposed to the Bacillus atrophaeus strain AD22 when exposed to the Bacillus subtilis strain 26D Alternaria alternate 36.20±1.99 22.50±2.30 Fusarium graminearum 32.05±5.31 25.9±4.21 Cladosporium cladosporioides 39.9±1.14 38.4±1.36

From the results presented in Table 1 it is evident that under the same experimental conditions the zones of inhibition of fungal phytopathogens in the Bacillus atrophaeus AD22 strain are larger than in the prototype strain Bacillus subtilis 26D, from which it follows that the declared bacterial strain Bacillus atrophaeus AD22 exhibits greater antagonistic activity towards the studied fungal phytopathogens: Fusarium graminearum , Alternaria alternata , Cladosporium cladosporioides , compared to the Bacillus subtilis 26D strain (producer of the biopreparation Fitosporin), which makes it a promising basis for the composition of biopreparations for the control of fungal phytopathogens.

To analyze the growth-promoting properties of the bacterial strain Bacillus atrophaeus AD22 in relation to soybeans, the following experiment was conducted.

Before germination, the En Argenta soybean seeds were washed in running tap water, then sterilized in a 70% ethyl alcohol solution (C ₂ H ₅ OH) for 1 minute, and then rinsed with tap water four times. The soybean seeds were then placed in sterile Petri dishes and inoculated with the test cultures: the culture liquid of the Bacillus atrophaeus AD22 bacterial strain and the culture liquid of the Bacillus subtilis 26D bacterial strain (prototype), diluted to a titer of 10 ⁷ CFU/ml, at a rate of 10 ml/kg of seeds (in the control - without treatment). The seeds were then kept in Petri dishes on moistened filter paper in a thermostat at a temperature of (28-30)°C. On the 1st, 2nd, 3rd and 7th days, the number of sprouted soybean seeds was counted and the length of the sprouts was measured; seeds whose sprouts were larger than or equal to the size of the seed were considered sprouted.

Data reflecting the proportion of germinated soybean seeds of the En Argenta variety depending on the treatment option are presented in Table 2.

Table 2. The effect of Bacillus atrophaeus AD22 and Bacillus subtilis 26D strains on the germination rate of soybean seeds of the En Argenta variety

Option for processing soybean seeds Proportion of sprouted seeds, % 1st day 2nd day 3rd day Day 7 Control (without turning) 22 35 49 60 Bacillus subtilis strain 26D (prototype) 21 39 50 61 Bacillus atrophaeus strain AD22 19 40 54 65

The data presented in Table 2 demonstrate that treating soybean seeds with bacterial suspensions slightly accelerates their germination on days 3 and 7. Thus, seeds treated with the Bacillus atrophaeus AD22 strain show an increase in their germination compared to untreated seeds (control) on day 3 by 10.2% and on day 7 by 8.3%. Moreover, the Bacillus atrophaeus AD22 bacterial strain has a stronger effect on the germination of soybean seeds compared to the Bacillus subtilis 26D strain, increasing the proportion of germinated seeds on day 3 by 8% and on day 7 by 6.5%. The effect of the Bacillus atrophaeus AD22 bacterial strain on the rate of soybean seed germination is a positive property, since earlier seed germination can give the plant an advantage in the use of soil resources.

Data reflecting the influence of the bacterial strain Bacillus atrophaeus AD22 and the bacterial strain Bacillus subtilis 26D (prototype) on the length of soybean seed sprouts of the En Argenta variety on the 3rd and 7th days depending on their treatment with the culture liquid of the corresponding bacterial strain, diluted to a titer of 10 ⁷ CoE/ml, with the calculation of 10 ml/kg of seeds (in the control - without treatment), are presented in Table 3.

Table 3. The effect of Bacillus atrophaeus AD22 and Bacillus subtilis 26D strains on the length of soybean seedlings of the En Argenta variety

Option for processing soybean seeds Length of sprouts, mm 3rd day Day 7 Control (without turning) 16.89±7.80 65.56±18.63 Bacillus subtilis strain 26D (prototype) 17.45±9.27 73.53±18.45 Bacillus atrophaeus strain AD22 23.18±7.66 73.89±15.23

From the data presented in Table 3, it follows that treatment of soybean seeds with bacterial suspensions increases the length of seedlings in comparison with the control on both the 3rd and 7th days.

Thus, in seeds treated with the Bacillus atrophaeus AD22 strain, the length of seedlings increased compared to untreated seeds (control) by 37.2% on the 3rd day and by 12.7% on the 7th day. Moreover, the Bacillus atrophaeus AD22 bacterial strain has a stronger effect on the elongation of soybean seedlings compared to the prototype strain Bacillus subtilis 26D, increasing the length of seedlings by 32.8% on the 3rd day and by 0.5% on the 7th day.

That is, the treatment of soybean seeds with the bacterial strain Bacillus atrophaeus AD22 promotes faster elongation of soybean seedlings on the 3rd day, which is a positive property, since faster growth of seedlings, as well as earlier germination of seeds, can give the plant an advantage in the use of soil resources.

To conduct vegetation experiments to evaluate the effectiveness of the Bacillus atrophaeus AD22 strain, a corresponding liquid biopreparation was obtained using the following method:

To obtain a stock culture of the bacterial strain Bacillus atrophaeus AD22, a liquid nutrient medium of the following composition was used: starch - 10 g/l; yeast extract - 1 g/l; K ₂ HPO ₄ - 0.5 g/l; MgSO ₄ ⋅7H ₂ O - 0.2 g/l; NaCl - 0.1 g/l. The pH of the mixture was 7.0.

