Application of Penicillium erlichii in glufosinate degradation and microbial inoculum

Abstract

The present invention belongs to the technical field of microbiology, and particularly relates to the application of Penicillium erlichii in degrading glufosinate and a microbial agent. The preservation number of the Penicillium erlichii is: CCTCC NO: M 2021635. The Penicillium erlichii provided by the present invention has a high tolerance concentration to glufosinate, and has the effect of accelerating growth under the stress of glufosinate. It can be used for biodegradation of glufosinate, and shows rapid and high efficiency in the metabolic degradation of glufosinate at a high concentration of 100 mg / L. After 3 days of cultivation, the degradation rate can reach 64.5%, showing good rapidity. After 7 days of cultivation, the degradation rate can be as high as over 90%, showing high efficiency in degradation. It has the potential to be developed for degrading glufosinate residues in natural environments such as soil and water bodies, as well as in foods such as tea, and has good application prospects in bioremediating environments, foods, and organisms contaminated with glufosinate residues.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and particularly to the application of Penicillium erichsenii in degrading glufosinate and a microbial agent. Background Art

[0002] Organophosphorus pesticides are widely used due to their characteristics such as a large variety, high efficacy, and broad control spectrum. In China, the production and usage amount of organophosphorus pesticides account for more than half of the total production and usage amount of pesticides. Glufosinate is a broad-spectrum contact and non-selective organophosphorus herbicide, which is a glutamine synthetase inhibitor and has characteristics such as high efficiency and low residue. It is widely used in crops such as fruits and vegetables, tea, and rice. Although glufosinate belongs to non-persistent pesticides, after long-term, large-scale, and random use, residues in water, soil, and agricultural products can still be caused, posing a serious threat to the safety of the agricultural ecological environment and the quality safety of agricultural products. Currently, countries around the world have set maximum residue limits for glufosinate in food. The regulatory value of the Codex Alimentarius Commission is 0.02 - 8 mg / kg, those of the United States and Japan are 0.05 - 6 mg / kg, that of Canada is 0.1 - 2 mg / kg, and that of the European Union is 0.1 - 5 mg / kg. The national food safety standard GB2763 - 2016 stipulates that the maximum residue amount of glufosinate in tomatoes, citrus fruits, and tea is 0.5 mg / kg, and the maximum residue amount in bananas and papayas is 0.2 mg / kg.

[0003] Camellia sinensis is a perennial evergreen cash crop. Herbicides residues in tea are the main pesticides residues in tea and also a factor seriously threatening the quality safety of tea. Research shows that among more than 800 tea samples nationwide in 2016, the average content of organophosphorus herbicide residues in the detected tea samples was 0.93 mg / kg, far higher than the national regulatory standards. Herbicides residues in tea mainly come from tea garden soil and irrigation water. Adopting reasonable and efficient technologies to reduce herbicides residues in tea garden soil is a feasible and effective method to improve the quality safety of tea. Therefore, developing efficient technical strategies for reducing glufosinate has become a key technical problem to be solved urgently.

[0004] Using microorganisms to degrade pesticide residues such as herbicides in soil, water bodies, tea, food, and the environment has advantages such as low cost, non-toxicity, and no secondary pollution, and is one of the most promising methods for reducing pesticide pollution. However, there are currently few germplasm resources for glufosinate degradation. Only a small amount of research has found that the bacterium Lactobacillus can be used to degrade glufosinate, and its highest degradation rate of glufosinate within 48 h is 56.21%. After 48 h, the degradation rate of glufosinate is slow, and it cannot degrade glufosinate quickly and efficiently, with limited potential for degrading and repairing glufosinate residues. Therefore, developing new germplasm resources is of great significance for efficiently treating glufosinate residue pollution. Summary of the Invention

[0005] In view of the problem of poor ability of forage grasses to degrade glyphosate in the prior art, the present invention provides the application of Penicillium erichsenii in degrading glufosinate-ammonium, and provides a microbial agent containing Penicillium erichsenii, aiming to solve a part of the problems in the prior art or at least alleviate a part of the problems in the prior art.

