Method for rapidly screening low-temperature-resistant and drought-resistant straw degrading bacteria

Abstract

The invention discloses a method for rapidly screening low-temperature-resistant and drought-resistant straw degrading bacteria. According to the screening method, metagenome sequencing analysis is carried out on a large number of collected bacterial source samples, core microorganism compositions and potential lignocellulose degradation functions in different bacterial source samples can be rapidly judged, bacterial source samples with lignocellulose degradation potential can be rapidly selected, low-efficiency bacterial source samples are removed, the screening efficiency is improved, and the screening cost is reduced. The low-temperature-resistant and drought-resistant target strain capable of degrading the straw is obtained through the steps of separation, screening and the like, and an effective method is provided for straw degradation in cold and arid regions such as Inner Mongolia under the low-temperature and drought conditions in winter.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of bio-agriculture, in particular to a method for rapidly screening low-temperature and drought-tolerant straw-degrading bacteria. BACKGROUND

[0002] Traditional microbial screening methods are cultivation followed by screening: first, select appropriate culture conditions to isolate target microorganisms from samples, and then select from them, such as plate culture method and liquid culture method. However, this method has certain limitations, and cannot cultivate specific types of microorganisms or meet the requirements of high-throughput screening.

[0003] Corn is an important food and economic crop widely planted around the world, and produces a large amount of straw waste every year, with an annual output of more than 300 million tons in China alone. 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 efficient decomposition of straw returned to the field. Using microorganisms to accelerate straw decomposition and solve the technical bottleneck of straw return to the field, improve soil fertility and improve soil quality, has important significance for the full use of straw resources, farmland fertility cultivation and sustainable development of agriculture.

[0004] 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 decomposition of straw and nutrient release.

[0005] Therefore, screening of low-temperature and drought-tolerant high-efficiency straw-degrading strains is the key to compounding straw decomposition agents and accelerating efficient in-situ decomposition of straw returned to the field in cold and dry areas. SUMMARY

[0006] The purpose of the present application is to provide a method for rapidly screening low-temperature and drought-tolerant straw-degrading bacteria.

[0007] The purpose of the present application is implemented by the following technical solutions.

[0008] A method for rapidly screening low-temperature and drought-tolerant straw-degrading bacteria, comprising the following steps: S1, collecting a bacterial source sample rich in lignocellulose in a cold and dry area; S2, performing macro-genome sequencing on the bacterial source sample, and obtaining the base sequence of the non-redundant gene set after processing the raw data obtained by sequencing; S3, align the base sequences of the non-redundant gene set genes of each of the bacterial source samples with the NR database to obtain the dominant bacterial phylum of each of the bacterial source samples, and take the bacterial source sample in which at least one of the top five dominant bacterial phyla in terms of sample abundance ratio belongs to the straw degradation dominant bacterial phylum as a first degradation potential bacterial source sample; S4, perform functional annotation analysis and carbohydrate enzyme analysis on the base sequences of the non-redundant gene set genes of each of the first degradation potential bacterial source samples using the KEGG database and the CAZy database, sort the lignocellulose degradation gene function abundance ratio and the lignocellulose degradation enzyme family function abundance ratio in each of the bacterial source samples, and take the bacterial source sample with the highest function abundance ratio as a second degradation potential bacterial source sample; S5, prepare a diluent solution from the second degradation potential bacterial source sample, and after culturing in a lignin culture medium and a carboxymethyl cellulose culture medium for 5-10 days, isolate and purify single colonies with a growth diameter of ≥2 mm and store them; S6, perform aniline blue fading ring and congo red fading ring determination on the purified strains in S5 to screen lignocellulose-degrading bacteria that can produce aniline blue fading rings and / or congo red fading rings; S7, perform low-temperature and drought tolerance screening on the lignocellulose-degrading bacteria to obtain degradation bacteria II; S8, inoculate the degradation bacteria II into a straw degradation culture medium, and after incubation at 15°C for 30 days, calculate the relative straw degradation rate; S9, the degradation bacteria II with a relative straw degradation rate of ≥20% are the low-temperature and drought-tolerant straw-degrading bacteria.

