Preparation method of trichoderma asperellum straw fermentation extracting solution and application of trichoderma asperellum straw fermentation extracting solution in oxytetracycline pollution remediation or crop growth promotion

Abstract

The invention relates to the field of environmental pollution treatment and microorganisms, and discloses a preparation method of a trichoderma asperellum straw fermentation extracting solution and application of the trichoderma asperellum straw fermentation extracting solution in oxytetracycline pollution remediation or crop growth promotion. The method comprises the following steps: 1) performing solid-state fermentation on a trichoderma asperellum T1 seed solution to obtain a fermentation product; and 2) drying the fermentation product, adding the dried fermentation product into the leaching solution for leaching, and filtering to obtain the trichoderma asperellum straw fermentation extracting solution rich in trichoderma asperellum spores, enzyme and reducing sugar. The invention finds that the trichoderma asperellum T-1 has new application of repairing oxytetracycline pollution and promoting crop growth, can efficiently degrade oxytetracycline at normal temperature especially in the aspect of repairing oxytetracycline pollution, and has practical application value. Furthermore, compared with a non-extracted trichoderma asperellum leavening and pure trichoderma spores, the trichoderma asperellum straw fermentation extracting solution obtained by a specific process has a better oxytetracycline pollution repairing effect and a better crop growth promoting effect respectively in comparison with the non-extracted trichoderma asperellum leavening and the pure trichoderma spores.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of environmental pollution control and microorganisms, and particularly relates to a preparation method of a Trichoderma asperellum straw fermentation extract and application of the Trichoderma asperellum straw fermentation extract in terramycin pollution remediation or crop growth promotion. BACKGROUND

[0002] As a typical broad-spectrum antibiotic, terramycin is widely used in aquaculture, livestock and poultry breeding and other fields due to its low production cost, high efficiency on specific pathogens, and convenient use. However, a large amount of unabsorbed terramycin is discharged into the soil environment through feces and sewage. The residue of terramycin in the soil can change the structure of soil microbial community, enzyme activity and other physicochemical properties, leading to the decline of soil quality, and even indirectly affecting human health.

[0003] In order to reduce the ecological safety risk, it is necessary to effectively remove terramycin from the soil. Microbial removal of antibiotics has the advantages of high efficiency and specificity, no secondary pollution, low cost and easy scaling, and is one of the core technologies for remediation of terramycin pollution. However, the degradation bacteria of terramycin often carry one or more antibiotic resistance genes, so that the input of degradation bacteria will increase the risk of environmental antibiotic resistance gene pollution to some extent. Therefore, it is very important to study how to repair terramycin pollution while controlling the input of exogenous bacterial antibiotic resistance genes for soil safety, reducing the health threat of animals and plants, and improving soil quality.

[0004] In recent years, the research on the removal of various types of antibiotics by different fungi has been a hot topic in this field. Suboh et al. found that a white rot fungus can effectively degrade terramycin in wastewater. Yu Wenli found that a marine Trichoderma has obvious removal effect on sulfamethoxazole. The research results of Xia et al. showed that the use of recombinant Trichoderma reesei for solid state fermentation under the condition of straw substrate can efficiently remove terramycin under laboratory conditions, but the temperature condition needs to be stabilized at 50℃, which is a great challenge for practical application. Overall, the current research on the remediation and removal of terramycin and other antibiotics by microorganisms is more focused on laboratory conditions or wastewater, and there is still a lack of research on the remediation and removal of antibiotics in soil with more complex conditions and components.

[0005] Trichoderma asperellum is a kind of biocontrol fungus and can be used for plant disease resistance. Whether Trichoderma asperellum can be used to develop new technology and products with soil terramycin pollution remediation and plant growth promotion is a topic worth exploring, but there is little relevant report at present. SUMMARY

[0006] The application provides a preparation method of a Trichoderma asperellum straw fermentation extract and application of the Trichoderma asperellum straw fermentation extract in olymycin pollution remediation or crop growth promotion.

[0007] The specific technical scheme of the application is as follows: Firstly, the application provides a preparation method of a Trichoderma asperellum straw fermentation extract, which comprises the following steps: 1) solid-state shallow fermentation: the solid-state fermentation substrate containing straw and bran is laid on a fermentation tank, Trichoderma asperellum seed liquid is inoculated, shallow fermentation is carried out, and fermentation product is obtained after the fermentation is completed.

