RU2193837C2 - Method for applying nitrogen-fixing bacteria into soil - Google Patents

Abstract

FIELD: agriculture. SUBSTANCE: method deals with applying bacterial preparation at the dose providing a hectare bacterial norm within 0,5-5,0•10¹² bac/ha. Moreover, one should conduct leaf feeding of bacterial preparation solution onto vegetating plants. The method enables to decrease financial and labor expenses for soil treatment. EFFECT: higher efficiency. 7 tbl

Description

 The invention relates to agriculture, specifically to methods for introducing bacterial nitrogen fertilizers into the soil for legumes and cereals, forming effective nitrogen-fixing plant-microbial complexes with nodule and associative bacteria.

There is a traditional method of introducing nitrogen-fixing bacteria into the soil, which consists in pre-sowing seed treatment with a bacterial preparation based on strains of nitrogen-fixing bacteria. For example, to treat soybean seeds, rhizotorfin is used - a mixture of a liquid culture of bacteria with peat containing 2.5 billion. nodule bacteria per 1 g of the drug. You can also use a liquid working culture of bacteria of standard concentration, usually with a titer of 3-5 • 10 ⁹ tank / ml. Seed treatment is carried out mainly by hand. For this purpose seeds are poured onto a tarpaulin of size 3 • 4 m per 1-2 hectares of sowing, i.e. 100-200 kg, moisten with water or an adhesive solution at the rate of 1% by weight of the seeds, mix the seeds by alternately raising the ends of the tarpaulin, spray with a solution of the bacterial preparation and mix again until the drug is evenly distributed on the surface of the seeds. Treated seeds are sown on moist soil the same day [1].

 The disadvantages of this method are the following. Firstly, it is necessary to process large masses of seeds in a short time before sowing. Pre-treatment of seeds before sowing is not possible due to the high or complete death of nitrogen-fixing bacteria. For example, it is known that nodule bacteria applied to the surface of soybean seeds die quickly: within 5-6 hours after treatment, their number is halved. Therefore, if the seeds were not sown during the day, they are again treated on the day of sowing. Secondly, manual seed processing is laborious and inefficient. Existing seed dressing machines do not provide the required processing quality, often mechanized processing leads to seed damage. Meanwhile, the mechanization of the introduction of nitrogen-fixing bacteria into the soil is dictated by an increase in the crops of legumes and cereals and the need to process large masses of seeds, which is 10-15% of the annual crop.

 The objective of the invention is to develop a simple and economical method of introducing nitrogen-fixing bacteria into the soil under legumes and cereals, eliminating the time-consuming operation of presowing treatment of seeds with a bacterial preparation and providing the ability to mechanize the process of applying bacterial fertilizers over large areas and in the best biological time.

 The problem is solved by the claimed method, according to which nitrogen-fixing bacteria enter the soil by foliar treatment of vegetative plants with a solution of the corresponding bacterial preparation.

When prescribing the consumption of the drug, one should proceed from the approved rule: success is achieved if the hectare consumption rate of bacteria is ensured in the range of 0.5-5 • 10 ¹² tank / ha, i.e. the titer of the drug should be considered. For example, in the prototype method, for the treatment of the hectare norm of soybean seeds, rhizotorfin with a titer of 2.510 ⁹ tank / g in a dose of 200 g is used. The same dose of the drug is sufficient for foliar treatment of 1 ha of vegetative soybean plants, i.e., hectare doses of the bacterial preparation in known and claimed method are almost the same.

 However, in essence, the methods are different from each other. In the prototype method, nitrogen-fixing bacteria are introduced into the soil with seeds, in the new method, they are applied to vegetative plants, excluding this laborious and inefficient seed treatment before sowing. A bacterial preparation in the form of a working solution is applied to plants by known methods: spraying or aerosols. Using aerosol technology of optimum dispersion, it is possible to process large sown areas in the best biological terms.

 The applicability of the claimed method and its advantages are confirmed by the following examples.

 Example 1. Vegetative experience with soy.

