A kind of pseudogymna, its liquid microbial agent and application

Abstract

The present invention relates to the field of industrial microbial technology, and specifically discloses a pseudogymnocystis, its liquid microbial agent and application. The pseudogymnocystis (Pseudogymnoascus sp) SDF-LT has a deposit number of CGMCC No.40106. The pseudogymnocystis SDF-LT provided by the present invention can be rapidly propagated in a low temperature range of 4 to 10°C, and the cellulase secreted by it still has a high enzyme activity at low temperatures; the straw degradation rates of the strain cultured at 10°C for 7d and 15d in a straw culture medium are 13.99% and 30.04% respectively; under 4°C conditions, its cellulase activity can still reach 171.8U / mL, and the filter paper enzyme activity can also reach 68.2U / mL, ensuring that abandoned agricultural straw can still be rapidly degraded under low temperature conditions, and can be applied to the return of straw to the fields in various regions, promoting the rapid return of straw to the fields in the low temperature seasons of autumn and winter.

Description

Technical Field

[0001] The invention relates to the technical field of industrial microorganisms, and in particular to a pseudogymna, a liquid microbial agent and an application thereof. Background Art

[0002] my country is a large agricultural producer with abundant straw resources. Crop straw contains rich nutrients such as nitrogen, phosphorus, and potassium, as well as a large amount of organic substances such as cellulose and hemicellulose. Returning straw to the field can not only increase the organic matter content of the soil, but also improve the soil structure and microbial characteristics, increase soil enzyme activity, and provide plants with the nutrients they need for growth, thereby increasing crop yields. In northern my country, the use of corn straw in wheat-corn rotation areas has shown many benefits for wheat growth and soil environment.

[0003] Under natural conditions, straw degradation is slow, and undegraded straw will also affect the sowing and growth of subsequent crops, causing seedling burns and increasing pests and diseases. Using microorganisms with straw-degrading properties to accelerate straw decomposition and solve the bottleneck problem of returning straw to the field is of great significance to the full utilization of straw resources, the cultivation of farmland fertility, and the sustainable development of agriculture. Straw decomposing agents are a type of microbial agents that can quickly decompose organic waste such as straw and convert the organic matter and nutrients such as phosphorus and potassium contained therein. Studies have found that applying straw decomposing agents on the basis of returning straw to the field can accelerate straw decomposition, change the number of soil microorganisms, and improve soil fertility, thereby promoting plant growth.

[0004] At present, the research on cellulose-degrading bacteria is mainly focused on normal and high temperature environments, and the research on low-temperature cellulose-degrading bacteria is relatively small. In particular, when the soil temperature is below 10°C, the degradation effect of microorganisms on straw is very poor. However, the winter in northern my country is cold and dry, with an average temperature basically below 10°C and low ground temperature. The use of general composting agents cannot quickly and thoroughly compost the straw, which can easily bring adverse effects to crops. Therefore, it is of great significance to find a microorganism or a new type of straw composting agent that can accelerate the rapid return of straw to the field under low temperature conditions. Summary of the invention

[0005] Aiming at the problem that the existing straw-degrading microorganisms have low cellulase activity in a low temperature environment and low efficiency in degrading straw, the present invention provides a Pseudomonas aeruginosa, a liquid microbial agent and an application thereof.

[0006] In order to solve the above technical problems, the technical solution provided by the present invention is:

[0007] A Pseudogymnoascus sp. SDF-LT, whose deposit number is CGMCC No.40106.

