RU2815318C1 - Strain of spore-forming bacteria bacillus thuringiensis species morrisoni, having insectoacaricidal, fungicidal and plant growth stimulating activity - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, namely to a strain of spore-forming bacteria Bacillus thuringiensis spp. morrisoni KA9, having insectoacaricidal, fungicidal and plant growth stimulating activity, suitable for organic farming and deposited in the Bioresource Centre of the All-Russian Collection of Industrial Microorganisms (BRC VKPM) of the National Research Centre "Kurchatov Institute" — GosNIIgenetics, under registration number VKPM B-14326, having insecticidal activity against coleopteran pests, acaricidal activity against spider mites, fungicidal activity, as well as stimulating plant growth. Invention can be used in biotechnology to produce a microbiological preparation used in agriculture.

EFFECT: invention widens the range of new environmentally safe strains of bacteria Bacillus thuringiensis, combining high insecticidal activity against insects of the order Coleoptera with acaricidal activity against the spider mite Tetranychus urticae, exhibiting fungicidal activity against phytopathogenic fungi and having a growth-stimulating effect on plants.

1 cl, 3 dwg, 5 tbl

Description

The invention relates to a strain of spore-forming bacteria Bacillus thuringiensis spp. morrisoni , which has insecticidal activity against coleopteran insect pests, acaricidal activity against spider mites, fungicidal activity, and also stimulates plant growth. The invention can be used in biotechnology to obtain a microbiological preparation used in agriculture.

The known strain Bacillus thuringiensis 16T100/18 (patent RU 2559548 C2) for combating the Colorado potato beetle. The disadvantage of the strain is its narrow spectrum of action.

A known bacterial strain is Bacillus thuringiensis ssp. kurstaki , which has insectoacaricidal activity against representatives of the orders Lepidoptera/ Coleoptera, Homoptera, Thysanoptera and Acariformes (patent RU 2278159 C1). The strain was obtained as a result of crossing by the Gonzalez method (Gonzalez JM, Brown BJ, Carlton BC 1982. Transfer of Bacillus thuringiensis plasmids cording for delta endotoxin among strains of B.thuringiensis and B.cereus . Proc.Natl.Acad.Sci.USA79^6951- 6955) of two parent strains of Bacillus thuringiensis ssp. kurstaki VKPM-V-1226 and Bacillus thuringiensis ssp. tenebrionis VKPM V-5081. In most countries, the use of such genetically modified organisms is limited or even banned due to potential risks to human health, on the one hand. On the other hand, in genetically modified microorganisms there is a possibility of loss of plasmids; the number of copies of plasmids in modified cells can vary significantly, which does not guarantee the stability of the properties of the Bacillus thuringiensis ssp strain. kurstaki .

The known strain is Bacillus thuringiensis var. darmstadiensis 56 as a multifunctional product for crop production (patent RU 2692655 C2). The strain exhibits entomocidal activity against coleopteran insects (Coleoptera), antifungal activity against phytopathogenic fungi and a growth-stimulating effect on plants. This strain, in addition to delta-endotoxin, synthesizes beta-exotoxin, which, although it has a wide spectrum of effects on insects, is unsafe for warm-blooded animals and humans.

A known strain of the genus Bacillus. thuringiensis ssp. morrisoni, Btm (V.S. Maslennikova, V.P. Tsvetkova, S.M. Nersesyan, E.V. Bedareva, G.V. Kalmykova, I.M. Dubovsky and L.A. Litvina / “The influence of tuber inoculation potatoes by bacteria of the genus Bacillus on the population of rhizosphere microorganisms"//"Vestnik NSAU" - 1(62)/2022, pp. 46-55.). The composition and structure of the soil microbial community were studied during pre-planting inoculation of potato tubers of the Tuleyevsky variety with the strain B. thuringiensis ssp. morrisoni with a titer of 2.7⋅10 ⁶ CFU/ml, which had a significant effect on ammonifier bacteria. This bacterial strain inhibited the development of fungi of the genera Fusarium and Penicillium. When using the strain B. thuringiensis vs. morrisoni in 2020, it had a multifunctional positive effect, and bacteria that assimilate mineral nitrogen developed more actively .

