Strain of low-temperature and drought-resistant straw-degrading bacteria and application thereof

Abstract

The application discloses a low-temperature and drought-resistant straw-degrading bacterium and application thereof. The application screens a low-temperature and drought-resistant straw-degrading bacterium, which is Stenotrophomonas maltophilia (Stenotrophomonas maltophilia) Stenotrophomonas maltophilia ) GF-Y18, has good abilities of producing exo-beta-1, 4-glucanase, laccase and lignin peroxidase, can effectively promote straw decomposition, and has certain low-temperature and drought resistance; the bacterium and the bacterium agent thereof can be used for degrading lignin and / or cellulose and / or straw in a low-temperature and drought environment.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of bio-agriculture, in particular to a low-temperature and drought-tolerant straw-degrading bacterium and its application. BACKGROUND

[0002] Corn is an important food and economic crop widely planted around the world, and produces a large amount of straw waste every year. These straws are rich in cellulose, hemicellulose and other available biomass resources. Straw degradation is a microbial enzymatic process, and screening and developing efficient straw-degrading bacteria is an effective way to promote the efficient decomposition of straw returned to the field. Using microorganisms to accelerate straw decomposition and solve the technical bottleneck of straw returning to the field can improve soil fertility and soil quality, and has important significance for the full utilization of straw resources, the cultivation of farmland fertility and the sustainable development of agriculture.

[0003] However, the high efficiency of the currently widely used straw-degrading microorganisms depends on suitable temperature and moisture conditions. In low-temperature or drought environments, the metabolic capacity of these microorganisms will decrease significantly, resulting in a sharp decrease in straw degradation efficiency, and even stagnation. The climate in the northern spring corn planting area is cold and dry, and the soil microbial activity and enzyme activity are low, which is not conducive to efficient straw decomposition and nutrient release.

[0004] Therefore, screening of low-temperature and drought-tolerant high-efficiency straw-degrading strains is the key to the development of straw decomposition agents and the acceleration of efficient in-situ decomposition of straw returned to the field in cold and dry areas. SUMMARY

[0005] The first object of the present application is to provide a low-temperature and drought-tolerant straw-degrading bacterium.

[0006] The second object of the present application is to provide a low-temperature and drought-tolerant straw-degrading bacterial agent.

[0007] The third object of the present application is to provide the application of the low-temperature and drought-tolerant straw-degrading bacterium (Stenotrophomonas maltophilia GF-Y18) or the low-temperature and drought-tolerant straw-degrading bacterial agent. Stenotrophomonas maltophilia Stenotrophomonas maltophilia The third object of the present application is to provide the application of the low-temperature and drought-tolerant straw-degrading bacterium (Stenotrophomonas maltophilia GF-Y18) or the low-temperature and drought-tolerant straw-degrading bacterial agent.

[0008] The first object of the present application is implemented by the following technical solutions.

[0009] The low-temperature and drought-tolerant straw-degrading bacterium is Stenotrophomonas maltophilia GF-Y18, which has a preservation number of CGMCC NO.35526, is preserved in the China General Microbiological Culture Collection Center, and has a preservation address of No.3, Beichen West Road, Chaoyang District, Beijing, a preservation date of August 5, 2025, and a classification name of Stenotrophomonas maltophilia. Stenotrophomonas maltophilia Stenotrophomonas maltophilia Stenotrophomonas maltophilia ​​

[0010] The second object of the present application is implemented by the following technical solutions.

[0011] A low-temperature and drought-resistant straw-degrading bacterial agent, comprising a low-temperature and drought-resistant straw-degrading bacterium and / or a fermentation liquor thereof.

[0012] The third object of the present application is implemented by the following technical solutions.

[0013] Application of the low-temperature and drought-resistant straw-degrading bacterium or the low-temperature and drought-resistant straw-degrading bacterial agent in degrading lignin and / or cellulose in a low-temperature and drought environment.

[0014] Application of the low-temperature and drought-resistant straw-degrading bacterium or the low-temperature and drought-resistant straw-degrading bacterial agent in degrading straw in a low-temperature and drought environment.

