SU1482942A1 - Method of primary extraction of effective strains of soya-bean tuber bacteria - Google Patents

Abstract

The invention relates to agricultural microbiology and can be used for the initial selection of effective strains of soybean nodule bacteria. The aim of the invention is to increase the reliability of the primary selection of effective strains of soybean nodule bacteria. The method consists in inoculation of sterile soybean seeds with the tested strains of bacteria RHIZOBIUM, cultivating plants in conditions that are sterile for the root system for 15–20 days. until the emergence of nodules, then transplanting plants into the soil with the natural population of soybean nodule bacteria and growing for 30–40 days, determining the dry mass of the aerial parts of plants and selecting effective strains according to the greatest increase in the dry mass of plants. The method allows, in comparison with the known laboratory, vegetative and field methods, to increase the reliability of the initial selection of effective strains of nodule bacteria for subsequent selection work with them and at the same time to reduce labor costs. 6 tab.

Description

The invention relates to agricultural microbiology and can be used for the initial selection of effective strains of soybean nodule bacteria.

The purpose of the invention is to increase the reliability of the primary selection of effective strains of soybean nodule bacteria.

The method consists in inoculation of sterile soybean seeds with the studied strains of Phizobium bacteria, cultivation of plants at stage 1 under sterile conditions for the root system for 15–20 days until nodules appear on the roots of plants, transplanted at stage 2 into plants with natural the population of soybean nodule bacteria and cultivation for 30–40 days, the determination of the dry mass of the aerial parts of plants and the selection of effective strains of bacteria by the greatest increase in the dry mass of plants.

Example. In a test tube with a diameter of 21 mm and a height of 200 mm put a strip of filter paper (width 200, height 150 mm), folded accordion and then rolled into a roll. A recess is made in the upper part of the roll to maintain moisture around the seeds. 20-30 ml are added to the tube.

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CO 4b 1C

nutrient medium for plants of the following composition, g / l: KtHPO 1.0; MgS04 1.0, CaS04 0.5, FeS04, H, BO ,, MnS04, and molybdenum salt traces.

The tube is closed and sterilized in an autoclave at 1 atm. 30 min. One sterile soybean seed is planted in a prepared test tube with filter paper and 1 ml of a suspension of soybean nodule bacteria containing 1 million cells / ml is added. B control tubes are added with 1 ml of sterile water. The tubes, closed with cotton plugs, are thermostated for 3-5 days at 25-27 ° C until germination of soybean seeds. They are then placed on racks with additional lighting. For shading the root system of plants, caps of thick paper or dark film are put on the tubes. On the day of the release of this lobe from the tube, the cotton plugs are removed, and around the stem the filter is placed with cotton wool. As required, he observed sterility; 10-20 ml of nutrient solution was additionally added to each tube. The soybean plant in test tubes with a sterile root system is grown for 15-30 days. It was found that for the formation of nodules on the roots due to artificial inoculation, it is enough to grow soybean plants in test tubes for 15–20 days. By this time, an average of 7-11 nodules per soybean root is calculated per plant. A shorter period of time for growing plants in test tubes (less than 15 days) does not fully ensure the formation of nodules on soybean roots. The accuracy of the data is checked by the absence of nodules on the roots of non-inoculated soybean plants. At least 15 soybean plants are grown in each variant.

At the second stage of the method, 15–20 soo-exact soybean plants with nodules on the roots are removed from the tube and immediately transplanted into the soil with a natural population of specific nodule bacteria. The previously infected soybean plants that have been transplanted into the soil have been resistant to secondary infection with nodule bacteria found in the soil for some time. This time is enough to show

the effectiveness of the studied strains of soybean nodule bacteria.

After the planting of soybean in the soil, primary watering is carried out. For 30–50 days, soybean plants are grown in soil with natural (field) or artificial (greenhouse) conditions. The efficiency of the tested strains of nodule bacteria is determined by the increase in the dry matter of the above-ground soybean mass. / In the experiment, strains of soybean nodule bacteria Rhizobium japonicum-646, 648a, 639a, TC-37, TD-55, AC-17 (Table 1.) Were studied.