[0008] The Trichoderma asperellum is Trichoderma asperellum T-1, which is classified and named as Trichoderma asperellum sp. Trichoderma asperellum The Trichoderma asperellum T-1 has been preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC 9722, the preservation date is September 24, 2014, and the preservation address is No. 1, Beichen West Road, Yard 3, Beijing Academy of Microbiology, Chinese Academy of Sciences.

[0009] 2) fermentation extraction: the fermentation product is dried, then a pH=4.5-5 extraction liquid is added for extraction; the obtained extract is filtered after the extraction, and the Trichoderma asperellum straw fermentation extract rich in Trichoderma asperellum spores, enzymes and reducing sugars is obtained, wherein the concentration of the Trichoderma asperellum spores is greater than or equal to 1×10 7

[0010] The Trichoderma asperellum T-1 is a new strain isolated and screened by the application team in the early stage. In the prior patent CN104450530A of the applicant, it is found that the strain has the effects of plant disease resistance and improvement of garbage compost quality. In the subsequent research, it is further found that the strain has the new uses of remediation of olymycin pollution and crop growth promotion, especially in the remediation of olymycin pollution, which can efficiently degrade olymycin at normal temperature (Trichoderma reesei needs at least 50 DEG C), and has more practical application value (it is difficult to increase the temperature of the environment (such as water body and soil) to 50 DEG C in practical application).

[0011] ​Further, the present application uses straw as the main raw material, and uses Trichoderma asperellum T-1 to perform solid-state fermentation. It is found that, after fermentation and extraction by a specific process, the obtained Trichoderma asperellum straw fermentation extract has better oxytetracycline pollution repair effect and crop growth promotion effect than the Trichoderma asperellum fermentation product without extraction and pure Trichoderma spores, and can be used in sprinkling irrigation, drip pipe and other systems.

[0012] Preferably, in step 1), the raw material of the solid-state fermentation substrate includes a mixture of straw bran and salt solution with a solid-liquid mass ratio of 1: (0.8-1.2).

[0013] Preferably, in step 1), the mass ratio of the bran of the straw is (2-4):1.

[0014] Preferably, in step 1), the salt solution contains: NaNO3 0.15-0.25wt%, CaCl2 0.01-0.02wt%, MgSO4·7H2O 0.02-0.04wt%, FeSO4·7H2O 0.0003-0.0007wt%, ZnSO4·H2O 0.0001-0.0002wt%, MnSO4·H2O 0.0001-0.0002wt%, CoCl2 0.00003-0.00007wt%, KH2PO4 0.1-0.2wt%.

[0015] Preferably, the optimal concentration range of the Trichoderma asperellum seed liquid is 10 6 ~10 7 g / mL, the inoculation amount is 8-12% (mass volume ratio, unit g / mL), the fermentation temperature is 23-32℃, and the Trichoderma spore production effect is best under the above conditions. The fermentation time is 3-5 days, and the fermentation period is relatively short.

[0016] Preferably, in step 2), the leaching solution includes: citric acid 4-6g / L, sodium citrate 6-7g / L.

[0017] The citric acid buffer solution can provide an environment suitable for cellulase hydrolysis, and effectively increase the content of soluble reducing sugar in the extract.

[0018] Preferably, in step 2), the optimal ratio (solid-liquid mass ratio) of the fermentation product to the leaching solution is 1:10 to 1:20. This ratio aims to balance the contradiction between the liquid yield and the spore concentration: too high solid-liquid ratio will dilute the spore concentration in the extract; on the contrary, too low solid-liquid ratio will increase the difficulty of solid-liquid separation and lead to a decrease in liquid yield.

[0019] Preferably, in step 2), the extraction temperature should be maintained at 25-35℃, and the optimal time is 20-30 hours. When setting the time, it is important to note that: too short an extraction time will result in insufficient extraction and a decrease in spore concentration; while too long a time will not only be time-consuming but will also increase the contamination rate of the extract due to increased exposure time to the culture medium.

[0020] Secondly, this invention provides the application of the Trichoderma echinococcosis straw fermentation extract obtained by the above preparation method in the remediation of oxytetracycline pollution: the Trichoderma echinococcosis straw fermentation extract is applied to an environment contaminated with oxytetracycline to degrade oxytetracycline at room temperature.