[0015] Advantages of the present application:

[0016] The present application screens a strain of Stenotrophomonas maltophilia (Stenotrophomonas maltophilia) Stenotrophomonas maltophilia ) GF-Y18, which is proved by experiments to have good abilities of producing exo-β-1, 4-glucanase, laccase and lignin peroxidase, can effectively promote straw decomposition, and has certain low-temperature and drought resistance. The decomposed straw can improve soil fertility and promote the growth of plants in the coming year. The present application discovers that Stenotrophomonas maltophilia GF-Y18 still has the ability of straw degradation under the conditions of low temperature and drought in winter in cold and arid regions such as Inner Mongolia, and provides a new strain resource for straw-degrading bacterial agents. BRIEF DESCRIPTION OF DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or prior art description will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0018] Figure 1 For the door level species relative abundance of the collected FL farmland soil sample;

[0019] Figure 2 For the door level species relative abundance of the collected FR forest soil and humus sample;

[0020] Figure 3 For the door level species relative abundance of the collected CS rotten straw sample;

[0021] Figure 4 For the door level species relative abundance of the collected CP compost and animal manure sample;

[0022] Figure 5Statistical results of macrogenomes of four types of samples based on KEGG database annotation; wherein, (a) is a KEGG annotation result histogram, (b) is a community barcode analysis diagram;

[0023] Figure 6 Annotation abundance of macrogenomes of four types of samples based on KEGG database; wherein, (a) is cellulose, (b) is hemicellulose, (c) is lignin;

[0024] Figure 7 Statistical results of macrogenomes of four types of samples based on CAZy database annotation; wherein, (a) is FL farmland soil sample, (b) is FR forest soil and humus sample, (c) is CS rotten straw sample, (d) is CP compost and animal manure sample;

[0025] Figure 8 Annotation abundance of macrogenomes of four types of samples based on CAZy database lignocellulose-degrading enzyme family;

[0026] Figure 9 Growth curve of low-temperature and drought-tolerant straw-degrading bacteria (Bacillus subtilis) GF-Y18; (a) is under different PEG6000 concentrations, (b) is under different NaCl concentrations; Stenotrophomonas maltophilia

[0027] Growth curve of low-temperature and drought-tolerant straw-degrading bacteria (Bacillus subtilis) GF-Y18; (a) is under different PEG6000 concentrations, (b) is under different NaCl concentrations; Figure 10 Stenotrophomonas maltophilia Growth curve of low-temperature and drought-tolerant straw-degrading bacteria (Bacillus subtilis) GF-Y18; (a) is under different PEG6000 concentrations, (b) is under different NaCl concentrations;

[0028] Figure 11 Stenotrophomonas maltophilia Growth curve of low-temperature and drought-tolerant straw-degrading bacteria (Bacillus subtilis) GF-Y18; (a) is under different PEG6000 concentrations, (b) is under different NaCl concentrations;

[0029] Figure 12 Growth curve of low-temperature and drought-tolerant straw-degrading bacteria (Bacillus subtilis) GF-Y18; (a) is under different PEG6000 concentrations, (b) is under different NaCl concentrations; Stenotrophomonas maltophilia DETAILED DESCRIPTION

[0030] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0031] The formula of the culture medium used in the embodiments is as follows:

[0032] ​​​Lignin medium: (NH4)2SO4 2 g / L, K2HPO4 1 g / L, KH2PO4 1 g / L, MgSO4 0.2 g / L, MnSO4 0.02 g / L, peptone 10 g / L, lignin 1 g / L, agar 18 g / L;

[0033] Carboxymethylcellulose sodium medium (CMC medium): (NH4)2SO4 2 g / L, K2HPO4 1 g / L, KH2PO4 1 g / L, MgSO4 0.2 g / L, MnSO4 0.02 g / L, peptone 10 g, CMC-Na 10 g / L, agar 18 g / L;

[0034] LB medium: peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, agar 18 g / L;

[0035] Aniline blue screening medium: 0.1 g / L aniline blue is added to the LB medium;

[0036] Enzyme production medium: urea 0.6 g / L, peptone 0.5 g / L, (NH3)2SO4 2.0 g / L, K2HPO4 1.0 g / L, MgSO4·7H2O 0.05 g / L, MnSO4·7H2O 0.016 g / L, ZnSO4·7H2O 0.017 g / L, CaCl2 0.02 g / L, NaCl 0.2 g / L;

[0037] Straw degradation medium: (NH4)2SO4 2 g / L, K2HPO4 2 g / L, MgSO4 0.05 g / L, CaCO3 2 g / L, NaCl 0.2 g / L, 20 mL of the above medium and 1 g of corn straw are added to a 20 mL triangular flask.