The closest analogue (prototype) is the Bacillus thuringiensis strain with growth-stimulating and fungicidal activity (Gorobey I.M., Kalmykova G.V., Davydova N.V., Andreeva I.V. “ Bacillus thuringiensis strains with growth-stimulating and fungicidal activity” / / Siberian Bulletin of Agricultural Science. 2018. T. 48. No. 6. P. 5-12. DOI: 10.26898/0370-8799-2018-6-1). The publication presents the results of studying the growth-stimulating and antifungal activity of the Bacillus thuringiensis ssp. morrisoni . A positive effect of the studied strain on the morphometric parameters of spring rapeseed and spring wheat seedlings was revealed. Treatment of spring rape seeds with B. thuringiensis contributed to a significant increase in root length. A tendency was revealed to reduce the number of spring wheat seedlings affected by root rot when seeds were treated with the strain B. thuringiensis ssp. morrisoni . The inhibitory activity of the B. thuringiensis strain on the 10th day of the experiment against Fusarium oxysporum varied from 68 to 83%, Alternaria alternata from 71 to 89%. The analysis showed that the studied strain stimulated the growth of spring rapeseed and spring wheat plants and exhibited antifungal activity.

The technical result of the claimed invention is the expansion of the range of new environmentally friendly strains of bacteria Bacillus thuringiensis , combining high insecticidal activity against insects of the order Coleoptera with acaricidal activity against the spider mite Tetranychus urticae , which exhibits fungicidal activity against phytopathogenic fungi and has a growth-stimulating effect on plants.

The specified technical result is achieved by obtaining a strain of spore-forming bacteria Bacillus thuringiensis ssp. morrisoni KA9, which has insectoacaricidal, fungicidal and plant growth-stimulating activity, suitable for organic farming and deposited in the Bioresource Center of the All-Russian Collection of Industrial Microorganisms (BRC VKPM) of the National Research Center "Kurchatov Institute" - GosNIIgenetika under registration number VKPM B-14326.

The invention is illustrated by the following graphics. In fig. Figure 1 shows the sequence of the KA9 strain sample stitched from sequenceograms. In fig. 2 shows a light microscopy photograph of a spore-crystalline suspensionBacillus thuringiensis ssp.morrisoni KA9 VKPM-14326. In fig. Figure 3 presents data from electrophoretic analysis in 10% SDS-PAGE of spore-crystalline mixtures: 1 -Bacillus thuringiensis ssp.morrisoni KA9 VKPM-14326, 2 -Bacillus thuringiensis ssp.aizawai,3 -Bacillus thuringiensis ssp.amagiensis.

Bacterial strain Bacillus thuringiensis ssp. morrisoni KA9 was isolated from natural samples (dead Colorado potato beetle larvae collected in a potato field in the Novosibirsk region in 2014) followed by multi-stage selection: to a suspension of the strain Bacillus thuringiensis ssp. morrisoni KA9 in Luria-Bertrani nutrient broth, 0.25 M sodium acetate was added and incubated for 4 hours at 30°C, stirring constantly. Then the samples were heated at 80°C for 15 minutes and spot-sown on agar media with different contents of amine nitrogen and glucose. The grown colonies were examined by light microscopy to determine the presence and synchronicity of the precipitation of parasporal crystals on all media. Selected variants were cultivated in liquid nutrient media to determine morphological uniformity and stability. The resulting strain of Bacillus thuringiensis ssp. morrisoni KA9 had morphological uniformity and formed large parasporal inclusions of different sizes, close to rectangular in shape, after cultivation in a liquid nutrient medium after 48 hours. The strain gave a positive flagellar antigen reaction with the H8ab type serum. The strain was deposited in the All-Russian Collection of Industrial Microorganisms under the number VKPM V-14326, which is confirmed by a certificate of deposit dated September 27, 2022 (Certificate attached).