[0009] Further, the bacterial source samples include one or more of farmland soil, forest soil, humus, rotten straw, animal manure, and compost.

[0010] Further, in S2, the processing specifically includes: performing data quality control on the raw data to cut off reads with a base quality value <20 and containing N to obtain high-quality sequences; assembling the high-quality sequences of different bacterial source samples to obtain long spliced sequences, performing ORF prediction on the long spliced sequences of different bacterial source samples, and translating genes with a nucleic acid length of ≥100 bp in the ORF prediction results into amino acid sequences; clustering the amino acid sequences obtained after prediction and translation of each bacterial source sample, taking the longest gene in each class as a representative sequence, constructing a non-redundant gene set, and obtaining the base sequences of the non-redundant gene set genes.

[0011] Further, the straw degradation dominant bacterial phylum includes the phyla Firmicutes, Actinobacteria, Verrucomicrobia, Fibrobacteres, Bacteroidetes, and Pseudomonadota.

[0012] Further, the S7 comprises the following steps: S71: inoculate the lignocellulose-degrading bacteria on aniline blue selection medium, gradient temperature culture for 1d, measure the ratio of bacterial colony diameter to fading circle diameter; inoculate the lignocellulose-degrading bacteria capable of producing fading circles at 5℃ in LB liquid medium containing polyethylene glycol 6000 (hereinafter referred to as PEG6000) and LB liquid medium containing NaCl respectively, and culture at 15℃ for 2d, measure the growth curve, and screen bacteria capable of growing in LB liquid medium containing PEG6000 and LB liquid medium containing NaCl after 2d of culture to obtain degrading bacteria I; S72, inoculate the degrading bacteria I screened in S71 in enzyme production medium, measure enzyme activity after gradient temperature culture for 1d; inoculate the degrading bacteria I screened in S71 in enzyme production medium containing PEG6000 and enzyme production medium containing NaCl respectively, and measure enzyme activity after 1d of culture at 15℃; S73, in the degrading bacteria I after 1d of culture in the enzyme production medium of S72, the enzyme activity of the degrading bacteria I cultured in 5℃ enzyme production medium, PEG6000-containing enzyme production medium and NaCl-containing enzyme production medium all meet any one of the following conditions ①-③: condition ① cellulase activity ≥1.0 U / mL, condition ② laccase activity ≥100 U / L, and condition ③ lignin peroxidase activity ≥35 U / L.

[0013] Further, the cellulase comprises endo-1, 4-beta-glucanase activity, exo-1, 4-beta glucanase activity and beta-glucosidase activity; and the condition ① specifically refers to that the endo-1, 4-beta-glucanase activity, the exo-1, 4-beta glucanase activity and the beta-glucosidase activity are all ≥1.0 U / mL.

[0014] Advantages of the present application: The present application provides a method for rapidly screening low-temperature and drought-resistant straw degrading bacteria, which can quickly determine the core microbial composition and potential lignocellulose degradation function in different bacterial source samples, quickly select bacterial source samples with potential lignocellulose degradation, eliminate low-efficiency bacterial source samples, improve the screening efficiency, and obtain target strains, thereby providing an effective method for straw degradation under low-temperature and drought conditions in winter in cold and arid regions such as Inner Mongolia. BRIEF DESCRIPTION OF DRAWINGS

[0015] In order to make the technical solutions in the embodiments of the present application or the prior art clearer, the accompanying drawings needed in the embodiments or prior art description will be briefly introduced. Obviously, the accompanying drawings in the following description only show some embodiments of the present application, and other drawings can be obtained by those skilled in the art without any creative effort.