[0006] The present invention is specifically realized through the following technical solutions:

[0007] The first aspect of the present invention provides the application of Penicillium erichsenii in degrading glufosinate-ammonium, and the preservation number of the Penicillium erichsenii is: CCTCC NO: M 2021635.

[0008] Further, the concentration of glufosinate-ammonium is 0 - 400 mg / L, and more preferably, the concentration of glufosinate-ammonium is 0 - 100 mg / L.

[0009] Further, the application includes the application in preparing a microbial preparation for degrading glufosinate-ammonium.

[0010] The second aspect of the present invention provides a microbial preparation for degrading glufosinate-ammonium, which includes the above-mentioned Penicillium erichsenii and acceptable excipients.

[0011] Further, the acceptable excipients include one or more of solvents, dispersants, stabilizers, wetting agents, emulsifiers, synergists, thickeners, defoamers, suspending agents, antioxidants, lyophilizing agents, preservatives, pH regulators, and protective agents.

[0012] The third aspect of the present invention provides the application of glufosinate-ammonium in promoting the growth of the mycelia of the above-mentioned Penicillium erichsenii.

[0013] Optionally, the concentration of glufosinate-ammonium is 50 - 400 mg / L.

[0014] The advantages and positive effects of the present invention are as follows:

[0015] The Penicillium erichsenii (CCTCC NO: M 2021635) provided by the present invention has a high tolerance concentration to glufosinate-ammonium, and has the effect of accelerating growth under the stress of glufosinate-ammonium. It can be used for the biodegradation of glufosinate-ammonium, and shows rapid and high efficiency in the metabolic degradation of glufosinate-ammonium at a high concentration of 100 mg / L. After 3 days of cultivation, the degradation rate can reach 64.5%, showing good rapidity. After 7 days of cultivation, the degradation rate can be as high as over 90%, showing high efficiency in degradation. It has the potential to be developed for degrading glufosinate-ammonium residues in natural environments such as soil and water bodies, as well as in foods such as tea, and has good application prospects in the bioremediation of environments, foods, and organisms contaminated with glufosinate-ammonium residues. Description of the Drawings

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0017] Figure 1 This is the mycelial growth status of Penicillium erlichii in the embodiments of the present invention on PDA solid medium containing different concentrations of glufosinate.

[0018] Figure 2 This is the mycelial growth status of the control fungus Metarhizium flavoviride in the embodiments of the present invention on PDA solid medium containing different concentrations of glufosinate.

[0019] Figure 3 This is the chromatogram of the detection of glufosinate content in the PDA solid medium after 1 day of cultivation of Penicillium erlichii in the embodiments of the present invention.

[0020] Figure 4 This is the chromatogram of the detection of glufosinate content in the PDA solid medium after 3 days of cultivation of Penicillium erlichii in the embodiments of the present invention.

[0021] Figure 5 This is the chromatogram of the detection of glufosinate content in the PDA solid medium after 7 days of cultivation of Penicillium erlichii in the embodiments of the present invention.

[0022] Figure 6 This is the curve graph of the change of the degradation rate of glufosinate by Penicillium erlichii over time in the embodiments of the present invention. Detailed implementation manners

[0023] In order to make the purpose, technical solutions and advantages of the present invention clearer, the following further details the present invention in conjunction with embodiments. The embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0024] Based on the information contained in the present invention, those skilled in the art can easily make various changes to the precise description of the present invention without departing from the spirit and scope of the appended claims. It should be understood that the scope of the present invention is not limited to the defined processes, properties or components, because these embodiments and other descriptions are only for illustrative purposes of specific aspects of the present invention. In fact, all various changes that those skilled in the art or related fields can obviously make to the embodiments of the present invention are covered within the scope of the appended claims.

