Cold area straw rapid decomposition biological agent as well as preparation method and application thereof

Abstract

The invention discloses a biological agent for rapidly decomposing straws in a cold area as well as a preparation method and application of the biological agent, and belongs to the technical field of microbial preparations. The invention relates to a biological agent for rapidly decomposing straws in a cold area. The biological agent is prepared from brevibacterium tumefaciens CGMCC (China General Microbiological Culture Collection Center) 1.10488, pichia pastoris CICC 32806, trichoderma longibrachiatum CICC 13054, aspergillus niger CICC 2151 and a carrier. The biological agent disclosed by the invention has the advantages of high cold resistance, high activity, high degradation speed and the like, has relatively high metabolic capability at low temperature, is suitable for straw decomposition treatment in cold and cool areas, and provides a new technical thought for crop product management in low-temperature areas.

Description

Technical Field

[0001] The invention relates to the technical field of microbial preparations, and in particular to a biological microbial agent for rapid decomposition of straw in cold regions, and a preparation method and application thereof. Background Art

[0002] Cold and cool areas usually refer to high-latitude and high-cold areas, with accumulated temperatures of 1900-2300℃, long and cold winters, short summers, and a frost-free period of only 90 days. The temperature changes greatly between day and night, and crop production is easily affected by low temperature damage, early frost and other disasters. When crops are harvested in autumn, the temperature is relatively low, which also affects the post-harvest management of crops.

[0003] Returning straw to the field is an important part of post-harvest management of crops. Its purpose is to use the residual materials of crops and return them to the field through deep plowing or mulching, so as to improve soil quality, increase soil fertility and retain moisture, reduce the application of chemical fertilizers, increase crop yields, and promote the virtuous cycle of agricultural ecosystems. Returning straw to the field includes direct return to the field, rapid decomposition return to the field and compost return to the field. Rapid decomposition return to the field accelerates the decomposition of crop straw through the action of microorganisms, shortening the process of converting straw into organic fertilizer that can be directly used by crops. At the same time, the microorganisms in the decomposed straw can also improve the soil microenvironment and provide a better growth environment for crops.

[0004] Due to the climatic characteristics of cold and cool areas, microorganisms are affected by the temperature during the process of returning straw to the field, their reproduction speed slows down, and the decomposition effect of straw decreases. Straw that cannot be completely decomposed remains in the soil, and its nutrients cannot be absorbed and utilized by crops, but will cause certain harm to the soil and crops. Existing straw composting agents are mostly made of microorganisms screened at room temperature, which are suitable for high temperature areas in the south, but not suitable for returning straw to the field in cold and cool areas. Therefore, it is necessary to provide a straw composting agent with strong cold resistance and suitable for cold and cool areas to solve the problem of difficult post-harvest management of crops in low temperature areas. Summary of the invention

[0005] The purpose of the present invention is to provide a biological agent for rapid decomposition of straw in cold and cool areas, and a preparation method and application thereof, so as to solve the problems existing in the above-mentioned prior art. The biological agent of the present invention has the advantages of strong cold resistance, high activity, fast degradation rate, etc., has strong metabolic capacity at low temperature, is suitable for straw decomposition in cold and cool areas, and provides a new technical idea for the management of crop products in low temperature areas.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The invention provides a biological microbial agent for rapid decomposition of straw in cold regions, comprising Brevibacterium frigoritolerans CGMCC 1.10488, Pichia pastoris CICC 32806, Trichoderma longibrachiatum CICC 13054, Aspergillus niger CICC 2151 and a carrier.

[0008] Preferably, the carrier comprises biochar, maltodextrin, betaine and calcium carbonate; the mass ratio of the biochar, maltodextrin, betaine and calcium carbonate is (20-40):(5-10):(3-6):(5-10).

[0009] Preferably, the ratio of the viable counts of Brevibacterium frostii CGMCC 1.10488, Pichia pastoris CICC 32806, Trichoderma longibrachiatum CICC 13054, and Aspergillus niger CICC 2151 is (3-5): (1-2): (1-3): (2-4).