[0038] The above medium is sterilized at 121℃, 0.1 MPa for 30 min before use.

[0039] Example 1

[0040] A method for rapidly screening low-temperature and drought-resistant straw-degrading bacteria, comprising the following steps one to five.

[0041] Step one, selection and collection of bacterial source samples

[0042] Collect bacterial source samples rich in lignocellulose in cold and arid regions (the cold and arid regions in the present application refer to the east four leagues of Inner Mongolia (Tongliao, Xing'an League, Chifeng, Hulunbuir) and the northeast region), farmland, forest soil, straw, compost and animal manure, etc.

[0043] Collection: Gently brush off the surface sediment, and use a sterile soil drill to drill 0-40 cm of farmland soil layer samples (denoted as FL), or use a sterile sampler to collect soil or humus in the forest (denoted as FR), rotten straw (denoted as CS), compost or cow and sheep manure (denoted as CP) into a sterile bag and transported back to the laboratory, and stored in a -86℃ freezer. In this embodiment, a total of 19 farmland soil layer samples (FL-1 ~ FL-19), 14 soil or humus samples in the forest (FR-1 ~ FR-14), 13 rotten straw samples (CS-1 ~ CS-13), and 7 compost or cow and sheep manure samples (CP-1 ~ CP-7) were collected.

[0044] Step two, metagenomic sequencing analysis

[0045] The above collected microbial source samples were sent to Shanghai Meiji Biomedicine Technology Co., Ltd. for metagenomic sequencing. The raw data obtained by sequencing was subjected to data quality control using Fastp software, and low-quality (base quality value < 20) and N-containing reads were cut off to obtain high-quality sequences required for subsequent analysis.

[0046] MEGAHIT software was used to assemble the sequences of different sequencing, and Prodigal v2.6.3 software was used to predict ORF of the splicing results. Genes with nucleic acid length greater than or equal to 100 bp were selected and translated into amino acid sequences. The amino acid sequences predicted and translated from all samples were clustered using CD-HIT software, and the longest gene in each class was taken as a representative sequence to construct a non-redundant gene set, and the base sequence of the non-redundant gene set gene was obtained.

[0047] The NR database was used to compare the base sequences of the non-redundant gene set genes of different microbial source samples to obtain the dominant bacterial phylum of each microbial source sample, and the KEGG database and CAZy database were used for functional annotation analysis and carbohydrate enzyme analysis of different microbial source samples. According to the species composition and function, the lignocellulose-degrading single strain was directionally screened.

[0048] The method provided by the application can quickly determine the core microbial composition and potential lignocellulose-degrading function in different microbial source samples, quickly select microbial source samples with potential lignocellulose-degrading function, eliminate low-efficiency microbial source samples, and improve the screening efficiency.

[0049] Figure 1As shown in the door level, the main advantage of FL sample phylum is concentrated in Actinomycetota (Actinomycetota), Pseudomonadota (Pseudomonadota) and Acidobacteriota (Acidobacteriota), Nitrososphaerota (Nitrososphaerota) and Bacteroidota (Bacteroidota) account for second.

[0050] As shown in the door level, the main advantage of FL sample phylum is concentrated in Actinomycetota (Actinomycetota), Pseudomonadota (Pseudomonadota) and Acidobacteriota (Acidobacteriota), Nitrososphaerota (Nitrososphaerota) and Bacteroidota (Bacteroidota) account for second. Figure 2

[0051] CS sample species relative abundance is shown in Figure 3 As shown in the door level, the main advantage of FL sample phylum is concentrated in Actinomycetota (Actinomycetota), Pseudomonadota (Pseudomonadota) and Acidobacteriota (Acidobacteriota), Nitrososphaerota (Nitrososphaerota) and Bacteroidota (Bacteroidota) account for second.

