Low-temperature-resistant and drought-resistant straw degrading bacterium and application thereof

Abstract

The invention discloses a low-temperature-resistant and drought-resistant straw degrading bacterium and application thereof. The low-temperature-resistant and drought-resistant straw degrading bacterium is obtained through screening, is stenotrophomonas maltophilia GF-Y18, has good capacity of producing exon-beta-1, 4-glucanase, laccase and lignin peroxidase, can effectively promote straw decomposition, has certain low-temperature-resistant and drought-resistant capacity, can be used for degrading straw, and can be applied to the field of straw degradation. The strain and the fungicide thereof can be used for degrading lignin and / or cellulose and / or straw in a low-temperature 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 efficient activity 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 objective of this invention is achieved by the following technical solution.

[0011] A low-temperature and drought-resistant straw-degrading microbial agent, comprising low-temperature and drought-resistant straw-degrading bacteria and / or their fermentation broth.

[0012] The third objective of this invention is achieved by the following technical solution.

[0013] Application of low-temperature and drought-resistant straw-degrading bacteria or straw-degrading bacteria agents in the degradation of lignin and / or cellulose in low-temperature and drought environments.

[0014] Application of low-temperature and drought-resistant straw-degrading bacteria or straw-degrading bacteria agents in degrading straw in low-temperature and drought environments.

[0015] Advantages of this invention: This invention screened and obtained a strain of Stenotrophomonas maltophilia ( Stenotrophomonas maltophilia The strain GF-Y18 has been experimentally proven to possess excellent production capabilities of exo-β-1,4-glucanase, laccase, and lignin peroxidase, effectively promoting straw decomposition. It also exhibits some resistance to low temperatures and drought. The decomposed straw can improve soil fertility and promote plant growth in the following year. This invention reveals that *Stenotrophomonas maltophilia* GF-Y18 retains its ability to degrade straw even under low-temperature and drought conditions in cold and arid regions such as Inner Mongolia, providing a new strain resource for straw-degrading inoculants. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 The relative abundance of species at the phylum level in FL farmland soil samples; Figure 2 Phylum-level relative abundance of species in soil and humus samples collected from FR forests; Figure 3 The relative abundance of species at the phylum level in the collected CS decayed straw samples; Figure 4 Phylogenetic relative abundance of species in collected CP compost and animal feces samples; Figure 5 Statistics on the KEGG database annotation results of metagenomics for four types of samples; where (a) is the histogram of KEGG annotation results and (b) is the community barcode analysis diagram; Figure 6 KEGG database annotation abundance of four types of sample metagenome; wherein, (a) is cellulose, (b) is hemicellulose, (c) is lignin; Figure 7 CAZy database annotation results statistics of four types of sample metagenome; 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; Figure 8 CAZy database lignocellulose-degrading enzyme family annotation abundance of four types of sample metagenome; Figure 9 Growth curve of low-temperature and drought-tolerant straw-degrading bacteria (Gluconacetobacter Stenotrophomonas maltophilia ) GF-Y18; (a) is under different PEG6000 concentrations, (b) is under different NaCl concentrations; Figure 10 Growth curve of low-temperature and drought-tolerant straw-degrading bacteria (Gluconacetobacter Stenotrophomonas maltophilia ) GF-Y18; (a) is under different temperatures, (b) is under different PEG6000 concentrations, (c) is under different NaCl concentrations; Figure 11 Growth curve of low-temperature and drought-tolerant straw-degrading bacteria (Gluconacetobacter Stenotrophomonas maltophilia ) GF-Y18; Figure 12 Growth curve of low-temperature and drought-tolerant straw-degrading bacteria (Gluconacetobacter Stenotrophomonas maltophilia ) GF-Y18; DETAILED DESCRIPTION

[0018] 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.