[0010] Preferably, the ratio of the cold-resistant Brevibacterium CGMCC 1.10488 to the biochar is 10 7-10 CFU:1g.

[0011] The present invention also provides a method for preparing the above-mentioned biological bacterial agent, comprising the following steps:

[0012] (1) respectively acclimating and culturing cold-resistant Brevibacterium CGMCC 1.10488, Pichia pastoris CICC 32806, Trichoderma longibrachiatum CICC 13054, and Aspergillus niger CICC 2151 at low temperature to obtain cold-resistant Brevibacterium liquid, Pichia pastoris liquid, Trichoderma longibrachiatum liquid, and Aspergillus niger liquid;

[0013] (2) mixing the cold-resistant Brevibacterium liquid, the Pichia pastoris liquid, the long-branched Trichoderma liquid, and the Aspergillus niger liquid according to the ratio of the number of live bacteria of (3-5): (1-2): (1-3): (2-4) to obtain a mixed bacterial liquid;

[0014] (3) adding biochar to the mixed bacterial solution and stirring the mixture evenly; after drying, adding maltodextrin, betaine and calcium carbonate and mixing the mixture evenly to obtain the biological bacterial agent.

[0015] Preferably, the low-temperature acclimation culture method of the cold-resistant Brevibacterium CGMCC 1.10488 is as follows: the cold-resistant Brevibacterium CGMCC 1.10488 is inoculated into a nutrient broth medium, cultured at 30°C for 24 hours, then cultured at 25°C for 48 hours, then cultured at 22°C for 48 hours, then cultured at 18°C ​​for 4 days, and then cultured at 15°C until the bacterial solution concentration is 10 8-9 CFU / mL.

[0016] Preferably, the low-temperature acclimation culture method of the Pichia pastoris CICC 32806 is: inoculate the Pichia pastoris CICC 32806 in a PDA liquid culture medium, culture at 25°C for 36 hours, transfer to 18°C ​​for 72 hours, and then transfer to 15°C for culturing until the bacterial liquid concentration is 10 8-9 CFU / mL.

[0017] Preferably, the low-temperature acclimation culture method of the long-branch Trichoderma CICC 13054 is: inoculate the long-branch Trichoderma CICC13054 in a PDA liquid culture medium, culture at 25°C for 36 hours, transfer to 18°C ​​for 72 hours, and then transfer to 15°C for culturing until the bacterial liquid concentration is 10 8-9 CFU / mL.

[0018] Preferably, the low-temperature acclimation culture method of Aspergillus niger CICC 2151 is: inoculating the Aspergillus niger CICC 2151 in a PDA liquid culture medium, culturing at 30°C for 24 hours, then culturing at 25°C for 48 hours, then culturing at 22°C for 48 hours, then culturing at 18°C ​​for 4 days, and then culturing at 15°C until the bacterial liquid concentration is 10 8-9 CFU / mL.

[0019] The present invention also provides an application of the above-mentioned biological agent or the biological agent prepared by the above-mentioned preparation method in low-temperature composting of straw. When the straw is composted at low temperature, the mass ratio of the biological agent to the straw is 1:(4-8).

[0020] The present invention discloses the following technical effects:

[0021] The present invention selects microbial strains with good cold resistance, and according to the climate characteristics of the cold and cool areas, performs low-temperature acclimation and cultivation on the microbial strains, thereby improving the survival and reproduction ability of the microbial strains at low temperatures; the microbial strains are matched with organic carriers such as biochar, maltodextrin, betaine, and calcium carbonate to provide colonization space and nutrients for the microbial strains, ensure the normal metabolic activity of the microbial strains at low temperatures, accelerate the decomposition of straw, and improve the straw degradation rate, thereby increasing the yield of crops. The biological microbial agent of the present invention has the advantages of strong cold resistance, high vitality, and fast degradation speed, and provides a new empirical method for straw waste treatment, postpartum management of crops, and green agricultural development in cold and cool areas. DETAILED DESCRIPTION

[0022] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0023] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0024] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0025] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0026] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0027] The sources of the strains used in the present invention are as follows: Brevibacterium frigoritolerans CGMCC1.10488 was purchased from China General Microbiological Culture Collection Administration Center; Pichia pastoris CICC32806, Trichoderma longibrachiatum CICC 13054, Aspergillus niger CICC 2151, Candida parapsilosis CICC 32907, Candida utilis CICC 31170, Trichoderma harzianum CICC 41290, and Aspergillus aculeatus CICC 2653 were all purchased from China Industrial Microbiological Culture Collection Administration Center.

