CN112301070B - Method for high-yield trichothecene of trichotheca rosea and application of trichotheca rosea in preparation of broadleaf weed herbicide - Google Patents

Abstract

The invention belongs to the technical field of microbial pesticides, and discloses a method for producing trichothecene with high yield by trichothecene roseum and application of the trichothecene roseum in preparation of broadleaf weed herbicides. The invention develops a trichothecene culture medium suitable for high-yield fermentation of trichothecene roseum, and the preservation number of the trichothecene roseum is CCTCC NO: m2019210. The culture medium has easily obtained components and low cost, greatly improves the yield of trichothecene, and improves the yield by 200 percent compared with the conventional culture medium. The invention realizes the industrial purpose of directly using the trichothecium roseum fermentation liquid for preparing the broadleaf weed herbicide, and is suitable for large-scale popularization.

Description

Method for high-yield trichothecene of trichotheca rosea and application of trichotheca rosea in preparation of broadleaf weed herbicide

Technical Field

The invention belongs to the technical field of microbial pesticides, and particularly relates to a method for producing trichothecene with high yield by trichothecene roseum and application of the trichothecene roseum in preparation of broadleaf weed herbicides.

Background

With the progress of social civilization and the improvement of public health awareness, the negative effects caused by the extensive, long-term and large-scale use of chemical herbicides are increasingly shown, and the chemical herbicides are attracted by global attention. The development of new biogenic herbicides using biologically active metabolites in natural organisms is an important approach and future development direction for herbicide development. The compounds have the characteristics of novel chemical structures, unique action targets, broad spectrum, low toxicity, low residue and small environmental influence. In addition, this approach to developing new herbicides from natural products has the advantages of directionality, short development cycle, and relatively low development cost relative to the 10 year, approximately $ 2.5 billion financial situation for developing a new chemical herbicide product. In recent years, the development of biogenic or novel herbicide lead compounds as natural products of microorganisms has attracted considerable interest to weed scientists and agrochemical chemists, the most successful being bialaphos (bialaphos) and glufosinate (phosphinothricin).

Regarding trichothecene rosea NBERC _49006, the applicant finds that an ethyl acetate extract of trichothecene rosea NBERC-49006 shows good seed germination inhibition effect in the process of screening a natural product with herbicidal activity, and separates an active compound trichothecene (Trichothecin) (Wumegayuan et al, research and development of natural products, 2019(31), 1772-containing 1776), and the article discloses that the inhibition capacity of the trichothecene (Trichothecin) on arabidopsis thaliana seeds is LC₅₀9.30ug/ml, but when Bermuda grass was tested for germination at a concentration of trichothecene of 32ug/ml, it was found to be unable to inhibit, indicating that it had an onset of concentration greater than 32ug/ml for Bermuda grass seeds or that it did not work for Bermuda grass seeds. However, in the actual production, there is no great reference to the action of arabidopsis thaliana, arabidopsis thaliana is a model plant and has the characteristic of high sensitivity in screening of herbicides, but the weeding experiments on weeds lack consistency and can possibly cause false positive results (Zhang Shi just et al, Hubei agricultural science, 2014 (53): 5371 and 5373), so that under the condition of considering the production cost and the weeding effect of biological weeding, a biological microbial inoculum which can be directly used for weeding after fermentation is still urgently needed to be developed.

The formulation of the fermentation medium is a very critical factor in the fermentation industry, which directly affects the yield of metabolites and the subsequent production costs. Although the existing fermentation culture media are many and have many successful paradigms, the formulation between different strains has no reference and reference significance due to the diversity of microbial strains. For example, for spinosad producing bacteria, the adopted optimized formula comprises 50g/L of glucose, 10g/L of maltose, 20g/L of cottonseed meal, 1g/L of ammonium sulfate, 0.2g/L of zinc sulfate, 15g/L of corn steep liquor, 2g/L of beef extract and 5g/L of calcium carbonate, and the yield is increased by 31.6% (Zhumingjun et al, pesticides 2009 (48): 728) and 731); for the fermentation of abamectin, the preferable formula is 12% of starch, 2.5% of bean cake powder, 1% of yeast powder, 0.004% of cobalt chloride, 0.08% of calcium carbonate and 1-2% of reducing sugar initial concentration (2008, Zhejiang industry university), and the culture medium formulas of the two are completely different.