Results of identification of the claimed bacterial strain Bacillus thuringiensis ssp. morissoni KA9 using sequencing of nucleotide sequences of the 16S rRNA gene of bacterial DNA.

Initial conditions: four genomic DNA samples

Brief work protocol:

• A DNA fragment of the 16S gene with a size of about 1500 bp was obtained using PCR and primers specific for the 16S rRNA gene in a preparative amount.

• The resulting DNA is purified from the components of the PCR reaction (primers, dNTPs, polymerase) using sorption on magnetic particles.

• The 16S rRNA gene fragment for each sample was sequenced on both sides using the Sanger method and analyzed on an ABI3130XL automatic capillary gene analyzer.

• Sequencegrams of all studied DNA samples were analyzed for homology/identity using the BLAST program of the GenBank database and the 16S ribosomal sequence database classifier - RDB.

In fig. 1 and the appendix shows the nucleotide sequence of SEQ ID NO:1 of the KA9 strain sample stitched from the sequencegrams.

Blast analysis of nucleotide sequences of the 16S rRNA gene, sample KA9:

No. Closest homolog Genbank number % homology 1 Bacillus thuringiensis YBT-1518 NC_022873 99.65 2 Bacillus cereus ATCC 14579 NC_004722 99.58

The claimed strain of Bacillus thuringiensis ssp. morrisoni KA9 VKPM-14326 does not produce water-soluble beta-exotoxin, but synthesizes insecticidal delta-endotoxin, consisting of a protein encoded by cry3A genes.

1. Cultivation of the strain.

To cultivate the strain, nutrient media were used: A, B, LB with the following composition, g/l:

Wednesday A:

Peptone “Difco” -7.0;

Pancreatic hydrolyzate of fish meal - 4.0;

Sodium chloride - 5.0;

Distilled water - 1000 ml.

Wednesday B :

Peptone “Difco” -7.0;

Pancreatic hydrolyzate of fishmeal - 4.0;

Sodium chloride - 5.0;

D-glucose - 5.0;

Distilled water - 1000 ml.

LB environment :

Tryptone “Difco” -10.0;

Yeast extract “Difco” -5.0;

Sodium chloride - 10.0;

Distilled water - 1000 ml.

To prepare agarized nutrient media, agar (1.2 wt%) was additionally added to the liquid media.

2. Cultural and morphological properties. Cultural and morphological characteristics of the strain indicating the environment: gram-positive, motile, flagella-forming rods measuring 0.7-1.2 × 2.5-4.0 µm. After 96 hours of growth on agar medium “A”, oval spores measuring 0.5 × 1.0 μm and parasporal inclusions close to rectangular in different sizes form (Fig. 1). On medium “A” the strain forms gray, large, dense, flat colonies with a fine-grained surface, the edges of the colonies are slightly wavy. On medium “B” the colonies are large, flat, slightly wrinkled with a dense whitish center, the edge is vaguely felt, gray, the surface is fine-grained. On Luria-Bertrani medium, large, flat, dense, white colonies with a wavy edge and a fine-grained surface are formed. In liquid nutrient media, growth is moderate and produces uniform turbidity. Optimum growth temperature +28°C.

In FIG. Figure 2 shows a light microscopy photograph of a spore-crystalline suspension of Bacillus thuringiensis ssp. morrisoni KA9 VKPM-14326.

3. Physiological and biochemical characteristics. Organic sources of nitrogen used by this microorganism: peptone, meat, fish and plant extracts. Soy, corn and fish bone meal, autolysates of yeast and casein, hydrolysates of soy and corn meal, feed yeast. The strain assimilates ammonium and nitrate forms of nitrogen, glucose and glycerol in the form of carbohydrate nutrition. Strain KA9 produces acetylmethylcarbinol, exhibits amylase and protease activities, and does not have urease and lecithinase activities. Salicin does not ferment, but it ferments mannose and sucrose to form acid. Strain KA9 is resistant to benzylpenicillin (10EA) and oxacillin (1 μg) and does not exhibit lysozyme activity. The strain does not produce beta exotoxin. Gives a positive flagellar antigen reaction with the standard serum of Bacillus thuringiensis spp. morissoni H-8ab.