The testing of these soybean strains by a laboratory-field method showed their substantially different efficacy in increasing the dry matter mass of the aerial parts of plants. So strains 648a and 639a give an increase in the dry matter of the above-ground mass of soybean by 2.1-2.5 times and are more effective in comparison with the known strain 646,

Determination of the duration of the laboratory and field stages of soybean cultivation in the method.

When developing the method, the occurrence of nodules on soybean roots was established at the first stage for 12–15 days after sowing inoculated seeds into test tubes.

However, some strains of nodule bacteria after the above period of time caused the formation of nodules on soybean roots in only 78-95% of the plants. With an increase in the first period to 20 days, nodules on soybean roots are noted in almost all plants. Therefore, the first stage of the process is limited to 15-20 days of growing soybeans in sterile tubes.

The optimal duration of growing soybean plants transplanted into the soil (the second stage of the method) is 30-40 days (Table 2). With an increase in the duration of growing soybean plants in the second stage of the process, the efficiency of artificial inoculation is noticeably reduced. The shorter duration of growing soybean plants does not provide an optimal manifestation of the effectiveness of the tested strains of nodule bacteria.

Comparative evaluation of the methods for the initial selection of effective strains of soybean nodule bacteria.

The primary selection of effective strains of the R. japonicum soybean bacteria is carried out by known laboratory and laboratory-field methods.

The description of the experiment according to the laboratory-field method is described above, using the soybean variety VNIIS-1.

In the analyzed methods have approximately the same number of strains of nodule bacteria of soybean with different efficacy. As a standard standard, use the known effective strain 646.

When using the known laboratory method with three types

Known laboratory method per- 10 substrates only in sand culture

The primary selection of soybean bacterial strains is the cultivation of inoculated plants in 1 liter vessels on sterile agar medium, sand culture or soil.

The agar medium is Prishnikova solution with 0.1 norm of nitrogen and the addition of 2% agar-agar. The same solution, but without agar-agar, is used for sand culture. The prepared vessels with the substrates are sterilized at 2 atm for 30 minutes. In vessels with sand and soil, humidity is maintained at 70% of the full moisture capacity. Soybean seeds are inoculated at the rate of 1 million bacterial cells per 1 seed. Use a grade of soy VNIIS-1. In each vessel, 4 plants are left that are grown for 30-40 days. Further cultivation of soybean plants is limited by the capacity of the vessels, as well as the high aggressiveness of natural Phizobium japonicum. The use of multi-liter vessels dramatically reduces the volume of the tested strains, which is unacceptable during the initial selection.

In the laboratory-field method of primary selection of effective strains of soybean nodule bacteria, the VNIIS-1 variety was also used. Seeds were inoculated at the rate of 1 million / ml of cells per one seed.

The proposed method consists of two stages - laboratory and field. Under laboratory conditions, bacterized soybean plants were grown in test tubes on filter paper to the nodule formation phase for 15–20 days. By growing soybean plants with a sterile root system in test tubes, it is possible to dramatically increase the number of test strains.

Comparing the known (laboratory) and laboratory-field methods of primary selection (Table 3), note the significant advantages of the latter.

It is possible to select the minimum number of strains of soybean nodule bacteria for subsequent trials in field trials. In vessels, where agar-agar and soil are used as a substrate, artificial inoculation of soybean seeds does not have a positive effect on plant productivity. On average, out of 45 tested

strains in a known manner only 2% increase the productivity of soybean plants by 25% in comparison with the control. At the same time, when testing these same strains using a laboratory-field method, 61% of bacterial cultures increase the productivity of soybean plants by more than 25% of the table. 3).

The method allows to identify strains that cause an increase in productivity.

soybean plants 2-2.5 times compared to control. This makes it possible to conduct the primary selection of the most effective :: strains of soybean nodule bacteria for subsequent final testing in field experiments (Table 4).