[0028] Example 1

[0029] A biological bacterial agent for rapid decomposition of straw in cold and cool areas, the preparation process is as follows:

[0030] (1) Brevibacterium frostii CGMCC 1.10488 was inoculated on LB medium (tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L) and activated at 30°C for 48 h. Pichia pastoris CICC 32806 was inoculated on malt extract agar medium (malt extract 1 L, agar 15 g) and activated at 30°C for 48 h. Trichoderma longifolia CICC 13054 and Aspergillus niger CICC 2151 were inoculated on modified Martin medium (peptone 5 g / L, yeast extract powder 10 g / L, glucose 10 g / L, potassium dihydrogen phosphate 1 g / L, magnesium sulfate 0.5 g / L, agar 15 g / L) and activated at 25°C for 4 d.

[0031] (2) Low temperature acclimation culture of cold-resistant Brevibacterium CGMCC 1.10488, Pichia pastoris CICC 32806, Trichoderma longifolia CICC13054 and Aspergillus niger CICC 2151:

[0032] A single colony of Brevibacterium frostii CGMCC 1.10488 was picked from the LB medium and inoculated into a nutrient broth medium (10 g / L peptone, 5 g / L sodium chloride, 3 g / L beef extract powder). After culturing at 30°C for 24 h, it was transferred to 25°C for 48 h, then transferred to 22°C for 48 h, then transferred to 18°C ​​for 4 days, and then transferred to 15°C until the bacterial solution concentration reached 10 8 CFU / mL.

[0033] A single colony of Pichia pastoris CICC 32806 was picked from the malt agar medium and inoculated into PDA liquid medium (potato 200 g / L, glucose 20 g / L), cultured at 25°C for 36 h, then transferred to 18°C ​​for 72 h, and then transferred to 15°C for culturing until the bacterial solution concentration reached 10 8 CFU / mL.

[0034] A single colony of Trichoderma longifolia CICC 13054 was selected from the modified Martin medium and inoculated into PDA liquid medium. The culture was carried out at 25°C for 36 h, then transferred to 18°C ​​for 72 h, and then transferred to 15°C for culturing until the bacterial solution concentration reached 10 8 CFU / mL.

[0035] A single colony of Aspergillus niger CICC 2151 was selected from the modified Martin medium and inoculated into PDA liquid medium. After culturing at 30°C for 24 h, it was transferred to 25°C for 48 h, then transferred to 22°C for 48 h, then transferred to 18°C ​​for 4 d, and then transferred to 15°C for culturing until the bacterial solution concentration reached 10 8 CFU / mL.

[0036] (3) Take 40 mL of the cold-resistant Brevibacterium liquid obtained in step (2), 10 mL of Pichia pastoris liquid, 20 mL of Trichoderma longifolia liquid, and 30 mL of Aspergillus niger liquid (i.e., the ratio of the number of live bacteria is 4:1:2:3) and mix them to obtain a mixed bacterial liquid. Add 40 g of biochar to the mixed bacterial liquid and stir at 200 rpm for 30 min. After drying the obtained mixture, add 8 g of maltodextrin, 5 g of betaine and 8 g of calcium carbonate, mix them evenly, and obtain a cold zone straw rapid decomposition biological bacterial agent.