[0016] Figure 1 Door level species relative abundance of the collected FL farmland soil sample; Figure 2 Door level species relative abundance of the collected FR forest soil and humus sample; Figure 3 Door level species relative abundance of the collected CS rotten straw sample; Figure 4 Door level species relative abundance of the collected CP compost and animal manure sample; Figure 5 KEGG database annotation result statistics of the metagenome of the four types of samples; wherein, (a) is a KEGG annotation result histogram, and (b) is a community barcode analysis diagram; Figure 6 KEGG database annotation abundance of the metagenome of the four types of samples; wherein, (a) is cellulose, (b) is hemicellulose, and (c) is lignin; Figure 7 CAZy database annotation result statistics of the metagenome of the four types of samples; wherein, (a) is the FL farmland soil sample, (b) is the FR forest soil and humus sample, (c) is the CS rotten straw sample, and (d) is the CP compost and animal manure sample; Figure 8 CAZy database lignocellulose-degrading enzyme family annotation abundance of the metagenome of the four types of samples; Figure 9 Growth curve of the low-temperature and drought-resistant straw-degrading bacteria (Bacillus subtilis) GF-Y18; (a) is under different PEG6000 concentrations, and (b) is under different NaCl concentrations; Stenotrophomonas maltophilia 30-day straw relative degradation rate of the low-temperature and drought-resistant straw-degrading bacteria (Bacillus subtilis) GF-Y18; (a) is under different temperatures, (b) is under different PEG6000 concentrations, and (c) is under different NaCl concentrations; Figure 10 Stenotrophomonas maltophilia Colony morphology of the low-temperature and drought-resistant straw-degrading bacteria (Bacillus subtilis) GF-Y18; Figure 11 Spore morphology of the low-temperature and drought-resistant straw-degrading bacteria (Bacillus subtilis) GF-Y18; Stenotrophomonas maltophilia Figure 12 Stenotrophomonas maltophilia ​​​Phylogenetic tree of GF-Y18. DETAILED DESCRIPTION

[0017] 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 a person of ordinary skill in the art without creative labor fall within the protection scope of the present application.

[0018] The formula of the culture medium used in the embodiments is as follows: Lignin culture medium: (NH4)2SO42 g / L, K2HPO41 g / L, KH2PO41 g / L, MgSO40.2 g / L, MnSO40.02 g / L, protein peptone 10 g / L, lignin 1 g / L, agar 18 g / L; Carboxymethyl cellulose sodium culture medium (CMC culture 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 culture 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 culture medium; Enzyme production culture 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 culture medium: (NH4)2SO42 g / L, K2HPO42 g / L, MgSO40.05 g / L, CaCO32 g / L, NaCl 0.2 g / L, 20 mL above culture medium and 1 g corn straw are added to a 20 mL triangular flask.

[0019] The above culture media are sterilized at 121℃, 0.1 MPa for 30 min before use.

[0020] Embodiment 1 A method for rapidly screening a low-temperature and drought-resistant straw degradation bacterium, comprising the following steps: S1, selection and collection of a bacterial source sample Collecting the 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) such as farmland, forest soil, straw, compost and animal manure.

[0021] When collecting: gently brush off the surface sediment, and drill 0-40 cm farmland soil layer samples (denoted as FL) using a sterile soil drill, or collect forest soil or humus (denoted as FR), rotten straw (denoted as CS), compost or cow and sheep manure (denoted as CP) using a sterile sampler, and then put them into a sterile bag and carry back to the laboratory, and store in a -86℃ freezer. In this embodiment, 19 farmland soil layer 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.

[0022] S2, macrogenomic sequencing analysis The collected samples are sent to Shanghai Meiji Biomedicine Technology Co., Ltd. for macrogenomic sequencing. The raw data obtained by sequencing are subjected to data quality control using Fastp software, and low-quality (base quality value <20) and N-containing reads in the data are cut off to obtain high-quality sequences required for subsequent analysis.

