BACILLUS AMYLOLIQUEFACIENS Bs 20 BACTERIAL STRAIN WITH COMPLEX BIOLOGICAL ACTIVITY

Abstract

FIELD: biotechnology.SUBSTANCE: strain of bacteria Bacillus amyloliquefaciens Bs 20, deposited on December 16, 2024 in the All-Russian Collection of Medicines under registration number VKPM B-14818.EFFECT: strain has complex biological activity, in particular antagonistic activity against phytopathogens, and increased nitrogen-fixing properties, which allows for an increase in sugar beet yield and a reduction in the application rates of nitrogen mineral fertilizers during its cultivation.1 cl, 6 dwg, 5 tbl, 3 ex

Description

[0001] The claimed invention relates to agricultural biotechnology in the field of plant growing and can be used as a producer of biopreparations in the cultivation of new domestically bred sugar beet hybrids to protect sugar beets from fungal diseases, optimize nitrogen nutrition of plants, and increase their yield. The bacterial strain Bacillus amyloliquefaciens Bs 20 was deposited on December 16, 2024, in the All-Russian Collection of Plant Genetic Materials (VKPM) under registration number VKPM B-14818. The strain exhibits complex biological activity, in particular antagonistic activity against phytopathogens, and enhanced nitrogen-fixing properties, which allows for an increase in sugar beet yield and a reduction in the application rate of nitrogen mineral fertilizers during its cultivation.

[0002] Although molecular nitrogen is the dominant component of the atmosphere (accounting for 75% by volume), its reserves in the soil are replenished by the nitrogen-fixing capacity of free-living and nodule microorganisms. Plants cannot directly assimilate atmospheric nitrogen. Sugar beet is a crop with high nutrient removal. Developing a powerful root system (up to 2 m deep), it utilizes nutrients from both the arable and subsoil layers. The nitrogen nutrition system in the cultivation of sugar beet can have a significant impact on the yield, sugar content and technological qualities of root crops (A.V. Goryainov, S.A. Iosifov, S.M. Zemtsov Nitrogen nutrition of sugar beet / / Sakhar. 2020. No. 7. URL: https: / / cyberleninka.ru / article / n / azotnoe-pitanie-saharnoy-svyokly).

[0003] Currently, many microbiological preparations for agriculture for various purposes are known: growth-stimulating, as well as suppressing the development of phytopathogenic bacteria and fungi.

[0004] A strain of bacteria, Bacillus amyloliquefaciens subsp. plantarum BS89, is known to enhance plant productivity and protect against diseases. The strain is deposited in the Departmental Collection of the All-Russian Research Institute of Agricultural Microbiology under accession number RCAM 03458. The strain exhibits high fungicidal activity against phytopathogenic fungi and bactericidal activity against phytopathogenic bacteria, as well as high growth-promoting activity against various agricultural crops, including wheat, barley, potatoes, cabbage, sugar beets, flax, and sunflower. This invention relates to biotechnology and agriculture.

[0005] A strain of Bacillus amyloliquefaciens bacteria is known, which has the ability to suppress the development of diseases of the sugar beet foliage by producing fungicidal substances into the nutrient medium during bacterial cultivation. It is deposited at the All-Russian Research Institute of Agricultural Microbiology under registration number RCAM 05515. The invention provides an expansion of the arsenal of means for combating diseases of cultivated plants.

[0006] A new strain of Bacillus amyloliquefaciens P20 is known to be used as an agent for combating phytopathogens. The invention relates to biotechnology and agriculture. The strain Bacillus subtilis I5-12 / 23, deposited in the State Collection of Microorganisms of the State Scientific Institution VIZR under the registration number Bacillus subtilis 93 VIZR, exhibits antagonistic activity against phytopathogenic fungi.

[0007] The bacterial strain Bacillus amyloliquefaciens subsp. plantarumbs 89 is proposed as a means of increasing plant productivity and protecting them from diseases (Russian Federation patent for invention No. RU 2599416 C1).

[0008] Russian Federation patent No. 2099947 for the invention "Biological product Fitosporin for plant protection against diseases" is known under application 96121980 with priority dated 11 / 15 / 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; C12R1:125, published on 12 / 27 / 1997. The basis of the biological product "Fitosporin" is the strain of bacteria Bacillus subtilis VNIISKhM 128 with a cell concentration of 10 9 -10 10 , 1 ml of saline solution in the amount of 92-98 vol.% and filler in the amount of 2-8 vol.