[0008] Pseudogymnoascus SDF-LT was screened out from the wheat topsoil in Leting County, Tangshan City, Hebei Province using the sodium cellulose-Congo red solid culture medium method. It was classified and named Pseudogymnoascus sp. and was deposited in the General Microbiology Center of the China Microorganism Culture Collection, abbreviated as CGMCC, on February 22, 2022. The strain collection number is CGMCC No. 40106, and the storage address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0009] The biological characteristics of the pseudogymna SDF-LT provided by the present invention are as follows: at 10° C., on a PDA solid culture medium, the initial colonies are white, nearly round colonies, and feathery, and a large number of rusty red granules are generated on the surface of the colonies in the later stage. Under an optical microscope, the conidiophores of the fungus are observed to be various short branches, translucent, smooth and upright, and the conidia are directly formed from the ends of the conidiophores and are elliptical.

[0010] The pseudogynocystis SDF-LT provided by the present invention can rapidly reproduce in a low temperature range of 4 to 10 DEG C, and the cellulase secreted by the pseudogynocystis SDF-LT still has a high enzyme activity at low temperature, so that the rapid decomposition of straw under low temperature conditions can be achieved; the straw degradation rates of the strain after being cultured in a straw culture medium at 10 DEG C for 7 days and 15 days are 13.99% and 30.04% respectively; under the condition of 4 DEG C, the cellulase activity can still reach 171.8U / mL, and the filter paper enzyme activity can also reach 68.2U / mL, so that the abandoned agricultural straw can still be rapidly degraded under low temperature conditions; and the pseudogynocystis SDF-LT provided by the present invention has strong adaptability to the region, can be applied to returning straw to the fields in various regions, and promotes the rapid return of straw to the fields in the low temperature seasons of autumn and winter.

[0011] The present invention also provides a liquid microbial agent, comprising the Pseudogymnoascus sp. SDF-LT.

[0012] The pseudogymna SDF-LT provided by the present invention can be widely used in straw degradation in various regions of China. It can be made into a liquid microbial agent for convenient use, and can be sprayed in the field or used in straw stacking and fermentation.

[0013] Preferably, the number of live bacteria of Pseudomonas in the liquid microbial agent is 2×10 7 ~7×10 8 Pieces / mL.

[0014] The preferred live bacteria count of Pseudogycetes can effectively reduce production costs while ensuring that the liquid microbial agent has a high ability to degrade straw.

[0015] The present invention also provides a method for preparing the liquid microbial agent, comprising the following steps:

[0016] The pseudogymna SDF-LT was inoculated into a PD liquid culture medium, and cultured at 10°C to 20°C and 180r / min to 200r / min for 5 to 6 days. The cultured bacterial solution was inoculated into a PDA solid culture medium, and cultured at 10°C to 20°C for 120h to 130h. The spores on the PDA solid culture medium were then washed off with sterile distilled water to ensure that the number of viable bacteria in the obtained microbial bacterial solution was ≥ 2×10 7 Pieces / mL.

[0017] Preferably, the PD culture medium comprises: 200 g potato, 20 g glucose, 3 mL gentamicin sulfate injection and 1000 mL water, and is sterilized at 121° C. for 20 min.

[0018] Preferably, the PDA culture medium comprises: 200 g potato, 20 g glucose, 15 g agar, 3 mL gentamicin sulfate injection and 1000 mL water, and is sterilized at 121° C. for 20 min.

[0019] Exemplarily, the inoculation volume of the Pseudomonas sp. SDF-LT accounts for 5% of the volume of the PD liquid culture medium.

[0020] The present invention also provides the use of the above-mentioned Pseudogymnoascus sp. SDF-LT in the preparation of cellulase. The method for preparing cellulase comprises the following steps: fermenting the Pseudogymnoascus sp. SDF-LT, collecting the fermentation product, and obtaining cellulase.

[0021] The present invention also provides the use of the above-mentioned Pseudogymnoascus sp. SDF-LT in the rapid return of crop straw to the field.

[0022] The present invention also provides application of the liquid microbial agent in the rapid return of crop straw to the field.

[0023] The present invention has no special requirements on the type of straw, and the straw may be wheat, corn, peanuts, soybeans and other types commonly grown in the north.