[0023] MEGAHIT software is used for assembling sequences of different sequencing, and Prodigal v2.6.3 software is used for ORF prediction of the assembly results. The genes with nucleic acid length greater than or equal to 100 bp are translated into amino acid sequences. The amino acid sequences predicted and translated from all samples are clustered using CD-HIT software, the longest gene in each class is taken as a representative sequence, a non-redundant gene set is constructed, and the base sequences of the non-redundant gene set genes are obtained.

[0024] S3, screening of first degradation potential microbial source sample The base sequences of the non-redundant gene set genes of different microbial source samples are subjected to species composition comparison using the NR database, the dominant phylum of each microbial source sample is obtained, and the KEGG database and CAZy database are used for functional annotation analysis and carbohydrate enzyme analysis of different microbial source samples. According to the species composition and function, lignocellulose-degrading single strains are directionally screened.

[0025] The method provided by the present application can quickly determine the core microbial composition and potential lignocellulose-degrading function in different microbial source samples, quickly select microbial source samples with lignocellulose-degrading potential, eliminate low-efficiency microbial source samples, and improve the screening efficiency.

[0026] 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.

[0027] 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

[0028] The relative abundance of CS sample species is shown in Figure 3 At the door level, the dominant phylum of CS sample is Pseudomonadota, Actinomycetota and Bacteroidota.

[0029] 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 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.

[0030] In the above four types of bacterial source samples, the dominant phylum of straw degradation is 3 in the top 10 of FL sample abundance, 4 in the top 10 of FR sample abundance, 3 in the CS sample, and 4 in the CP sample, 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.

[0031] S4, second degradation potential bacterial source sample screening

[0032] 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.

[0033] 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.

[0034] 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).

[0035] 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.

[0036] Based on 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 microbial source sample.

[0037] S5, isolation and purification of strains The CP samples collected above, i.e., compost and animal manure samples, are used as microbial source samples for strain isolation and screening.

[0038] 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 a concentration gradient of 10 -1 ~10 -8 The diluted solution is taken and spread on lignin medium and carboxymethyl cellulose medium, respectively, and placed in a constant temperature incubator at 15°C for 5-10 d. The utilization of cellulose and lignin by the strains is evaluated according to the diameter (i.e., growth rate) of the colonies on the carboxymethyl cellulose sodium medium and the lignin medium. Strains with a single colony diameter of ≥2 mm are selected and subjected to continuous streaking on carboxymethyl cellulose sodium medium and lignin medium for isolation and purification. The obtained 71 strains are stored in a refrigerator at 15°C.

[0039] S6, screening of lignocellulose-degrading bacteria Aniline blue fading circle determination is performed: the selected single bacteria are inoculated on aniline blue screening medium and cultured in a constant temperature incubator at 15°C for 1 d. The colony diameter (d) and the fading circle diameter (D) are measured.

[0040] Congo red fading circle 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 d. 1% Congo red staining solution is spread on the plates, and after staining for 1 h, the plates are washed with 1 mol / L NaCl solution for 30 min. The colony diameter (d) and the fading circle diameter (D) are measured.

[0041] The 71 lignocellulose-degrading bacteria (denoted as GF-Y1-GF-Y71) screened from the 7 CP samples are subjected to aniline blue fading circle and Congo red fading circle determination according to the above method. The results are shown in Table 1: there are a total of 48 strains that can produce fading circles on aniline blue screening medium, 3 strains that can produce Congo red fading circles, and a total of 2 strains that can fade on both aniline blue screening medium and Congo red staining medium. Among them, there is 1 strain (GF-Y18) with a ratio (D / d) of the transparent circle (D) to the colony diameter (d) on the aniline blue screening medium of ≥5.00, and the ratio is 5.40.

[0042] Table 1 Discoloration results of different strain selection medium

[0043] Note: - means no discoloration, + means discoloration.