[0019] The formula of the culture medium used in the embodiments is as follows: Lignin culture medium: (NH4)2SO42g / L, K2HPO41g / L, KH2PO41g / L, MgSO40.2g / L, MnSO40.02g / L, protein peptone 10g / L, lignin 1g / L, agar 18g / L; Carboxymethylcellulose sodium medium (CMC medium): (NH4)2SO42 g / L, K2HPO41 g / L, KH2PO41 g / L, MgSO40.2 g / L, MnSO40.02 g / L, protein peptone 10 g, CMC-Na 10 g / L, agar 18 g / L; LB medium: protein peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, agar 18 g / L; Aniline blue screening medium: 0.1 g / L aniline blue is added to the LB medium; Enzyme production medium: urea 0.6 g / L, protein peptone 0.5 g / L, (NH3)2SO42.0 g / L, K2HPO41.0 g / L, MgSO4·7H2O 0.05 g / L, MnSO4·7H2O 0.016 g / L, ZnSO4·7H2O 0.017 g / L, CaCl20.02 g / L, NaCl 0.2 g / L; Straw degradation medium: (NH4)2SO42 g / L, K2HPO42 g / L, MgSO40.05 g / L, CaCO32 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.

[0020] The above medium is sterilized at 121℃, 0.1 MPa for 30 min before use.

[0021] Example 1 A method for rapidly screening low-temperature and drought-tolerant straw-degrading bacteria includes the following steps one to five.

[0022] Step one, selection and collection of bacterial source samples The bacterial source samples rich in lignocellulose are collected from the 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.

[0023] When collecting, gently brush off the surface sediments, and use a sterile soil drill to drill 0-40 cm farmland soil layer samples (marked as FL), or use a sterile sampler to collect forest soil or humus (marked as FR), rotten straw (marked as CS), compost or cow and sheep manure (marked as CP) into a sterile bag and take back to the laboratory, and store in a-86℃ freezer. In this embodiment, 19 farmland soil samples (FL-1 ~ FL-19), 14 forest soil or humus samples (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) are collected.

[0024] Step two, metagenomic sequencing analysis The above collected bacterial 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.

[0025] MEGAHIT software was used to assemble the sequences of different sequencing, and Prodigal v2.6.3 software was used to predict ORF of the assembly 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 was obtained.

[0026] NR database was used to compare the base sequences of the non-redundant gene set of different bacterial source samples to obtain the dominant bacterial phylum of each bacterial source sample, and KEGG database and CAZy database were used to analyze the function annotation and carbohydrate enzyme of different bacterial source samples. According to the species composition and function, the lignocellulose-degrading single strain was screened.

[0027] The method provided by the application can quickly determine the core microbial composition and potential lignocellulose-degrading function in different bacterial source samples, quickly select bacterial source samples with potential lignocellulose-degrading function, eliminate low-efficiency bacterial source samples, and improve the screening efficiency.

[0028] Figure 1 As shown, at the phylum level, the main dominant bacterial phylum of the FL sample is concentrated in Actinomycetota (Actinomycetota), Pseudomonadota (Pseudomonadota) and Acidobacteriota (Acidobacteriota), Nitrososphaerota (Nitrososphaerota) and Bacteroidota (Bacteroidota) account for the second.

[0029] As Figure 2 shown, at the phylum level, the dominant bacterial phylum of the FR sample is Pseudomonadota (Pseudomonadota), Actinomycetota (Actinomycetota) and Acidobacteria, Verrucomicrobia (Verrucomicrobia) and Bacteroidota (Bacteroidota) account for the second.

[0030] The relative abundance of species of the CS sample is shown in Figure 3At the phylum level, the dominant phyla of CS samples were Pseudomonadota, Actinomycetota and Bacteroidota.

[0031] By Figure 4 It can be seen that at the phylum level, the dominant species of CP samples are mainly concentrated in Pseudomonadota, Actinomycetota, Bacteroidota and Bacillota (Bacillota).

[0032] Firmicutes usually cooperates with Actinomycetota, Verrucomicrobia and Fibrobacteres in the process of degrading residual plants; Bacteroidetes also has significant cellulose degradation ability; Actinomycetota is also a common low-temperature straw degrading bacteria; Pseudomonadota is a kind of bacteria widely existing in natural environment, which has strong straw degradation ability.

[0033] Among the four types of bacterial source samples, the top ten abundance of FL sample accounts for 3, the top ten abundance of FR sample accounts for 4, the top ten abundance of CS sample accounts for 3, and the top ten abundance of CP sample accounts for 4, which indicates that the four types of bacterial source samples have certain potential for degrading straw. The four types of bacterial source samples are used as the first degradation potential bacterial source sample.