[0024] The pseudogymna SDF-LT provided by the present invention is particularly suitable for degrading corn stalks, degrading cellulose, hemicellulose and lignin in the stalks into small molecular nutrients that are completely absorbed by crops, improving the composting effect, shortening the composting time, and continuously promoting the rapid return of stalks to the fields even in cold and dry winters, thereby realizing the resource utilization of agricultural waste under low temperature conditions, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is the hydrolysis circle produced by the pseudogymna SDF-LT of the present invention on the sodium carboxymethyl cellulose-Congo red solid culture medium;

[0026] Figure 2 The colony morphology of the Pseudomonas sp. SDF-LT of the present invention is shown in FIG.

[0027] Figure 3 The morphology of conidiophores of Pseudomonas sp. SDF-LT observed under an optical microscope;

[0028] Figure 4 The morphology of conidia of Pseudogycetes SDF-LT observed under an optical microscope;

[0029] Figure 5 The phylogenetic tree of Pseudomonas sp. SDF-LT constructed based on ITS gene sequence similarity in Example 1;

[0030] Figure 6 This is a comparison chart of the cellulase and filter paper enzyme activities of Pseudomonas sp. SDF-LT at 4°C and 10°C in Example 2 of the present invention, wherein Figure A is the cellulase activity result and Figure B is the filter paper enzyme activity result;

[0031] Figure 7 This is a graph showing the results of the filter paper disintegration test in Example 4 of the present invention. DETAILED DESCRIPTION

[0032] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0033] The culture medium used in the examples is:

[0034] PDA medium (1L): 200g potato, 20g glucose, 15g agar, 3mL gentamicin sulfate injection, 1000mL water, natural pH, sterilize at 121℃ for 20min before use.

[0035] PD liquid culture medium (1L): 200g potato, 20g glucose, 3mL gentamicin sulfate injection, 1000mL water, natural pH, sterilize at 121℃ for 20min before use.

[0036] Sodium cellulose-Congo red medium: sodium carboxymethyl cellulose 5g, (NH4)2SO4 2g, KH2PO41g, MgSO4·7H2O 0.5g, NaCl 0.5g, NaNO3 1g, peptone 0.5g, yeast extract 0.5g, Congo red 0.2g, agar 12g, water 1000mL, pH 7.0, 121℃, high pressure sterilization for 20min before use.

[0037] Straw liquid culture medium: Use a 100mL triangular flask, put 2g of 1cm long corn straw and 30mL of nutrient solution in the bottle. Nutrient solution formula:

[0038] Urea 3.0g, (NH4)2SO4 6.0g, peptone 3.0g, CaCl2 0.1g, MgSO4·7H2O0.5g, K2HPO41.0g, NaCl 0.1g, FeSO4·7H2O 0.05g, MnSO4·7H2O 0.016g, ZnSO4·7H2O 0.014g, CoCl2·6H2O 0.037g, distilled water 1000mL, natural pH, sterilize at 121℃ for 30min before use.

[0039] Liquid enzyme production culture medium: (NH4)2SO4 4.0g, MgSO4·7H2O 0.5g, K2HPO4 2g, beef extract 5g, peptone 10g, sodium carboxymethyl cellulose 10g, natural pH value, dilute to 1000mL, sterilize at 121℃ for 20min for later use.

[0040] Filter paper strip culture medium: (NH4)2SO4 2g, K2HPO4 2g, MgSO4·7H2O 0.05g, CaCO32g, NaCl 0.2g, 1 filter paper strip of about 1cm×6cm per bottle, sterilize at 121℃ for 20min and set aside.

[0041] Commercially available straw composting agents: The straw composting agent produced by Luoyang Ouke Biotechnology Co., Ltd. has an effective live bacteria count of >500 million / g. The effective bacteria are Bacillus subtilis and Trichoderma harzianum. It contains complex bacteria such as lactic acid bacteria and yeast, and the dosage form is powder.