[0044] S7, screening of low-temperature and drought-resistant straw-degrading bacteria The strains producing discoloration rings on the aniline blue screening medium and / or the strains producing discoloration rings on the Congo red medium were inoculated on the aniline blue screening medium, respectively, and cultured at 5℃, 10℃, 15℃, 20℃ and 25℃ for 1d, respectively, to measure the diameters (d) of the bacterial rings and the diameters (D) of the discoloration rings, and to determine the low-temperature adaptability.

[0045] The strains producing discoloration rings in the low-temperature environment after the low-temperature adaptability determination (in this embodiment, referring to the strains producing discoloration rings at 5℃) were inoculated in the LB liquid medium and cultured at 15℃ and 160 r / min for 24h to prepare the standard bacterial suspension with OD 600 value of 1.0. The standard bacterial suspension was inoculated in five kinds of LB liquid medium containing different concentrations of PEG6000 (5%, 10%, 15%, 20%, 25%) and five 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 to monitor the growth curve in real time by using a growth curve analyzer. The bacteria that at least one concentration of PEG6000 meets OD 600 ≥1.0 and at least one concentration of NaCl meets OD 600 ≥1.0 after 48h of culture were screened, that is, the degrading bacteria I were obtained.

[0046] The degrading bacteria I were inoculated in the LB liquid medium to prepare the bacterial suspension with a concentration of 1×10 7 CFU / mL.

[0047] The bacterial suspension of each degrading bacteria I (one strain GF-Y18 was screened in this embodiment) was inoculated in the enzyme-producing 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).

[0048] The bacterial suspension of each degrading bacteria I (one strain GF-Y18 screened in this embodiment) was inoculated into five enzyme-producing culture mediums containing different concentrations of PEG6000 (5%, 10%, 15%, 20%, and 25%) and five enzyme-producing 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.

[0049] 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.

[0050] 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.

[0051] S8, degradation effect analysis of the degrading bacteria 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 (one strain GF-Y18 screened in this 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 until the weight was constant, 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.

[0052] 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.

[0053] S9. Low-temperature and drought-resistant straw-degrading bacteria were screened and obtained. 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.

[0054] Table 2. Bleaching zone ratio (D / d) of strains at different temperatures

[0055] 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.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] 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.

[0060] Table 3 Enzyme activities of strain GF-Y18 under different temperatures

[0061] Note: The same column data after different lowercase letters represent significant difference (p<0.05), the same below.

[0062] Table 4 Enzyme activities of strain GF-Y18 under different PEG6000 concentrations

[0063] Table 5 Enzyme activities of strain GF-Y18 under different NaCl concentrations

[0064] After 30 days of culture, the relative degradation rate of the straw of strain GF-Y18 gradually increased with the increase of temperature, as shown in Figure 10 a, the relative degradation rate of the straw of strain GF-Y18 at 15℃ was 27%, indicating that a strain of low-temperature and drought-tolerant straw-degrading bacteria had been screened.

[0065] As shown in Figure 10 a, at 15℃ to 25℃, the relative degradation rate of the straw of strain GF-Y18 could be maintained at more than 20%, and at 5℃, the relative degradation rate of the straw was still 14%, further indicating that strain GF-Y18 had the ability to degrade straw at low temperature. As shown in Figure 10 b, when the content of PEG6000 was 20% or less, the relative degradation rate of the straw of strain GF-Y18 could be maintained at about 20%, and at 25%, the relative degradation rate of the straw was still 16%; as shown in Figure 10 c, the relative degradation rate of the straw of strain GF-Y18 under 0.6 mol / L NaCl was the highest at 19%, and under 0.8 mol / L and below of NaCl, the relative degradation rate of the straw was more than 10%, further indicating that strain GF-Y18 had the ability to degrade straw under drought stress. The above test results showed that strain GF-Y18 could degrade straw under low temperature and drought conditions.

[0066] 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.