The resulting liquid nutrient medium was poured into 200 ml 750 ml shaking flasks and sterilized at 1 atm for 30 minutes. The flasks were then inoculated with a sterile microbiological loop from a test tube with a nutrient agar slant (the medium composition is indicated above) containing a pure culture of Bacillus atrophaeus AD22. The flasks were then placed on a shaker (120 rpm) and cultured for 72 hours at 28°C. Thus, a stock culture of Bacillus atrophaeus AD22 with a bacterial titer of approximately 15⋅10 ⁸ CFU/ml was obtained in the flasks. The stock culture was stored in the refrigerator for up to 1 month at 4-6°C for subsequent inoculation in bottles or fermenters.

Work culture

For industrial cultivation of the Bacillus atrophaeus AD22 bacterial strain, a working culture was prepared in bottles or fermenters using a medium similar to the one described above. The inoculation rate of the stock culture was 2-5%, and the cultivation time was 72 hours at 28-30°C.

The efficacy of a product based on the Bacillus atrophaeus AD22 bacterial strain, obtained using the above method, on soybean plants of the 'OAK Prudence' variety was assessed in a microfield trial in the summer of 2023 in the Voronezh Region. Fitosporin, a product based on the prototype strain Bacillus subtilis 26D, was used for comparison.

During the experiment, the influence of biopreparations based on Bacillus atrophaeus AD22 and Bacillus subtilis 26D strains, as well as methods of treating seeds and plants of the OAK Prudence soybean variety on crop yield was assessed.

The field experiment was carried out in five replicates using the split plot method and included the following variants:

By the composition of the solution for treatment

1.1 - control (tap water);

1.2 - Bacillus subtilis 26D preparation;

1.3 - preparation Bacillus atrophaeus AD22.

According to the method of bacterization (processing) of plants:

2.1 - seed treatment with a 10% solution of the preparation at a rate of 10 l/t;

2.2 - spraying of vegetative plants in the budding phase-

flowering with 1% solution of the biopreparation at a rate of 100 l/ha;

2.3 - combined treatment of seeds and vegetative plants.

The soil of the experimental plot is chernozem-like, medium loamy in composition, with an arable layer of 20 cm and a humus content of approximately 3.5%. The preceding crop is soybeans, grown without fertilizer.

Soybean seeds were sown at a rate of 600,000 viable seeds per hectare with a row spacing of 20 cm. Seed treatment with biopreparations was carried out immediately before sowing, and spraying of vegetative plants was carried out during the budding-flowering period.

The yield was measured by plots using bin scales, followed by grain weighing and drying. The following data were obtained as a result of the study, and are presented in Table 4.

Table 4. The effect of biopreparations based on Bacillus atrophaeus AD22 and Bacillus subtilis 26D strains, as well as methods of treatment with them, on the yield of soybean variety ‘OAK Prudence’

Composition for
processing Processing method Productivity,
t/ha Increase to
control,
t/ha (%) Control (water) Seed inoculation 1.42 - Spraying during vegetation 1.40 - Combined 1.41 - Preparation
Fitosporin
( Bacillus subtilis strain 26D
prototype) Seed inoculation 1.56 0.14 (9.9%) Spraying during vegetation 1.52 0.12 (8.6%) Combined 1.62 0.21 (14.9%) Biopreparation on
based on the Bacillus atrophaeus strain AD22) Seed inoculation 1.64 0.22 (15.5%) Spraying during vegetation 1.54 0.13 (9.3%) Combined 1.70 0.29 (20.6%)

The results of the experiment presented in Table 4 showed that the use of biological preparations in relation to the soybean variety "OAK Prudence" based on the strains Bacillus subtilis 26D and Bacillus atrophaeus AD22 in the form of seed treatment before sowing, spraying of vegetative plants, and also in combination when treating seeds and vegetative plants, increased the yield of this soybean crop.

At the same time, the yield of plants treated with preparations based on the strains Bacillus subtilis 26D and Bacillus atrophaeus AD22 on seeds increased by 9.9% and 15.5%, respectively; when treated with preparations on the leaves, the yield increased by 8.6% and 9.3%, respectively; and when combined with biological preparations, the yield increased by 14.9% and 20.6%, respectively.

Moreover, the use of the drug obtained in the above-mentioned manner based on the bacterial strainBacillus atrophaeusAD22 through seed inoculation and combined seed and foliar treatment provides more significant increases in the yield of soybeans of the OAK Prudence variety (by 8%), compared to the use of the drug Fitosporin - the producer strainBacillus subtilis26D - prototype.

Thus, it has been experimentally proven that the new strain of Bacillus atrophaeus AD22 bacteria has a positive effect on the rate of soybean seed germination, promoting faster seedling elongation, which gives soybean plants an advantage in the use of soil resources.

It has also been experimentally shown that the use of a biopreparation based on the Bacillus atrophaeus AD22 strain with any method of treatment increases the yield of soybean crops, and more effectively than with the same methods of treatment with the Fitosporin preparation based on the prototype strain Bacillus subtilis 26D.

In addition, it has been experimentally confirmed that the new strain of bacteria Bacillus atrophaeus AD22 has fungicidal activity against phytopathogenic fungi.

Claims (1)

The endophytic bacterial strain Bacillus atrophaeus AD22, deposited in the Network Bioresource Collection in the field of genetic technologies for agriculture on June 13, 2023, under the number RCAM 06418, as a means of increasing the productivity of soybean plants and protecting them from phytopathogenic fungi.