Strain of microorganism azotobacter vinelandii for preparing biopreparation for control of wheat root rots and enhancing quality and yield of harvest

FIELD: agricultural microbiology.

SUBSTANCE: the strain Azotobacter vinelandii IB 4 is obtained by analytical selection of natural isolates by method for selection of producers eliciting rather high antagonistic activity with respect to fungal phytopathogens. The strain is deposited in collection of microorganisms of Biology Institution UNTS RAN at number Azotobacter vinelandii IB 4. For preparing the preparation the strain is grown in nitrogen-free nutrient medium at 28-30oC for 60 h under aeration condition up to the titer value 1010 CFU/ml. The strain elicits high nitrogenase activity and ability for producing cytokinins that simulate the growth and development of plants.

EFFECT: valuable properties of microorganism strain.

7 tbl, 7 ex

 

The invention relates to agricultural Microbiology and concerns of the strain, which has antagonistic activity against the causative agents of root rot of wheat increases wheat yield and protein content in grain. The new strain can be implemented in microbiological industries for production of a biological product for protection from diseases and increase yield and grain quality of cereal crops.

A known strain of Pseudomonas sp. CMPM-348, used to receive the drug while protecting grass from common root rot pathogen Helminthosporium sativum [1]. However, this strain is not effective to suppress root rot of cereals caused by a complex of fungi Fusarium sp. and Helminthosporium sativum.

Known strains of Pseudomonas putida, suppressing the growth of the phytopathogenic fungus Fusarium oxysporum [2], and Pseudomonas putida BKM-D suppressing the growth of fungi of the genus Fusarium [3]. A disadvantage of the known strains is a narrow range of antagonistic action.

Known strain of Azotobacter chroococcum N-3173 designed for bacterial fertilizers [4], increasing the yield of barley. The disadvantage is the lack of antagonism against phytopathogenic fungi.

Closest to the claimed is a strain of Azotobacter chroococcum 92 [5], which are able to inhibit the growth of some phytopathogenic fungi. The lack of strain is narrow when ECTR antagonistic action and relatively low productivity.

An object of the invention is the isolation of a new natural strain with a broad spectrum of antagonistic action on pathogens of fungal diseases of wheat, which have high productivity and nitrogenase activity, the ability to increase the yield and quality of grain.

This object is achieved by identifying and exploiting strain of Azotobacter vinelandii IB 4, selected from a sample of arable land, selected in Salavat district Republic of Bashkortostan.

The strain is kept and maintained in the Collection of microorganisms, Institute of biology, Ufa scientific center of RAS, has a collection number Azotobacter vinelandii IB 4.

1. Cultural and morphological characteristics.

A. On agar nutrient medium (40), having the composition, g/l: KN2RHO4- 0,2; CNRO4- 0,8; CaSO4*2H2O - 0,1; MgSO4*7H2O - 0,2; Fl3- 0,01; PA2Moo4- 0,01; yeast extract and 0.5; sucrose 20 g; agar - 1,5%; distilled water 1000 ml, pH 7.4, culture forms a round colonies with a diameter of 3-4 mm, with a smooth shiny surface, convex, transparent, with smooth edges, soft mucous consistency. 24 hour cell growth - small sticks 0,5*0,7 µm, slightly movable; at 48-72 hour - have cysts.

b. On nutrient agar medium Burke, having the composition, g/l: K2NRA4- 0,8; sub> 2RHO4- 0,2; MgSO4to 0.3; NaCl To 0.2; CaSO4- 0,1; Fe2(MoO4) is 0.01; glucose - 10 g; distilled water to 1000 ml; agar-agar and 1.5%; pH of 6.8 to 7.0, culture forms a round colonies 1-2 mm in diameter, with a smooth shiny surface, convex, transparent, colorless, homogeneous consistency.

C. On solid nutrient medium with benzalkonium sodium as the sole carbon source growth is absent. When making benthological sodium in the environment in a quantity of 0.15% there are small colonies of 1-2 mm

d. Liquid nutrient medium Fedorov with molasses, having the composition, g/l: K2NRA4to 0.3; Sanro4- 0,2; MgSO4- 0,3; K2SO4to 0.2; NaCl To 0.5; FeCl3- 0,01; caso3- 5,0; molasses - 40 g; a mixture of trace elements 1 ml; pH of 6.8 to 7.0, culture forms a round colonies with a diameter of 4 mm, convex, with smooth edges, cream color, mucous consistency.

that is, In a nutrient medium the CCA of the colonies are round with a diameter of 5 mm, flat, opaque, ivory, buttery soft texture. Pigment does not form. 24 hour growth - small sedentary sticks with obtuse ends, for 48 h to form cysts.

f. The nutrient medium Ashby with mannitol as carbon source colony oily translucent 1-2 mm in diameter, texture stretching. 24-48 hour cell growth - small sticks of 0.6 μm, C is rounded ends, laboudigue.