[0009] Russian Federation patent No. 2528058 is known for the invention "Bacillus amyloliquefaciens strain of bacteria with fungicidal and bactericidal action, and a biological preparation based on it for protecting grain plants from diseases caused by phytopathogenic fungi" according to application 2013125726 with priority dated 04.06.2013 in the name of FSUE GosNIIGenetika, IPC A01N 63 / 02, A01C 1 / 06, C12N 1 / 20, C12R 1 / 07, publication dated 10.09.2014. The strain of bacteria Bacillus amyloliquefaciens VKPM B-11475, which has fungicidal and bactericidal action, is described. Bacillus amyloliquefaciens OPS-32 bacterial strain for producing a complex-action biopreparation for protecting agricultural plants from phytopathogenic fungi, stimulating their growth and increasing yields.

[0010] A new strain of Bacillus amyloliquefaciens subsp. plantarum AB101 is described, which is characterized by high efficiency against the root rot disease ("black scab") in potatoes caused by Rhizoctonia solani.

[0011] An antifungal drug, Bacteriocin, has been developed for the treatment of fungal and other microbial and viral infections. It is obtained from the spores of Bacillus amyloliquefaciens subsp. plantarumAB101 bacteria, which have a strong effect against gram-positive bacteria.

[0012] 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" is known under application No. 2012151104 with priority dated November 29, 2012, in the name of the All-Russian Research Institute of Agricultural Microbiology of the Russian Academy of Agricultural Sciences. The strain was deposited in the collection of the All-Russian Research Institute of Agricultural Microbiology of the Russian Academy of Agricultural Sciences on November 14, 2011, under number RCAM 00876 as a means of increasing plant productivity and protecting them from phytopathogenic microorganisms. This strain has high fungicidal and bactericidal activity.

[0013] The high growth-stimulating activity of the Bacillus subtilis 8A strain and the biopreparation based on it was also established, leading to increased plant yields. Experiments confirmed that the strain's effectiveness is due to the ability of its bacteria to create a microbial-plant system with plants by colonizing the rhizosphere and root system, and can be used in agriculture. Due to the fact that bacterial strains of the taxonomic group Bacillus amyloliquefaciens subsp. plantarum are deposited in collections geographically remote from the applicant (including in other countries), and also that some publications do not specify the bacterial strain titer required for its use in the biopreparation, the Bacillus subtilis 8A bacterial strain, available to the applicant and deposited in the collection of the All-Russian Research Institute of Agricultural Microbiology, was selected as the prototype.

[0014] Many strains of these bacteria enhance plant growth and inhibit pathogens, significantly improving plant health. They colonize plant roots and reduce plant diseases through both direct and indirect effects.

[0015] The closest strain to the claimed invention is the rhizospheric bacteria strain Bacillus amyloliquefaciens subsp. plantarumBS89. The strain of rhizospheric bacteria Bacillus amyloliquefaciens subsp. plantarumBS89 was isolated from the roots of winter wheat of the Lira variety, growing in the chernozem soils of the Krasnodar Territory of the Russian Federation. The strain was deposited in the Departmental Collection of Beneficial Microorganisms for Agricultural Purposes (RCAM) of the All-Russian Research Institute of Agricultural Microorganisms on July 9, 2015, under the number RCAM 03458 as a means of increasing plant productivity and protecting them from phytopathogenic microorganisms. Experiments have shown that the bacterial strain Bacillus amyloliquefaciens subsp. plantarumBS89 increases plant yields and exhibits antagonistic properties against pathogens of agricultural crops, in particular:

[0016] - winter and spring wheat - against powdery mildew (Erysiphe graminis), brown rust (Puccinia recondita), fusarium root rot (Fusarium culmorum), basal bacteriosis (Pseudomonas syringae);

[0017] - spring barley - against seed mold (Penicillium, Alternaria), root rot (Bipolaris sorokiniana), dark brown spot (Drechlera sorokiana);

[0018] - white cabbage and cauliflower - against vascular bacteriosis (Xanthomonas campestris), black leg (Rhizoctonia solani), pythium root rot (Pythium irregulare);

[0019] - potatoes - against late blight (Phytophtora infestans), rhizoctonia (Rhizoctonia solani), fusarium wilt (Fusarium oxysporum);

[0020] - sugar beet - against rootworm (Pythium debarianum, Phoma betae), cercospora leaf spot (Cercospora beticola).