[0042] Example 1

[0043] 1. Initial screening of Pseudogycetes

[0044] Samples were collected from wheat plough soil in Leting County, Tangshan City. The strains were separated using PDA medium by dilution plating method. A total of 100 strains with different colony morphologies were obtained. The isolated bacteria were streaked and purified multiple times to obtain pure culture strains and preserve them. The 42 purified strains were inoculated on sodium cellulose-Congo red medium and cultured at 10°C. The colony diameter (d) and transparent circle diameter (D) were measured after 5 days of culture, and the D / d value was calculated.

[0045] Pseudogycetes SDF-LT with a deposit number of CGMCC No.40106 can grow on sodium carboxymethylcellulose-Congo red medium and produce a transparent hydrolysis zone with a diameter of more than 3.5 cm. Figure 1 As shown in the figure, the D / d value was 2.6 at 10℃, indicating that the bacteria had a high straw degradation ability and good low temperature adaptability.

[0046] 2. Rescreening of Pseudogycetes

[0047] The strains with straw degradation ability obtained in the initial screening were inoculated into PDA medium and cultured in a shaking incubator at 10°C for 72 h. Then 1 mL of the bacterial solution was aspirated and inoculated into the straw medium respectively. The medium was placed in an incubator at 10°C for culture and shaken twice a day. At 7 and 15 days of culture, the straw in the bottle was rinsed 3 times with distilled water and dried to constant weight. The weight of the corn straw in each treatment was weighed and the degradation rate was calculated. Each treatment was repeated 3 times.

[0048] Straw degradation rate (%) = (W0-W1) / W0×100%

[0049] W0 is the weight of the straw in the culture medium before inoculation; W1 is the weight of the straw after drying after the culture is completed.

[0050] When the pseudogymna SDF-LT with the deposit number CGMCC No.16280 was cultured at 10℃, the straw degradation rates were 13.99% and 30.04% after 7 and 15 days of culture, respectively.

[0051] Morphological observation

[0052] The biological characteristics of Pseudogycetes SDF-LT are as follows: at 10°C, on PDA solid medium, the initial colonies are white, nearly round, and feathery, and a large number of rusty red particles are generated on the surface of the colonies in the later stage, such as Figure 2 As shown. Under an optical microscope, the conidiophores of the fungus are short branches of various shapes, translucent, smooth and upright. Conidia are formed directly from the ends of the conidiophores and are oval in shape, as shown in Figure 1. Figure 3-Figure 4 According to the characteristics of colony morphology and conidia structure, the strain SDF-LT was preliminarily identified as Pseudogycetes by comparing and analyzing with the Manual of Fungal Identification and Chinese Fungi.

[0053] Molecular Biology Identification

[0054] The fungal genomic DNA extraction kit (Beijing Solebow Technology Co., Ltd.) was used to extract the genomic DNA of the strain SDF-LT as a PCR amplification template, and the fungal universal primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTT ATTGATATGC-3') were used to amplify the ITS region rDNA sequence of the strain. The ITS sequence was amplified according to the reaction conditions (94℃5min, 94℃1min, 53℃1min, 72℃1min, 33 cycles, 72℃7min) to obtain a 559bp amplification product, which was sequenced. The ITS sequence of the strain is sequence 3 in the sequence table. The determined sequences were compared and analyzed by BLAST in the database of the National Center of Biotechnology (NCBI), and finally a phylogenetic tree was constructed with the help of the Neighbor-Joining method of MEGA 5.1 (such as Figure 5 The phylogenetic status of the strain was determined, and combined with the morphological characteristics of the strain, it was determined that the strain was Pseudogymnoascus sp. SDF-LT.