Soybean seeds of Omskaya 8 variety were grown in vegetation vessels containing 7 kg of washed quartz sand, calcined to create sterile conditions. Pryanishnikov’s nutrient mixture was added to the vessels with 0.1 norm nitrogen composition (based on 1 kg of soil or 1 liter of water): NH ₄ NO ₃ - 24 mg, CaHPO ₄ - 172 mg, FeCl ₃ • 6H ₂ O - 25 mg, CaSO ₄ • 2H ₂ O - 344 mg, MgSO ₄ • H ₂ O - 123 mg, KCl - 160 mg. In addition to these salts, the nutrient mixture was supplemented with a solution of trace elements. The humidity in the vessels was maintained at a level of 70% of the total moisture capacity by daily watering by weight. 8-10 seeds were sown in each vessel, followed by 5 well-developed plants. In the experiment with different variants of introducing nodule bacteria, strain 2940 Bradyrhizobium japonicum from the culture collection of the All-Russian Research Institute of Agricultural Microbiology of the Russian Academy of Agricultural Sciences was used. Repeat 4-fold options. In the control variant K _{1, the} seeds were inoculated during sowing, i.e., the seeds were traditionally treated with a bacterial strain. In the second control variant K ₂ bacteria were not introduced at all. In three experimental variants (O ₁ , O ₂ , O ₃ ), bacterization was carried out by spraying at different intervals after germination. Moreover, in the O ₁ variant, spraying was performed a week after germination (phase of one non-leafed leaf). In this case, the bacteria hit the plants and soil. In option O _2, spraying was carried out 2 weeks after germination (phase of two leaves, one of which was triple). In option O _3, spraying was also carried out 2 weeks after germination, but the soil in the vessels was covered to isolate from bacteria entering the soil. The titer of the bacterial preparation was 10 ⁷ tank / ml. The consumption rate of a liquid preparation is 20 ml / m ² . The bacterial solution was applied using a Kwasar hand sprayer.

 During the experiment, attention was paid to the growth and development of soybeans, as well as the color of the experimental and control plants was recorded as an indicator of the nitrogen supply obtained by nitrogen fixation of nodules that arose during different methods of introducing bacteria. At the end of the experiment, 2 months after sowing (June 15 - August 15), the roots of the plants were washed from the sand, the number and biomass of nodules were calculated, and the nature of their location on the roots in each experiment was noted. Nitrogenase (nitrogen-fixing) activity was analyzed by the acetylene method. The elements of soybean productivity (the number and weight of beans) were determined, plant samples were taken for protein analysis, determined by the method of Buzun and others. Quantitative data are shown in tables 1-4.

During visual observation 48 days after sowing, it was noted that in variant K ₁ , the plants are normally developed, green in color; in option K ₂ - the color of the leaves of the plants is yellow; in the O ₁ variant, the plants are also green, in appearance they do not differ from the type of plants in the K ₁ variant; in variant O ₂ - the color of the leaves is yellowish, noticeably different from the green color of variants K ₁ and O ₁ ; in O ₃ - the leaves also have a yellow color, indicating a lack of nitrogen.

55 days after planting, plants in variants O ₁ , O ₂ and O ₃ are green in contrast to yellow leaves in variant K ₂ . Thus, if 48 days after planting, the plants in variants O ₂ and O ₃ were still yellow, then a week later the leaves turned green, which indicates the flow of nitrogen from nodules formed on the roots of the treated plants.

When studying the root system of soybean plants 60 days after planting, it was found that nodules were completely absent on the roots of soybean in variant K ₂ . The yellow color of such plants indicated a lack of nitrogen. In variant K _1, nodules formed on the main root and provided a green color for the plants. In variants O ₁ , O _2, and O _3, nodules formed later on the lateral roots.

 Thus, the nature of the location of nodules on the roots of plants grown from seeds inoculated with bacteria during sowing differed from plants treated with bacteria according to the claimed method (1-2 weeks after germination in the phase of 1-2 leaves). Nodules during seed inoculation were located on the main root, while spraying the leaves on the lateral roots, which indicates their later formation.

In the table. 1 shows quantitative data on the nitrogen-fixing activity of soybean nodules. Soybean nodules in all experimental vessels had significantly higher nitrogenase activity. It is 24-35% higher compared to control K ₁ . Thus, the nitrogen-fixing activity of soybean nodules formed during the application of a bacterial culture during spraying of plants at the beginning of the growing season is significantly higher than during inoculation of seeds, and the higher the earlier the spraying is performed. In our experiments, nodules during treatment 1 week after germination had higher activity compared to plants treated 2 weeks after germination.

The higher nitrogenase activity of nodules in the O ₁ , O _2, and O ₃ variants is explained by their later formation. The nodules in the K ₁ control apparently had already passed the peak of high activity by the time of measurement.

In the table. 2 shows the number and weight of soybean nodules according to the experimental options. When spraying leaves and soil surface (O ₁ and O ₂ ) the number and fresh weight of nodules is significantly greater than the control K ₁ .