[0021] To obtain a stock culture of the bacterial strain Bacillus amyloliquefaciens subsp. plantarumBS89, a liquid nutrient medium, KSB (potato-sucrose broth), was used. For this purpose, a potato broth was first prepared by cutting 200 g of peeled potatoes into slices and boiling them in 800 ml of distilled water for 20 minutes. Then, the broth was filtered through a cotton-gauze filter, 10 g of sucrose was added to it, and the pH of the mixture was brought to 7.0.

[0022] Environmental problems require the development of new methods for improving and stabilizing agricultural production, particularly environmentally friendly systems for increasing crop yields. One such approach is the use of biotechnological solutions based on rhizobacteria strains.

[0023] The objective of the invention is to identify a strain with complex biological activity, in particular antagonistic activity against phytopathogens, and increased nitrogen-fixing properties.

[0024] The technical result consists in increasing the yield of sugar beet and the quality of root crops, reducing the dose of nitrogen mineral fertilizers during its cultivation.

[0025] The proposed strain was selected as a result of extensive search and selection work from rhizobacteria isolated from the rhizoplane of sugar beet at the experimental plot of the All-Russian Research Institute of Sugar Beet and Sugar named after A.L. Mazlumov (VNIISS) (Voronezh Region, VNIISS settlement). Subsequent screening for antagonistic and nitrogen-fixing activity, stimulation of sugar beet seed growth and development, colonization ability, and other properties revealed the most active strain, classified as Bacillus amyloliquefaciens according to 16s rRNA sequence analysis.

[0026] Cultural and morphological characteristics

[0027] Cells are gram-positive, motile rods, peritrichous. Size: 0.5-0.7 × 2.5-3.0 µm. Spores are oval. Colonies are round, with a scalloped edge, the center is distinguished by a crater and is noticeably raised. The surface is rough, with a noticeable tendency to wrinkle. Matte, opaque, rough, yellowish-gray.

[0028] Cultivation

[0029] The cells grow well on simple nutrient media: meat-peptone agar, meat-peptone wort, Hottinger agar, and L-broth. The optimum growth temperature is 30-40°C.

[0030] Cells grow at temperatures from 20 to 45°C with an optimal pH of 6.8 to 7.5. Sucrose and D-glucose, D-fructose, and lactose are used as carbon sources. Nitrogen sources include both mineral salts and organic matter.

[0031] They produce biologically active metabolites, including antibiotics and broad-spectrum enzymes that suppress the growth of pathogenic and opportunistic bacterial and fungal microflora, and cytokinins. The latter has been proven by a test method to reduce chlorophyll decomposition in the dark. [Zurochka A.V. Bacteria as producers of cytokine-like substances / A.V. Zurochka / / Russian Journal of Immunology. - 2017. - Vol. 11 (20) No. 3. - P. 374-376].

[0032] Methods of propagation and storage conditions

[0033] Cells are grown and maintained on meat-peptone agar and meat-peptone wort at 28°C. Store in the refrigerator on slant agar at 4-5°C with periodic subculture (once every 6 months).

[0034] Culture does not dissociate.

[0035] The strain is non-pathogenic for warm-blooded animals.

[0036] The strain does not inhibit, and in some cases stimulates, the growth of soil microflora. This has been demonstrated for representatives of the soil microbial community: ammonifiers, oligoisophylls, cellulolytics, nitrogen immobilizers, phosphorus-mobilizing microorganisms, etc.

[0037] Bacillus amyloliquefaciens strain (VKPM number B-14818 dated 12 / 16 / 2024) is able to actively take root and function in the rhizoplane, rhizosphere, phylloplane of sugar beet, and colonize the surface of the root system. (Shagaev A.A., Zhuravleva A.S., Dmitrieva E.N., Belov A.A., Markvichev N.S. Features of colonization of the cucumber root system by Pseudomonas fluorescens bacteria / / Advances in Chemistry and Chemical Technology. 2018. No. 12 (208). URL: https: / / cyberleninka.ru / article / n / osobennosti-kolonizatsii-kornevoy-sistemy-ogurtsa-bakteriyami-pseudomonas-fluorescens.

[0038] Positive properties of the strain

[0039] Example 1

[0040] The influence of the bacilli's culture medium on their antagonistic properties was determined under laboratory conditions. The following media were used: classic MPS (meat-peptone wort) and Czapek, L-broth, and L-broth+wort (Table 1).