[0034] As Figure 5 As shown in (a), the KEGG metabolic pathway annotation includes 46 metabolic pathways, among which metabolism accounts for the highest proportion in the test sample. In the second type of metabolism metabolic pathway, global and overview maps, carbohydrate metabolism and amino acid metabolism account for the highest proportion. As shown in (b), in the third type of KEGG metabolic pathway annotation, in the global overview pathway, metabolic pathways, biosynthesis of secondary metabolism and microbia metabolism in diverse environments account for the highest proportion. Figure 5

[0035] Figure 6 The figure shows the lignocellulose degradation genes annotated in the KEGG database of each sample, among which the gene abundance of CP sample accounts for the highest proportion in cellulose, hemicellulose and lignin, and the overall is CP>CS>FR>FL.

[0036] ​Carbohydrate enzymes were analyzed based on the CAZy database. As shown in Figure 7 FIG. 1, the FL sample, the FR sample, the CS sample, and the CP sample have 303815, 269406, 344129, and 209652 genes, respectively, annotated to 559, 553, 574, and 552 enzyme families of six functional classes of glycoside hydrolases (GHs), glycosyl transferases (GTs), polysaccharide lyases (PLs), carbohydrate esterases (CEs), auxiliary activities (Aas), and carbohydrate-binding modules (CBMs).

[0037] The enzyme families of each sample annotated in the CAZy database involved in lignocellulose degradation are shown in FIG. 2. Figure 8 As shown in FIG. 2, GH represents glycoside hydrolase family, CBM represents carbohydrate-binding module, and AA represents auxiliary enzyme class. For example, GH9 represents glycoside hydrolase family 9. It can be seen that the abundance ratio of the CP sample is the highest, and the overall trend is CP>CS>FR>FL.

[0038] Based on the above KEGG and CAZy annotation results, it can be seen that the CP sample has the highest potential for decomposing straw, and the CP sample is used as a second degradation potential microbial source sample.

[0039] Step three, isolation and screening of straw-degrading strains The CP sample, i.e., the compost and animal manure sample, collected above is used as a microbial source sample for isolation and screening of strains.

[0040] 10 g of the microbial source sample is added to a 250 mL flask with small glass beads, 90 mL of sterile water is added, and the flask is shaken (120 r / min) in a constant temperature shaking incubator at 28°C for 30 min. The sample suspension is diluted with sterile water to form a concentration gradient of 10 -1 ~10 -8 The diluted solution is taken and spread on a lignin culture medium and a carboxymethyl cellulose culture medium, respectively, and placed in a constant temperature incubator at 15°C for 5-10 days. The diameter of the colonies (i.e., the growth rate) on the carboxymethyl cellulose sodium culture medium and the lignin culture medium is used to evaluate the degradation and utilization of cellulose and lignin by the strains. Strains with a single colony diameter of ≥2 mm are selected and subjected to continuous streaking on the carboxymethyl cellulose sodium culture medium and the lignin culture medium for isolation and purification. The obtained 71 strains are stored in a refrigerator at 15°C.

[0041] Aniline blue fading circle determination was performed: the screened single bacteria were inoculated on aniline blue screening medium and cultured in a 15℃ constant temperature incubator for 1 day, and the bacterial colony diameter (d) and fading circle diameter (D) were measured.

[0042] Congo red fading circle determination was performed: the screened single bacteria were inoculated on CMC medium and lignin medium respectively and cultured in a 15℃ incubator for 5-7 days, 1% Congo red staining solution was spread on the plate, and after staining for 1 hour, the plate was washed with 1 mol / L NaCl solution for 30 minutes, and the bacterial colony diameter (d) and fading circle diameter (D) were measured.

[0043] A total of 71 lignocellulose-degrading bacteria (denoted as GF-Y1-GF-Y71) screened from 7 CP samples were subjected to aniline blue fading circle and Congo red fading circle determination according to the above method, and the results are shown in Table 1: a total of 48 strains can produce fading circles on aniline blue screening medium, 3 strains can produce Congo red fading circles, and a total of 2 strains can fade on both aniline blue screening 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 screening medium is GF-Y18, and the ratio is 5.40.