In the screening of the fermentation medium, the components which have more remarkable influence are a carbon source, a nitrogen source, inorganic salts and some trace elements. The carbon source, nitrogen source, inorganic salt and the like which can be used for industrial production at present are very various, and particularly, the carbon source and the nitrogen source are generally natural components which have great influence on the product yield and also cause great randomness and workload in the process of screening the formula. For example, on the basis of previous work in Song Yuan et al, more than 170 culture medium formulas are further screened to obtain an ideal culture medium formula (Song Yuan et al, J. China antibiotic, 2001 (26): 176-. In addition, since the active metabolites are secondary metabolites and are not essential compounds for the microorganism, the large-scale metabolism of the microorganism generally has certain difficulty, such as the optimization of fermentation media such as spinosad, lovastatin and the like, and the yield of the microorganism is increased by 17-56% (Wubo et al, J. China antibiotic, 2007(07): 409-.

Disclosure of Invention

The invention provides a culture medium suitable for high-yield trichothecene of trichothecene roseum fermentation, wherein the preservation number of the trichothecene roseum is CCTCC NO: m2019210.

The invention also aims to provide a method for producing trichothecin with high yield by fermenting trichothecene roseum, wherein the preservation number of the trichothecene roseum is CCTCC NO: m2019210.

The last object of the invention is to provide a culture medium for fermenting trichothecin roseum NBERC _49006 to produce trichothecin with high yield or an application of the method in preparing biological herbicides.

In order to achieve the purpose, the invention adopts the following technical measures:

a culture medium suitable for high-yield trichothecene fermentation of trichothecene roseum NBERC-49006 comprises: 10-20g/L of starch, 2-5g/L of glucose, 2-5g/L of soybean meal, 0.5-1g/L of yeast extract, 1-1.25g/L of monopotassium phosphate, 0.5-0.625g/L of magnesium sulfate, and the balance of water, wherein the preservation number of the trichotheca rosea is CCTCC NO: m2019210.

A method for producing trichothecin with high yield by fermentation of trichotheca rosea comprises the following steps: m2019210 trichotheca rosea is inoculated in the culture medium for fermentation culture.

The protection scope of the invention also includes the application of the culture medium or the culture method in the preparation of broadleaf weed herbicides; the weeds comprise: herba Erigerontis, herba Amaranthi Tricoloris, Achyranthis radix, herba Nepetae Laciniatae, herba Commelinae, herba Ixeritis Denticulatae, herba Bidentis Bipinnatae, herba Xanthii, herba Convolvuli Japonicae, and Ecliptae herba.

Compared with the prior art, the invention has the following advantages:

1. the invention solves the problem that the herbicide can not be prepared directly by trichotheca roseum NBERC _49006 fermentation liquor at present; according to the existing fermentation formula and the proportion of the existing components, the highest concentration of trichothecene in the fermentation liquor can only reach 99 mu g/mL, which is far lower than the effective concentration of the compound in practical use, and the requirements of practical production and application can not be met at all. After the culture medium is used for fermentation culture, the concentration of a target product in fermentation liquor can approach 300 mug/mL, and is increased by 200%, which is the height which is extremely difficult to achieve in the field of fermentation.

2. The fermentation liquor obtained by the method can be directly used for removing broadleaf weeds (horseweed herb, amaranthus retroflexus, achyranthes bidentata, alternanthera philoxeroides, digitaria canadensis, sowthistle tasselflower herb, sticktight, xanthium sibiricum, Convolvulus arvensis and eclipta) in the seedling stage, and has great application prospect.

