CN109294928B

CN109294928B - Microbial soil activating microbial inoculum and preparation method and application thereof

Info

Publication number: CN109294928B
Application number: CN201811237799.0A
Authority: CN
Inventors: 贡国鸿; 吴跃进; 吴正岩; 吴丽芳
Original assignee: Hefei Institutes of Physical Science of CAS
Current assignee: Hefei Institutes of Physical Science of CAS
Priority date: 2018-10-23
Filing date: 2018-10-23
Publication date: 2022-02-11
Anticipated expiration: 2038-10-23
Also published as: CN109294928A

Abstract

The invention discloses a microbial soil activating inoculant, a preparation method and an application thereof, relates to the field of microbial application, and is provided based on the problems of slow soil digestion and low efficiency when straws are returned to a field in situ, wherein the microbial soil activating inoculant is prepared from Geotrichum candidum GGC105(Geotrichum candidum) and Bacillus subtilis GBS118(Bacillus subtilis) according to the proportion of (20-30): (70-80) volume ratio, the invention has the advantages that: the two strains have the characteristics of mutual antagonism, stability and durability and strong adaptability; the straw decomposition can be strengthened, the functions of phosphate and potassium decomposition are achieved, the soil is effectively activated, and the supply of available phosphate and potassium is increased; the beneficial microorganism quantity of the soil is improved, and the absorption and rapid decomposition capability of the soil to the straws and the like which are difficult to utilize in a short time and the biomass and the mineral substances are increased.

Description

Microbial soil activating microbial inoculum and preparation method and application thereof

Technical Field

The invention relates to the field of microorganism application, in particular to a microorganism soil activation microbial inoculum and a preparation method and application thereof.

Background

China is a big agricultural country and a big straw country, and according to statistics, the yield of straws in China is about 7-8 hundred million tons every year, wherein direct combustion and feed account for six crops, two crops are discarded and burned, and less than 15% of straws are directly returned to the field. The crop straw is used as an important part of an agricultural ecological chain, and relates to the problems of substance conversion and energy circulation in an agricultural production system, soil fertility and environmental safety. In China, the nitrogen, phosphorus and potassium contents in the straws produced every year account for eight percent of the annual fertilizer application amount. After a large amount of straws are taken out from an agricultural ecological system, the problems of soil structure deterioration and soil fertility reduction are obvious and the sustainable development of agriculture is restricted, wherein the defects of soil organic matters are aggravated, and the loss and proportion imbalance of nutrient elements of nitrogen, phosphorus and potassium are caused.

The main reasons for the low rate of direct straw return are: firstly, the investment of manpower and corresponding mechanical equipment is insufficient; secondly, in the farmland returning the whole amount of straws to the field, compared with the farmland not returning the straws to the field in the first few years, the farmland returning the whole amount of straws to the field is easy to cause the dead seedlings of the succeeding crops, bad root rot and growth, unstable yield and obvious reduction, thereby influencing the enthusiasm of straw returning to the field of growers; thirdly, the defects of the in-situ treatment technology of partial straws show that the adaptability of the strain is not strong when the straws are treated by the microbial agent, so that the decomposition effect of the straws cannot meet the requirements.

The method improves the quantity and activity of microorganisms in beneficial soil in cultivated land, especially improves the quantity and activity of microorganisms which have the functions of promoting decay and decomposing straw biomass and mineral substances which are difficult to degrade in a short period, and is a key link and effective measure for solving the problem of straw returning and improving soil fertility. The content of cellulose and hemicellulose in crop straws is about 35-40%, and the content of lignin is 6-12%, and the lignin and the hemicellulose wrap the cellulose, so that the degradation of the cellulose is limited. Therefore, if the straw digestibility is to be improved, the degradation of the lignin of the straw is firstly realized, and the rigid structure of the straw is damaged. The breakdown of lignin requires the synergistic action of multiple enzymes, the major degrading enzymes are 3: lignin peroxidase (LiP), manganese peroxidase (MnP) and laccase (Lac). The construction and screening of the microbial strains capable of secreting the lignin degrading enzyme for application to crop straws is beneficial to the degradation of the lignin of the straws, so that the biodegradation process of cellulose and hemicellulose in the straws is effectively promoted.

