SU922104A1 - Method for preparing inoculum for seed of leguminous plants - Google Patents

Description

(5) METHOD FOR OBTAINING INOCULATOR FOR SEEDS. - I., The invention relates to organic fertilizers with the addition of cultures of bacteria and can be used in agriculture. A known method of half inoculation for seeds of legumes, according to which, in order to intensify the process and improve the quality of the preparation, the layer of peat crumb is fluidized and at the same time periodically sprayed culture liquid at a temperature of a layer of peat crumb 30-35 C to reach the final moisture JS-tO tU. The disadvantage of this method is that mixing peat with a liquid culture and drying the preparation at a temperature of 30-35 C leads to a significant death of nodule bacteria. The device is contaminated with mold fungi, since the preparation is obtained without observing aseptic conditions when drying on non-sterile peat and on thermal peat. BOBY CULTURES - .2,,. sterilization. A preparation with a low titer is obtained due to the death of bacteria in the process of applying the culture to peat. There is also known a method for producing inoculum for leguminous seeds, including using crushed peat, soil and their mixtures as a substrate carrier, adding to the substrate carrier of the source of carbohydrates, minerals and activated carbon, neutralization of the mixture obtained with chalk, sterilization, seed inoculation of the culture of nodule bacteria, mechanical mixing, packing in plastic bags, bringing the end LfTetanus product to humidity TS0-60% and rearing nodule bacteria at a temperature 20ZOS for three-five days. In soybean nutritional supplements include ammonium and phosphoric acid salts in the amount of 0,., Molasses or glucose in the amount of 8-10% 2.

J 92210

The known method has the disadvantage that the titer of the inoculum is not high enough, there are many sroflora in the composition of the preparation and it is difficult to store it for more than three months. five

The purpose of the invention is to increase the titer of inocula nta, reduce the content of extraneous microflora in it and increase the shelf life of the inoculant.

The goal is achieved by the fact that according to the method, sterilization is carried out after packaging in plastic bags with ionizing radiation at a dose of 2.6-6.0 Mrad, inoculation of nopota np bacteria is produced by. p) of packing the packing under aseptic conditions with a hollow needle, the hole from the needle is sealed with adhesive tape, the moistening is carried out in two stages — first, before packing, to carry 25–0%, and then after packing, to 0–60, bags are filled inoculum for 40-80% of their total

Substrate

Source contamination

volume, and rearing is carried out at a temperature of 3 to 3 days.

If necessary, a substance selected from the group consisting of glycerin, dextrin sorbitol, hydrol is added to the carrier substrate.

Work on an industrial plant with a dose rate of 3 {O rad / se to a one-time load of 0.8-1.0 tons of substrate, using various peat and soil, found that the optimal doses that ensure the sterility of the substrates necessary for the preparation of the preparation are. doses of 2.0-6.0 Mrad depending on the initial contamination (dissemination) of the substrate and the required shelf life of the drug (Table T),

In tab. 1 shows an effective sterilizing dose, depending on the initial contamination of peat or soil, spore microorganisms.

Table 1

Effective sterilimn / g zazu inga dose, Mrad

Sod podzolist

Preliminary incubation of the substrate with humidity at a temperature of 20–37 ° C for three to five days provokes the germination of spore forms of fungi and bacteria, resulting in effective sterilization up to approximately 30–50, which is of significant economic importance. in the production of nodule bacteria on the considered substrates, 55 tons.

When substrates are irradiated at the Industrial Electron Accelerator, it has been established that the dose of beta rays does not exceed the biological / Ohm equivalent of the dose of gamma rays to obtain an equal sterilizing effect.

Comparative data on the production of peat preparation of nodule bacteria on peat sterilized by various methods are presented in Table. 2.,

As can be seen from the table. 2. radiation method of sterilization of peat, in contrast to the heat, provides dos592210A "

sufficiently high amounts of the tuberous number of extraneous micro-mayor bacteria in the preparation, even after the organism (there are hundreds of times less than six months of storage, and the minimum with the thermal method

Thermal (in autoclave) at 2.0 atm 1 h

10.50

at 2.0 at 2 h. Tubercular ba 2, 3.17 at 1.0 at 1 h g. - 3.10 at 1.5 at 3 h at 2.0 at 1 h 1, 3.35 rd (And 2 , 0 at 2 h 1 times 2.95 at 2.0 at 2 h 3 times 2.47 at 2.0 at 3 h 1 times 3.00 at 2.0 at 5 h 1 times 2.57 Radiation gamma rays (2.5 mrad)

gamma rays (2.0-6.0 hell)

beta rays (2.5-5.0 Mrad)

Table 2

118.0

6.80

6.00

14.8712.00

0.55

0.83.