Drawings

FIG. 1 is a comparison of the efficacy of Tricholoma roseum active extract, Trichothecin and commercial herbicide glyphosate after 14 days of treatment on Alternanthera philoxeroides;

wherein: CK is sterile water containing 0.1% Tween-80, and original concentration of active extract contains Trichothecin250 μ g/mL. FIG. 2 is a comparison of trichotheca rosea activity extracts, Trichothecin and commercial herbicide glyphosate after 14 days of treatment against a commelina communis exemplary;

wherein CK is sterile water containing 0.1% Tween-80, and original concentration of active extract contains Trichothecin250 μ g/mL.

Detailed Description

The present invention will be further described in the following examples to provide those skilled in the art with a more complete understanding of the present invention, but the present invention is not limited to the following examples. The technical schemes of the invention are conventional schemes in the field if not particularly stated; the reagents or materials, if not specifically mentioned, are commercially available.

Example 1:

acquisition and physiological and biochemical properties of Trichothecium roseum (Trichothecium roseum) NBERC _ 49006:

the trichotheca rosea is obtained by separating and purifying soil in suburb of salining in Hubei province, fermenting the strain, extracting with ethyl acetate to obtain an ethyl acetate extract, and finding that the ethyl acetate extract can completely inhibit the germination of arabidopsis seeds by adopting a method for inhibiting the germination of the seeds. The species was identified as trichothecium roseum (trichothecium roseum) by morphological identification and 18SrDNA alignment.

To this end, the applicant obtained a single-ended strain of Trichothecium roseum (Trichothecium roseum) which was sent to the collection of the chinese type culture collection on 27 days 3 and 2019 under the taxonomic designation: trichothecium roseum (Trichothecium roseum) NBERC _49006 with the preservation number of CCTCC NO: m2019210, the preservation address is Wuhan university in Wuhan, China.

The conidiophores of the strain are upright, do not bend and branch, most of terminal conidiophores are 1, the conidiophores are in an oblong shape or a pear shape, the spore bases have a nipple bulge, the back surface of a flat plate is in a light yellow color after 1-week PDA flat plate culture, the front surface of the flat plate is in a pink color, and the aerial silks are thick.

Example 2:

fermentation of trichothecium roseum (trichothecium roseum) NBERC _ 49006:

(1) preparation of a culture medium:

potato dextrose agar medium (PDA): 200g/L of potato, 20g/L of glucose, 20g/L of agar and natural pH value;

fermentation medium a: 6.25g/L of maltose, 6.25g/L of malt extract, 1g/L of yeast extract, 0.625g/L of peptone, 1.25g/L of monopotassium phosphate, 0.625g/L of magnesium sulfate and the balance of water, and the pH value is natural. The liquid content of the 500mL Erlenmeyer flask was 100 mL.

Fermentation medium B: 20g/L of starch, 5g/L of glucose, 5g/L of soybean meal, 1g/L of yeast extract, 1.25g/L of monopotassium phosphate, 0.625g/L of magnesium sulfate and the balance of water, wherein the natural pH value is 100mL of liquid filling volume of a 500mL triangular flask.

Fermentation medium B-1: 10g/L of starch, 2g/L of glucose, 2g/L of soybean meal, 0.5g/L of yeast extract, 1g/L of monopotassium phosphate, 0.5g/L of magnesium sulfate, natural pH value and 100mL of liquid filling volume in a 500mL triangular flask.

Fermentation medium B-2: 15g/L of starch, 3.75g/L of glucose, 3.75g/L of soybean meal, 0.75g/L of yeast extract, 1.25g/L of monopotassium phosphate, 0.625g/L of magnesium sulfate and the balance of water, and the pH value is natural.

Fermentation medium C: 20g/L of starch, 5g/L of glucose, 10g/L of soybean meal, 2g/L of yeast extract, 1.25g/L of monopotassium phosphate, 0.625g/L of magnesium sulfate and the balance of water, wherein the natural pH value is 100mL of liquid filling volume of a 500mL triangular flask.

Fermentation medium D: 20g/L of starch, 5g/L of glucose, 7.5g/L of soybean meal, 1.5g/L of yeast extract, 1.25g/L of monopotassium phosphate, 0.625g/L of magnesium sulfate and the balance of water, wherein the natural pH value is 100mL of liquid filling volume of a 500mL triangular flask.