4. Molecular genetic properties. SDS-PAGE electrophoresis of the spore-crystal mixture of strain KA9 showed the presence of an insecticidal Coleoptera-specific protein in the 70 kDa region (Fig. 2). The results of PCR analysis showed the presence of the cry3 gene in the DNA of the strain. Strain KA9 contained the acyl-homoserine lactonase (aiiA) gene, which is responsible for the control of signaling molecules that regulate the population density (QS) of phytopathogens. In fig. Figure 3 shows electrophoretic analysis in 10% SDS-PAGE of spore-crystalline mixtures: 1 - Bacillus thuringiensis ssp. morrisoni KA9 VKPM-14326, 2 - Bacillus thuringiensis ssp. aizawai, 3 - Bacillus thuringiensis ssp. amagiensis,

5. Biological properties of the strain

The strain exhibits insecticidal activity against first-third instar larvae of the Colorado potato beetle Leptinotarsa decemlineata , acaricidal activity against the spider mite Tetranychus urticae , suppresses the growth of phytopathogenic fungi: Fuzarium oxisporum , Alternaria alternata , Rhizoctonia solani , Bipolaris sp. , increases potato yield. The strain combines a growth-stimulating effect on spring rapeseed and spring wheat with fungicidal activity against the phytopathogenic fungi Fusarium oxysporum and Alternaria alternata .

The invention is illustrated by the following examples

Example 1. Cultivation of the Bacillus thuringiensis strain VKPM B-1432. To cultivate the strain, 750 ml Erlenmeyer flasks were used, which were filled with 100 ml of sterile medium “A”. The medium was prepared with distilled water, the pH was adjusted to 7.0, poured into 100 ml flasks and sterilized in an autoclave at a pressure of 1.1 atm at a temperature of 121°C for 15 minutes. After sterilization, the flasks with the medium they contained were cooled to room temperature. Sterility and pH were controlled by sterile sampling of the medium. The flasks were inoculated by adding 1 ml of inoculum prepared as follows: a bacterial culture collected with a loop from agar medium “A” was placed in a standard test tube with 5 ml of sterile saline solution. The contents of the tube, after checking for the absence of foreign microflora, were used as inoculum.

The strain was grown at a temperature of +28-30°C with shaking (220 rpm) for 72 hours. During the cultivation process, control was carried out for the absence of foreign microflora using a light-optical method or visual control of the morphology of colonies that grew after sowing the culture liquid of the strain on a Petri dish with agar nutrient medium “A”. After 72 hours, the presence and size of crystals were assessed using light microscopy. The titer of CFU (colony forming units - bacterial cells and spores) was also determined, which on medium “A” was 1.0×10 ⁹ CFU/ml.

Example 2. Evaluation of the insecticidal activity of the proposed strain in laboratory conditions.

The assessment of insecticidal activity for larvae of the Colorado potato beetle ( Leptinotarsa decemlineata ) of the first and second instars was carried out in laboratory conditions. To do this, 1.5% starvation agar was poured into Petri dishes, onto which 2 separated potato leaves (4-5 ^cm2 in area) were placed. 40 μl of a spore-crystalline suspension of the Bacillus thuringiensis ssp strain was applied to each leaf. morissoni VKPM B-14326 with a titer of 1.0 x10 ⁷ spores/ml, spore-crystalline suspension of the strain Bacillus thuringiensis ssp. thuringiensis with a titer of 5.0×10 ⁸ spores/ml; saline solution and the drug Bitoxibacillin were used as a control.

As follows from the results presented in Table 1, the spore-crystalline suspension of the proposed strain VKPM B-14326 has high insecticidal activity against the larvae of the Colorado potato beetle ( Leptinotarsa decemlineata ), which differs from the Bt ssp strain. thuringiensis is a producer of bitoxybacillin, which has no activity against this insect. The effect of the drug bitoxibacillin is due to the presence of β-exotoxin, synthesized during vegetative growth in the nutrient medium.