[0021] In previous reports, crude enzyme solutions obtained by fermentation extraction of Trichoderma reesei in existing technologies only showed good oxytetracycline removal capabilities at 50°C. However, the Trichoderma echinosporum straw fermentation extract developed in this invention can efficiently promote the removal and degradation of oxytetracycline at room temperature, and can be applied to various environments contaminated with oxytetracycline, thus possessing greater practical application value.

[0022] Preferably, the oxytetracycline pollution is water or soil pollution.

[0023] Current research on the remediation and removal of oxytetracycline and other antibiotics by microorganisms focuses more on laboratory conditions or wastewater. Research on the remediation and removal of antibiotic pollution in soil with more complex conditions and compositions is still insufficient. The Trichoderma echinosporum straw fermentation extract of this invention has been verified to be highly effective in remediating oxytetracycline-contaminated soil.

[0024] Finally, this invention provides the application of the Trichoderma hygroscopicis straw fermentation extract obtained by the above preparation method in crop growth promotion.

[0025] The fermented extract of Trichoderma hygroscopicum obtained by this invention contains abundant soluble fermentation products, including enzymes, reducing sugars and other substances. According to comparative experiments, its growth-promoting effect on plants is better than that of pure Trichoderma hygroscopicum spores.

[0026] Preferably, the crop is sorghum.

[0027] Through experiments, this invention has found that, compared with pure Trichoderma spore liquid treatment, Trichoderma echinosporum T-1 straw fermentation extract has a more significant effect on promoting the growth of sorghum, and has a better effect in four different sorghum varieties.

[0028] Compared with the prior art, the beneficial effects of the present invention are: (1) The present invention found that Trichoderma echinosporum T-1 has the effect of remediating oxytetracycline pollution. It can efficiently degrade oxytetracycline at room temperature and has practical application value (it is difficult to raise the ambient temperature in practical applications). In addition, this strain is a fungus and does not carry antibiotic resistance genes of bacteria. Therefore, it will not introduce antibiotic resistance genes into the soil while remediating oxytetracycline pollution.

[0029] (2) This invention uses straw as the main raw material and uses Trichoderma echinosporum T-1 for solid-state fermentation. The results show that after fermentation and extraction through a specific process, the obtained Trichoderma echinosporum straw fermentation extract is more effective in remediating oxytetracycline pollution than the unextracted Trichoderma echinosporum fermentation product.

[0030] (3) The fermented extract of Trichoderma hygroscopica straw obtained by this invention contains abundant soluble fermentation products, including enzymes, reducing sugars and other substances. According to the experimental comparison, the growth-promoting effect on plants is better than that of pure Trichoderma hygroscopica spores.

[0031] (4) The Trichoderma hygroscopicum T-1 fermentation substrate used in this invention has a wide range of applications, including various crop straws, and can effectively realize the resource utilization of agricultural waste. Attached Figure Description

[0032] Figure 1 The graph shows the degradation results of tetracycline by three different Trichodermas (JB: Trichoderma echinosporum; LS: Trichoderma reesei; HZ: Trichoderma harzianum). Figure 2 The degradation results of oxytetracycline by three different Trichodermas are shown in the figure (JB: Trichoderma echinosporum; LS: Trichoderma reesei; HZ: Trichoderma harzianum). Figure 3 The figure shows the removal results of oxytetracycline in water by different Trichoderma acicularis extracts; Figure 4 Figure 1 shows the removal results of oxytetracycline from soil by Trichoderma straw fermentation under different treatments; where: granulation: solid fermentation (unextracted); W: unfermented straw substrate (extracted); CK: control, sterile water. Figure 5 The results of the removal of oxytetracycline from water by components of Trichoderma echinosporum fermentation extract; Figure 6 The results of the removal of oxytetracycline from soil by the components of Trichoderma acicularis fermentation extract; Figure 7 Photographs of Trichoderma hydathoides T-1 fermenting on different agricultural and forestry waste substrates; Figure 8 The images show the growth-promoting effects of Trichoderma echinosporum fermentation and pure Trichoderma spores on sorghum. Among them: straw: extract of Trichoderma echinosporum T-1 straw fermentation; Trichoderma: pure Trichoderma echinosporum T-1 spore liquid; substrate soil: sterile water control. Detailed Implementation

[0033] The present invention will be further described below with reference to embodiments.