Fermentation medium E: 20g/L of starch, 5g/L of glucose, 5g/L of fish meal, 1g/L of yeast extract, 1.25g/L of monopotassium phosphate, 0.625g/L of magnesium sulfate and the balance of water, wherein the natural pH value is 100mL of liquid filling volume of a 500mL triangular flask.

Fermentation medium F: 20g/L of starch, 5g/L of glucose, 5g/L of soybean meal, 1g/L of peptone, 1.25g/L of monopotassium phosphate, 0.625g/L of magnesium sulfate and the balance of water, wherein the natural pH value is 100mL of liquid filling volume of a 500mL triangular flask.

(2) Preparing a seed solution: inoculating Tricholoma roseum (Trichothecium roseum) NBERC _49006 strain in eggplant-shaped bottle (PDA culture medium), culturing at 28 deg.C for 7d, washing with sterile water containing 0.1% Tween-80, and making into 1.0 × 10⁷Bacterial suspension per mL for use.

(3) And (3) shake flask culture: inoculating the bacterial suspension into the above fermentation culture medium according to the inoculation amount of 5%, performing shaking culture at 28 deg.C and 120rpm for 8 days to obtain fermentation liquid,

(4) measuring the content of Trichothecin in the fermentation liquor: and (3) adding 4 times of methanol solution into 5mL of fermentation liquor, performing ultrasonic treatment for 5 minutes, centrifuging to obtain a supernatant, and detecting the content of Trichothecin. The detection method comprises the following steps: mass spectrometer: waters XEVO TQD, ion source: ESI, mass spectrometry conditions: ESI +, capillary voltage (voltage capillary), 3.5 kv; desolventization temperature (desolvation temperature): at 450 ℃; nitrogen flow rate (gas flow): 1000L/h. In the MRM ion monitoring mode, the parent ion: 330; daughter ions: 109, 187; cone voltage (cone): 30V; collision energy (collision): 12V. Chromatography apparatus: waters Acquity UPLC, column: waters Acquity UPLC BEH c 181.7um, 2.1 × 100 mm; chromatographic conditions are as follows: the column temperature is 40 ℃; the flow rate is 0.45 ml/min; binary gradient elution: water + 0.2% acetic acid; acetonitrile + 0.2% acetic acid; 0-0.2min, 95% A; 0.2-4.2min, 95% A-100% B; 4.2-5.2min, 100% B; 5.2-5.70min, 100% B-95% A; 5.70-7.00, 95% A. The sample size was 2 uL.

By utilizing the method, the strain inoculated in the fermentation medium A is detected, and the content of Trichothecin in the fermentation liquor is 99 mu g/mL; the content of Trichothecin in the fermentation liquor of the strain inoculated in the fermentation medium B is 291.5 mu g/mL.

And (4) conclusion: the fermentation medium B is used for fermenting trichothecium roseum (Trichothecium roseum) NBERC _49006, the raw materials are cheap and easy to obtain, the production cost is low, the yield of an active compound Trichothecin in fermentation liquor can be remarkably increased, the highest yield can be close to 300 mu g/mL, and the requirement of practical application can be completely met. Adopting the same culture condition, inoculating the strain in a fermentation medium B-1, wherein the content of Trichothecin in the fermentation liquor is 265.3 mu g/mL; the strain inoculated into the fermentation medium B-2 has the content of Trichothecin in the fermentation liquor of 286.8 mu g/mL. And adopting the same culture condition, inoculating the strain in the fermentation medium C, wherein the Trichothecin is not detected in the fermentation liquor; the strain is inoculated in the fermentation medium D, and the content of Trichothecin in the fermentation liquor is 15.8 mu g/mL; the strain is inoculated in the fermentation medium E, and the content of Trichothecin in the fermentation liquor is 65.4 mu g/mL; the content of Trichothecin in the fermentation liquid of the strain inoculated in the fermentation medium F is 203.6 mu g/mL.