[0037] Example 2

[0038] A biological bacterial agent for rapid decomposition of straw in cold and cool areas, the preparation process is as follows:

[0039] (1) Brevibacterium frostii CGMCC 1.10488 was inoculated on LB medium (tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L) and activated at 30°C for 48 h. Pichia pastoris CICC 32806 was inoculated on malt extract agar medium (malt extract 1 L, agar 15 g) and activated at 30°C for 48 h. Trichoderma longifolia CICC 13054 and Aspergillus niger CICC 2151 were inoculated on modified Martin medium (peptone 5 g / L, yeast extract powder 10 g / L, glucose 10 g / L, potassium dihydrogen phosphate 1 g / L, magnesium sulfate 0.5 g / L, agar 15 g / L) and activated at 25°C for 4 d.

[0040] (2) Low temperature acclimation culture of cold-resistant Brevibacterium CGMCC 1.10488, Pichia pastoris CICC 32806, Trichoderma longifolia CICC13054 and Aspergillus niger CICC 2151:

[0041] A single colony of Brevibacterium frostii CGMCC 1.10488 was picked from the LB medium and inoculated into a nutrient broth medium (10 g / L peptone, 5 g / L sodium chloride, 3 g / L beef extract powder). After culturing at 30°C for 24 h, it was transferred to 25°C for 48 h, then transferred to 22°C for 48 h, then transferred to 18°C ​​for 4 days, and then transferred to 15°C until the bacterial solution concentration reached 10 8 CFU / mL.

[0042] A single colony of Pichia pastoris CICC 32806 was picked from the malt agar medium and inoculated into PDA liquid medium (potato 200 g / L, glucose 20 g / L), cultured at 25°C for 36 h, then transferred to 18°C ​​for 72 h, and then transferred to 15°C for culturing until the bacterial solution concentration reached 10 9 CFU / mL.

[0043] A single colony of Trichoderma longifolia CICC 13054 was selected from the modified Martin medium and inoculated into PDA liquid medium. The culture was carried out at 25°C for 36 h, then transferred to 18°C ​​for 72 h, and then transferred to 15°C for culturing until the bacterial solution concentration reached 10 9 CFU / mL.

[0044] A single colony of Aspergillus niger CICC 2151 was selected from the modified Martin medium and inoculated into PDA liquid medium. After culturing at 30°C for 24 h, it was transferred to 25°C for 48 h, then transferred to 22°C for 48 h, then transferred to 18°C ​​for 4 d, and then transferred to 15°C for culturing until the bacterial solution concentration reached 10 8 CFU / mL.

[0045] (3) Take 50 mL of the cold-resistant Brevibacterium liquid obtained in step (2), 1 mL of Pichia pastoris liquid, 1 mL of Trichoderma longifolia liquid, and 30 mL of Aspergillus niger liquid (i.e., the ratio of the number of live bacteria is 5:1:1:3) and mix them to obtain a mixed bacterial liquid, add 50 g of biochar to the mixed bacterial liquid, and stir at 200 rpm for 30 min. After the obtained mixture is dried, 10 g of maltodextrin, 12 g of betaine and 20 g of calcium carbonate are added, and mixed evenly to obtain a cold zone straw rapid decomposition biological bacterial agent.

[0046] Example 3

[0047] A biological bacterial agent for rapid decomposition of straw in cold and cool areas, the preparation process is as follows:

[0048] (1) Brevibacterium frostii CGMCC 1.10488 was inoculated on LB medium (tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L) and activated at 30°C for 48 h. Pichia pastoris CICC 32806 was inoculated on malt extract agar medium (malt extract 1 L, agar 15 g) and activated at 30°C for 48 h. Trichoderma longifolia CICC 13054 and Aspergillus niger CICC 2151 were inoculated on modified Martin medium (peptone 5 g / L, yeast extract powder 10 g / L, glucose 10 g / L, potassium dihydrogen phosphate 1 g / L, magnesium sulfate 0.5 g / L, agar 15 g / L) and activated at 25°C for 4 d.

[0049] (2) Low temperature acclimation culture of cold-resistant Brevibacterium CGMCC 1.10488, Pichia pastoris CICC 32806, Trichoderma longifolia CICC13054 and Aspergillus niger CICC 2151:

[0050] A single colony of Brevibacterium frostii CGMCC 1.10488 was picked from the LB medium and inoculated into a nutrient broth medium (10 g / L peptone, 5 g / L sodium chloride, 3 g / L beef extract powder). After culturing at 30°C for 24 h, it was transferred to 25°C for 48 h, then transferred to 22°C for 48 h, then transferred to 18°C ​​for 4 days, and then transferred to 15°C until the bacterial solution concentration reached 10 9 CFU / mL.