[0025] For a better understanding of the present invention and not as a limitation on its scope, all numbers representing amounts, percentages and other numerical values used in the present invention should be understood to be modified by the word "about" in all cases. Therefore, unless otherwise specified, the numerical parameters listed in the specification and the appended claims are approximate values, which may vary according to the different desired properties to be obtained. Each numerical parameter should at least be regarded as obtained according to the significant figures reported and by means of the conventional rounding method. In addition, the meanings of the terms "comprising", "including", "containing", "having" and the like are non-restrictive, that is, other steps and other components can be added without affecting the result.

[0026] In order to make the above objects, features and advantages of the present invention more obvious and understandable, the present invention will be described in detail below.

[0027] Penicillium ehrlichii is a fungus belonging to Ascomycota, Eurotiomycetes, Eurotiales, Aspergillaceae, Penicillium. Fungi of the genus Penicillium have been isolated as endophytic fungi of various plants, and have antagonistic biological activities against many plant pests and diseases, and are also applied in the fields of enzyme production, drug research and development, and remediation of soil pollution. Plant endophytes are microbial communities that coexist harmoniously with plants and are an important microbial resource with potential development value. In recent years, the research on tea tree endophytes has mainly focused on the isolation methods, distribution patterns, identification of dominant strains in different regions, biological functions, etc. However, there are few reports on the screening and application of pesticide-degrading bacteria, and no research on the degradation of glufosinate by tea tree endophytes has been reported.

[0028] An embodiment of the present invention provides an application of Penicillium ehrlichii in degrading glufosinate based on a strain of tea tree endophyte Penicillium ehrlichii screened in the early stage. The preservation number of the Penicillium ehrlichii is: CCTCC NO: M 2021635, and it was preserved in the China Center for Type Culture Collection on May 31, 2021. Address: Wuhan University, Wuhan, China. The name of the culture is Penicillium ehrlichii Z19.

[0029] Penicillium erlichii (CCTCC NO: M 2021635) provided by the present invention grows well on PDA medium containing up to 4 mg of glufosinate (final concentration in the medium is 400 mg / L), and the mycelial growth rate is faster than that of the control group, indicating that it has a high tolerance concentration to glufosinate. Moreover, at a high concentration of 100 mg / L, the glufosinate content on the PDA medium can be reduced by more than 90% on the 7th day, indicating that this strain can efficiently degrade glufosinate and has the potential to be developed for degrading glufosinate residues in natural environments such as soil and water bodies, as well as in foods such as tea. It has good application prospects in bioremediating environments, foods, and organisms contaminated with glufosinate residues.

[0030] Optionally, the concentration of glufosinate is 0 - 400 mg / L, preferably 0 - 100 mg / L.

[0031] Optionally, the above-mentioned application includes the application in the preparation of a microbial preparation for degrading glufosinate.

[0032] Another embodiment of the present invention is a microbial preparation for degrading glufosinate, which includes Penicillium erlichii as described above and acceptable excipients.

[0033] Acceptable excipients refer to components that do not interfere with the biological activity and efficacy of Penicillium erlichii and do not have obvious toxicity to organisms (including humans or animals), the environment (including soil and water bodies), and foods at the concentrations at which they are applied. They include any one or at least two combinations of solvents, dispersants, stabilizers, wetting agents, emulsifiers, synergists, thickeners, defoamers, suspending agents, antioxidants, lyophilizing agents, preservatives, pH regulators, protective agents, etc. The use of the above components in microbial preparations is well-known in the art. For example, solvents or dispersants may include sterile water, physiological saline, common media such as PDA medium, and also mixtures composed of these solvents or dispersants. For example, antioxidants may include benzoic acid, ascorbic acid, 2,6-di-tert-butyl-4-methylphenol, sodium sulfite, or sodium bisulfite, either alone or in any combination. For example, pH regulators may include sodium carbonate, sodium bicarbonate, phosphoric acid, dipotassium hydrogen phosphate, sodium hydroxide, ammonia water, either alone or in any combination. For example, protective agents may include lyophilization or cryopreservation protective agents such as glycerol, sodium alginate, and polyvinyl alcohol. Through the aforementioned acceptable excipients, Penicillium erlichii of the present invention can be prepared into solid preparations or liquid preparations.