[0052] As shown in the door level, the main advantage of FL sample phylum is concentrated in Actinomycetota (Actinomycetota), Pseudomonadota (Pseudomonadota) and Acidobacteriota (Acidobacteriota), Nitrososphaerota (Nitrososphaerota) and Bacteroidota (Bacteroidota) account for second. Figure 4

[0053] Firmicutes usually cooperates with Actinomycetota, Verrucomicrobia and Fibrobacteres in the process of degrading residual plants; Bacteroidota also has significant cellulose degradation ability; Actinomycetota is also a common low-temperature straw degradation bacteria; Pseudomonadota is a kind of bacteria widely existing in natural environment, which has strong straw degradation ability.

[0054] In the above four types of bacterial source samples, the abundance of the top ten in the straw degradation advantage phylum accounts for 3, the abundance of the top ten in the FR sample accounts for 4, the abundance of the top ten in the CS sample accounts for 3, and the abundance of the top ten in the CP sample accounts for 4, which indicates that the four types of bacterial source samples have certain potential for straw degradation. The four types of bacterial source samples are used as the first degradation potential bacterial source sample.

[0055] As shown in the door level, the main advantage of FL sample phylum is concentrated in Actinomycetota (Actinomycetota), Pseudomonadota (Pseudomonadota) and Acidobacteriota (Acidobacteriota), Nitrososphaerota (Nitrososphaerota) and Bacteroidota (Bacteroidota) account for second. Figure 5 ​​As shown in (a), the KEGG metabolic pathway annotation includes 46 metabolic pathways, with metabolism accounting for the highest proportion in the tested samples. Among the second category of metabolic pathways, global and overview maps, carbohydrate metabolism, and amino acid metabolism have the highest proportions. Figure 5 As shown in (b), in the third type of KEGG metabolic pathway annotation, among the global overview pathways, metabolic pathways, biosynthesis of secondary metabolism, and microbia metabolism in diverse environments account for the highest proportion.

[0056] Figure 6 The gene abundance of lignocellulose degradation genes annotated in the KEGG database for each sample is shown. Among them, the gene abundance of CP is the highest in cellulose, hemicellulose and lignin samples, and the overall order is CP > CS > FR > FL.

[0057] Analysis of carbohydrate enzymes was performed based on the CAZy database. For example... Figure 7 As shown, FL, FR, CS, and CP samples have 303,815, 269,406, 344,129, and 209,652 genes annotated to 559, 553, 574, and 552 enzyme families across six major functional classes: glycoside hydrolases (GHs), glycosyl transferases (GTs), polysaccharide lyases (PLs), carbohydrate esterases (CEs), auxiliary redox enzymes (Aas), and carbohydrate-binding modules (CBMs).

[0058] The families of lignocellulose-degrading enzymes annotated in the CAZy database for each sample are as follows: Figure 8 As shown in the figure, GH represents the glycoside hydrolase family, CBM represents the carbohydrate binding module, and AA represents the coenzyme family; for example, GH9 belongs to the glycoside hydrolase 9 family. It can be seen that the CP sample has the highest abundance, with the overall order being CP > CS > FR > FL.

[0059] According to the above KEGG and CAZy annotation results, among the four sample types, the CP sample has the highest potential to decompose straw, and the CP sample is used as the second degradation potential bacterial source sample.

[0060] Step three, isolation and screening of straw degrading strains

[0061] The CP samples collected above, i.e. compost and animal manure samples, are used as bacterial source samples for strain isolation and screening.

[0062] 10 g of the bacterial source sample is added to a 250 mL triangular flask with small glass beads, 90 mL of sterile water is added, and the sample suspension is shaken in a constant temperature shaking incubator at 28°C (120 r / min) for 30 min. The sample suspension is diluted with sterile water to a concentration gradient of 10 -1 ~10 -8 The diluted solution is taken and spread on lignin culture medium and carboxymethyl cellulose culture medium, respectively, and placed in a constant temperature incubator at 15°C for 5-10 days. The diameter of the bacterial colony on the carboxymethyl cellulose sodium culture medium and the lignin culture medium, i.e. the growth rate, is used to evaluate the degradation and utilization of cellulose and lignin by the strains. Strains with a single colony diameter ≥ 2 mm are selected and subjected to continuous streaking on carboxymethyl cellulose sodium culture medium and lignin culture medium for isolation and purification. The obtained 71 strains are stored in a refrigerator at 15°C.