[0055] The strain SDF-LT was deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration, abbreviated as CGMCC, on February 22, 2022. The strain deposit number is CGMCC No.40106, and the deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0056] Example 2

[0057] The Pseudogycetes SDF-LT stored at -80℃ was inoculated into PD culture medium at a 5% inoculation rate, and cultured at 10℃ and 180r / min for 5 days. The cultured Pseudogycetes liquid was inoculated into liquid enzyme-producing culture medium at a 10% inoculation rate, and cultured at 180r / min, 4℃ or 10℃ for 6 days, and then centrifuged at 10000r / min and 4℃ for 10min. The supernatant was taken as the crude enzyme liquid, and the cellulase and filter paper enzyme activities of the crude enzyme liquid were tested.

[0058] 1. Cellulase Activity Assay

[0059] The carboxymethyl cellulase (CMCase) activity of Pseudogycota was determined by 3,5-dinitrosalicylic acid method (DNS method).

[0060] Take 1.0mL of the above crude enzyme solution and add 4.0mL CMC buffer, react in a 50℃ water bath for 20min, then take it out, add 3mL DNS colorimetric solution, color in a boiling water bath for 10min, then cool, compare at 490nm, measure its OD value, compare with the standard glucose curve, and calculate the glucose amount (m1) from the OD value. Add the crude enzyme solution in the control tube after 20min in the water bath, and measure the glucose amount (m2) of the crude enzyme solution. Calculate the enzyme activity of Pseudomonas aeruginosa under the above conditions (unit: U / mL) by the following formula:

[0061] U = (m1-m2) / 20*dilution factor.

[0062] 2. Filter Paper Enzyme Activity Assay

[0063] Take 0.5mL of the above crude enzyme solution, add 1mL of acetic acid-sodium acetate buffer solution with pH of 4.8 and 0.1mol / L or 1mL of citric acid-sodium citrate buffer solution, then add a filter paper strip of about 1cm×6cm, keep the enzymatic reaction at 50℃ for 1h, add 3mL of DNS colorimetric solution (the amount used for the standard curve is 1.5mL), boil in water bath for 10min, cool to room temperature with running water, measure absorbance at 540nm, and at the same time, use the crude enzyme solution inactivated after boiling at 100℃ for 10min as a control to deduct the background. According to the absorbance, find out the corresponding glucose content from the glucose standard curve, and calculate the enzyme activity value according to the following formula based on the number of grams of glucose generated.

[0064] X=(W * N * 1000) / (T * M)

[0065] X: Enzyme activity of filter paper enzyme, unit: U / mL

[0066] W: The concentration of glucose obtained from the glucose standard curve, in μg;

[0067] N: Total multiple of enzyme solution dilution;

[0068] T: reaction time, h;

[0069] M: volume of sample, mL.

[0070] The results are as follows Figure 6 As shown, Figure A is the result of cellulase activity, and Figure B is the result of filter paper enzyme activity. The cellulase activity of Pseudogycetes SDF-LT can reach 456.38U / mL at 10℃, and its cellulase activity is 171.8U / mL at 4℃; the filter paper enzyme activity of Pseudogycetes SDF-LT can reach 101.6U / mL at 10℃, and its filter paper enzyme activity is 68.2U / mL at 4℃.

[0071] Example 3

[0072] This embodiment provides a liquid bacterial agent of Pseudomonas aeruginosa, and the preparation method thereof comprises the following steps:

[0073] The pseudogymna SDF-LT with the collection number of CGMCC No.16280 stored at -80℃ was inoculated into PD liquid medium and cultured at 10℃ and 180r / min for 5 days. Then, 200μL of the cultured bacterial solution was inoculated into PDA solid medium and cultured at 10℃ for 120h. Then, the spores on the PDA solid medium were washed off with sterile distilled water and counted by blood cell plate. The number of viable bacteria in the obtained microbial bacterial solution was 2×10 7 Pieces / mL.

[0074] Example 4

[0075] Filter paper disintegration test

[0076] The bacterial solution prepared in Example 3 was inoculated into the filter paper strip culture medium at a 10% inoculation rate, and the filter paper was placed at 4°C and 10°C, respectively. After 20 days, the disintegration of the filter paper was observed. The results are as follows: Figure 7 As shown in the figure, it can be seen that it still has a good ability to hydrolyze cellulose at a low temperature of 4°C.