Disclosure of Invention

The invention solves the technical problems of slow soil digestion and low efficiency when the straws are returned to the field in situ.

The invention adopts the following technical scheme to solve the technical problems:

description of the strains: geotrichum candidum GGC105(Geotrichum candidum) used in the present invention, the preservation date: 13/9/2018, depository: china Center for Type Culture Collection (CCTCC), preservation number: CCTCC M2018618, preservation address: eight Lopa in Wuchang region of Wuhan city, Hubei province; bacillus subtilis GBS118(Bacillus subtilis), preservation date: 13/9/2018, depository: china Center for Type Culture Collection (CCTCC), preservation number: CCTCC M2018617, preservation address: eight-path Lojia mountain in Wuchang region of Wuhan city, Hubei province.

The invention provides a microbial soil activation microbial inoculum, which consists of Geotrichum candidum GGC105(Geotrichum candidum) and Bacillus subtilis GBS118(Bacillus subtilis), wherein the preservation number of the Geotrichum candidum GGC105(Geotrichum candidum) is CCTCC M2018618, is separated from dead branches of small trees in Hefei-Dong-Wen island and has laccase enzyme activity; the preservation number of the Bacillus subtilis GBS118(Bacillus subtilis) is CCTCC M2018617, is separated from the soil of a mixed-fertilizer island cultivated land, and has protease activity, phosphate solubilizing and potassium solubilizing functions.

The invention also provides a preparation method of the microbial soil activation microbial inoculum, which comprises the following preparation steps:

(1) separately culturing Geotrichum candidum GGC105(Geotrichum candidum) and Bacillus subtilis GBS118(Bacillus subtilis), wherein the viable count of Geotrichum candidum reaches (1-2.2) x 10 after the single culture is finished⁷cfu/mL, the viable count of the bacillus subtilis reaches (1-2.8) x 10⁸cfu/mL；

(2) Mixing the Geotrichum candidum GGC105(Geotrichum candidum) culture and a Bacillus subtilis GBS118(Bacillus subtilis) culture according to the volume ratio of (20-30) to (70-80) to prepare the microbial soil activating agent.

Preferably, Geotrichum candidum GGC105(Geotrichum candidum) is inoculated to a PDA medium to prepare a slant strain, and then inoculated to a liquid seed medium for secondary seed culture.

Preferably, the liquid seed culture medium consists of 2% of glucose, 0.2% of peptone, 0.3% of yeast extract, 0.5% of ammonium sulfate and the balance of water.

Preferably, the culture temperature of the Geotrichum candidum GGC105(Geotrichum candidum) in the liquid seed culture medium is 28-30 ℃, and the culture time is 48-96 h.

Preferably, after a slant strain is prepared by inoculating Bacillus subtilis GBS118(Bacillus subtilis) to a beef extract peptone medium, the slant strain is inoculated to an LB liquid medium for secondary seed culture.

Preferably, the culture temperature after the inoculation in the LB liquid culture medium is 30-33 ℃, and the culture time is 24-48 h.

The invention also provides application of the microbial soil activation microbial inoculum in straw in-situ field recovery.

Preferably, the microbial soil activation agent is uniformly sprayed on the surfaces of the straws, turned over, turned into the soil, leveled and sown in the afterreap crops.

Preferably, the straw is corn straw or wheat straw.