12,139,66 Pea nodule bacteria, pieces U, 47 6.80 2.90 108.0 Pea nodule bacteria pcs., 2.57.008,855.63 7.00 .8,855.630, Soybean nodule bacteria, pcs. 6116 soybeans, pieces 6 (6, 00 187.0, I. ... ..... .... The inoculum obtained from nodule bacteria by the proposed method consists of the following operations: preparation of inoculum - liquid culture of nodule bacteria preparation of substrate substrate) (grinding, neutralization with chalk to pH-6.8-7.2, wetted or not packed into non-transparent (plastic bags for microorganisms, sterilization of the packed carrier substrate with gamma or beta radiation at a dose of 2.6-6., O, inoculating the substrate with a liquid culture by injecting it directly into the packaging; Pouring the outside of the ulium in bags in a drum mixer for 3-, 10 minutes to bring the final moisture content of the inoculum to kQ-60% (the bag is filled to 0-80 from their spin) and glue the hole from the needle with adhesive tape, growing under aseptic conditions. inoculum nTA at a temperature of three to five days Example 1. Preparation for soybean: A liquid culture for inoculation of a sterile carrier substrate was prepared by the submerged culture method of soybean nodule bacteria in flasks and five-liter bottles on a rocking chair, and also in 100 l fermenters on medium are as follows of the composition,%: corn steep liquor 0, 0; Baker's yeast 0.05-0.1-, (NNch) 2.SOit-0.1-, KHjPOi 0, 025, - 0.025; MgO :, 0.02, glucose 0.5-1.0; molasses - 1.0 Caco or CaCli .. - 0.02-0.01. The culture was grown for three days. Peat with a humidity of 20-25 or soil with a moisture of 10 was ground, 3 10 technical chalk was added, 3-25 technical activated carbon was added, then the mixture of peat with coal and chalk was moistened to ЗЗ- + О, packed in plastic bags of 0.35 -0.85 kg (based on the preparation of 2–5 hectare portions of the drug, when filled to 0–80 volumes. The bags were sealed, packaged for three to five days at a temperature, and sterilized with gamma radiation at a dose of 2.6 Mrad on an industrial gamma device. Sterile peat under aseptic conditions (in boxing) was inoculated with a culture containing carbohydrate sources (glucose, sucrose, mixtures thereof, lactose, dried milk serum, milk powder or molasses) or protective substances (glycerin, dectri, sorbitol, hydrol) by injection directly into the bag, Injection was performed by needle, Protective substances were introduced in the amount of 1-10% by weight of the dry substrate, depending on the shelf life of the drug. Liquid culture was made from the calculation of the initial content of nodule soy bacteria in the preparation 5.0-0.1 billion / g, depending on the shelf life of the drug and humidity in the range of 50-60 for the peat and LO for soil preparation. The puncture was sealed with adhesive tape and the contents of the bags were stirred mechanically in special mixers for 3-10 minutes. The preparation for soybean was stored at a temperature of 10-U4. Using the proposed method allows obtaining peat preparation of nodule bacteria with a titer of 1025 billion bacteria per 1 g. Results of experiments on obtaining peat, soil and peat soil preparations of nodule bacteria according to the proposed method are listed in Table 3. Peat soil soil 3: 1 1: 1 1: 3 When used in northern hectares

9.84

8.67

6.48

11.65 9.50

1.3 Peat-Pskov Peat-Neman Peat-Pskov Soil-Shchebekinsko Soch-Leningradsk Soil-Kuznetsk Shelf life, months T ------- 2 6 12 I 2 6 926 9 Number of soybean nodule bacteria, billion / g 12 , 60 ,. 10.87 8.27 10.50 5.83 2.7 12.7 11, “UE, 80 13.0 8.42 3.85 8.80 2.26 1.67 3.70 2.87 1.65 16.00 11.70 9.00 11.40 5.20 2.64-13, 40 11.64. 9.73 12,254,202,35 10,20 5,173.30. . 14.83 11.72 7.4J 12.404.002.07 11.10 5.002.18 Tagging. The number of extraneous microorganisms in all cases did not exceed 0.5 of the number of nodule bacteria. This method is suitable for soybean. The results of the production time of .1977 produced 11.3 thousand experimental batches of the drug are given in the second portions of the peat preparation. Table. 4. Orff Titer of soybean nodule bacteria. The number of constant bacteria 1 3 I 6 I 9 I 12 12 Table 3. -. , in t and b and c a 4 billion / hronney microflora. from nodule. Period of storage j mon 11, Effect of different moisture on growth and survival