[0041] Table 1. Effect of cultivation medium on the antagonistic properties of Bacillus amyloliquefaciens cultures20

[0042] Nutrient media Bacillus amyloliquefaciens 20 Alternaria alternata Capek 38 MPS 49 L-broth 39 L-broth + wort 40 Fusarium avenaceum Capek 10 MPS 30 L-broth 15 L-broth + wort 0 Fusarium oxysporum Capek 8 MPS 12 L-broth 18 L-broth + wort 11

[0043] Observations of the diameter of the phytopathogen growth inhibition zone showed that for both strains, MPS and L-broth were the optimal media for producing active antagonists. The ability of microorganisms to suppress the growth and development of plant pathogens is widely used in biological control [Stognienko, O.I. Updated list of sugar beet diseases and pathogens / O.I. Stognienko, E.S. Herr / / Sugar beet. - 2020. - No. 10. - P. 21-23].

[0044] This method for combating agricultural plant diseases reduces the use of chemical pesticides and improves the environmental sustainability of agriculture. Suppression of pathogen growth and development can occur in various ways: through competition with pathogens for nutrients; through the release of antimicrobial substances; through the induction of the plant defense system, etc. Most of the antagonist microorganisms used are also capable of colonizing the plant surface, creating a "barrier" to pathogen penetration. Suppression of pathogen growth by Bacillus amyloliquefaciens20 is shown in Figs. 1-3.

[0045] Example 2

[0046] Nitrogen fixation activity was studied using two nutrient media:

[0047] Ashby's medium (KH2PO4- 0.1 g / l; MgSO4- 0.2 g / l; K2HPO4- 0.2 g / l; NaCl - 0.2 g / l; CaCO3- 5.0 g / l; sucrose - 20.0 g / l; distilled water - up to 1000 ml).

[0048] Fedorov's medium with molasses (K2HPO4 - 0.3 g / l; CaHPO4 - 0.2 g / l; MgSO4 - 0.3 g / l; K2SO4 - 0.2 g / l; NaCl - 0.5 g / l; FeCl3 - 0.01 g / l; CaCO3 - 5.0 g / l; molasses - 40 g; a mixture of trace elements - 1 ml; distilled water - up to 1000 ml). The determination was carried out by the acetylene method on an FGH-1 device at the Federal Scientific Center for Legumes and Cereal Crops (Table 2).

[0049] Table 2 - Determination of nitrogen-fixing activity of strains on various nutrient media Bacillus amyloliquefaciens20

[0050] Nutrient medium Strains AF, nM C2H4 / h mcg N2 / ml / h Wednesday Ashby Bacillus amyloliquefaciens 20 478.38 1.75 Fedorov's Wednesday Bacillus amyloliquefaciens 20 493.39 1.82

[0051] Bacillus amyloliquefaciens20 is able to colonize the surface of plants, creating a “barrier” to the penetration of pathogens inside.

[0052] The study of the colonizing ability of microorganism strains was carried out under laboratory conditions in 20 cm long agronomic test tubes.

[0053] The results of laboratory studies showed that Bacillus amyloliquefaciens20 is able to colonize the roots of sugar beet.

[0054] Colonization of the surface of sugar beet roots with rhizobacteria strains on the 5th day of cultivation (Fig. 4 - control) (Fig. 5 - Bacillus amyloliquefaciens20).

[0055] Figure 5 shows that the seedling roots are coated with a gel of colonizing bacteria, Bacillus amyloliquefaciens. This suggests that the bacteria are also capable of interacting with the above-ground parts of plants.

[0056] Spraying the foliar apparatus of sugar beets with Bacillus amyloliquefaciens 20. At the early stage of spraying, preventive, the number of spore-forming bacteria on the leaf surface increased to 0.97 million CFU per 1 cm 2 (in control -0.14). This confirms the survival of Bacillus amyloliquefaciens 20 on the leaf surface. The effect of the introduction of Bacillus amyloliquefaciens 20 strains on the number of spore-forming bacteria in the sugar beet phylloplane (thousands of CFU per 1 cm 2 ) is shown in Fig. 6.

[0057] Example 3

[0058] To obtain a suspension, the Bacillus amyloliquefaciens20 strain (VKPM number B-114821 dated 16.12.2024) is grown for 3 days in a liquid meat-peptone wort medium. For this, the cells are washed from the slants with 1 ml of sterile saline and added to flasks with 250 ml of medium. Cultivation is carried out on a shaker at 28°C and 180 rpm for 3 days. The resulting culture is inoculated into 1 L flasks containing liquid MPS medium at a rate of 10% of the volume of the nutrient medium. Cultivation is carried out on a shaker at 28°C and 180 rpm for 3 days. The suspension has a titer of 10 10 CFU / ml.