[0044] Table 1 Fading results of different strains on selection medium

[0045] Note: - indicates no fading, + indicates fading.

[0046] Step four, screening of low-temperature and drought-resistant straw-degrading bacteria The strains producing fading circles on the aniline blue screening medium and / or the strains producing fading circles on the Congo red medium were inoculated on aniline blue screening medium respectively, and cultured at 5℃, 10℃, 15℃, 20℃ and 25℃ for 1 day, and the bacterial colony diameter (d) and fading circle diameter (D) were measured to determine the low-temperature adaptability.

[0047] The strains producing fading circles in low-temperature environment after the low-temperature adaptability determination (in this embodiment, it refers to the strains producing fading circles at 5℃) were inoculated in LB liquid medium and cultured at 15℃ and 160 r / min for 24 h to prepare OD 600Standard bacterial suspension with 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°C and 200 r / min for 48 h. The growth curve was monitored in real time by using a growth curve analyzer. The bacteria that met the requirement of OD 600 ≥1.0 at least at one concentration of the above-mentioned screening concentrations of PEG6000 and OD 600 ≥1.0 at least at one concentration of the above-mentioned screening concentrations of NaCl were screened out, and the degradation bacteria I was obtained.

[0048] The degradation bacteria I was inoculated into LB liquid medium to prepare a bacterial suspension with a concentration of 1×10 7 CFU / mL.

[0049] The bacterial suspension of each degradation bacteria I (one strain GF-Y18 was screened out in this example) was inoculated into enzyme production medium at an inoculation amount of 10% by volume ratio, and cultured at 5°C, 10°C, 15°C, 20°C and 25°C for 1 d (at a rotation speed of 180 r / min).

[0050] The bacterial suspension of each degradation bacteria I (one strain GF-Y18 was screened out in this example) was inoculated into 5 kinds of enzyme production medium containing different concentrations of PEG6000 (5%, 10%, 15%, 20%, 25%) and 5 kinds of enzyme production medium containing different concentrations of NaCl (0.2, 0.4, 0.6, 0.8, 1.0 mol / L) at an inoculation amount of 10% by volume ratio, and cultured at 15°C and 180 r / min for 1 d.

[0051] Determination of enzyme activity: the activities of endo-1,4-β-glucanase (C X ), exo-1,4-β-glucanase (C1) and β-glucosidase (C B ) were determined by using DNS method; the activity of laccase (Lac) was determined by using ABTS method; and the activity of lignin peroxidase (Lip) was determined by using veratryl alcohol method. The enzyme activity screening requirements were as follows: ① the cellulase activity of the strain was ≥1.0 U / mL, i.e. the activities of endo-1,4-β-glucanase, exo-1,4-β-glucanase and β-glucosidase were ≥1.0 U / mL; ② the activity of laccase was ≥100 U / L; and ③ the activity of lignin peroxidase was ≥35 U / L.

[0052] Degrading bacteria I that meet any one of the above enzyme activity screening requirements ①-③ at low temperature (5℃), at least one of the above screening concentrations of PEG6000, and at least one of the above screening concentrations of NaCl are considered degrading bacteria II.

[0053] Degrading bacteria II were inoculated into LB liquid medium to prepare a concentration of 1×10⁻⁶. 7 CFU / mL bacterial suspension. Bacterial suspensions of each degrading bacterium II (one strain, GF-Y18, was selected in this example) were inoculated into the straw degradation medium at an inoculation rate of 10% by volume. The medium was incubated statically at 15℃ for 30 days. The straw residue was washed three times repeatedly with sterile water, dried in a 60℃ oven until constant weight, weighed, and the relative degradation rate of the straw was calculated. Relative degradation rate of straw = (M1-M2) / M1×100% (where M1 is the initial straw mass, and M2 is the mass of the degraded straw). Straws with a relative degradation rate of ≥20% are considered low-temperature and drought-resistant straw-degrading bacteria.

[0054] 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.

[0055] Step 5: Screening for low-temperature and drought-resistant straw-degrading bacteria 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.

[0056] Table 2. Bleaching zone ratio (D / d) of strains at different temperatures

[0057] 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.