2. Physiological and biochemical characteristics.

Strain is an aerobic microorganism, the optimal growth temperature of 28°C.

The sources of carbon: very good metabolizes glucose, sucrose, molasses; absorbs well mannitol, maltose, xylose, galactose, mannose, glycerin, fructose, ethanol; weakly absorbs rhamnose, dextrin, mesoinositol, lactose; not absorbed: sorbitol, arabinose. Weakly increases in MPA.

Grows well on medium with acetic acid sodium as the sole carbon source.

The sources of nitrogen: growing on nitrogen-free media, while maintaining high productivity not less than 1*1010CFU/ml does Not die when heated at 50°C for 15 minutes

Strain resistant to ampicillin (700 μg/ml), kanamycin (400 mg/ml). Sensitive to rifampicine (100 μg/ml), chloramphenicol (100 µg/ml).

The strain does not possess phytopathogenic activity, what svidelstvuet lack of maceration on the slices of potatoes when applied to them by the injection of living cells of strain.

The strain is stored on the doorposts with the environment-40 in the refrigerator subculture on fresh posts in 2-4 months, and freeze-dried state.

The strain is identified by the identifier Bergey Manual of Determinative Bacteriology.

Example 1. To compare the productivity of a new strain of Azotobacter vinelandii IB 4 with a known strain Aotobacter chroococcum 92 were grown in flasks on a shaker at 180-200 rpm for 60 hours, at a temperature of 28°With liquid nutrient medium of the following composition, %: molasses - 3,0;2NRA4- 0,03; Sanro4- 0,02; NaCl - 0,05; caso3- 0.5; K2SO4- 0,02. The results are shown in table 1 and show a higher productivity of the proposed strain on the strain of the prototype. The high value of the productivity of the proposed strain noted when growing on other nutrient media used for the cultivation of microorganisms of the genus Azotobacter (table 2).

Example 2. The study of the antagonistic activity of a new strain of Azotobacter vinelandii IB 4.

On Petri dishes with agar medium the CCA (grated potatoes - 200 g; glucose, 20 g; agar 20 g; water, water - 1 l) plated solid lawn suspension of spores of mushroom phytopathogen. Then in the same Cup prick plant bacteria strain Azotobacter vinelandii IB 4. Cup incubated at 28°C for 3 days. Accounting is carried out on areas of the absence or suppression of fungal growth around the colony of the strain. The results are shown in table 3 and indicate a broad spectrum of antagonistic action of a new strain.

Example 3. The new strain was tested in small-plot experiment in the experimental field of the Institute of biology, Ufa branch of RAS on the efficiency of biological protection against complex pathogens of root rot of wheat and to increase the quality and quantity of the giving birth on wheat varieties “Reaper” with the natural level of contamination of seed. The day before sowing the wheat was treated with culture fluid (with a titer of 4*109CFU/ml) strain of Azotobacter vinelandii IB 4 at a rate of 1 ml culture fluid per 1 kg of grain, which corresponds to the average of 3.2*104living cells to 1 grain. The control was treated with sterile tap water. In the course of the experiment were used land plots 4 m2in triplicate for each option, any repetition of all variants were randomized to each other. As standards took known commercial biological products “agate-25” in the amount of 0.04 kg/t and mitosporic” - 0.5 kg/so the protein Content in grain was evaluated according to GOST 10846-91. The results of the experiment are given in table 4 and indicate a greater efficiency of the new strain of Azotobacter vinelandii IB 4, manifested in enhancement of the degree of biological protection of wheat from root rot, increasing its yield and the increase in protein content in the grain.

Example 4. The strain of Azotobacter vinelandii IB 4 has a sufficiently high nitrogenase activity, which was determined by acetylene method. The results are shown in table 5, this table shows nitrogenase the activity of some strains of bacteria of the genus Azotobacter, proposed for use under various crops [6-9].

Some microorganisms living in resof the re plants, able to synthesize cytokinins. Cytokinins are a natural compound involved in the hormonal regulation of plant ontogeny, and are characterized by their ability to induce the division of plant cells and are the most active among the open astragulus substances. The formation of the inventive strain of cytokinins in the amount of 118 ng in 1 ml of culture fluid was detected by indirect competitive enzyme-linked immunosorbent assay [10]. To confirm the presence of growth stimulating activity in the proposed strain experiments were carried out on bean plants (examples 6 and 7).

Example 6. The seeds of haricot Kharkiv Belokamennaya 45 were soaked for 3 hours in 20 ml of the diluted culture fluid strain of Azotobacter vinelandii IB 4 with title 107CFU/ml Control variant were treated in sterile tap water. Each option was taken on 12 seed beans. The plants were grown for 15 days after appearance of the first shoots. The results are shown in table 6.