[0059] In 2023-2025, in the Voronezh region, at the experimental site of the Federal State Budgetary Scientific Institution "All-Russian Research Institute of Soil Science named after A.L. Mazlumov"), in sugar beet crops in the fallow link of the grain-fallow-row crop rotation with alternating crops: fallow - winter wheat - sugar beet - barley, against the background of no mineral fertilizers applied, Bacillus amyloliquefaciens 20 (VKPM number B-14818 dated 12 / 16 / 2024) with a titer of 10 was introduced into the foliar apparatus 10 CFU / ml. The working fluid consumption rate is 200 l / ha. Seeds of the diploid sugar beet hybrid RMS 503 were used for sowing.

[0060] The prevalence of Alternaria in the control group was 16.67%. Spraying crops with the Bacillus amyloliquefaciens 20 strain reduced the prevalence of the disease by almost 25-30%, while not affecting the intensity of disease development.

[0061] The disease leads to an increase in reducing sugars, reducing the utilization of sugar in overall metabolism. Cercospora leaf spot increases transpiration, leading to a decrease in root weight.

[0062] The intensity of development and prevalence of cercospora in sugar beet crops was not significant, therefore Bacillus amyloliquefaciens20 had virtually no effect on this disease.

[0063] Viral yellows. The disease appears mid-season and appears on older leaves as yellowing, beginning at the leaf margin and progressing toward the center. Gradually, all leaves turn yellow. The disease reduces root crop yield and sugar content, as well as increases the content of molasses: nitrogenous substances and ash compounds.

[0064] The spread and development of jaundice was inhibited by preventive treatment of crops with Bacillus amyloliquefaciens20 (Table 3).

[0065] Table 3 - Distribution of leaf diseases 2023-2025

[0066] Option Alternaria Cercospora leaf spot Jaundice Fomoz Powdery mildew P,% R,% P,% R,% P,% R,% R,% P,% R,% Control 10,33 0,53 6,30 0,33 20,77 5,47 14,3 5,00 0,60 Bacillus amyloliquefaciens 20 8,33 0,40 5,27 0,27 16,03 4,47 12,01 4,00 0,57

[0067] P - prevalence of the disease; R - intensity of disease development.

[0068] The effectiveness of using Bacillus amyloliquefaciens20 in controlling leaf diseases of sugar beet has been proven.

[0069] Preventive spraying of sugar beet crops with Bacillus amyloliquefaciens20 contributed to an increase in the chlorophyll content in the leaves to 572 c.u., and the leaf blade area to 191.0 cm 2 The photosynthetic productivity coefficient increased to 5.61 (in the control 3.40) (Table 4).

[0070] Table 4 - Effect of Bacillus amyloliquefaciens on the Photosynthetic Productivity Index (PPI) of Sugar Beet in 2024-2025.

[0071] Option Processing time N-tester readings Average leaf area, cm2 KPF Control 520 111,5 3,40 Bacillus amyloliquefaciens 20 preventively 572 191,0 5,61 Bacillus amyloliquefaciens 20 + in 2 weeks 551 179,2 5,82

[0072] Two-time spraying with Bacillus amyloliquefaciens contributed to the maximum increase in chlorophyll content in sugar beet leaves to 561 c.u., and the leaf blade area increased to 179.2 cm 2 The photosynthetic productivity coefficient was 5.82.

[0073] Treating foliar tissue with pathogen-antagonist microorganisms, Bacillus amyloliquefaciens20, increased sugar beet yields. This is due to the introduced species successfully colonizing the phylloplane of developing plants, optimizing the microbial community structure, and reducing foliar tissue diseases (Table 5).

[0074] Table 5 - Sugar beet productivity 2023-2025

[0075] Experimental variant Number of treatments Productivity Sugar content Sugar harvesting To extract. t / ha ±d % ±d t / ha ±d Control 21,7 17,4 3,78 79,79 Bacillus amyloliquefaciens 20 1 28,9 7,2 16,8 -0,6 4,85 1,07 78,72 Bacillus amyloliquefaciens 20 2 34,4 12,7 17,0 -0,4 5,83 2,09 80,23

[0076] K extract. - coefficient of extraction of sucrase from root crops during processing at the plant.

[0077] A single treatment of sugar beet crops with Bacillus amyloliquefaciens20 increased yield by 7.2 t / ha and sugar yield by 1.07 t. A double treatment increased yield by 12.7 t / ha. Although sugar content decreased slightly, sucrose recovery during root processing increased, as K recovery reflects improved technological quality of the root crops.

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