[0067] GF-Y18 was identified: (1) Morphological characteristics: GF-Y18 was streak inoculated on LB solid culture medium, and incubated at 15℃ for 24 h, and the shape, size, color, edge, transparency and other morphological characteristics of the colony were observed. As shown in Figure 11 , strain GF-Y18 was white, round, with convex surface and smooth edge.

[0068] (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 Sunred Bioengineering 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 Figure 12 , the strain GF-Y18 was identified as Stenotrophomonas maltophilia. Stenotrophomonas maltophilia .

[0069] A low-temperature and drought-resistant straw degrading bacterium was screened: Stenotrophomonas maltophilia (Stenotrophomonas maltophilia) Stenotrophomonas maltophilia ) GF-Y18, which is currently preserved in the General Microbial Center of the Chinese Microbial Strain Preservation Management Committee, located at No. 1, Beichen West Road, Yard 3, Institute of Microbiology, Chinese Academy of Sciences, Beijing Chaoyang District, and its preservation number is CGMCC NO. 35526, and the preservation date is August 5, 2025.

[0070] Table 6 PCR reaction system

[0071] Table 7 PCR reaction procedure

[0072] Example 3 A low-temperature and drought-resistant straw degrading bacterial agent, which comprises a low-temperature and drought-resistant straw degrading bacterium (i.e. Stenotrophomonas maltophilia (Stenotrophomonas maltophilia) Stenotrophomonas maltophilia ) GF-Y18) and / or a fermentation broth thereof.

[0073] The low-temperature and drought-resistant straw degrading bacterial fermentation broth, i.e. the fermentation broth of Stenotrophomonas maltophilia (Stenotrophomonas maltophilia) Stenotrophomonas maltophilia ) GF-Y18, comprises the following steps: (1) Inoculation: inoculating the low-temperature and drought-resistant straw degrading bacterium, i.e. Stenotrophomonas maltophilia (Stenotrophomonas maltophilia) Stenotrophomonasmaltophilia ) GF-Y18 was inoculated on LB medium and incubated at 15°C for 2 days to obtain activated bacteria; (2) Fermentation: the activated bacteria were inoculated into LB medium and incubated at 15°C in a 180 rpm shaker for 2 days; (3) Filtration: the mycelium in the bacterial solution after 2 days of shaker culture was filtered out to obtain a filtrate, which was a crude metabolite stock solution, i.e. a Streptomyces amyqisoli (S. amyqisoli) GF-Y18 fermentation broth. Stenotrophomonas maltophilia ) GF-Y18 fermentation broth.

[0074] The above merely describes the preferred embodiments of the present application and is not intended to limit the present application. 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 method for rapidly screening low-temperature and drought-resistant straw-degrading bacteria, characterized in that, It includes the following steps: S1. Collect fungal samples rich in lignocellulose from cold and arid regions; S2. Perform metagenomic sequencing on the bacterial sample, and process the raw data obtained from the sequencing to obtain the base sequence of the non-redundant gene set. S3. Using the NR database, the species composition of the non-redundant gene set genes of each of the bacterial source samples is compared to obtain the dominant bacterial phylum of each bacterial source sample. The bacterial source sample with at least one of the top five dominant bacterial phyla in terms of sample abundance belonging to the dominant bacterial phylum of straw degradation is regarded as the first degradation potential bacterial source sample. S4. Using the KEGG and CAZy databases, perform functional annotation analysis and carbohydrate enzyme analysis on the base sequences of the non-redundant gene sets of each of the first potential degradation bacterial source samples. Sort the functional abundance ratio of lignocellulose degradation genes and the functional abundance ratio of lignocellulose degradation enzyme families in each bacterial source sample, and select the bacterial source sample with the highest functional abundance ratio as the second potential degradation bacterial source sample. S5. Prepare a dilution using the second degradation potential bacterial source sample, and culture it on lignin medium and carboxymethyl cellulose medium for 5-10 days. Then, isolate, purify and preserve single colonies with a growth diameter ≥2 mm. S6. The purified strains in S5 are subjected to aniline blue bleaching zone and Congo red bleaching zone determination to screen for lignocellulose degrading bacteria that can produce aniline blue bleaching zone and / or Congo red bleaching zone. S7. The lignocellulose-degrading bacteria are screened for low temperature and drought resistance to obtain degrading bacteria II; S8. The degrading bacteria II were inoculated into the straw degradation medium and cultured statically at 15°C for 30 days. The relative degradation rate of straw was then calculated. S9. The degrading bacteria II with a relative straw degradation rate of ≥20% is the low-temperature and drought-resistant straw degrading bacteria.