The invention has the beneficial effects that:

(1) the microbial soil activation microbial inoculum prepared by the invention is prepared from Geotrichum candidum GGC105(Geotrichum candidum) and bacillus subtilis, and the two strains have the characteristics of mutual antagonism, stability, durability and strong adaptability;

(2) the microbial soil activation microbial inoculum prepared by the invention can strengthen straw decomposition, has the functions of phosphate and potassium decomposition, effectively activates soil and increases the supply of available phosphate and potassium;

(3) the microbial soil activating microbial inoculum prepared by the invention can quickly adapt to the soil environment, improve the quantity of beneficial microorganisms in soil, and increase the capability of the soil in absorbing and quickly decomposing the short-term difficultly-utilized biomass and mineral substances such as straws and the like;

(4) the microbial soil activation microbial inoculum prepared by the invention can secrete laccase, cellulase, hemicellulase and other degradation enzyme systems, effectively promotes the degradation of straw cellulose, hemicellulose and lignin and the humification of other organic matters, can improve the soil fertility, improves the soil structure, has the soil improvement effect and reduces the application amount of chemical fertilizers.

Drawings

FIG. 1 shows isolation and cultivation of Geotrichum candidum (Geotrichum candidum);

FIG. 2 shows the effect of the corn straw sprayed with the microbial inoculum in situ and returned to the field on the yield of the succeeding wheat;

geotrichum candidum (Geotrichum candidum) GGC105, preservation date: 13/9/2018, depository: china Center for Type Culture Collection (CCTCC), preservation number: CCTCC M2018618; bacillus subtilis GBS118, deposit date: 13/9/2018, depository: china Center for Type Culture Collection (CCTCC), preservation number: CCTCC M2018617.

Detailed Description

The present invention will be described in detail with reference to preferred embodiments thereof for the purpose of providing a better understanding and appreciation for the structural features and advantages achieved thereby.

Example 1

Isolation screening of Geotrichum candidum GGC105

(1) Sampling: selecting a white rotten wood sample from a forest;

(2) separation: adding the sample obtained in the step (1) into sterile physiological saline, uniformly oscillating, and coating the sample on a GU-PDA separation flat plate after gradient dilution; the GU-PDA separation plate culture medium is prepared by preparing PDA culture medium, collecting peeled potato 200g, cutting into small pieces, adding appropriate amount of water, boiling for 30min, filtering with gauze, adding glucose 20g and KH into the filtrate₂PO₄3g，MgSO₄1.5g, agar 15-20g and trace vitamin B₁Heating to dissolve, fixing the volume to 1000mL, and adjusting the pH value to 6.5. Sterilizing at 115 deg.C for 30min, cooling to 60 deg.C, adding filtered and sterilized guaiacol-ethanol solution to make the final concentration of guaiacol be 4mmol/L, and pouring into flat plate;

(3) selecting bacterial colonies which generate brown color-changing circles around the bacterial colonies, repeatedly scribing on a separation flat plate until pure bacterial strains are obtained, and storing the pure bacterial strains in a refrigerator at 4 ℃ on a PDA inclined plane;

(4) preparation of a filter paper strip culture medium: first, the preparationInorganic salt solution containing KH per 1000mL₂PO₄1g，MgSO₄·7H₂O 0.3g，CaCl₂0.1g，NaCl 0.1g，FeCl₃0.01g，NaNO₃2.5g, pH 7.0, adding 5mL of the inorganic salt solution into a test tube with the diameter of 1.5cm and the length of 15cm, adding a piece of filter paper with the length of 6cm multiplied by 1cm, vertically standing in the test tube with a part exposed out of the liquid level, adding a silica gel plug, and sterilizing at 121 ℃ for 30min for later use;

(5) preparation of liquid enzyme production medium (g/L): ammonium tartrate 0.2, glucose 10, KH₂PO₄2，MgSO₄·7H₂O 1.5g，CaCl₂0.1, 10mL of trace element solution, 0.5mL of vitamin solution, DMS (2, 2-dimethyl succinate) was added to a final concentration of 20mmol/L, pH 4.5.