[0063] Aniline blue fading ring determination is performed: the selected single bacteria are inoculated on aniline blue selection medium and cultured in a constant temperature incubator at 15°C for 1 day. The diameter of the bacterial colony (d) and the diameter of the fading ring (D) are measured.

[0064] Congo red fading ring determination is performed: the selected single bacteria are inoculated on CMC medium and lignin medium, respectively, and cultured in a 15°C incubator for 5-7 days. 1% Congo red staining solution is spread on the plate and stained for 1 hour. Then the plate is washed with 1 mol / L NaCl solution for 30 minutes. The diameter of the bacterial colony (d) and the diameter of the fading ring (D) are measured.

[0065] The 71 lignocellulose-degrading bacteria (denoted as GF-Y1-GF-Y71) selected from the 7 CP samples are subjected to aniline blue fading ring and Congo red fading ring determination according to the above method. The results are shown in Table 1: there are a total of 48 strains that can produce fading rings on aniline blue selection medium, 3 strains that can produce Congo red fading rings, and a total of 2 strains that can fade on both aniline blue selection medium and Congo red staining medium. Among them, the strain with a ratio (D / d) of the transparent circle (D) to the colony diameter (d) ≥ 5.00 on the aniline blue selection medium is 1 (GF-Y18), and the ratio is 5.40.

[0066] Table 1 Fading results of different strain selection media

[0067]

[0068] Note: - means no fading, + means fading.

[0069] Step four, screening low temperature and drought resistant straw degrading bacteria

[0070] The above strains producing fading rings on aniline blue screening medium and / or strains producing fading rings on Congo red medium were inoculated on aniline blue screening medium, respectively, and cultured at 5℃, 10℃, 15℃, 20℃ and 25℃ for 1d, and the diameters (d) of bacterial rings and fading rings (D) were measured to determine the low temperature adaptability.

[0071] The strains producing fading rings in low temperature environment after the low temperature adaptability determination (in this embodiment, it means that the strains can produce fading rings at 5℃) were inoculated in LB liquid medium and cultured at 15℃ and 160 r / min for 24h to prepare standard bacterial suspension with OD 600 value of 1.0. The standard bacterial suspension was inoculated into 5 kinds of LB liquid medium containing different concentrations of PEG6000 (5%, 10%, 15%, 20%, 25%) and 5 kinds of LB liquid medium containing different concentrations of NaCl (0.2, 0.4, 0.6, 0.8, 1.0 mol / L) at 10%, and cultured at 15℃ and 200 r / min for 48h. The growth curve analyzer was used to monitor the growth curve in real time. The bacteria with OD 600 ≥1.0 at least at one concentration of the above screening concentration of PEG6000 and OD 600 ≥1.0 at least at one concentration of the above screening concentration of NaCl were screened, that is, degrading bacteria I was obtained.

[0072] The degrading bacteria I was inoculated into LB liquid medium to prepare bacterial suspension with a concentration of 1×10 7 CFU / mL.

[0073] The bacterial suspension of each degrading bacteria I (one strain GF-Y18 was screened in this embodiment) was inoculated into enzyme production medium at a volume ratio of 10%, and cultured at 5℃, 10℃, 15℃, 20℃ and 25℃ for 1d (at a speed of 180 r / min).

[0074] The bacterial suspension of each degrading bacteria I (a strain GF-Y18 screened in the embodiment) was inoculated into five enzyme production culture mediums containing different concentrations of PEG6000 (5%, 10%, 15%, 20%, and 25%) and five enzyme production culture mediums containing different concentrations of NaCl (0.2, 0.4, 0.6, 0.8, and 1.0 mol / L) at an inoculation amount of 10% by volume, and cultured at 15°C and 180 r / min for 1 day.