[0051] A single colony of Pichia pastoris CICC 32806 was picked from the malt agar medium and inoculated into PDA liquid medium (potato 200 g / L, glucose 20 g / L), cultured at 25°C for 36 h, then transferred to 18°C ​​for 72 h, and then transferred to 15°C for culturing until the bacterial solution concentration reached 10 9 CFU / mL.

[0052] A single colony of Trichoderma longifolia CICC 13054 was selected from the modified Martin medium and inoculated into PDA liquid medium. The culture was carried out at 25°C for 36 h, then transferred to 18°C ​​for 72 h, and then transferred to 15°C for culturing until the bacterial solution concentration reached 10 9 CFU / mL.

[0053] A single colony of Aspergillus niger CICC 2151 was selected from the modified Martin medium and inoculated into PDA liquid medium. After culturing at 30°C for 24 h, it was transferred to 25°C for 48 h, then transferred to 22°C for 48 h, then transferred to 18°C ​​for 4 d, and then transferred to 15°C for culturing until the bacterial solution concentration reached 10 9 CFU / mL.

[0054] (3) Take 30 mL of the cold-resistant Brevibacterium liquid obtained in step (2), 20 mL of Pichia pastoris liquid, 10 mL of Trichoderma longifolia liquid, and 40 mL of Aspergillus niger liquid (i.e., the ratio of the number of live bacteria is 3:2:1:4) and mix them to obtain a mixed bacterial liquid, add 30 g of biochar to the mixed bacterial liquid, and stir at 200 rpm for 30 min. After drying the obtained mixture, add 10 g of maltodextrin, 3 g of betaine and 5 g of calcium carbonate, mix them evenly, and obtain a cold zone straw rapid decomposition biological bacterial agent.

[0055] Comparative Example 1

[0056] The only difference from Example 1 is that Pichia pastoris CICC 32806 is replaced by Candida utilis CICC 31170.

[0057] Comparative Example 2

[0058] The only difference from Example 1 is that Pichia pastoris CICC 32806 is replaced by Candida parapsilosis CICC 32907.

[0059] Comparative Example 3

[0060] The only difference from Example 1 is that Trichoderma longibrachiatum CICC 13054 is replaced by Trichoderma harzianum CICC 41290.

[0061] Comparative Example 4

[0062] The only difference from Example 1 is that Aspergillus niger CICC 2151 is replaced by Aspergillus aculeatus CICC 2653.

[0063] Comparative Example 5

[0064] The only difference from Example 1 is that the low temperature acclimation culture process of Brevibacterium refractory CGMCC 1.10488, Pichia pastoris CICC 32806, Trichoderma longifolia CICC 13054 and Aspergillus niger CICC 2151 is omitted: a single colony of Brevibacterium refractory CGMCC 1.10488 is cultured at 30°C until the bacterial solution concentration is 10 8 CFU / mL, a single colony of Pichia pastoris CICC32806 was cultured at 25°C until the bacterial solution concentration was 10 8 CFU / mL, a single colony of Trichoderma longifolia CICC 13054 was cultured at 25°C until the bacterial solution concentration was 10 8CFU / mL, a single colony of Aspergillus niger CICC 2151 was cultured at 30°C until the bacterial solution concentration was 10 8 CFU / mL.

[0065] Comparative Example 6

[0066] The only difference from Example 1 is that the culture temperature and culture time of low-temperature acclimation culture of Pichia pastoris CICC 32806, Trichoderma longibrachiatum CICC 13054 and Aspergillus niger CICC 2151 are adjusted to the culture temperature and culture time of low-temperature acclimation culture of Brevibacterium frostii CGMCC1.10488.

[0067] Comparative Example 7

[0068] The only difference from Example 1 is that betaine is omitted.

