RU1805849C - Bacterial strain pseudomonas putida for production of preparation for plant growth promotion and plant protection from fusarium and erwinia - Google Patents

Abstract

Usage: can be used to obtain a bacterial preparation to stimulate plant growth and protect plants from fungi of the genus Fusarium and Erwinia bacteria. SUMMARY OF THE INVENTION: The bacterial strain Pseudomonas putida BKM B-1743D was isolated from the rhizosphere of wild-growing cereals in the Armenian SSR. It grows well on various media, including synthetic ones, under aeration conditions at 30 ° C, pH 7.0-7.5. To obtain the preparation, the strain is grown on BCH to a concentration of 10 cells / ml. The drug has the ability to suppress the growth of phytopathogenic fungi of the genus Fusarium, stimulates plant growth, and has the ability to dissolve mineral phosphates. 5 tab.

Description

The invention relates to biotechnology, and relates to a new strain of bacteria Pseudomonas putida, which can be used to obtain a bacterial preparation for protecting plants from phytopathogenic fungi of the genus Fusarium and bacteria of the genus Erwinla or to stimulate plant growth.

The aim of the invention was to obtain a new strain of Pseudomonas putida having the ability to inhibit the growth of phytopathogenic fungi of the genus Pseudomonas and Erwinia bacteria and stimulate plant growth.

The strain Pseudomonas putida BS1380 was isolated from the wild

cereals in the Armenian SSR using selective King B and $ 1 media. The main selection criteria were stimulation of plant growth and suppression of the growth of phytopathogenic fungi of the genus Fusarium and bacteria of the genus Erwinia.

The composition of the medium of King B (g / l):

peptone - 20

glycerin (ml) - 10

MgS04x7H20-1.5

agar - 15

The composition of the medium SI (g / l)

sucrose - 10

glycerin (ml) - 10

casein hydrolyzate - 5

Mansoz-1

00

about with

Yu

with

MgS04x7H20-1

KzHPCM - 2.3

sarcosyl - 1.2

trimethoprim (mg) - 20

agar - 15.

The strain Pseudomonas putida BS1380 was deposited in the All-Union Collection of Microorganisms at the IBPM RAS under number В К В-1743Д.

Strain P.putida VShV-11743 has the following properties:

Culture l morphological features

The cells are rod-shaped, measuring (2.2-3.2) x (0.6-0.9) microns, motile, peritrichi, gram-negative, do not form spores.

Cell sizes are given for a one-day culture grown on MPA at 30 degrees.

The strain grows well on the following media: BCH (m sopeptone broth). Kinga B: peptone - 20 g / l, glycerin - 10 ml / l, MgSCM x NaO - 1.5 g / l; on a minimal synthetic medium M9 of the following composition: 2НР04 - 7 g / l, КН2РСЦ - 3 g / l. NH4CI - 1 g / l, glucose - 1 g / l. On m sopeptone agar, the colonies are smooth, shiny, with smooth edges.

Turbidity, sediment and a yellow-green fluorescent pigment are observed in the m sopeptone broth.

On standard media, Kinga produces a yellow-green fluorescent pigment, phenazine pigments do not form.

Physiological and biochemical characteristics

Obligatory aerob, temperature optimum of growth 28-30 ° С, does not grow at 41 ° С, optimum pH 7.0-7.5.

It reduces nitrates, does not dilute gelatin, does not peptone milk, does not use trehalose and d-xylose as carbon sources, and utilizes ethyl alcohol.

Assimilates ammonium and nitrate nitrogen. It uses d-glucose, d-fructose, sucrose, maltose, lactose, acetate, propinate, butyrate as a carbon source. succinate, pyruvate, fumarate, benzoate, lactate, citrate, catechol, protocatechoate.

The oxidase reaction is positive.

Arginine dehydrolase is present.

The storage conditions of the strain:

The bacterial strain Pseudomonas putida VKM B-1743D is stored on plates, braids with MPA, or with King B medium at 5-7 ° C. Reseeding on fresh environments - once a month c. It can be stored for at least 1 year in 15% glycerol at -20-70 ° C, in a freeze-dried state with sugar-gelatin agar. or semi-fluid

agar medium under vaseline oil of the following composition:

Nutrient Broth (Difco) - 4 g / l NaCI - 5 g / l

agar - b g / l pH - 6.8

The strain P.putida VKM B-1743D is not pathogenic and has a genetic label - resistance to rifampicin (100-200 μg / ml),

0 which allows to control its survival on the roots of plants.