[0075] The enzyme activities were determined: the endo-1, 4-beta-glucanase (C X ) activity, the exo-1, 4-beta-glucanase (C1) activity, and the beta-glucosidase (C B ) activity were determined by the DNS method; the laccase (Lac) activity was determined by the ABTS method; and the lignin peroxidase (Lip) activity was determined by the veratryl alcohol method. The enzyme activity screening requirements were as follows: ① the cellulase activity of the strain was ≥1.0 U / mL, that is, the endo-1, 4-beta-glucanase activity was ≥1.0 U / mL, the exo-1, 4-beta-glucanase activity was ≥1.0 U / mL, and the beta-glucosidase activity was ≥1.0 U / mL; ② the laccase activity was ≥100 U / L; and ③ the lignin peroxidase activity was ≥35 U / L.

[0076] The degrading bacteria I with the enzyme activity meeting any one of the above enzyme activity screening requirements ①-③ at low temperature (5°C), at least one of the above screening concentrations of PEG6000, and at least one of the above screening concentrations of NaCl was the degrading bacteria II.

[0077] The degrading bacteria II was inoculated into an LB liquid culture medium to prepare a bacterial suspension with a concentration of 1×10 7 CFU / mL. The bacterial suspension of each degrading bacteria II (a strain GF-Y18 screened in the embodiment) was inoculated into a straw degradation culture medium at an inoculation amount of 10% by volume, and cultured at 15°C for 30 days. The residue of the straw was repeatedly washed with sterile water for 3 times, dried in an oven at 60°C to constant weight, weighed, and the relative degradation rate of the straw was calculated. The relative degradation rate of the straw = (M1-M2) / M1×100% (wherein M1 is the initial straw mass, and M2 is the straw mass after degradation). The strain with a relative degradation rate of the straw ≥20% was the straw degrading bacteria resistant to low temperature and drought.

[0078] The bacteria were statically cultured at 5℃, 10℃, 15℃, 20℃, and 25℃ for 30 days. Bacterial suspensions of each degrading bacterium II were inoculated at a 10% (v / v) into five different straw degradation media containing different concentrations of PEG6000 (5%, 10%, 15%, 20%, and 25%) and statically cultured at 15℃ for 30 days. Bacterial suspensions of each degrading bacterium II were also inoculated at a 10% (v / v) into five different straw degradation media containing different concentrations of NaCl (0.2, 0.4, 0.6, 0.8, and 1.0 mol / L) and statically cultured for 30 days. This was used to evaluate the degradation ability of the strains under low temperature and / or drought conditions.

[0079] Step 5: Screening for low-temperature and drought-resistant straw-degrading bacteria

[0080] After the above steps, a strain with a ratio of transparent zone (D) to colony diameter (d) (D / d) ≥ 5.00 was obtained. Its D / d value was 5.40 at 15℃, as shown in Table 2. It can grow in the range of 5~25℃ and is numbered GF-Y18.

[0081] Table 2. Bleaching zone ratio (D / d) of strains at different temperatures

[0082]

[0083] like Figure 9 As shown, strain GF-Y18 can grow under both 25% PEG6000 and 0.8 mol / L NaCl conditions, and its OD value is higher under 10% PEG6000 and 0.4 mol / L NaCl conditions. 600 Maintaining a stable value above 2.0 indicates that it can tolerate drought.

[0084] Table 3 shows the enzyme activities of strain GF-Y18 at different temperatures. The highest activities of endo-1,4-β-glucanase, exo-1,4-β-glucanase, and β-glucosidase in strain GF-Y18 were observed at 5℃, with values ​​of 2.23 U / mL, 2.05 U / mL, and 3.05 U / mL, respectively. Laccase activity was highest at 25℃ (384.81 U / L), and lignin peroxidase activity was highest at 15℃ (54.23 U / L). All enzyme activities of strain GF-Y18 met the enzyme activity screening requirements at 5℃, indicating that strain GF-Y18 possesses the ability to degrade cellulose and lignin at low temperatures.