[0077] Application Example 1

[0078] This example is a straw pot sandbag method experiment to simulate the return of corn straw to the field. A square flower pot with a diameter of 10 cm and a height of 10 cm is used. After the bottom of the pot is covered with soil, 1.5 g of crushed corn straw is put into a 7 cm × 7 cm nylon bag and placed in the middle of the flower pot. Then, 2 × 10 viable bacteria prepared in Example 3 are added. 7 3mL of bacterial solution with a concentration of 100 μg / mL was added, and then covered with soil to adjust the soil moisture to about 60%. Each treated flower pot was placed in a dark environment at 4°C for cultivation. Three experimental groups were set up: the bacterial solution prepared in Example 3 was used as the example group, the commercially available straw decomposing bacterial agent was used as the control group, and the blank group was used without adding bacterial agent. Three parallel samples were set up in each experimental group, and each parallel sample had 12 flower pots. Three pots were randomly selected from each parallel sample for each sampling to measure the dry weight of straw and calculate the straw degradation rate. The sample was taken once every 15 days until the end of the 45th day. The results are shown in Table 1.

[0079] Table 1 Degradation rate of straw in flower pots at different treatment times

[0080]

[0081] In summary, the Pseudogycetes with a preservation number of CGMCC No. 40106 provided by the present invention can significantly accelerate the decomposition of straw in the soil, especially under low temperature conditions (4° C.), and still has a strong ability to accelerate the decomposition of straw. It is particularly suitable for straw degradation in cold northern regions and has high practical value.

[0082] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent substitution or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A Pseudogymnoascus sp. SDF-LT, characterized in that: Its deposit number is CGMCC No.40106.

2. A liquid microbial agent, characterized in that: The method comprises the Pseudogymnoascus sp. SDF-LT according to claim 1.

3. The liquid microbial agent according to claim 2, characterized in that: The number of live bacteria of Pseudomonas in the liquid microbial agent is 2×10 7 ~7×10 8 Pieces / mL.

4. The method for preparing the liquid microbial agent according to claim 2 or 3, characterized in that: The steps include: The Pseudogymnoascus sp. SDF-LT was inoculated into a PD liquid culture medium, and cultured at 10°C to 20°C and 180 r / min to 200 r / min for 5 to 6 days. The cultured bacterial solution was inoculated into a PDA solid culture medium, and cultured at 10°C to 20°C for 120 h to 130 h. The spores on the PDA solid culture medium were then washed off with sterile distilled water to ensure that the number of viable bacteria in the obtained microbial bacterial solution was ≥ 2×10 7 Pieces / mL.

5. The method for preparing the liquid microbial agent according to claim 4, characterized in that: The PD liquid culture medium comprises: 200 g of potato, 20 g of glucose, 3 mL of gentamicin sulfate injection and 1000 mL of water, and is sterilized at 121° C. for 20 min.

6. The method for preparing the liquid microbial agent according to claim 4, characterized in that: The PDA solid culture medium comprises: 200 g of potato, 20 g of glucose, 15 g of agar, 3 mL of gentamicin sulfate injection and 1000 mL of water, and is sterilized at 121° C. for 20 min.

7. Use of the Pseudogymnoascus sp. SDF-LT according to claim 1 in the preparation of cellulase, characterized in that: The method for preparing cellulase comprises the following steps: fermenting the Pseudogymnoascus sp. SDF-LT, and collecting the fermentation product to obtain cellulase.

8. Use of the Pseudogymnoascus sp. SDF-LT according to claim 1 in the rapid return of crop straw to fields.

9. Use of the liquid microbial agent according to any one of claims 2 to 3 in the rapid return of crop straw to fields.

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