Example 3:

the application of trichothecene in preparing broadleaf weed herbicide comprises the following steps:

(1) trichothecin (reference for preparation method: research and development of natural products, 2019(31), 1772-1776) is prepared into a solution with the concentration of 10mg/mL by ethanol, and then sterile water containing 0.1 percent of Tween-80 is added to be sequentially diluted to 500 mu g/mL, 250 mu g/mL, 125 mu g/mL, 62.5 mu g/mL and 31.25 mu g/mL.

(2) Planting seedlings of five weeds (erigeron breviscapus, amaranthus retroflexus, achyranthes bidentata, alligator alternanthera and digitaria communis) in a cube plastic pot with the side length of 7cm, and spraying a sample (90 mL/m) to the seedlings by adopting a spraying method when the seedlings grow to 8-10 leaves²) Sterile water containing 0.1% of Tween-80 is used as a blank control, 5 times of blank and each concentration are selected, the experiment is repeated for 3 times, the temperature is 28 ℃, the illumination intensity is 8000lx, the experiment result is observed after 5 days, and the inhibition rate is calculated.

(3) The calculation method comprises the following steps:

the inhibition efficiency was recorded as 0-5: 0 indicates plant health (no significant phytotoxicity), 1 indicates withering of less than 1/3 leaves, 2 indicates withering of 1/3-1/2 leaves, 3 indicates withering of 1/2-2/3 leaves, 4 indicates withering of more than 2/3 leaves, and 5 indicates withering or death of the whole plant.

The inhibition rate is calculated according to the following formula:

inhibition rate ═ Σ (inhibition efficiency × number of weeds exhibiting corresponding inhibition efficiency)/(5 × number of all weeds subjected to observation of results)

As a result: the Trichothecin can obviously inhibit the growth of broad-leaved weed erigeron breviscapus, amaranthus retroflexus, alternanthera philoxeroides, achyranthes bidentata, eclipta alba and dactylus communis seedlings, wherein the erigeron breviscapus is resistant weed, the alternanthera philoxeroides is invasive weed, common herbicide is difficult to control, the growth inhibition effect of the Trichothecin adopted as the herbicide is enhanced along with the increase of concentration, when the concentration is 250 mu g/mL, the growth inhibition rates of the erigeron breviscapus, amaranthus retroflexus, achyranthes bidentata, alternanthera philoxeroides and dactylus communis seedlings are respectively 84.0%, 100%, 77.3%, 80.0% and 80.7%, when the concentration is continuously increased to 500 mu g/mL, the five broad-leaved weeds are basically killed, and the leaves are seriously curled and yellowed and finally turned into dark brown, which indicates that the seedlings are subjected to very serious toxicity, and the specific result is shown in Table 1.

TABLE 1 inhibition of five weeds by Trichothecin

^*0.1% Tween-80 in sterile water

Example 4:

comparison of the effect of trichlothecin as a herbicide with the commercial herbicide glyphosate:

five weed seedlings were subjected to herbicidal growth test and the inhibition rate was measured using the commercial herbicide glyphosate in the same manner as in example 3.

As a result: the glyphosate concentrations were 82.7%, 100%, 78.7%, 56.0% and 18.7% inhibition of the growth of erigeron breviscapus, amaranthus retroflexus, achyranthes bidentata, alternanthera philoxeroides and commelina communis seedlings, respectively, at a concentration of 250 μ g/mL, and the inhibition of the growth of erigeron breviscapus, amaranthus retroflexus and achyranthes bidentata was 100% and the inhibition of the growth of erigeron breviscapus and commelina communis was 81.3% and 28% as the concentrations continued to rise to 500 μ g/mL, with the results shown in table 2; the weeding effect of Trichothecin on erigeron breviscapus, amaranthus retroflexus and achyranthes bidentata is equivalent to that of glyphosate, and the weeding effect on pistacia canescens and commelina communis is stronger than that of the glyphosate.