[0034] The advantages of the microbial preparation for degrading glufosinate compared with the prior art are the same as those of the application of Penicillium erlichii in degrading glufosinate as described above, and will not be elaborated here.

[0035] Another embodiment of the present invention provides the application of glufosinate in promoting the mycelial growth of Penicillium erlichii as described above.

[0036] Optionally, the concentration of glufosinate is 50 - 400 mg / L.

[0037] For the experimental methods without specific conditions noted in the following examples, they are generally carried out according to the conditions recommended by the manufacturer.

[0038] Example

[0039] 1. Growth test of Penicillium ehrlichii in a medium containing glufosinate

[0040] The preservation number of Penicillium ehrlichii in this example is: CCTCC NO: M2021635, which was preserved at the China Center for Type Culture Collection on May 31, 2021. Address: Wuhan University, Wuhan, China. Culture name: Penicillium ehrlichii Z19.

[0041] Weigh a certain mass of glufosinate standard product (Shanghai Yuanye Bio-Technology Co., Ltd., CAS: 77182 - 82 - 2) and dissolve it in sterilized ultrapure water to make a glufosinate stock solution of a certain concentration, and place it in a 4°C refrigerator for later use. Add different volumes of the glufosinate stock solution to the PDA medium to prepare PDA solid media (experimental groups) with glufosinate contents of 4 mg, 2 mg, 1 mg, and 0.5 mg respectively, corresponding to final concentrations of 400 mg / L, 200 mg / L, 100 mg / L, and 50 mg / L respectively, and set a negative control (CK) without glufosinate.

[0042] The formula of the PDA medium is as follows: 200 grams of potato, 20 grams of glucose, 1000 milliliters of distilled water, and 15 - 20 grams of agar are added when preparing the solid medium.

[0043] Inoculate a 5 - mm Penicillium ehrlichii mycelial disc in the center of each PDA solid medium (plate) containing glufosinate at different final concentrations, and culture it in a constant - temperature incubator. Observe and take pictures of the mycelial growth status every day. At the same time, use a vernier caliper to measure the growth diameter (mm) of each colony, and calculate the mycelial growth rate. The growth promotion rate of glufosinate on mycelial growth is calculated by the following formula:

[0044] Growth promotion rate (%) = [(Dc - Dt) / (Dt - 5)]×100%, where Dc and Dt are the average diameters (mm) of the fungal colonies in the experimental group and the control group respectively.

[0045] The results are as Figure 1As shown in Table 1, on the PDA medium containing glufosinate, the mycelial growth of Penicillium ehrlichii was faster and better, and the colony diameter was larger, indicating that at the tested high-dose glufosinate concentration, glufosinate did not have a toxic effect on the strain Penicillium ehrlichii, which also shows that this strain can tolerate high concentrations of glufosinate. In addition, all four tested concentration gradients showed a certain effect of promoting mycelial growth.

[0046] Table 1 Effects of different concentrations of glufosinate on the mycelial growth of the strain Penicillium ehrlichii on the 4th and 6th days

[0047]

[0048] However, Metarhizium flavoviride (the Metarhizium flavoviride strain was isolated and preserved by this laboratory), which is also in the Ascomycota phylum, could not grow normally on the medium containing 50 - 400 mg / L glufosinate and its growth was inhibited. The results are as Figure 2 shown in Table 2. On the PDA solid medium containing glufosinate, the mycelial growth of Metarhizium flavoviride was significantly inhibited, the colony diameter was significantly smaller compared to the control, and there was a phenomenon of poor vegetative growth and a shift to reproductive growth and sporulation (the center of the colony turned yellow and sporulation structures appeared), indicating that at the tested glufosinate concentration of 50 - 400 mg / L, glufosinate had a certain toxic effect on Metarhizium flavoviride.