Example 7. Studied the ability of the proposed strain to stimulate root formation in cuttings of beans. To do this, cuttings 15-day-old bean plants were placed in water with the addition of the culture liquid obtained by fermentation in a nutrient medium With 40, the titer of the resulting suspension 107CFU/ml of the control Cuttings R is Steny were placed in sterile tap water. The number of cuttings in each case is 10, the length of experience 12 days. After finishing the experiment was counted formed roots. The results are shown in table 7.

The results of the experiments (table 6, 7) indicate the presence of certain growth-stimulating activity of a new strain, expressed as in improving the development of bean plants, and in a considerable degree of klubenkoobrazovaniya on the roots of these plants and stimulate the formation of roots on cuttings.

Thus, isolated a new strain of Azotobacter vinelandii IB 4, which has high productivity, broad spectrum antagonistic effects on fungal phytopathogens. Pre-inoculation of wheat these microorganisms can increase its yield and protein content in grain. The strain has the ability to produce cytokines, showing growth-stimulating activity. Listed useful properties allow it to be used to combat fungal diseases of wheat and increase its yield.

References

1. USSR author's certificate No. 1464468, class a 01 N 63/00, 1988.

2. U.S. patent No. 4714614, CL 424-93, 1987.

3. Patent RU No. 1805849, class a 01 N 63/00, 1993.

4. USSR author's certificate No. 1359272, CL 05 F 11/08, 1985.

5. USSR author's certificate No. 922105, CL 05 F 11/08, 1982.

6. Patent RU No. 2074159, CL 05 F 11/08, 1997.

7. atent RU # 2074158, CL 05 F 11/08, 1997.

8. Patent RU No. 2074157, CL 05 F 11/08, 1997.

9. Patent RU No. 2076088, CL 05 F 11/08, 1997.

10. By YOB, Veselov HE, Karavaiko N.N., Guli-ass old Testament, Cherezova H.E., A.R. Mustafina, Moshkov IE, Kulaeva O.N. Immunoassay system for detection of cytokines //plant Physiology. - 1990. - V.37, # 1. - S-199.

Table 1
StrainThe fermentation time, hThe number of viable cells (CFU), bn/ml
Azotobacter vinelandii IB 46093
Azotobacter chroococcum 92 (prototype)602,3

Table 2

The titer of microorganisms Azotobacter vinelandii IB 4 under cultivation on different nutrient media
The number of viable cells (CFU)/ml
Nutrient medium24 hours48 h60 h
S-401·1076,5·1082,2·1011
Fedorov4·1071·1093·1011
Ashby6,5·1082,2·1011 3,3·109
The CCA2,6·1086·1099·1010

Table 3

Antagonistic action of strains of Azotobacter vinelandii IB 4 and the prototype strain on fungal phytopathogens
Kind of phytopathogenic fungiThe diameter of the zone of inhibition of growth of the fungus, mm
Strains
Azotobacter chroococcum 92 (prototype)Azotobacter vinelandii IB 4
Fusarium culmorum-20
Fusarium gibbosum-19
Fusarium graminesrum-15
Fusarium nivale-9
Fusarium semitectum-14
Fusarium solani29
Fusarium avenaceum-19
Fusarium moniliforme-12
Bipolaris sorokiniana420
Penicillium funiculosum-14

Table 5

Nitrogenase asset is ity of the proposed strain of Azotobacter vinelandii IB 4 and its analogues
CultureNitrogenase activity, Μg N2/ml/hour
z IB 40,656
Az. chroococcum 35 [6]is 0.260
Az. chroococcum ø 25 [7]0,450
Az. chroococcum C 40 [8]0,110
Az. chroococcum B 1 [9]0,220

Table 6

The results of model experiments to study the effect of a new strain Az IB 4 on the development of plants beans
OptionThe average plant height, cmThe average weight of roots*, gThe number of nodules formed, larger than 2 mm, PCs
Control21,819,602
Azotobacter vinelandii IB 423,9515,6046
* figures are given on a wet weight

Table 7

The results of the experiment in the stimulation of rooting in cuttings of beans
OptionThe number of dead plants by the end of the experience, %The number of cuttings that formed roots, PCThe total number of roots per 10 cuttings beans,PC
Control8217
Azotobacter vinelandii IB 41793

The strain of bacteria Azotobacter vinelandii, deposited in the collection of microorganisms, Institute of biology, Ufa scientific center of RAS number Azotobacter vinelandii IB 4, to obtain the drug against diseases of wheat caused by fungal phytopathogens, improving the quantity and quality of the harvest.



 

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