Wherein the trace element solution (g/L): nitrilotriacetic acid 1.5, MgSO₄·7H₂O 3.0，MnSO₄0.5，NaCl 1.0，FeSO₄·7H₂O 0.1，CoSO₄0.1，CaCl₂0.082，ZnSO₄0.1，CuSO₄·5H₂O 0.01，KAl(SO₄)₂0.01，H₃BO₃0.01，Na₂MoO₄0.01；

Wherein the vitamin solution (mg/L): biotin 2, folic acid 2, thiamine hydrochloride (vitamin B)₁)5, riboflavin (vitamin B)₂)5, vitamin B₆Hydrochloride 10, vitamin B₁₂0.1, 5 percent of nicotinic acid, 5 percent of DL-calcium pantothenate, 5 percent of p-aminobenzoic acid and 5 percent of lipoic acid;

(6) and (3) laccase activity detection: 0.1mol/L acetate buffer (pH5.0) containing 0.5mmol/L of LABTS (2, 2-azino-bis (3-ethylbenzothiazole-6-sulfonic acid)) was prepared: the 0.1mol/L acetic acid solution is 1L solution containing glacial acetic acid (17.5mol/L)5.72mL, the 0.1mol/L sodium acetate solution is 1L solution containing 8.2g sodium acetate, the sodium acetate solution is slowly added into the acetic acid solution, stirring is continuously carried out, pH and measurement are carried out until the pH is 5.0, namely 0.1mol/L acetic acid buffer solution (pH5.0), 0.027g ABTS is weighed and dissolved in 100mL0.1mol/L acetic acid buffer solution;

(7) preparation of crude enzyme solution: inoculating slant strains into a 150mL triangular flask containing 50mL liquid enzyme production culture medium, performing shake culture at 28 ℃ and 150rpm for 5-10d, centrifuging the fermentation liquor at 5000rpm/min for 10min, and taking supernatant to obtain crude enzyme solution;

(8) and (3) enzyme activity determination: 0.5mmol/LABTS of 0.1mol/L acetic acid buffer solution (pH5.0)2mL, adding 1mL of crude enzyme solution, starting the reaction, measuring the change of absorbance at 420nm, preheating the buffer solution and the crude enzyme solution to 37 ℃, mixing the reaction, and defining the amount of 1 mu mol of product generated in 1min as 1 enzyme activity unit.

According to the separation and screening method and the laccase enzyme activity determination method, as shown in figure 1, a decay fungus strain which can decompose fiber substances and has laccase activity is separated and screened, the number of the decay fungus strain is Geotrichum candidum GGC105, and the laccase activity of the decay fungus strain reaches 16-35 mu/mL.

Example 2

The preparation method of the microbial soil activation microbial inoculum comprises the following preparation steps:

(1) inoculating Geotrichum candidum GGC105(Geotrichum candidum) obtained by separation on a PDA culture medium, preparing slant strains at 28 ℃, and then inoculating the slant strains on a liquid seed culture medium for secondary seed culture, wherein the liquid seed culture medium consists of 2% of glucose, 0.2% of peptone, 0.3% of yeast extract, 0.5% of ammonium sulfate and the balance of water; culturing at 28 deg.C for 48 hr until viable count reaches (1.0-1.5) x 10⁷cfu/mL；

(2) Inoculating Bacillus subtilis GBS118(Bacillus subtilis) to a beef extract peptone culture medium, culturing at 30 ℃ to prepare slant strains, inoculating an LB liquid culture medium to perform secondary seed culture, wherein the LB liquid culture medium comprises 0.5% of yeast extract, 1.0% of peptone, 1.0% of NaCl and the balance of water, and culturing with the 30 ℃ liquid for 24 hours until the viable count reaches (1.0-2.0) multiplied by 10⁸cfu/mL；

(3) Mixing the Geotrichum candidum GGC105(Geotrichum candidum) culture and a Bacillus subtilis GBS118(Bacillus subtilis) culture according to the volume ratio of 20:80 to prepare the microbial soil activating agent.