3.8

2.6

28.0 The effect of sterilization dose on the quality of the preparations obtained is presented in Table. 6 in comparison with the quality of preparations obtained by a known method (Table 7). As can be seen from the data table. 6 and 7, the quality of preparations for peas, clover, alfalfa is significantly increased by Hc sterile peat, and CB preparations for lupine and soybeans can be obtained exclusively on substrates subjected to radiation sterilization. Radiation sterilization is the most expensive process of drug manufacturing technology. Despite this, it is beneficial to apply radiation sterilization. the purpose of the floor is a high-quality drug. Radiation metal sterilization with proper control of the technological process of production of the drug pos2,

3.3

2.0

1.5 of prenobic bacteria of soybeans and peas, while stuck in Table 5. ..TableZ 9221 g almost works without processing. For reasons of economy, it is necessary to apply a possible minimal dose of radiation without reducing the quality of the preparation. Experimental data show that such a dose is in the range of 2.6-6% Mrad and depends on the initial contamination of substrates and the species composition of the microflora. In tab. 6 shows data on the quality of some preparations prepared on substrates, sterilized at various doses of radiation. In tab. 7 shows the growth and survival of nodule bacterium in preparations prepared on non-sterile and sterile peat .. Table 6

1L1L VD

CM

n 1l

oo

VO

40

f

OL

en

.

M

in

. 0

oo

in

s

“B

Jt

. I

about

u

CO

01

Ik

in

CM

cho

1L

l

-

"

u

about

oo

about

4oo

"I

“B

01

vO

cho

01

CO

er

"

k

Frequency converter

Jf

r

01

oo

01

-at v

01

"K

Ih

"

in

"

"K

in

what

-9rn

00

m

r

in

four

"

01

k

oo

oo

CO

in

m

r

about

k o u

x -4

is Ia about "to V

o179221

The data obtained indicate that the dose of 1.0 Mrad is unacceptable.

The introduction of carbohydrate sources and protective substances in carrier substrates .5

In natural substrates, such as peat or soil, there is a small amount of digestible carbohydrate, which provides the energy requirement of nodule bacteria for short-term storage of preparations up to a maximum of three months. In practice, however, it is necessary to store drugs for at least 6-9 months. In this connection, the methods of application of angle 5, 83.0 without additives were added6.6 16.38.7 Glucose12, CI, 79 ,. Sucrose 11,0 15, A8,2 Lactose 10,0 12.67,7 Dry milk 9,7 Dry milk 18,09,1 serum 11,8 12,878,7 Glycerin 10,7 10,27; 8 Sorbitol 9,0 U, 39,0 Hydrol 10,9 16,211.0 Dextrin12.0 15.710.5 Molasses11.0 The data obtained indicate that the addition of various energy sources has a significant

18

OC

Levodov and protective substances in packages with sterile substrates, Experi-. mentally select suitable sources and determine their optimal concentrations.

In tab. Figure 8 presents the results of the influence of various available and relatively inexpensive energy sources on the development of a culture bank in peat. Similar results were obtained for soil and peat / soil mixture. in these cases, the absolute values of the titers are somewhat lower, in connection with the high content of inertial soil in the soil - the mineral parts of the soil.

Table 8 2,12,81,01, 5 5,07,68,85,06,2 12,85,69, 9 11,96,88,86,57,3 8,95,67,8. S6, I.O7.311.05.87.0 10.05.89.05 26.8.8.8.8C, B5.7 / 3.53.8 11.27.89.3 5. 13.8- 8.10,866,17,7 12,38,08,8. B, 6.0 influence on the content of CB in the preparation during long-term storage. In general, from the experimental data, it follows that 199 all tested sources have almost the same effect on the titers of nodule bacteria. The choice of one or another source of energy. during production, it will be determined by its availability and economic conditions. The practice of obtaining experimental batches of the drug indicates the preference of using molasses, dextrin, and I glycerol. ) and the contents of the bag were mixed in rotating drums - 60 rpm for min.