Table 1 Death of first and second instar larvae of the Colorado potato beetle ( Leptinotarsa decemlineata ) under the influence of spore-crystalline suspensions of Bt ssp. morissoni VKPM B-14326 (titer 1.0×10 ⁷ spores/ml) and Bt ssp. thuringiensis (titer 5.0×10 ⁸ spores/ml) and the drug Bitoxibacillin in the laboratory Strain/drug Death of larvae, % on the 3rd day Control 0 Bitoxibacillin 93.3±2.9 Bt ssp. thuringiensis* 0 Bt ssp. morissoni VKPM V-14326 100

Note: * - Bitoxibacillin producer, isolated during sieving of the drug

Example 3. Acaricidal activity of the claimed strain in laboratory conditions.

Laboratory experiments to evaluate the acaricidal effect of the spore-crystalline suspension of the proposed strain VKPM B-14326 were carried out using the cup method, including applying the working suspension to the feed substrate with further replanting of the tested mites Tetranychus urticae . 100 μl of a spore-crystalline suspension of the Bacillus thuringiensis ssp strain was applied to the food plant. morissoni VKPM B-14326 with a titer of 1.0×10 ⁸ spores/ml. Ticks of the same age were selected for the experiment; variants of laboratory experiments were carried out in 3-5 fold repetitions. The number of spider mites tested is from 25 to 95 individuals per replicate. The number of surviving ticks was counted on the 3rd and 5th day after the start of the experiment. Calculation of biological effectiveness was carried out using the Abbott formula.

The results of the laboratory experiment are shown in Table 2.

The data in Table 2 indicate the high acaricidal effectiveness of the proposed strain VKPM B-14326 against Tetranychus urticae ticks. The survival rate of mites 5 days after treatment with the spore-crystalline suspension was 17%, while in the control the number increased by 28%.

table 2 The effect of a spore-crystalline suspension of the strain Bacillus thuringiensis ssp. morissoni VKPM B-14326 (titer 1.0×10 ⁸ spores/ml) against the spider mite Tetranychus urticae Option Initial sample of individuals Number of living individuals, % of the original number, by day Biological effectiveness on the 5th day, % 3 4 5 6 Control 45 43 96 48 107 25 33 132 32 128 40 60 150 59 148 124 128 Bt ssp. morissoni VKPM V-14326 75 40 53 15 20 80 25 31 15 19 95 40 42 20 21 75 46 61 7 9 65 15 23 10 15 42 17 83.0

Example 4. Evaluation of the fungicidal activity of the proposed strain in laboratory conditions

The fungicidal activity of microorganisms was determined by the agar block method. Phytopathogenic test cultures of the causative agent of early blight of spring rape , Alternariaa aternata, and the most common causative agent of fusarium blight in agricultural crops, Fusarium oxysporum, were cultivated on Czapek's medium for 6 days. The inventive strain VKPM B-14326 was sown in a continuous “lawn” on the surface of agar medium “B”, and cut out disks of phytopathogenic fungi were placed in the center of the Petri dish. Petri dishes were incubated in a thermostat at 25-28°C for 7-10 days. Experiments to determine fungicidal activity were carried out in quadruple repetition.

Antagonistic activity of strain VKPM B-14326 were assessed by the diameter of the phytopathogenic fungus in the experimental variants in comparison with the control. Inhibitory activity (IA) was calculated using the formula IA = (Dk - Do)/ Dk × 100, where Dk is the diameter of the colony in the control variant, cm; Before - the diameter of the colony in the experimental version, see. The results obtained are shown in Table 3.