[0058] The enzyme activities of strain GF-Y18 at different temperatures are shown in Table 3. The endo-1,4-beta-glucanase activity, exo-1,4-beta-glucanase activity and beta-glucosidase activity of strain GF-Y18 were the highest when cultured at 5℃, being 2.23 U / mL, 2.05 U / mL and 3.05 U / mL, respectively. The laccase activity was the highest when cultured at 25℃, being 384.81 U / L. The lignin peroxidase activity was the highest when cultured at 15℃, being 54.23 U / L. The enzyme activities of strain GF-Y18 cultured at 5℃ all met the requirements of enzyme activity screening, indicating that strain GF-Y18 had the ability to degrade cellulose and lignin at low temperature.

[0059] The enzyme activities of strain GF-Y18 at 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 when cultured at 5% PEG6000, being 2.32 U / mL, 2.10 U / mL, 1.48 U / mL, 338.64 U / L and 49.68 U / L, respectively. The enzyme activities all met the requirements of enzyme activity screening at 15% PEG6000 concentration, and the cellulase activity met the requirements of enzyme activity screening at 25% PEG6000 concentration, indicating that strain GF-Y18 had the ability to degrade cellulose and lignin under drought stress, and had strong cellulose degradation ability.

[0060] The enzyme activities of strain GF-Y18 at 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 at 0.6 mol / L, being 1.90 U / mL and 1.64 U / mL, respectively. The beta-glucosidase activity was the highest when cultured at 0.2 mol / L, being 2.65 U / mL. The laccase activity was the highest when cultured at 0.4 mol / L, being 220.08 U / L. The lignin peroxidase activity was the highest when cultured at 0.6 mol / L, being 39.58 U / L. The enzyme activities all met the requirements of enzyme activity screening at 0.4-0.8 mol / L NaCl concentration, and the cellulase activity and lignin peroxidase activity all met the requirements of enzyme activity screening at 1.0 mol / L NaCl concentration, indicating that strain GF-Y18 had the ability to degrade cellulose and lignin under drought and saline-alkali stress.

[0061] 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.

[0062] Table 3 Enzyme activity of strain GF-Y18 at different temperatures

[0063] Note: The same column data after different lower case letters represent significant difference (p<0.05), the same below.

[0064] Table 4 Enzyme activity of strain GF-Y18 at different PEG6000 concentrations

[0065] Table 5 Enzyme activity of strain GF-Y18 at different NaCl concentrations

[0066] After 30 d of culture, the relative degradation rate of straw of strain GF-Y18 gradually increased with the increase of temperature, as shown in Table 3, the relative degradation rate of straw of strain GF-Y18 at 15℃ was 27%, indicating that a strain of low-temperature and drought-tolerant straw-degrading bacteria has been screened. Figure 10 aAs shown in Table 3, the relative degradation rate of straw of strain GF-Y18 at 15℃ was 27%, indicating that a strain of low-temperature and drought-tolerant straw-degrading bacteria has been screened.

[0067] As shown in Table 4, the relative degradation rate of straw of strain GF-Y18 could be maintained at more than 20% at 15℃-25℃, and the relative degradation rate of straw was still 14% at 5℃, further indicating that strain GF-Y18 had the ability to degrade straw at low temperature. Figure 10 aAs shown in Table 4, the relative degradation rate of straw of strain GF-Y18 could be maintained at more than 20% at 15℃-25℃, and the relative degradation rate of straw was still 14% at 5℃, further indicating that strain GF-Y18 had the ability to degrade straw at low temperature. Figure 10 bAs shown in Table 4, the relative degradation rate of straw of strain GF-Y18 could be maintained at about 20% when the content of PEG6000 was 20% or less, and the relative degradation rate of straw was still 16% when the content of PEG6000 was 25%; as shown in Table 4, Figure 10 cAs shown in Table 4, the relative degradation rate of straw of strain GF-Y18 was highest at 19% under 0.6 mol / L NaCl, and the relative degradation rate of straw was more than 10% under 0.8 mol / L and below concentration of NaCl, further indicating that strain GF-Y18 had the ability to degrade straw under drought stress. The above test results show that strain GF-Y18 can degrade straw under low temperature and drought conditions.