To inoculate the seeds, the biomass of cells of the F.putida strain VKM B-1743D grown in King's liquid medium is used

5 in or in m sopeptone broth (MPB) on a rocking chair at 30 ° C for 16-18 hours. Seed treatment is carried out at a rate of 106-10 cells per 1 g of seed.

Examples 1-3 showing

0 antagonism of F.putida strain VKM B-1743D against phytopathogenic fungi of the genus F.usarium.

PRI me R 1. The strain P.putida VKM B-1743D grown on medium BCH to

5 densities 10 cells / ml. 1 ml of overnight culture is pelleted using a Millipore syringe onto a Millipore 0.45 μm nitrocellulose filter. The filter is placed on a cup with wort agar or Chapek medium containing

0 previously introduced mycelium. Phytopathogenic fungus Fusarium moniiiformi. The cup is incubated at room temperature (25 ° C) for 3-4 days and zones of inhibition of fungal infection are visually recorded.

5 growth around the filters. Zones of growth inhibition of fungal mycelium by P. Putida strain VKM B-1743D around the filter range from 12 to 17 mm.

PRI me R 2. Carry out similarly

0 example 1, but Pusarium heterospprum is used as an indicator phytopathogenic fungus. Zones of growth inhibition of fungal mycelium of F.heterosporum strain F.putida VKM B-1743D are from 10 to

5 15mm.

Example 3. Carried out analogously to example 1. using Fusarium solani as an indicator phytopathogenic fungus. Mycelial growth inhibition zones

F.solani strain F.putida VKM B-1743D ranged from 10 to 15 mm.

Examples 4-6 demonstrating the antagonism of the P.putida strain VKM B-1743D against phytopathogenic bacteria of the genus 5 and Erwinia.

PRI me R 4. A strain of P.putida VKM B-1743D is streaked onto an MPA plate and grown at 30 ° C for 24 hours. Then, a suspension of cells is applied to the surface of the dish using a spray gun.

After 5 days of plant growth in a climate chamber at 25 ° C, the sizes of control (untreated) and experimental plants are compared (Fig. 4). The height of the experimental plants on the 5th day averages 103 ± 6.5 mm, and the control - 50 ± 15 mm.

Example 9. Carry out analogously to example 8, using, in contrast to example 8, seeds of cabbage of the June variety. The germination rate of the test plants also exceeds the germination rate of the control plants. The height of the experimental plants on day 7 is 50 ± 12 mm, and that of the control plants is 30 ± 8 mm.

As can be seen from examples 8 and 9, the proposed strain of Ps. putida VSh V-1743D has the ability to stimulate the growth of radish and cabbage under hypnotic biotic conditions. Thus, in the previous examples, two types of stimulation of plant growth are shown: indirect or indirect stimulation due to suppression of soil phytopathogens; direct stimulation of plant growth in the absence of phytopathogens (hypnotic biotic conditions), apparently, due to the direct effect of the strain on the plant. One of the mechanisms of direct stimulation is, apparently, the ability of the strain to dissolve mineral phosphates. Plant growth stimulation consists of indirect stimulation (by suppressing phytopathogens) (Example 7) and direct stimulation in the absence of phytopathogens (Example 8.9).

Try it on. Demonstrating the ability of the P.putida strain VKM B-1743D to dissolve mineral phosphates. A method is used to identify the dissolution of mineral phosphates by Erwinia herbicola bacteria. A medium is prepared containing 2% purified agar Difco, 1% glucose, 0.1% MgS04.0.1% NaCI and 0.5% NH / iCI. Hydroxylapatite (HAP) prepared as follows is used as a source of insoluble phosphate. HAPs are autoclaved, washed 10 times with sterile bidistilled water, dried and triturated under sterile conditions until a homogeneous powdery powder is obtained. 5 g of HAP powder is added to 100 ml of the mineral medium, the pH is adjusted to 7.2 (III NaOH), and 10 ml of this medium are layered in Petri dishes per 10 ml of pre-poured 1% purified agar. In a medium thus prepared, the content of water-soluble phosphates is less than 1 mm. Cultures in

the stationary phase of growth is plated on plates with the indicated medium, and after 48 hours, the HAP dissolution zones are recorded.

Six strains of bacteria of the genus Erwinia used as a positive control dissolve HAP very poorly. Strains of E.coll B and P.putida 40a are not capable of dissolving HAP and growth on this medium. The strain P.putida VKM B-1743D grows well and forms transparent dissolution zones on this medium. HAP.