TABLE 2 inhibition of five weeds by glyphosate

^*0.1% Tween-80 in sterile water

Example 5:

comparison of control efficacy of trichotheca rosea NBERC _49006 active extract, Trichothecin and the commercial herbicide glyphosate on alternanthera philoxeroides and dactylicaba canadensis:

preparation of trichotheca pinicola NBERC-49006 active extract: 100ml of fermentation broth were shake-flask fermented according to the method in example 2 using the fermentation recipe in fermentation medium B. After fermentation, the supernatant and the mycelia were separated by centrifugation. Extracting the mycelium with methanol twice, filtering and mixing the methanol extractive solutions, and recovering methanol to obtain mycelium extract. Dissolving the mycelium extract with small amount of ethanol, adding fermentation supernatant to obtain active extract solution containing Trichothecin250 ug/mL and 0.1% Tween-80.

The Trichothecin and the commercial herbicide glyphosate are prepared into a 10mg/mL concentration solution by ethanol, and then are added with sterile water containing 0.1 percent of Tween-80 to be sequentially diluted to 500 mu g/mL, 250 mu g/mL, 125 mu g/mL, 62.5 mu g/mL and 31.25 mu g/mL; the active extract solution was formulated to contain Trichothecin 250. mu.g/mL, 125. mu.g/mL, 62.5. mu.g/mL, 31.25. mu.g/mL, and 15.625. mu.g/mL in this order. Seedlings were planted in square plastic pots 7cm on a side, and when the seedlings grew to 10 leaves, samples (90 mL/m) were sprayed on the seedlings by spraying²) Sterile water containing 0.1% of Tween-80 is used as a blank control, 5 times of blank and each concentration are selected, the experiment is repeated for 3 times, the temperature is 28 ℃, the illumination intensity is 8000lx, and the overground fresh weight of the weeds is weighed after 14 days.

The inhibition rate calculation method comprises the following steps: after spraying for 14d, the above-ground fresh weight of the weeds was weighed. Inhibition ∑ (blank control mean above-ground fresh weight-treated mean above-ground fresh weight)/blank control mean above-ground fresh weight × 100%.

As a result: by comparing table 3, fig. 1 and fig. 2, at the same concentration, the trichothecium roseum active extract has weaker control effect on alternanthera philoxeroides and dactylicapnos canadensis than that of Trichothecin, but has no statistical difference, and is stronger than that of glyphosate. The control effect of the trichotheca rosea active extract on alternanthera philoxeroides is equivalent to that of Trichothecin250 mu g/mL and glyphosate 500 mu g/mL, namely 54.5 percent, 56.5 percent and 56.3 percent respectively, while the control effect on the dactylicapnos canadensis is obviously stronger than that of the glyphosate 500 mu g/mL, and the inhibition rates are 72.6 percent and 50.4 percent respectively.

TABLE 3 comparison of the control of Alternanthera philoxeroides and Trichothecin against glyphosate, a commercial herbicide, on Alternanthera philoxeroides and Commelina communis

^*0.1% Tween-80 in sterile water^**The concentration of the active extract is expressed by the active compound Trichothecin, namely the test sample contains the Trichothecin250, 125, 62.5, 31.25 and 15.625 mu g/mL respectively

The invention obtains active extract from trichotheca rosea and Trichothecin adopts different concentrations as herbicide, except being applied to the broad-leaved weeds of erigeron breviscapus, amaranthus retroflexus, achyranthes bidentata, alternanthera philoxeroides and digitaria canadensis, the invention has better weeding effect on other common various broad-leaved weeds, such as ixeris sonchifolia, sticktight, xanthium sibiricum, bindweed, eclipta and the like.

Claims (3)

1. A culture medium suitable for high-yield trichothecene of trichothecene roseum fermentation comprises the following components: 10-20g/L of starch, 2-5g/L of glucose, 2-5g/L of soybean meal, 0.5-1g/L of yeast extract, 1-1.25g/L of monopotassium phosphate, 0.5-0.625g/L of magnesium sulfate, the balance of water and natural pH.

2. A method for producing trichothecin with high yield by fermentation of trichotheca rosea comprises the following steps: m2019210 trichotheca rosea is inoculated in the culture medium of claim 1 for fermentation culture.

3. Use of the medium of claim 1 or the method of claim 2 for the preparation of a herbicide for broad-leaved weeds: herba erigerontis, Amaranthus retroflexus, Achyranthis radix, herba Nepetae Laciniatae or herba Commelinae.

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