[0085] The enzyme activities of strain GF-Y18 under different PEG6000 concentrations are shown in Table 4. The endo-1,4-beta-glucanase activity, exo-1,4-beta-glucanase activity, beta-glucosidase activity, laccase activity and lignin peroxidase activity of strain GF-Y18 were the highest under 5% PEG6000, which were 2.32 U / mL, 2.10 U / mL, 1.48 U / mL, 338.64 U / L and 49.68 U / L, respectively. All enzyme activities under 15% PEG6000 concentration and cellulase activity under 25% PEG6000 concentration met the requirements of enzyme activity screening, indicating that strain GF-Y18 had the ability to degrade cellulose and lignin under drought stress, and the cellulose degradation ability was strong.

[0086] The enzyme activities of strain GF-Y18 under different NaCl concentrations are shown in Table 5. The endo-1,4-beta-glucanase activity and exo-1,4-beta-glucanase activity of strain GF-Y18 were the highest when cultured under 0.6 mol / L, which were 1.90 U / mL and 1.64 U / mL, respectively. The beta-glucosidase activity was the highest under 0.2 mol / L, which was 2.65 U / mL. The laccase activity was the highest when cultured under 0.4 mol / L, which was 220.08 U / L. The lignin peroxidase activity was the highest when cultured under 0.6 mol / L, which was 39.58 U / L. All enzyme activities under 0.4-0.8 mol / L NaCl concentration and cellulase activity and lignin peroxidase activity under 1.0 mol / L NaCl concentration met the requirements of enzyme activity screening, indicating that strain GF-Y18 had the ability to degrade cellulose and lignin under drought and saline-alkali stress.

[0087] It is shown that strain GF-Y18 can still produce cellulose-degrading enzymes and lignin-degrading enzymes to degrade cellulose and lignin under low temperature and drought conditions, and maintain growth and metabolism.

[0088] Table 3 Enzyme activities of strain GF-Y18 under different temperatures

[0089]

[0090] Note: Different lowercase letters after the same column data indicate significant difference (p<0.05), the same below.

[0091] Table 4 Enzyme activities of strain GF-Y18 under different PEG6000 concentrations

[0092]

[0093] Table 5 Enzyme activities of strain GF-Y18 under different NaCl concentrations

[0094]

[0095] After 30 days of culture, the relative degradation rate of the straw of the strain GF-Y18 gradually increased with the increase of temperature, as shown in Figure 10 a, the relative degradation rate of the straw of the strain GF-Y18 at 15℃ was 27%, indicating that a strain of low-temperature and drought-resistant straw-degrading bacteria has been screened.

[0096] As shown in Figure 10 a, at 15℃-25℃, the relative degradation rate of the straw of the strain GF-Y18 can be maintained at more than 20%, and at 5℃, the relative degradation rate of the straw is still 14%, further indicating that the strain GF-Y18 has the ability to degrade straw at low temperature. As shown in Figure 10 b, when the content of PEG6000 is 20% or less, the relative degradation rate of the straw of the strain GF-Y18 can be maintained at about 20%, and at 25%, the relative degradation rate of the straw is still 16%; as shown in Figure 10 c, the relative degradation rate of the straw of the strain GF-Y18 under 0.6 mol / L NaCl is the highest at 19%, and under 0.8 mol / L and below of NaCl, the relative degradation rate of the straw is more than 10%, further indicating that the strain GF-Y18 has the ability to degrade straw under drought stress. The above test results show that the strain GF-Y18 can degrade straw under low temperature and drought conditions.

[0097] Example 2

[0098] According to the procedure of Example 1, a strain GF-Y18 which exhibits high enzyme activity and degradation rate under low temperature and drought environment is screened.

[0099] The GF-Y18 is identified, and the identification steps and results are as follows.

[0100] (1) Morphological characteristics: the GF-Y18 is streak inoculated on LB solid culture medium, and incubated at 15℃ for 24 h, and the colony shape, size, color, edge, transparency and other morphological characteristics are observed. As shown in Figure 11 , the strain GF-Y18 is white, round, convex on the surface, and smooth on the edge.