[0069] Test Example 1

[0070] Indoor straw degradation experiment:

[0071] 5g (dry weight) of sterilized corn stalks were cut into 1-2cm lengths, placed in a culture dish, and the water content was adjusted to 60% to obtain a substrate. 1g of each of the biological agents of Examples 1-3 and Comparative Examples 1-7 were taken, dissolved in 3mL of sterile water, sprayed on the corn stalks in the culture dish, and cultured at 15°C for 30d. After the culture was completed, the undecomposed residues in the culture dish were picked out, dried in an oven at 50°C and weighed, and the straw degradation rate was calculated as (5g-residue weight) / 5g×100%. The calculation results of the straw degradation rate of each group of biological agents are shown in Table 1.

[0072] Table 1 Straw degradation rate of each group of biological agents

[0073] Group Straw degradation rate Example 1 15.67％ Example 2 15.83％ Example 3 15.00％ Comparative Example 1 8.83％ Comparative Example 2 11.83％ Comparative Example 3 9.33％ Comparative Example 4 11.00％ Comparative Example 5 10.33％ Comparative Example 6 12.50％ Comparative Example 7 13.17％

[0074] As can be seen from Table 1, the bio-microorganisms of Examples 1-3 have the best degradation effect on corn stalks. In contrast, Comparative Examples 1-4 adjusted the types of microorganisms in the bio-microorganisms, resulting in a decrease in the degradation effect on corn stalks, indicating that the types of microorganisms have a direct impact on the degradation effect of corn stalks; Comparative Example 5 omitted the low-temperature acclimation process, resulting in a decrease in the degradation effect of the bio-microorganism on corn stalks, indicating that low-temperature acclimation is an important means to adjust the metabolic capacity of microorganisms, so that they can adapt to a specific environment and maintain a high survival and reproduction capacity; Comparative Example 6 acclimated different microorganisms according to the same low-temperature acclimation process, resulting in a certain degree of decrease in the degradation effect of the bio-microorganism on corn stalks, indicating that there are differences in the acclimation methods of different microorganisms, and the acclimation methods will affect the metabolic capacity of microorganisms; Comparative Example 7 omitted betaine, resulting in a certain degree of decrease in the degradation effect of the bio-microorganism on corn stalks, indicating that betaine can be beneficial to the metabolic activity of microorganisms at low temperatures to a certain extent.

[0075] Test Example 2

[0076] Outdoor straw degradation experiment:

[0077] A field that had been planted with corn for three consecutive years was selected in Tieling, Liaoning as the experimental field. After the corn was harvested, the corn stalks were collected and crushed until the stalk length was less than 5 cm. Two months before corn planting, the experimental field was divided into 9 experimental areas, each with an area of ​​30 m 2 , respectively, the blank control group, the embodiment 1 group, and the comparative examples 1-7 groups, each group respectively sprinkled 30kg of crushed straw, the embodiment 1 group and the comparative examples 1-7 groups respectively spread the biological agents of the embodiment 1 and the comparative examples 1-7, and the spreading amount was 4kg. After spreading the biological agents, the biological agents and the straw were turned into the soil, and the blank control group turned the straw into the soil. After 2 months, corn "Liangyu 99" was sown, and the sowing density was 6000 plants / mu. The field management method was the same as the local management method. The corn yield of each test area was counted, and the results are shown in Table 2.

[0078] Table 2 Corn yield in each experimental area

[0079]

[0080]

[0081] As can be seen from Table 2, compared with the blank control group, the corn yields of the other test areas were all increased after the application of the biological agent, among which the corn yield of the Example 1 group was the highest, and the corn yields of the comparative examples 1-7 groups were lower than those of the Example 1 group, which was consistent with the results of the indoor straw degradation experiment. It shows that the degree of straw degradation / maturity has a significant effect on the yield of crops, and the biological agent of the present invention can accelerate the decay process of straw in the soil of cold and cool areas, increase the beneficial nutrients in the soil, and thus improve the yield and quality of crops.