Table 3 Effect of spore-crystalline strain suspension
Bt ssp . morissoni VKPM B-14326 (titer 1.0×10 ⁶ spores/ml) for the growth of phytopathogenic test cultures Option Colony diameter, cm Inhibitory activity, % R.solani Day 7 10 7 10 Control 9.5 9.5 - - Bt ssp. morissoni VKPM V-14326 1.2 1.2 88 88 Al. alternata Day 7 10 7 10 Control 8.8 8.9 - - Bt ssp. morissoni VKPM V-14326 1.7 2.1 81 76 F. oxysporum Day 7 10 7 10 Control 9.0 9.0 - - Bt ssp. morissoni VKPM V-14326 1.5 1.5 83 83

The results presented in Table 3 indicate that strain VKPM B-14326 has antagonistic activity against phytopathogenic fungi, which most often cause plant diseases.

Example 5. Growth-stimulating effect

The ability of the strain to have a growth-stimulating effect was studied in laboratory experiments on spring rape and soybeans. When assessing the growth-stimulating effect, the roll method was used according to GOST 123038-84. Morphometric parameters were taken into account on the 7th day of the experiment.

To conduct a field experiment, spring rapeseed and soybean seeds were treated with a spore-crystalline suspension of the Bt ssp strain on the eve of sowing. morissoni VKPM V-14326. The seeds of the control variant were soaked in distilled water. The sowing and recording area of the plot was 36 ^m2 , 3-fold repetition, placement in 1 tier. The yield of spring rapeseed and soybean seeds was taken into account during their ripening period. The yield of green mass of spring rape was taken into account in the flowering phase, soybean in the pod filling phase, with a frame on an area of 0.25 m ² in 2 plots.

The experimental results are presented in Table 4.

Table 4 Growth-stimulating activity of the spore-crystalline suspension of the strain
Bt ssp. morissoni VKPM V-14326 (titer 1.0×10 ⁶ spores/ml) against spring rape Culture % to control Root length Seedling height Seed yield Biological yield Spring rape 290 140 127 143 Soybeans 115 129 108 138

Laboratory and field tests presented in Table 4 showed that seed treatment with a spore-crystalline suspension of the Bt ssp. morissoni VKPM -14326 had a positive effect on the growth of plant roots and seedlings, which confirms the growth-stimulating properties of the strain, and in field experiments, seed treatment increased the yield of tested forage crops.

Field experiments were also carried out on potatoes to evaluate the growth-promoting effect of the Bt ssp strain. morissoni VKPM-14326. To conduct the experiment, potato tubers were sprayed with a spore-crystalline suspension before planting, and in the control - with water. The dried tubers were planted in the ground and grown using the usual method. After 10 weeks of growth, the number and height of stems, the weight of plants were measured, and after harvesting the potatoes, the resulting yield was determined. The experimental results are shown in Table 5.

The results of the experiment, presented in Table 5, demonstrate the growth-stimulating effect of the strain on the tops and yield of potatoes when sprayed on tubers. The control variant is inferior in height, number of stems and especially in plant weight at the 10th week of recording. Pre-sowing spraying of potato tubers with a spore-crystalline suspension of the strain increases the yield by 58%.

Table 5 Effect of culture fluid based on strain
Bt ssp. morissoni VKPM V-14326 (titer 1.0×10 ⁶ spores/ml) on the growth and development of potato tops and potato yield Option Accounting for 10 weeks Productivity, % to control Plant weight, g Height of stems, cm Number of stems, pcs. Control 292 60.5 2.8 - Bt ssp. morissoni
VKPM V-14326 1285 77 3.7 158

Thus, examples 2-5 confirm the achievement of the claimed technical result, namely: obtaining a new environmentally friendly strain of bacteria Bacillus thuringiensis ssp. morissoni VKPM-14326, which combines high insecticidal activity against insects of the order Coleoptera with acaricidal activity against the spider mite Tetranychus urticae , exhibiting fungicidal activity against phytopathogenic fungi and having a growth-stimulating effect on plants.

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Claims (1)

A strain of spore-forming bacteria Bacillus thuringiensis ssp. morrisoni KA9, which has insectoacaricidal, fungicidal and plant growth stimulating activity, is suitable for organic farming and deposited in the Bioresource Center of the All-Russian Collection of Industrial Microorganisms (BRC VKPM) of the National Research Center "Kurchatov Institute" - GosNIIgenetika under registration number VKPM B-14326.

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