[0049] Table 2 Effects of different concentrations of glufosinate on the mycelial growth of the strain Metarhizium flavoviride on the 5th and 10th days

[0050]

[0051] 2. Test on the degradation ability of Penicillium ehrlichii to glufosinate

[0052] Prepare 10 mL of PDA solid medium containing 100 mg / L glufosinate (experimental group), pour the medium into a sterile 9 cm sterilized petri dish, and the content of glufosinate in the medium is 1 mg. Set up an equal amount of sterilized ultrapure water added as a negative control (CK).

[0053] Inoculate a 5-mm Penicillium ehrlichii cake in the center of each PDA solid medium and culture it in a constant-temperature incubator. Take the experimental group and control group PDA plates on the 1st, 3rd, and 7th days respectively. Add ultrapure water for extraction. After homogenizing, vortexing, and centrifuging the medium, perform liquid chromatography-mass spectrometry (LC-MS) detection. The results are shown in Table 3.

[0054] The LC-MS detection conditions are as follows: I. Liquid phase conditions, chromatographic column: Agilent ZORBAX Eclipse Plus C18 (2.1×100 mm 1.8-Micron); column temperature: 35°C; mobile phase: 0.5% formic acid solution (A), methanol (B); flow rate: 0.2 mL / min; mobile phase gradient: 0 - 2 min, 80% A; 2 - 3 min, 20% A; 3 - 6 min, 20% A; 6 - 7 min, 80% A. II. Mass spectrometry conditions: electrospray ionization source (ESI); data acquisition uses negative ion scanning and multiple reaction monitoring mode (MRM); desolvation temperature 600°C; ion source temperature 150°C, desolvation flow rate 1000 L / h. Spray voltage 5500 v, nebulizing gas pressure 344.5 kPa.

[0055] Table 3 Degradation rate of glufosinate by Penicillium ehrlichii at different growth times

[0056]

[0057] As can be seen from Table 2, Penicillium ehrlichii can degrade glufosinate in the PDA solid medium well at different growth times. And as the growth time prolongs, the content of glufosinate in the medium gradually decreases, and the degradation efficiency gradually increases. Figures 3 - 5 LC-MS detection chromatograms of glufosinate content in the experimental group and control group on the 1st, 3rd, and 7th days are shown respectively. These results indicate that the metabolic degradation of glufosinate by Penicillium ehrlichii shows an obvious time-dose effect (see Figure 6 ). In addition, the metabolic degradation of glufosinate by this strain also shows rapid and high efficiency. After 3 days of culture, the degradation rate can reach 64.5%, showing good rapidity. After 7 days of culture, the degradation rate of glufosinate can be as high as over 90%, showing high efficiency of degradation.

[0058] The above research results indicate that Penicillium ehrlichii has a good function of tolerating and degrading glufosinate, and can be used to degrade glufosinate in the environments such as soil, water body, and food, with potential development and application prospects.

[0059] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. Application of Penicillium erichsenii in degrading glufosinate, characterized in that, the preservation number of the Penicillium erichsenii is: CCTCC NO: M 2021635.

2. Application of Penicillium erichsenii according to claim 1 in degrading glufosinate, characterized in that, the concentration of the glufosinate is 0 - 400 mg / L.

3. Application of Penicillium erichsenii according to claim 2 in degrading glufosinate, characterized in that, the concentration of the glufosinate is 0 - 100 mg / L.

4. Application of Penicillium erichsenii according to claim 1 in degrading glufosinate, characterized in that, the application includes the application in preparing a microbial preparation for degrading glufosinate.

5. Application of glufosinate in promoting the growth of Penicillium erichsenii hyphae, characterized in that, the preservation number of the Penicillium erichsenii is: CCTCC NO: M 2021635.

6. Application of glufosinate according to claim 5 in promoting the growth of Penicillium erichsenii hyphae, characterized in that, the concentration of the glufosinate is 50 - 400 mg / L.

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