In the table. Figure 3 shows data on productivity elements (the number and weight of beans) of two-month-old soybeans according to the experimental options. It can be seen that in all experimental variants with different terms and methods of spraying with nodule bacteria, the number of beans is not lower, and in the variant with earlier spraying, O ₁ is 37% higher than in the control with traditional seed inoculation during sowing (variant K ₁ ). The raw and dry weight of the beans in all experimental versions, with the exception of O ₃ , was 1.6-2 times higher than the control version of the prototype.

In the table. 4 shows data on the protein content (% by dry weight) in the leaves of the beans (pods) and soybean nodules, depending on the timing and methods of introducing nodule bacteria. The data obtained reflect the dynamics of protein accumulation and redistribution into economically important plant organs. It was shown that in nodules the protein content in all variants was the same. In the leaves of the experimental variants O ₁ and O ₂ accumulated 2% more protein than in the control K ₁ prototype. In beans of an experimental version of an earlier period of spraying with nodule bacteria (O ₁ ), 3% more protein was accumulated in comparison with the prototype K ₁ . In a variant with a later period of introducing bacteria by spraying (О ₂ ), the beans were less developed and the bulk of the protein was contained in the leaves. The data on option O ₃ from tables 2, 3, and 4 indicate the unpromising isolation of the soil path of penetration of nodule bacteria.

 Thus, under the controlled conditions of the growing experiment, it was reliably established that nodule bacteria introduced by spraying the plants enter the soil and penetrate the roots, forming nitrogen-fixing nodules, which in their activity exceed the control variant using traditional seed treatment with bacterial culture during sowing. The functioning of nodules increases the protein content in the plant, which results in a significant increase in the yield of beans.

 Field experiments on soybean and wheat crops were performed on plots located on the experimental fields of the Siberian Research Institute of Agriculture and Chemicalization SB RASHN.

 Example 2. Field experiments with soy.

The experiments were conducted on plots with an area of 25 m ² in 4-7 short repetition. Experience options included:
a) traditional presowing treatment (inoculation) of soybean seeds with a culture of nodule bacteria, similar to that used in the growing experiment. To treat 10 kg of soybean seeds, 50 ml of bacterial culture with a titer of 10 ⁹ tank / ml was used. With this flow rate, the hectare norm of bacteria (for 100 kg of seeds) is 0.5 • 10 ¹² tank / ha.

b) foliar application of the same nodule bacteria by spraying soybean plants in a 2-leaf phase with a solution of a bacterial preparation with a titer of 10 ⁸ tank / ml. The consumption rate of the drug is 1.0 ml / m ² , which provides a hectare rate of bacteria 10 ¹² tank / ha. To obtain a working solution, the bacterial preparation was diluted with water in a ratio of 1:20. Spraying was carried out with a Kwasar backpack sprayer at a rate of solution consumption of 20 ml / m ² .

 c) sowing seeds without any treatment (control option).

 A measure of the effectiveness of field experiments was the soybean seed crop. Harvesting was carried out by the Sampo-500 combine with subsequent weighing of each repetition and sampling to determine the moisture and weediness of seeds.

 The results of the experiments are shown in tables 5 and 6.

 In the table. 5 shows the soybean crop and the mass of 1000 grains in all 3 variants of the experiment. It is seen that the increase in the soybean seed yield and the mass of 1000 grains with foliar application of nodule bacteria are greater than in other variants of the experiment. This indicates the supply of additional nitrogen to the plants as a result of the functioning of nodules formed by spraying soybean plants with a culture of nodule bacteria.

 From the table. Figure 6 shows that the weight of nodules per 1 plant and the weight of one nodule in sprinkled plots is greater than in the control variant, which is consistent with an increase in soybean yield in the experimental plots. Thus, an increase in soybean seed yield and weight of nodules in the treated plots indicates the effectiveness of the claimed method of foliar application of nitrogen-fixing bacteria into the soil.

 Example 3. Field experiments with wheat.

Three varieties of hexaploid soft spring wheat were used: strong early ripe "Novosibirsk 22", valuable mid-ripening "Lyuttsens 25" and strong mid-ripe "Novosibirsk 89". Field plots had an area of 25 m ² in 4-7-fold repetition in the experiment and control.

 For the preparation of the bacterial preparation, pure cultures of associative bacteria of 3 genera were used: Azospirillum lipoferum 137 (preparation Azorin), Arthrobacter mysorens 7 (preparation Mizorin) and Flavobacterium sp. L-30 (drug Flavobacterin). The preparations were obtained from the All-Russian Research Institute of Agricultural Microbiology of the Russian Academy of Agricultural Sciences (St. Petersburg, Pushkin) and propagated at the Vector NGO (Koltsovo, Novosibirsk).