[0068] Example 2 According to the procedure of Example 1, a strain GF-Y18 was screened which showed high enzyme activity and degradation rate under low temperature and drought environment.

[0069] GF-Y18 was identified, and the identification steps and results are as follows.

[0070] (1) Morphological characteristics: GF-Y18 was streaked on LB solid medium, and incubated at 15℃ for 24 h, and the colony shape, size, color, edge, transparency and other morphological characteristics were observed. As shown in Table 1, Figure 11As shown, strain GF-Y18 is white, round, with a convex surface and a smooth edge.

[0071] (2) Sequencing identification: The genomic DNA of strain GF-Y18 was extracted using a Genomic DNA Extraction Kit for Bacteria, and the bacterial universal primers 27F (5'-GAGAGTTTGATCCTGGCTCAG-3', see sequence listing SEQ ID No. 1) and 1492R (5'-TACGGCTACCTTGTTACGAC-3', see sequence listing SEQ ID No. 2) were used to amplify the 16S rRNA gene of bacteria. 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 Sunred Bioengineering Co., Ltd., and the sequence is shown as SEQ ID No. 3. The sequencing results were spliced using Contig Express, and the spliced sequence of the 16S rRNA gene of the strain was compared with the known sequences in the NCBI database (https: / / www.ncbi.nlm.nih.gov / ) using BLAST. The phylogenetic tree was constructed using the Neighbor-Joining method in MEGA 11.0 software. As shown in Figure 12 , strain GF-Y18 was identified as Stenotrophomonas maltophilia. Stenotrophomonas maltophilia ).

[0072] A low-temperature and drought-tolerant straw-degrading bacterium was screened: Stenotrophomonas maltophilia (Stenotrophomonas maltophilia) Stenotrophomonas maltophilia Stenotrophomonas maltophilia ) GF-Y18, which is currently preserved in the General Microbial Center of the China Microbial Strain Preservation Management Committee, located at No. 1, Beichen West Road, Beijing City, Chaoyang District, Institute of Microbiology of the Chinese Academy of Sciences, with a preservation number of CGMCC NO. 35526 and a preservation date of August 5, 2025.

[0073] Table 6 PCR reaction system

[0074] Table 7 PCR reaction procedure

[0075] Example 3 A low-temperature and drought-tolerant straw-degrading bacterial agent, which comprises a low-temperature and drought-tolerant straw-degrading bacterium (i.e., Stenotrophomonas maltophilia (Stenotrophomonas maltophilia) Stenotrophomonas maltophilia ) GF-Y18) and / or a fermentation broth thereof.

[0076] wherein the low-temperature and drought-tolerant straw-degrading bacterial fermentation broth, i.e., Stenotrophomonas maltophilia (Stenotrophomonas maltophilia) Stenotrophomonas maltophilia Stenotrophomonas maltophilia) GF-Y18 fermentation liquor, a preparation method thereof comprises the following steps (1)-(3).

[0077] (1) inoculation: inoculate the low-temperature and drought-resistant straw degrading bacteria, i.e. the Xanthomonas translucens into LB culture medium, and culture at 15°C for 2 days to obtain activated bacteria; Stenotrophomonas maltophilia Stenotrophomonas maltophilia (2) fermentation: inoculate the activated bacteria into LB culture medium, and culture in a 15°C, 180 rpm shaker for 2 days; (3) filtration: filter the mycelium in the bacterial liquid after 2 days of shaker culture to obtain a filtrate, which is a crude metabolite stock solution, i.e. the Xanthomonas translucens ) GF-Y18 fermentation liquor. ​

[0078] The above merely describes preferred embodiments of the present application but should not be used 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. Low-temperature and drought-resistant straw-degrading bacteria, characterized in that, The low-temperature and drought-resistant straw-degrading bacteria is *Stenotrophomonas maltophilia* (… Stenotrophomonas maltophilia GF-Y18, with accession number CGMCC NO.35526.

2. A low-temperature and drought-resistant straw-degrading microbial agent, characterized in that, It includes the low-temperature and drought-resistant straw-degrading bacteria as described in claim 1 and / or their fermentation broth.

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

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

Images