Example 11. The effect of a preparation based on the P.putida strain VKM B-1743D on plants infected with phytopathogenic fungi of the genus Fusarlum.

The seeds of greenhouse cucumbers and Black Prince tomatoes are inoculated in suspension of the strain, as described above. Soil infected with phytopathogenic fungi is preliminarily prepared. . In a sterile soil (sterilized by autoclaving for 1 hour at 1 atm.), A mixture of 5-day-old 5-day-old mycelium of phytopathogenic fungi Fusarium heterosporum, F.moniliformj, F.solani is applied at a rate of 100 ml of suspension per 500 g of sterile the soil.

Seeds of cucumbers and tomatoes inoculated with bacterial suspension and control. uninoculated, planted in pots with infected soil in 12 replicates. After 1.0–20 days of growth, the diseased plants with typical signs of root rot and fusarium infection are counted in the climatic chamber.

The data of this experiment are presented in table.3.

As can be seen from Table 3, treatment of seeds of cucumbers and tomatoes of P.putida VKM B-1743D strains under conditions of artificial phytopathogenic background reduces the incidence of plants by 82 and 63%, respectively. A protective effect is achieved by efficiently colonizing plant roots with cells of the P.putida VKM B-1743D strain and suppressing this strain of Fusarium fungi. The strain P.putida AKM B-1743D showed high liveability on plant roots. At the end of the experiment, the strain titer averaged 106 cells per gram of root.

Example 12. Use of a preparation based on the P.putida strain VKM B-1743D for protecting cabbage under conditions of a natural phytopathogenic background.

Cabbage seeds of varieties Slava, Podarok. Dauerweis is treated with a suspension (10 cells / ml) of phytopathogenic bacteria Erwlnia carotovora var. carotovora. After incubation of the plates for 24 hours at 30 ° C, zones of inhibition of growth of phytopathogenic bacteria around the line of the strain P.putida BKM B-1743D are visually recorded. Figure 2 shows the inhibition of the growth of the phytopathogenic bacterial strain Erw. carotovora var. carotovora by several antagonist strains from the collection of a laboratory of plasmid biology, IBPM RAS, including the P.putida strain BKM B-1743D. Zones of growth inhibition of phytopathogenic bacteria Erv.Carotovora var. carotovora strain P.putida BKM B-1743D are 8-12 mm, which indicates the antagonism of the strain P.putida BKM B-1743D against this type of phytopathogenic bacteria.

Example 5. Carry out analogously to example 4, using strain Erw.carotovora var.atroseptica as indicator phytopathogenic bacteria. The growth inhibition zones of these phytopathogenic bacteria by the P.putida BKM B-1743D strain are 10–13 mm, which shows the antagonistic potential of the P.putida BKM B-1743D strain with respect to the phytopathogenic bacteria Erw. carotovora var.atroseptica.

Example 6. Carried out analogously to example 4, using phytopathogenic bacteria Erv.herbicola as an indicator culture. Zones of growth inhibition of Erv.herbicola by P.putida BKM strain. B-1743D is 10-12 mm, which also indicates the antagonism of the P. putida BKM B-1743D strain against this type of phytopathogenic bacteria.

An example demonstrating the ability of the P.putida BKM B-1743D strain to inhibit the growth of phytopathogens in the rhizosphere of cucumbers.

Greenhouse cucumber seeds are inoculated in a suspension of strain cells, as described above. The suspension is prepared as follows: 250 ml of King B sterile medium or MPB are poured into 750 ml flasks; a stationary culture of the strain in the amount of 1-2 ml is introduced into it. Cultivation was carried out at 30 ° C on a shaker (250 rpm) for 12-16 hours to a final titer (3-5) x 10 cells / ml. If necessary, the cell suspension is diluted with Na-phosphate buffer (pH 7.0) or tap water. The soil with a natural phytopathogenic background, taken from the foci of death of cucumbers from root rot, is preliminarily prepared. The treated and control seeds are untreated into the infected soil - untreated seeds. After 2 weeks of growth in a climate chamber at 25 ° C, the sizes of the experimental and control plants are compared. As can be seen from Table 1, seed treatment with cells of the strain P. putida BKM B-1743D leads to a twofold increase in all growth parameters of cucumbers under phytopathogenic conditions.