[0034] Example 1 (1) Test materials Oxytetracycline was purchased from Sigma-Aldrich, with a purity >99%. Potatoes were sourced from the local market in Hangzhou. Straw and bran were from Jiangxi Kenuo Biotechnology Co., Ltd. The soil was red-yellow soil, sourced from Pujiang County, Jinhua City, Zhejiang Province. All other chemical reagents were from Sinopharm Chemical Reagent Co., Ltd.

[0035] PDA culture medium components: 200g potato, 20g glucose, 1L water.

[0036] Solid-state PDA tablet: 200g potato, 20g glucose, 20g agar, 1L water.

[0037] Solid-state fermentation medium composition: Straw:wheat bran mass ratio = 3:1; Salt solution: NaNO3 0.2%, CaCl2 0.015%, MgSO4·7H2O 0.03%, FeSO4·7H2O 0.0005%, ZnSO4·H2O 0.00016%, MnSO4·H2O 0.00014%, CoCl2 0.00005%, KH2PO4 0.15%. Solid-liquid ratio 1:1.

[0038] Extraction solution composition (citric acid buffer): 5g citric acid monohydrate, 6.76g sodium citrate, 1L water.

[0039] (2) Material preparation method (2.1) Seed liquid preparation Laboratory-preserved Trichoderma echinococcus T-1 (accession number CGMCC 9722), Trichoderma reesei, and Trichoderma harzianum spore plates were eluted with sterile water and transferred to fresh solid PDA plates at an inoculation volume of 100 μl. The plates were then incubated at 28°C for 3 days. After the spores had completely covered the plates, they were eluted with sterile water to maintain a spore concentration of 1 × 10⁻⁶. 7 Prepare seed culture at a concentration of 100 cells / mL.

[0040] (2.2) Preparation method of straw fermentation extract Solid-state fermentation was performed using Trichoderma hygroscopic seed culture, with an initial inoculum concentration of 1×10⁻⁶. 7 A 10% inoculum of *Trichoderma* cells / mL was added to a solid-state fermentation medium and fermented for 3 days. After fermentation, the culture was air-dried. Four treatment methods for the fermented product were designed, including: 20h-extract (the extract was added at a solid-liquid mass ratio of 1:10 and extracted for 20 hours to obtain the fermentation extract), 2h-extract (the extract was added at a solid-liquid ratio of 1:10 and extracted for 2 hours to obtain the fermentation extract), water-extract (sterile water was added at a solid-liquid ratio of 1:10 and extracted for 20 hours to obtain the fermentation-sterile water extract), and *Trichoderma* solid-state fermentation product (solid powder fermentation product without extraction treatment). The product was filtered using four layers of gauze.

[0041] (3) Repair and removal methods (3.1) Removal test of tetracycline and oxytetracycline by three Trichoderma species This experiment used liquid PDA medium as the system, adding tetracycline and oxytetracycline to a final concentration of 1 mg / L, respectively, and inoculating with 0.1% of a solution containing 1×10⁻⁶ mg / L. 7 The seed solution was prepared at 0.1% per mL to remove tetracycline and oxytetracycline during spore germination on PDA medium. Four treatments were designed: CK (treatment with 0.1% sterile water), JB (treatment with 0.1% *Trichoderma echinococcus* seed solution), LS (treatment with 0.1% *Trichoderma reesei* seed solution), and HZ (treatment with 0.1% *Trichoderma harzianum* seed solution), with three replicates for each treatment.

[0042] The samples were cultured in a constant temperature shaker at 28℃ and 180rpm in the dark, and samples were taken at 0 and 3 days.

[0043] (3.2) Removal test of oxytetracycline in water This experiment used the fermentation extract of *Trichoderma echinococcus* straw as the experimental subject to investigate its removal of oxytetracycline from water. The final concentration of oxytetracycline in the water was 1 mg / L. Three treatments were designed: CK (with 10% sterile water), T (with 10% *Trichoderma echinococcus* straw fermentation extract), and W (with 10% unfermented solid culture medium extract), with three replicates for each treatment.

[0044] The samples were cultured in a constant temperature shaker at 28℃ and 180rpm in the dark, and samples were taken at 0 and 1 day.