O - the number of KB after inoculation (initial)

Example 2: Preparation for lupine (Etorf Noy).,

Peat, 0) with a moisture content of 28% was ground, neutralized with chalk (5%) to pH = 6.9, moistened to 37.8%, and packaged in plastic bags of 0.8 kg each with a filling factor of 0.6. The packages were sealed, packed in cardboard boxes, kept in those, for three days at a temperature and sterilized at a dose of 6.0 Mrad,

Table 9

Sterile peat was inoculated under aseptic conditions in boxing with a liquid culture of RhJzoblurn lupini pcs. 36 with a titer of 9.0 billion / ml, to which 5% Ulicerin was added. A 200 ml liquid culture was injected into a bag with sterile peat. The hole from the injection was sealed with a polychloroforine film, the contents of the bags were stirred mechanically in a rotating drum (CtO rpm) for 10 min. The ne {initial content of lupine CB in the wet preparation is compounded. For better mixing of the carrier substrate with the liquid culture, the bag is filled with kO-80% of the volume. The storage temperature of drugs has a significant effect on the preservation of CBs in them. At high temperatures, the bacteria die off more intensively. In this work, this issue was given serious attention and the effect of temperature on nodule bacteria was studied in detail. It turned out that lupine and soybeans belonging to the group of so-called slow-growing ones are better preserved during storage of the drug after inoculation at temperatures of fast-growing nodule bacteria (peas, clover, alfalfa, etc.) are better preserved at temperatures of 3-5 ° C. In tab. E shows the survival rate of KB at various storage temperatures.

lt 1.77 billion / g, glycerin 2.0 by weight of dry peat. Humidity of the drug - 50. The drug was stored at the rate of Lupine, billion / g

Shelf life, months

15.3

10.7

1.77 3. Preparation for soy. Example (soil). moisture And, 5 The soil ((, 0) was crushed, neutralized with chalk (6%) to, 8, 25 activated carbon was drained, moistened to 26.3, packed in 0.3 kg plastic bags at Q% fillings The bags were sealed, packed in cardboard boxes, kept for four days at temperature and sterilized with O Mrad. The sterile soil was inoculated under aseptic conditions with a liquid culture.

The number of KB soybeans, billion / g

Period of storage, month

8.9

5.8

1.5

7.2

, Example f. Drug for soybean (soil-peat),

The procedure is 10–15 ° C (Table 10). In tab. fO is the peat preparation for lupine.

Table 10

The number of extraneous microflora in% of the number of KB of lupine

0.1

9,

7.5

Table 11

The number of extraneous microflora, in the number of KB soybeans

6,

1.5

1.2

The soil (, 5) with a moisture content of 15 and peat (pHs, 7) was milled using a neutral Rh i zob i urn up i n i pc. with a titer of 10.0 billion / ml, in which 13 lactose was added. A liquid culture in the amount of 60 ml was injected into a bag with sterile soil. The hole was removed from the injection site with adhesive tape, the contents of the bags were stirred mechanically in a rotating drum (kO rpm) for 3 minutes. The initial CB content of soybean in the -moist preparation was 1.5 billion / g, lactose 3.1% by weight of dry soil. The moisture content of the drug is 39.8%. The preparation was stored at a temperature of lO-IS C (table, 11). In table. Figure 11 shows the soil preparation for soybean. | Melised (5) to, 0 and 15% of activated carbon added. The mixture of soil with peat (1: 1), chalk and coal was moistened until it was packaged in plastic bags of 0.33 kg when filled with kO%. The bags were sealed, packed in cardboard boxes, kept at a temperature for five days and sterilized at 2.5 Mrad. ; The sterile substrate was inoculated under aseptic conditions with a culture of Rhizoblumjaponlcum pcs. 6k (with a title of 8.5 billion / ml, to which 3% was added

The number of soybean nodule bacteria, billion / g

Period of storage, month

1.6 13.3 10.5 7.0

Example 5. Preparation for clover (Eorph Noe). .

Peat (, 8) with a moisture content of 30% was ground, neutralized with chalk (10%) to 1, moistened to 0,3 0.3%, was filled into 0.8 kg plastic bags {calculated as 5 hectares of the preparation with filling factor 0 6). The bags were sealed, packed in cardboard boxes, kept for four days at a temperature and sterilized at a dose of 2.5 Mp /).

Sterile peat under aseptic conditions was inoculated with liquid poftfthlzobJum-trlfotH pcs. BtB with 92

Table 12

The number of extraneous microflora, in the number of nodule bacteria

1.8

.7

5, 2.5

a titer of 7.6 billion / ml to which 15% dextrin was added. A 200 ml liquid culture was injected into a bag with sterile peat. The hole from the injection was sealed with adhesive tape, the contents of the packages were stirred mechanically in a rotating drum CiO rpm) for 10 minutes. The initial content of clover CB in a wet preparation was 1.3 billion / g, dextrin 6.2% of the weight of dry peat. Humidity of the drug 51, 8%. The drug was stored at a temperature (table. 13). In tab. 13 shows a clover drug preparation. 2 hydrols. A 70 ml liquid culture was introduced by injection into a bag with a sterile substrate, the injection hole was sealed with adhesive tape, the contents of the bags were stirred mechanically in a rotating drum (0 rpm), and for -k minutes. The initial content of soybean nodule bacteria in the wet preparation was 1.6 billion / g, hydrola 1, Q% by weight of the dry substrate. The moisture content of the drug is 52.5%. The preparation was stored at a temperature of 10–15 ° C (Table 12). Table 12 shows the soil-peat preparation for soybean.