These data are given in Table 4 for a comparative analysis of the effectiveness of laboratory-field and

a known method of conducting the primary selection.

Known strain 646 is not effective when tested in a known laboratory method with substrates

agar-agar, sand, soil. The use of the new method of the effectiveness of this strain is confirmed.

Strains 648a, 639a, TC-37, TD-55, AC-17, tested in a known manner, are poorly effective or not effective. When tested by their laboratory-field method, these strains show high efficiency. So, strains 648a and 639a

increase the increase in dry matter aboveground mass of soybeans 2.1-2.5 times.

Comparing the results obtained by laboratory and laboratory-field

by means of the primary selection of promising strains, the significant advantages of the latter have been established. The method allows a dramatic increase in the effectiveness and reliability of the initial selection of new strains of soybean nodule bacteria, it is less laborious, it makes it possible to select strains not only in the summer (field), but also in the winter (greenhouse) periods, which accelerates the selection process.

Table 5 shows the results of the vegetation experiment to assess the effectiveness of soybean rhizobia strains. In comparison with the laboratory-field method, in which for these strains there are significant differences in the effectiveness of different strains from - "- 9 to + 257%, in the vegetative experiment on the increase of phytomass, the effectiveness of the strains is much closer (from -13 to +11% the difference in relation to the control). On the basis of the grain productivity, the changes in comparison with the control are -2 to + 8% .g

Labor costs during the vegetation experiment (40 Wagner vessels) 30 make up (without laying out and taking into account the crop) 96 man-days, whereas with the laboratory-field method (40-variant experiment) 24 man-days for the whole work.

When revealing the effectiveness of rhizobia strains in the field experiment (10 variant scheme, total area of 50 m2), total costs reach 120 man-days when taking into account grain to productivity and 60-70 man-days when taking into account the growth rate and reliability of the data obtained compared to laboratory -field experience is also significantly lower (Table 6). 45

Thus, the laboratory-field method of the primary selection of effective strains of soybean nodule bacteria allows, in comparison with known methods, to increase the objectivity of selection and reduce labor costs.

Claims (1)

Invention Formula The method of primary selection of effective strains of soybean nodule bacteria, including the inoculation of sterile plant seeds with the tested strains of bacteria of the genus Khizobium, growing plants under sterile conditions for the root system, determining the dry matter mass of the aerial part of plants, followed by the greatest increase in the dry weight of plants , characterized in that, in order to increase the reliability of the method, growing inoculated plants under sterile conditions is carried out for 15-20 days, then asthenia transplanted into the soil with a population of natural soybean root nodule bacteria and grown 30-40 days. T a to l and c one Strains Mass of above-ground part of soy,% of control costly 178 257 219 145 109 148 table 2 control 6390 TS-31 AS-37 TS-37 TD-73 AS-17 AS-20 AS-26-ZD-4 19.1 18.6 21.2 20.4 19.9 20.0 21.1 18.8 19.1 20.0 100 87 111 107 104 105 110 98 100 105 NSR 1.6 g / vessel That person 3 Table 4 9.6 9.4 10.5 10.3 9.8  9.6 10.4 9.5 9.5 9.7 100 98 109 107 102 100 108 99 99 101 9.5 9.2 10.7 10.1 10.1 10.4 10.7 9.3 9.6 10.3 5 x 0.5 g 6.8 7.6 7.2 6.6 6.2 5.5 6.2 7.6 7.3 Above-ground dry weight, g 6.8 1-and the phase of fruit formation,% to control 100 Yield soybeans, t / ha 18.03 18.13 19.01 17.13 17.79 18.15 16.6318.1318.5517.5% to control 100 112 106 97 91 81 91 112 107 100 100 105 95 99 101 103 100 103 97 NCP05. 2.33 c / ha (13%) x 0.81 c (4.5%) T a b l and c a 6 91 81 91 112 107 7.13 17.79 18.15 16.6318.1318.5517.5 95 99 101 103 100 103 97