Example 3

(1) the separated geotrichum candidum GGC is subjected to GGC105(Geotrichum candidum) is inoculated on a PDA culture medium, after a slant strain is prepared at 30 ℃, the slant strain is inoculated on a liquid seed culture medium for secondary seed culture, and the liquid seed culture medium consists of 2 percent of glucose, 0.2 percent of peptone, 0.3 percent of yeast extract, 0.5 percent of ammonium sulfate and the balance of water; culturing at 30 deg.C for 96 hr until viable count reaches (1.0-2.2) × 10⁷cfu/mL；

(2) Inoculating Bacillus subtilis GBS118(Bacillus subtilis) to a beef extract peptone culture medium, culturing at 30 ℃ to prepare slant strains, inoculating an LB liquid culture medium to perform secondary seed culture, wherein the LB liquid culture medium comprises 0.5% of yeast extract, 1.0% of peptone, 1.0% of NaCl and the balance of water, and culturing with a 33 ℃ liquid for 48h until the viable count reaches (1.0-2.8) × 10⁸cfu/mL；

(3) Mixing the Geotrichum candidum GGC105(Geotrichum candidum) culture and a Bacillus subtilis GBS118(Bacillus subtilis) culture according to the volume ratio of 30:70 to prepare the microbial soil activating agent.

Example 4

The decomposition of the microbial soil activation microbial inoculum on the straws under the laboratory conditions is as follows:

taking 1kg (dry weight) of wheat straw, wherein the length of the straw is about 10cm, placing the wheat straw in a plastic box with a cover, adjusting the water content to be 40-50%, adding 50mL of microbial soil activating agent, and uniformly stirring; adding 50mL of water in contrast, adding no microbial inoculum, performing decay culture at 28-30 ℃ with a cover, turning over once every day at regular time, and judging decay effect according to color, smell and crude fiber content.

The experimental results are as follows: after one month of culture, the straw added with the microbial inoculum is dark brown, is not shaped, has sapropel, has no peculiar smell, has a little mellow fragrance, and has 8.5 percent of crude fiber; the contrast is gray brown, part is not molded, the smell of mildew is fishy, and the crude fiber content is 15.1 percent. The straw decomposition degree of the added activating microbial inoculum is obviously higher than that of a control, which indicates that the microbial soil activating agent has obvious straw decomposition promoting effect.

Example 5

The application of the microbial soil activation microbial inoculum on the corn straws in the test field comprises the following steps:

the returning time is selected to be at the end of 9 months and at the beginning of 10 monthsWhen the corn is harvested, the succeeding crop is wheat; pulverizing corn stalk into 5-15cm length with harvester and pulverizer; area of each test field was 66m²Applying fertilizer conventionally; uniformly spraying a microbial soil activation microbial inoculum on the surface of fresh straws in the field, wherein the using amount of the microbial soil activation microbial inoculum is 1-3% of the weight of the fresh straws; then ploughing, wherein the ploughing depth is more than 23cm, the broken straws are ploughed below a 10cm soil layer, and rotary ploughing and harrowing are carried out for 2 times.

The experimental results are as follows: as shown in figure 2, the wheat harvest shows that under the condition of returning the whole corn straws to the field, the yield of the wheat sprayed with the microbial soil activation fungicide is improved by about 77 percent compared with the yield of the wheat not sprayed with the fungicide, and the adverse effect on the yield reduction of the succeeding crops under the condition of returning the whole straws to the field in situ is effectively relieved.

Example 6

The application of the microbial soil activation microbial inoculum to the wheat straws in the test field comprises the following steps:

returning to the field in 6 months and harvesting wheat in the beginning, wherein the succeeding crop is corn; pulverizing wheat straw into 5-15cm length with harvester and pulverizer; area of each test field was 66m²Applying fertilizer conventionally; uniformly spraying the composite microbial soil activation microbial inoculum on the surface of fresh straws in the field, wherein the using amount of the composite microbial soil activation microbial inoculum is 1-3% of the weight of the fresh straws; then ploughing, wherein the ploughing depth is more than 23cm, the broken straws are ploughed below a 10cm soil layer, and rotary ploughing and harrowing are carried out for 2 times.