The number of CB clover, billion / g

. Period of storage, month

,.,,.13

11.3

12.9 example. Drug for pea (soil). The soil (, 8) with a moisture content of 17, was ground, neutralized with chalk (7) to 2 was added 25% activated carbon, moistened to 27.7% was packaged in 0.3 kg plastic bags, when filled with kO%. The bags were sealed, packed in cardboard boxes, kept for three days at a temperature of 37 ± tlC and sterilized at a dose of 0 Mra. The sterile soil was inoculated with a liquid culture Rh {zobi urn legumosorum 50) in aseptic conditions.

-Number of peas KB, billion / g

Period of storage, month

0.63 10.0 8.3

Example 7. The preparation for cervine (soil-peat). 55

The soil (pH-7.0) with a moisture content of 11.8% and peat (pH-6.6) with a moisture content of 26% were ground, brought with chalk (3%) to

Table 13

The number of nodTOpoHHefi microflora, in% of the number of KB clover

12

12

b ,.

10.8

1.2

Table}

The number of extraneous microflora in% of the number of pea nodule bacteria

I 12 I 24

2k

12

--J-L ...

5.0 2.1

1.7 3.5

0.5

The pH was 7.2, 15% activated carbon was added, the mixture of soil with peat (1: 1) was moistened with chalk and coal to 35%, and it was packaged in plastic bags of 0.8 kg with a filling of 60%. Packages with a titer of 5.0 billion / ml, in which 30% molasses was added. A 60 ml liquid culture was introduced by injection into a bag with sterile soil, the opening from the injection was sealed with a PVC film, the contents of the bags were stirred mechanically in a rotating drum (0 rpm) for 3 minutes. The initial content of pea nodule bacteria in the wet preparation was 0.63 billion / g, molasses 7.5% bt of dry soil weight. Humidity - drug - 39%. Store the drug at a temperature of 3-5C; (tab.). In tab. 1 A pea soil preparation is available.

279221

sealed, packed in cardboard boxes, kept at a temperature of five days and sterilized at a dose of 2.5 Mrad.

  .five

The sterile substrate was inoculated under aseptic conditions with a liquid culture of Rhizobiummetiloti pcs. k2S with a titer of 11.0 billion / ml, to which 25 dry whey was added, 10 Liquid culture in the amount of 200 ml was injected into a bag with steril Amount of nodule alfalfa bacteria, billion / g

Period of storage, month

Qit28

nym substrate, the hole from the injection; glued with adhesive tape, the contents of the bags were stirred mechanically in a rotating drum rpm) for 8 minutes. The initial CB content of alfalfa in the wet preparation was 2.0 billion / f-, whey 10 by weight of the dry substrate. The moisture content of the drug is k8%. The drug was stored at a temperature (table. 15). In tab. 15 shows the soil-peat preparation for alfalfa.

. . , T a b l and c a 15

The number of extraneous microflora,% of the number of nodule bacteria

Claims (2)

Claim . 1. A method of producing an inoculant for leguminous seeds, including the use of ground peat, soil and mixtures thereof as a substrate carrier, introduction of a source of carbohydrates, mineral, into the substrate 40. substances and activated carbon, neutralization of the resulting mixture with chalk, sterilization, packaging in packaging impermeable to microorganisms, inoculation with a culture of tuber-, new bacteria, bringing the final product moisture to 40-60%, mechanical stirring and growing of nodule bacteria at a temperature 50 re 20-30 ° С for three to five days, characterized in that, in order to increase the titer of the inoculant, reduce the content of extraneous microflora in it and increase the term 55 VNIIPI Order 2495/32 under conditions by piercing the package with a hollow needle and sticking the puncture site with adhesive tape, the packages are filled with 40-80% of their volume and then the packages are placed in drum mixers for 3–3 min and the culture is grown until the finished product is obtained. 2. The method of pop. 1, characterized by the fact that protective substances selected from the group consisting of glycerin, dextrin, sorbitol, and hydrol are added to the substrate carrier. * Sources of information taken into account during the examination 1. USSR copyright certificate No. 477150, cl. C 05 F 11/08, 1973. 2. USSR copyright certificate No. 592807, cl. C 05 F 11/08, 1976 (prototype). Circulation 699 Subscription Branch of the PPP Patent, Uzhgorod, Project 4,