[0045] (3.3) Soil remediation removal test This experiment used red and yellow soil from Pujiang County as the soil material. Oxytetracycline was added and mixed evenly to simulate contaminated soil. The initial contamination concentration was 10 mg / kg, and the soil moisture content was controlled at about 60% of the maximum moisture content. The experiment was designed with 6 treatments, namely 4 different fermentation products, and sterile water control CK and W (unfermented solid culture medium extract), with a dosage of 10% and 3 replicates for each treatment.

[0046] The cells were incubated in a 28°C incubator in the dark. Water was added with sterile water and aeration was performed every 3 days using the weight difference method. Samples were taken on days 0 and 3 of incubation.

[0047] (3.4) Removal of oxytetracycline from extract supernatant and precipitate To investigate the removal effects of soluble substances and insoluble substances such as spores and mycelium in the extract on oxytetracycline, three treatments were designed: U (10% extract supernatant), D (10% extract precipitate), and CK (10% sterile water). Removal tests were conducted in soil and water, respectively, under the same culture conditions.

[0048] (4) Testing and Analysis Methods After each sampling, soil samples were pre-extracted for oxytetracycline using solid-phase extraction; both liquid and soil samples were analyzed for antibiotic residues in the soil using high-performance liquid chromatography (HPLC). The removal rate was calculated using the following formula: Y = (C0 - C t ) / ( C0) ×100%; where C0 is the antibiotic content detected in the sample at day 0; C t To detect antibiotic content in samples taken at regular intervals.

[0049] (5) Results and Analysis (5.1) Removal of tetracycline and oxytetracycline by three types of Trichoderma Figure 1 The figure shows the degradation of tetracycline by three different Trichoderma species; the different lowercase letters in the figure represent the different treatments. P The difference was statistically significant at the <0.05 level. Figure 1 It can be seen that in the treatment group with added Trichoderma echinosporum seed liquid, the degradation rate of tetracycline was significantly higher. P <0.05) was higher than the control and the treatment group with added Trichoderma harzianum seed solution; Trichoderma echinosporum and Trichoderma reesei did not significantly degrade tetracycline ( P <0.05) difference; the degradation of tetracycline by Trichoderma harzianum and Trichoderma reesei was not significantly different from the control ( P <0.05) difference. *Trichoderma echinococcus* showed the highest degradation rate of tetracycline at 10.01%, followed by *Trichoderma reesei* with a degradation rate of 7.96%. In summary, the results indicate that *Trichoderma echinococcus* can significantly ( P <0.05) promotes the degradation of tetracycline, and the degradation effect is significant. P <0.05) is superior to Trichoderma harzianum.

[0050] Figure 2 The degradation of oxytetracycline by three different Trichoderma species is shown in the figure; different lowercase letters in the figure represent the degradation of oxytetracycline by different treatments. P The difference was statistically significant at the <0.05 level. Figure 2 It can be seen that in the treatment group with added Trichoderma echinococcosis seed liquid, the degradation rate of oxytetracycline was significantly higher. P <0.05) was higher than that of *Trichoderma reesei*, *Trichoderma harzianum*, and the control. The degradation of oxytetracycline by *Trichoderma reesei* and *Trichoderma harzianum* was not significantly different compared to the control. P <0.05) difference. The degradation rate of oxytetracycline by *Trichoderma echinococcus* was 61.96%. In summary, *Trichoderma echinococcus* can significantly ( P <0.05) promotes the removal of oxytetracycline, and the removal effect is significant. P <0.05) is superior to Trichoderma reesei and Trichoderma harzianum.

[0051] (5.2) Biochemical properties of fermentation products under different treatments Table 1 shows the biochemical properties of the fermentation products under different treatments, where different lowercase letters indicate differences between different treatment groups. P The difference was statistically significant at the <0.05 level. As shown in Table 1, under the same extraction time, the protein content obtained by extracting the fermentation broth using the extract solution was significantly higher (…). P <0.05) was higher than that obtained by using water extraction of fermentation products and other treatments; when using extraction solution, the protein content, spore count, and viable cell count obtained after 20 hours of extraction were all significantly higher ( P <0.05) was higher than that of the treatment with extraction for only 2 hours; when the extract was used for 2 hours, the viable count was significantly ( P <0.05) lower than other treatment groups; when the fermentation product was directly pulverized without extraction, the spore count was significantly lower ( P <0.05) lower than other treatment groups.