[0082] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A biological agent for rapid decomposition of straw in cold and cool areas, characterized in that: The invention comprises Brevibacterium frigoritolerans CGMCC 1.10488, Pichia pastoris CICC 32806, Trichoderma longibrachiatum CICC 13054, Aspergillus niger CICC 2151 and a vector.

2. The biological agent according to claim 1, characterized in that: The carrier comprises biochar, maltodextrin, betaine and calcium carbonate; the mass ratio of the biochar, maltodextrin, betaine and calcium carbonate is (20-40):(5-10):(3-6):(5-10).

3. The biological agent according to claim 1, characterized in that: The ratio of the number of live bacteria of the cold-resistant Brevibacterium CGMCC 1.10488, Pichia pastoris CICC 32806, Trichoderma longifolia CICC 13054, and Aspergillus niger CICC 2151 is (3-5): (1-2): (1-3): (2-4).

4. The biological agent according to claim 2 or 3, characterized in that: The ratio of the cold-resistant Brevibacterium CGMCC 1.10488 to the biomass charcoal is 10 7-10 CFU:1g.

5. A method for preparing the biological agent according to any one of claims 1 to 4, characterized in that: The steps include: (1) respectively acclimating and culturing cold-resistant Brevibacterium CGMCC 1.10488, Pichia pastoris CICC 32806, Trichoderma longibrachiatum CICC13054, and Aspergillus niger CICC 2151 at low temperature to obtain cold-resistant Brevibacterium liquid, Pichia pastoris liquid, Trichoderma longibrachiatum liquid, and Aspergillus niger liquid; (2) mixing the cold-resistant Brevibacterium liquid, the Pichia pastoris liquid, the long-branched Trichoderma liquid, and the Aspergillus niger liquid according to the ratio of the number of live bacteria of (3-5): (1-2): (1-3): (2-4) to obtain a mixed bacterial liquid; (3) adding biochar to the mixed bacterial solution and stirring the mixture evenly; after drying, adding maltodextrin, betaine and calcium carbonate and mixing the mixture evenly to obtain the biological bacterial agent.

6. The preparation method according to claim 5, characterized in that: The low-temperature acclimation culture method of the cold-resistant Brevibacterium CGMCC1.10488 is as follows: the cold-resistant Brevibacterium CGMCC1.10488 is inoculated into a nutrient broth medium, cultured at 30°C for 24 hours, then cultured at 25°C for 48 hours, then cultured at 22°C for 48 hours, then cultured at 18°C ​​for 4 days, then cultured at 15°C until the bacterial solution concentration is 10 8-9 CFU / mL.

7. The preparation method according to claim 5, characterized in that: The low temperature acclimation culture method of the Pichia pastoris CICC 32806 is as follows: the Pichia pastoris CICC32806 is inoculated into a PDA liquid culture medium, cultured at 25°C for 36 hours, then cultured at 18°C ​​for 72 hours, and then cultured at 15°C until the bacterial liquid concentration reaches 10 8-9 CFU / mL.

8. The preparation method according to claim 5, characterized in that: The low-temperature acclimation culture method of the long-branch Trichoderma CICC13054 is as follows: the long-branch Trichoderma CICC13054 is inoculated into a PDA liquid culture medium, cultured at 25°C for 36 hours, transferred to 18°C ​​for 72 hours, and then transferred to 15°C for culturing until the bacterial liquid concentration is 10 8-9 CFU / mL.

9. The preparation method according to claim 5, characterized in that: The low-temperature acclimation culture method of Aspergillus niger CICC2151 is as follows: the Aspergillus niger CICC2151 is inoculated into a PDA liquid culture medium, cultured at 30°C for 24 hours, then cultured at 25°C for 48 hours, then cultured at 22°C for 48 hours, then cultured at 18°C ​​for 4 days, then cultured at 15°C until the bacterial liquid concentration is 10 8-9 CFU / mL.

10. Use of the biological agent according to any one of claims 1 to 4 or the biological agent prepared by the preparation method according to any one of claims 5 to 9 in low-temperature decomposing straw, characterized in that: When the straw is decomposed at low temperature, the mass ratio of the biological agent to the straw is 1:(4-8).

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