[0101] (2) Sequencing identification: The genomic DNA of the strain GF-Y18 was extracted by using the bacterial genomic DNA extraction kit, and the universal primer for bacteria was 27F (5'-GAGAGTTTGATCCTGGCTCAG-3', see sequence listing SEQ ID No. 1) and 1492R (5'-TACGGCTACCTTGTTACGAC-3', see sequence listing SEQ ID No. 2) to amplify the 16S rRNA gene of bacteria, and the PCR reaction system is shown in Table 6, and the reaction procedure is shown in Table 7. The amplified PCR product was stored at 10°C. The PCR product was sequenced by Shanghai Sunway Biotech Co., Ltd., and the sequence is shown as SEQ ID No. 3. The sequencing results were spliced by Contig Express, and the spliced sequence of the 16S rRNA gene of the strain was compared with the known sequence in the NCBI database (https: / / www.ncbi.nlm.nih.gov / ) using BLAST. The phylogenetic tree was constructed by using the Neighbor-Joining method in MEGA 11.0 software. As shown in Table 8, the strain GF-Y18 was identified as Stenotrophomonas maltophilia. Figure 12 . Stenotrophomonas maltophilia . .

[0102] A low-temperature and drought-resistant straw-degrading bacterium was screened, i.e., Stenotrophomonas maltophilia (Stenotrophomonas maltophilia) Stenotrophomonas maltophilia Stenotrophomonas maltophilia ) GF-Y18, which is currently preserved in the General Microbiological Center of the Chinese Microorganism Strain Preservation Management Committee, located at No. 1, Beichen West Road, Yard 3, Institute of Microbiology, Chinese Academy of Sciences, Beijing, Chaoyang District, with the preservation number CGMCC NO. 35526 and the preservation date of August 5, 2025.

[0103] Table 6 PCR reaction system

[0104]

[0105] Table 7 PCR reaction procedure

[0106]

[0107] Example 3

[0108] A low-temperature and drought-resistant straw-degrading bacterial agent includes a low-temperature and drought-resistant straw-degrading bacterium (i.e., Stenotrophomonas maltophilia (Stenotrophomonas maltophilia) Stenotrophomonas maltophilia ) GF-Y18) and / or a fermentation liquor thereof.

[0109] The low-temperature and drought-resistant straw-degrading bacterial fermentation liquor, i.e., the Stenotrophomonas maltophilia (Stenotrophomonas maltophilia) Stenotrophomonas maltophilia Stenotrophomonas maltophilia ) GF-Y18 fermentation liquor, and the preparation method thereof includes the following steps (1)-(3).

[0110] (1) Inoculation: low-temperature and drought-resistant straw-degrading bacteria, i.e. Stenotrophomonas maltophilia (S. maltophilia) GF-Y18, is inoculated on LB medium and cultured at 15°C for 2 days to obtain activated bacteria; Stenotrophomonas maltophilia Stenotrophomonas maltophilia

[0111] (2) Fermentation: the activated bacteria are inoculated into LB medium and cultured in a 15°C, 180 rpm shaker for 2 days;

[0112] (3) Filtration: the mycelium in the bacterial solution after 2 days of shaker culture is filtered out to obtain a filtrate, which is a crude metabolite stock solution, i.e. S. maltophilia (S. maltophilia) GF-Y18 fermentation liquor. ​

[0113] The above merely describes preferred embodiments of the present application and is not intended to limit the present application, and any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.​​

Claims

1. A low-temperature and drought-tolerant straw-degrading bacteria, characterized in that, The low-temperature and drought-resistant straw-degrading bacteria is Stenotrophomonas maltophilia (Stenotrophomonas maltophilia) Stenotrophomonas maltophilia ) GF-Y18, and the preservation number is CGMCC NO. 35526.

2. A low temperature and drought tolerant straw degrading bacterial agent, characterized in that, It comprises the low-temperature and drought-resistant straw-degrading bacteria as claimed in claim 1 and / or the fermentation liquor thereof.

3. The use of the low-temperature and drought-resistant straw-degrading bacteria as claimed in claim 1 or the low-temperature and drought-resistant straw-degrading bacteria agent as claimed in claim 2 in degrading lignin and / or cellulose in a low-temperature and drought environment.

4. The use of the low-temperature and drought-resistant straw-degrading bacteria as claimed in claim 1 or the low-temperature and drought-resistant straw-degrading bacteria agent as claimed in claim 2 in degrading straw in a low-temperature and drought environment.

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