In the example, it was shown that treatment of cucumber seeds with a suspension of strain cells leads to an increase in cucumber growth under phytopathogenic conditions. Those. The suppressive effect of inoculation with P.putida BKM B-1743D strain under natural conditions is shown. In the analysis, this soil shows a high background of phytopathogenic fungi and bacteria with the predominance of Fusarium growth fungi. Inoculated and uninoculated seeds of Teplichny cucumbers are sown not only in soil with a natural phytopathogenic background, but also in sterile soil (sterilized by autoclaving for 1 hour, at 1 atm). In this case, the following data are obtained, presented in table.2.

A comparison of these data with the data of Table 1 shows that soil sterilization leads to an effect comparable to the effect of bacterization of seeds under phytopathogenic conditions. Bacterization of seeds under sterile soil conditions gives a slight stimulating effect. This indicates that the main stimulation mechanism in this experiment is the suppression of soil phytopathogens by the P.putida strain VKM-1743D.

Examples 8.9 demonstrate the ability of the P.putida BKM B-1743D strain to stimulate plant growth under hypnotic biotic conditions.

PRI me R 8. The seeds of the radish varieties Teplichny processed for 30 minutes 10% H202 solution and checked for sterility by standing on MPA plates. Non-sterile seeds forming bacterial or fungal growth zones on MPA are discarded. Treatment of sterile seeds with P.putida strain BKM B-1743D was carried out as in Example 7. Seeds were seeded in a test tube with sterile medium of the following composition: (per 1 liter).

CaCla x 2 HaO. 0.44 g CMOs. 1.9 g K. 0.83 mg Ca (MO3) 2 x 6Had 0.036 mg KH2P04 0.17 g KZOz 6.2 mg MazMo04 x 2H20 0.25 mg MgS04x7H20 0.37 g MnS04x4H20 22 gm CuSQ4x5H20 0.025 mg ZnSO4 x7H20 8.6 mg FeSCM. 16.5 mg No. 2x EDTA 16.7 mg

ziya strain VKM B-1743D, as described above. Subsequently, three treatments of vegetative plants are carried out in the following growth phases: first - 5-7 real leaves; the second is the heading of a head of cabbage: the third is of the middle head of head. The treatment is carried out with a suspension of cells with a density of 10 cells / ml with an average inoculation load of 3x10 cells per plant. The experiment was carried out under conditions of high natural phytopathogenic background, including fungi of the genus Fusarium. The experimental results are presented in table 4.

Treatment of seeds and vegetative plants with the strain preparation leads to a decrease in cabbage disease, in vivo by 17%.

Example 13. Bacteria of the genus Erwinia are the main causative agent of potato root rot.

The effect of processing potatoes with the VKM-V1743D strain was also tested in vivo, i.e. on a natural basal background of soil pathogens.

Research was conducted on Nevsky potato. The tubers were sprayed. The rate of consumption of the bacterial suspension of density-.IO10 cells / ml was 0.5 l

per 1 ton of planting material. Planting material weighing 3.6 tons was planted on a pilot plot of 0.4 ha.

The final indicator is the yield of potatoes from the experimental plot. Comparison data of the crop from the experimental and control plots of the same area are presented in Table 5.

Thus, the strain Pseudomonas putida VKM B-1743D has the ability to suppress the growth of phytopathogenic fungi: Fusarium moniliformi, Fusarium heterosporum, Fusarium solani and bacteria Erwinia carotovora var. carotovora, Erwlnia carotovora var. atrosptica, Erwinia herbicola. The strain is non-toxic, non-pathogenic to plants, has a stimulating effect on plant growth, reduces the incidence of infection with phytopathogenic fungi of the genus Fusorium, has the ability to dissolve mineral phosphates, which allows the use of this strain to create a drug based on it intended to stimulate plant growth and protect them from fungi of the genus Fusarium and bacteria of the genus Erwinia.

Claims (1)

The claims The bacterial strain Pseudomonas putida VKM B-1743 D for the preparation of a preparation used to stimulate plant growth and protect plants from fungi of the genus Fusarium and Erwinia bacteria. The effect of presowing treatment of cucumber seeds with P.putida strain VKM B-1743D on plant growth Table 1 The effect of presowing treatment of seeds of cucumbers with P.putlda strain KM V-1743D on plant growth in sterile the soil The protective effect of the strain P. putida BKM B-1743D under artificial phytopathogenic background The protective effect of the strain P.putida BKM B-1743D under natural phytopathogenic background table 2 Table 3 Table 4 Table 5