[0052] Table 1: Biochemical properties of fermentation products under different treatments (5.3) Removal of oxytetracycline from water by fermentation extract Figure 3 The figure shows the removal of oxytetracycline from water by Trichoderma echinococcosis fermentation extract. Different lowercase letters in the figure represent the differences between different treatment groups. P The difference was statistically significant at the <0.05 level. Figure 3 It can be seen that the fermentation extract of Trichoderma echinosporum significantly reduced the removal rate of oxytetracycline. P <0.05) was higher than that of the control and unfermented straw extract groups, and the removal rate of oxytetracycline by unfermented straw extract was significantly higher ( P <0.05) higher than the control. Among them, the removal rate of oxytetracycline by the *Trichoderma echinococcus* fermentation extract reached 79.82%, while the removal rate of oxytetracycline by the unfermented solid culture medium extract was only 14.64%, and the control had the lowest removal rate of 5.99%. In summary, the *Trichoderma echinococcus* fermentation extract can significantly ( P <0.05) Promotes the removal of oxytetracycline from water bodies.

[0053] (5.4) Application of Trichoderma straw fermentation products in the remediation of soil oxytetracycline pollution Figure 4 The figure illustrates the removal of oxytetracycline from soil by Trichoderma straw fermentation under different treatments. Different lowercase letters in the figure represent differences between different treatment groups. P The difference was statistically significant at the <0.05 level. Figure 3 It can be seen that when the straw fermentation product is not subjected to leaching treatment, its removal rate of oxytetracycline in the soil is significantly higher. P<0.05) lower than that of unfermented straw extract; after 20 h of extraction treatment of Trichoderma straw fermentation product, the removal effect of oxytetracycline in soil was significant ( P <0.05) was superior to the control and unfermented straw extract; at the same time, the extraction of fermented material for 20 h significantly reduced the removal of oxytetracycline in the soil. P <0.05) is superior to the treatment of extraction for only 2 hours; the removal rate of oxytetracycline by extraction of fermentation broth for 20 hours can reach 60.96%, while the removal rate of oxytetracycline by sterile water extraction of fermentation broth for 20 hours is 57.81%. In summary, unextracted Trichoderma fuciformis straw fermentation broth cannot promote the removal of oxytetracycline in soil, while the fermentation extract can significantly ( P <0.05) Promotes the removal of oxytetracycline from the soil.

[0054] (5.5) Removal of oxytetracycline from water and soil by extract components Figure 5 The figure shows the effect of Trichoderma echinosporum fermentation extract on the removal of oxytetracycline from water. Different lowercase letters in the figure represent the differences between different treatment groups. P The difference was statistically significant at the <0.05 level. Figure 5 It can be seen that for the removal of oxytetracycline from water, precipitation of the extract plays a major role, and its removal rate is significant. P <0.05) was higher than that of the control and the extract supernatant, with a removal rate of 67.60%; while the removal rate of oxytetracycline in the extract supernatant was significantly higher ( P <0.05) was higher than the control, with a removal rate of 10.53%. In summary, both the supernatant and precipitate in the extract can be significantly ( P <0.05) promotes the removal of oxytetracycline from water, but the precipitation of the extract plays a major role in the removal of oxytetracycline.

[0055] Figure 6 The figure shows the effect of Trichoderma echinosporum fermentation extract on the removal of oxytetracycline from soil; different lowercase letters in the figure represent the differences between different treatment groups. P The difference was statistically significant at the <0.05 level. Figure 6 It can be seen that for the removal of oxytetracycline from soil, precipitation of the extract plays a major role, and its removal rate of oxytetracycline is significantly higher than that of (…). P <0.05) The supernatant of the extract and the control showed a removal rate of 55.10%; the supernatant of the extract showed a significant removal rate of oxytetracycline ( P The concentration of oxytetracycline in the extract was <0.05%, lower than the control, with a removal rate of only 29.08%. This indicates that under more complex environmental conditions, the supernatant of the extract alone is unlikely to significantly promote the removal of oxytetracycline, while the precipitated substances in the extract can still promote its removal. In conclusion, the insoluble substances in the extract have a significant effect on the removal of oxytetracycline in both water and soil environments. PThe oxytetracycline showed a promoting effect of <0.05%, and its removal in both water and soil was significant. P <0.05) is higher than the soluble substances in the extract.