2. The method for rapidly screening low-temperature and drought-resistant straw-degrading bacteria according to claim 1, characterized in that, The bacterial source samples include one or more of the following: farmland soil, forest soil, humus, rotten straw, animal manure, and compost.

3. The method for rapidly screening low-temperature and drought-resistant straw-degrading bacteria according to claim 1, characterized in that, In step S2, the processing specifically involves: performing data quality control on the original data, cutting out reads with a base quality value <20 and containing N to obtain high-quality sequences; assembling the high-quality sequences from different bacterial sources to obtain spliced ​​long sequences; performing ORF prediction on the spliced ​​long sequences from different bacterial sources, translating genes with a nucleic acid length ≥100 bp in the ORF prediction results into amino acid sequences; clustering the predicted and translated amino acid sequences from each bacterial source sample, taking the longest gene from each cluster as the representative sequence, constructing a non-redundant gene set, and obtaining the base sequences of the genes in the non-redundant gene set.

4. The method for rapidly screening low-temperature and drought-resistant straw-degrading bacteria according to claim 1, characterized in that, The dominant bacterial phyla for straw degradation include Firmicutes, Actinobacteria, Verrucous Microbes, Fibrobacteria, Bacteroidetes, and Pseudomonas.

5. The method for rapidly screening low-temperature and drought-resistant straw-degrading bacteria according to claim 1, characterized in that, S7 includes the following steps: S71: The lignocellulose-degrading bacteria were inoculated onto aniline blue selection medium and cultured at gradient temperatures for 1 day. The ratio of the bacterial zone diameter to the fading zone diameter was measured. The lignocellulose-degrading bacteria that could produce fading zones at 5℃ were inoculated into LB liquid medium containing PEG6000 and NaCl, respectively, and cultured at 15℃ for 2 days. Bacteria that could grow in both LB liquid medium containing PEG6000 and LB liquid medium containing NaCl after 2 days of culture were screened to obtain degrading bacteria I. S72. The degrading bacteria I obtained from S71 are inoculated into an enzyme-producing medium and cultured at a gradient temperature for 1 day, and then the enzyme activity is measured. The degrading bacteria I obtained from S71 are inoculated into enzyme-producing mediums containing PEG6000 and NaCl, respectively, and cultured at 15°C for 1 day, and then the enzyme activity is measured. S73. Among the degrading bacteria I after 1 day of culture in the enzyme-producing medium of S72, the degrading bacteria I whose enzyme activity meets any one of the following conditions ①-③ under the conditions of 5℃ enzyme-producing medium, enzyme-producing medium containing PEG6000, and enzyme-producing medium containing NaCl is the degrading bacteria II: Condition ① Cellulase activity ≥1.0 U / mL, Condition ② Laccase activity ≥100 U / L, Condition ③ Lignin peroxidase activity ≥35 U / L.

6. A method for rapidly screening low-temperature and drought-resistant straw-degrading bacteria according to claim 5, characterized in that, The cellulase includes endo-1,4-β-glucanase activity, exo-1,4-β-glucanase activity, and β-glucosidase activity; the specific condition ① is: endo-1,4-β-glucanase activity, exo-1,4-β-glucanase activity, and β-glucosidase activity are all ≥1.0 U / mL.

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