The experimental results are as follows: as shown in Table 1, the corn harvest shows that the yield of the corn sprayed with the microbial soil activation fungicide is improved by about 20 percent compared with the yield of the corn not sprayed with the fungicide under the condition of returning the wheat straws to the field in full, and the adverse effect on the yield reduction of the succeeding crops under the condition of returning the straws to the field in full in situ is effectively relieved.

TABLE 1 influence of wheat straw-sprayed inoculant on yield of succeeding corn after returning to field in situ

The above is only a preferred embodiment of the present invention, the protection scope of the present invention is not limited to the above examples, and various process schemes which are not different from the concept of the present invention are within the protection scope of the present invention.

Claims

1. A microbial soil activation microbial inoculum is characterized in that: GGC105 (from Geotrichum candidum)Geotrichum candidum) And Bacillus subtilis GBS118(Bacillus subtilis) Composition of said Geotrichum candidum GGC 105: (Geotrichum candidum) The preservation number of (A) is CCTCC M2018618, and the Bacillus subtilis GBS118 (B)Bacillus subtilis) The preservation number of (A) is CCTCC M2018617, the viable count of the geotrichum candidum GGC105 reaches (1-2.2) x 10⁷cfu/mL, the viable count of the bacillus subtilis GBS118 reaches (1-2.8) x 10⁸cfu/mL, the culture volume ratio of the Geotrichum candidum GGC105 to the Bacillus subtilis GBS118 is (20-30): (70-80).

2. A preparation method of a microbial soil activation microbial inoculum is characterized by comprising the following steps: the preparation method comprises the following preparation steps:

(1) geotrichum candidum GGC105(Geotrichum candidum) And Bacillus subtilis GBS118(Bacillus subtilis) Respectively and independently culturing until the viable count of Geotrichum candidum reaches (1-2.2) × 10⁷cfu/mL, the viable count of the bacillus subtilis reaches (1-2.8) x 10⁸cfu/mL；

(2) GGC105 (of Geotrichum candidum)Geotrichum candidum) Culture and Bacillus subtilis GBS 118: (Bacillus subtilis) The cultures are mixed according to the volume ratio of (20-30) to (70-80) to prepare the microbial soil activation microbial inoculum.

3. The method for preparing a microbial soil activating agent according to claim 2, wherein: geotrichum candidum GGC105(Geotrichum candidum) Inoculating to PDA culture medium to prepare slant strain, and inoculating to liquid seed culture medium for secondary seed culture.

4. The method for preparing a microbial soil activating agent according to claim 3, wherein: the liquid seed culture medium consists of 2 percent of glucose, 0.2 percent of peptone, 0.3 percent of yeast extract, 0.5 percent of ammonium sulfate and the balance of water.

5. The method for preparing a microbial soil activating agent according to claim 3, wherein: said Geotrichum candidum GGC105(Geotrichum candidum) The culture temperature in the liquid seed culture medium is 28-30 ℃, and the culture time is 48-96 h.

6. The method for preparing a microbial soil activating agent according to claim 2, wherein: bacillus subtilis GBS118(Bacillus subtilis) Inoculating the strain to a beef extract peptone culture medium to prepare a slant strain, and then inoculating the slant strain to an LB liquid culture medium to perform secondary seed culture.

7. The method for preparing a microbial soil activating agent according to claim 6, wherein: the culture temperature after the inoculation in LB liquid culture medium is 30-33 ℃, and the culture time is 24-48 h.

8. The use of the microbial soil activation inoculant according to claim 1 in straw return in situ.

9. The application of the microbial soil activation inoculant in straw in-situ return to fields as claimed in claim 8, wherein: uniformly spraying a microbial soil activation microbial inoculum on the surface of the straw, turning over the straw, turning the straw into the soil, leveling, and sowing the afterreap crops.

10. The application of the microbial soil activation inoculant in straw in-situ return to fields as claimed in claim 8, wherein: the straw is corn straw or wheat straw.