[0056] (5.6) Fermentation substrate profile of Trichoderma hygroscopicum T-1 Spores were washed from PDA plates cultured for 4 days with 0.1% Tween 80 solution. The spore solution was then evenly sprayed onto solid fermentation media of different types of agricultural and forestry waste, covered with gauze, and kept at 28°C. The substrate was observed daily to ensure it remained moist but not watery when squeezed. After 7 days of culture, observations showed that... Figure 7 As shown, Trichoderma echinosporum T-1 has a broad fermentation substrate spectrum, and can grow on a variety of agricultural and forestry wastes, including wheat straw, rice straw, bamboo powder, sugarcane bagasse, and cotton straw, indicating that it has a wide range of agricultural and forestry waste degradation capabilities.

[0057] (5.7) Growth-promoting effect of Trichoderma hygroscopicum T-1 straw fermentation broth The extract of Trichoderma harzianum T-1 straw fermentation product (straw; concentration 1×10⁻⁶) was validated on four different sorghum varieties (108, HYZ, wh, BTX). 7 1 spore / ml, diluted 10 times and then poured into the substrate soil), pure Trichoderma echinosporum T-1 spores (Trichoderma: cultured on PDA medium, spores obtained by washing with sterile water, concentration 1×10⁻⁶). 7 (Spores / ml, diluted 10 times, then poured into the substrate soil). Results are as follows: Figure 8 As shown in the figure, compared with the pure Trichoderma spore liquid treatment and the control treatment, the extract of Trichoderma echinosporum T-1 straw fermentation product had a significant growth-promoting effect on sorghum, and was effective in all four different sorghum varieties.

Claims

1. A method for preparing a Trichoderma asperellum straw fermentation extract, characterized by: The method comprises the following steps: 1) solid state shallow fermentation: Trichoderma asperellum seed liquid is inoculated into a solid state fermentation substrate containing straw and bran to perform shallow fermentation, and a fermentation product is obtained after the fermentation is completed; the Trichoderma asperellum is Trichoderma asperellum T-1, and the preservation number is CGMCC9722; 2) Fermentation extraction: after drying the fermentation product, add the leaching liquid with pH=4.5-5 to carry out leaching, and after leaching, filter the obtained leaching product to obtain the straw fermentation extraction liquid of Trichoderma asperellum rich in Trichoderma asperellum spores, enzymes and reducing sugar, wherein the concentration of Trichoderma asperellum spores is ≥1×10 7 / ml.

2. The production method according to claim 1, characterized by: In step 1), raw materials of the solid state fermentation substrate include a straw-bran mixture and a salt solution, and the solid-liquid mass ratio is 1: (0.8-1.2).

3. The production method according to claim 2, characterized by: In step 1), The mass ratio of the straw to the bran is (2-4):

1. The salt solution contains NaNO3 0.15-0.25 wt%, CaCl2 0.01-0.02 wt%, MgSO4·7H2O 0.02-0.04 wt%, FeSO4·7H2O 0.0003-0.0007 wt%, ZnSO4·H2O 0.0001-0.0002 wt%, MnSO4·H2O 0.0001-0.0002 wt%, CoCl2 0.00003-0.00007 wt%, and KH2PO4 0.1-0.2 wt%.

4. The production method according to claim 1, wherein: In step 1), the concentration of the Trichoderma asperellum seed liquid is 10 6 ~10 7 mL, the access amount is 8-12% by volume mass ratio, the fermentation temperature is 23-32℃, and the fermentation time is 3-5 days.

5. The production method according to claim 1, wherein: In step 2), the leaching solution contains citric acid monohydrate 4-6 g / L and sodium citrate 6-7 g / L.

6. The production method according to claim 1 or 5, characterized by: In step 2), the solid-liquid mass ratio of the fermentation product to the leaching solution is 1:10-1:

20.

7. The production method according to claim 1 or 5, characterized by: In step 2), the temperature of the leaching is 25-35 ℃, and the leaching time is 20-30 h.

8. The use of the straw fermentation extract of Trichoderma asperellum obtained by the method according to any one of claims 1 to 7 for the remediating of oxytetracycline pollution, characterized in that: The Trichoderma asperellum straw fermentation extract is applied to an oxytetracycline-polluted environment to degrade the oxytetracycline at room temperature.

9. Use according to claim 8, wherein: The oxytetracycline pollution is water or soil.

10. Application of the Trichoderma asperellum straw fermentation extract obtained by the preparation method of any one of claims 1-7 to crop growth promotion.

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