Spraying of the plots was carried out with a Kwasar backpack sprayer at a rate of flow of working fluid of 20 ml / m ² . The treatment time is at the beginning of the growing season, in the tillering phase. The consumption rate of a mixture of 3 strains of associative bacteria is 1.0 ml / m ² with a titer of 10 ⁸ tank / ml with the addition of 0.1-0.2 ml / m ^{2 of a} 2% solution of salts of 6 trace elements in equal proportions: zinc, molybdenum, cobalt, boron, manganese and iodine for better plant growth and ensuring normal conditions for the functioning of bacteria. Such a consumption of the bacterial preparation provides a hectare rate of associative bacteria of 10 ¹² tank / ha.

 Control - no treatment.

 The results of the experiments were determined by grain yield. Cleaning was carried out by the Sampo-500 combine. Grain samples were weighed, with an accuracy of 50 g immediately after mowing the plot directly on the combine with sampling for moisture and weeds. The results of the experiments are given in table.7. Test options included the precursor for stubble (wheat for wheat without fertilizer) and steam. The increase in the yield of wheat cultivars Novosibirsk 22 and Lutescene 25, sprinkled with associative bacteria in the tillering phase, is reliable. There is no increase for a couple.

 Variety Novosibirsk 89 turned out to be immune to nitrogen-fixing bacteria: there is no yield increase for both stubble and steam.

 Thus, the introduction of nitrogen-fixing bacteria on grain crops makes sense against a poor background, in this case, on wheat stubble without fertilizers, and does not yield returns on the steam field. This can be explained by the lack of nitrogen in the grain precursor, which was supplemented by the introduced nitrogen-fixing bacteria and thereby ensured an increase in yield. The steam field wheat crops are apparently immune to nitrogen-fixing bacteria due to the presence of available mineral nitrogen accumulated in the steam field in the soil.

 According to the literature, publications from the beginning of 1980 [2] it is known that in associative systems: soil - bacteria - plants, the decisive role belongs to the plant. It was experimentally established on 40 varieties of spring, winter and durum wheat from the world collection of VIR that only a quarter of the studied varieties could receive nitrogen supplied by associative bacteria. This ability is genetically determined and is associated with the nis trait in both wheat and other cereal crops, including barley, millet, millet, sorghum, and rice. In field experiments, the ability to associative nitrogen fixation was 8-50 times different in cereals. The dependence of associative nitrogen fixation on ploidy of wheat was also established. In diploid wild and cultivated wheat species, the ability for associative nitrogen fixation is up to 4 times greater than in tetra- and hexaploid forms. Modern varieties are tetra- and hexaploid forms of wheat, since an increase in ploidy is accompanied by an increase in yield, but there is a decrease in protein content. Nevertheless, in modern varieties of hexaploid wheat, aimed at obtaining the maximum yield, the ability to associative nitrogen fixation is not lost [3]. Apparently, the strategy for providing cereals with “biological” nitrogen is to select crops for the nis trait mentioned above and to conduct a wide screening of varieties that are responsive to the introduction of nitrogen-fixing bacteria.

 So, the conducted vegetation and field experiments on the application of the claimed method on soy and wheat showed that the foliar application of nitrogen-fixing bacteria on the leaves and soil surface is more effective than the traditional treatment of seeds with these bacteria before sowing. Moreover, the claimed method is less time-consuming, its implementation is carried out using known and affordable mechanized devices: sprayers, aerosol generators. When using, for example, an optimal dispersion generator [4], it is possible to process large areas (1-5 thousand ha per night shift) in the best biological terms, which makes this method economically viable and promising.

Literature
1. Recommendations for the rational use of rhizotorfin under soy in the south of Ukraine. All-Russian Research Institute of Agricultural Microbiology. Simferopol - 1985.

 2. Noisy V.K., Sidorova K.K. and others. - Biological fixation of nitrogen. Novosibirsk The science. Sib. Separation. 1991, p. 271.

 3. Cheremisov B.M. Enhanced nitrogen fixation is a new direction in the selection of wheat and other non-leguminous field crops. Review.- Agricultural Biology, 1988, 6 pp. 43-49.

 4. RF patent 950266, 1993. (A.C. 950266, 1982, BI 30).

Claims (1)

The method of introducing nitrogen-fixing bacteria into the soil for legumes and cereals, including the use of a bacterial preparation in a dose that provides a hectare norm of bacteria in the range of 0.5-5.0 • 10 ¹² tank / ha, characterized in that the solution of the bacterial preparation is applied by the foliar method vegetative plants.

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