CN112280709A

CN112280709A - Streptomyces and streptomyces secondary metabolite Nanchangmycin and preparation method and application thereof

Info

Publication number: CN112280709A
Application number: CN202011156262.9A
Authority: CN
Inventors: 赵军伟; 向文胜; 韩丽媛; 王相晶; 于明莹
Original assignee: Northeast Agricultural University
Current assignee: Northeast Agricultural University
Priority date: 2020-10-26
Filing date: 2020-10-26
Publication date: 2021-01-29
Anticipated expiration: 2040-10-26
Also published as: CN112280709B

Abstract

The invention provides Streptomyces and a secondary metabolite Nanchangmycin of the Streptomyces and a preparation method and application thereof, belongs to the field of microorganisms, aims to effectively inhibit the growth of weeds and reduce the problem of environmental pollution caused by chemical herbicides, and provides a Streptomyces (Streptomyces sp), the strain number is NEAU-H4, the preservation number is CCTCC NO. M2020572, the strain is used for preparing the effective components of the herbicide to treat the weeds, the strain has an efficient weed control effect on the weeds, the strain can produce Nanchangmycin for inhibiting the growth of the weeds, and a high-quality material is provided for researching and developing environment-friendly Streptomyces inocula.

Description

Streptomyces and streptomyces secondary metabolite Nanchangmycin and preparation method and application thereof

Technical Field

The invention belongs to the field of microorganisms, and particularly relates to streptomyces and a secondary metabolite Nanchangmycin of the streptomyces as well as a preparation method and application of the product.

Background

Farmland weeds refer to wild plants grown in the farmland that are not intentionally grown by man. It is estimated that there are approximately 250 weeds that harm major food crops worldwide, with the most serious weeds including green bristlegrass, purslane, goosegrass, and the like. The farmland weeds can evolve gradually along with the change of natural conditions and the agricultural development process, have the characteristics of strong adaptability, wide propagation mode, large seed setting amount and the like, not only can influence the environment, but also has serious influence on economy and production. The control of farmland weeds typically includes agricultural control, chemical control and biological control. Compared with the artificial mechanical weeding, the chemical herbicide has higher working efficiency and is more timely, but causes environmental pollution and harms people and livestock, so that the search for the microbial herbicide which is more environment-friendly becomes a great trend. The development of novel microbial products which can gradually replace chemical pesticides has important significance for realizing the sustainable development of the coordination of the yield and the quality safety of agricultural products and the protection of agricultural ecological environment.

Disclosure of Invention

The invention aims to effectively inhibit the growth of weeds and reduce the problem of environmental pollution caused by chemical herbicides, and provides streptomyces, application thereof and a method for removing weeds.

In order to solve the technical problem, the invention provides Streptomyces, wherein the strain number of the Streptomyces sp is NEAU-H4, the Streptomyces sp is preserved in China center for type culture Collection (CCTCC for short), and the preservation number is CCTCC NO. M2020572.

The invention also provides a streptomyces secondary metabolite Nanchangmycin, which is prepared by the streptomyces as claimed in claim 1.

The invention also provides a preparation method of the streptomyces secondary metabolite Nanchangmycin, which comprises the following specific steps:

1) inoculating Streptomyces sp.NEAU-H4 spore to glucose yeast extract liquid culture medium to obtain seed solution, and culturing the seed solution;

2) inoculating the seed liquid obtained in the step 1) into cottonseed fermentation liquid to obtain seed fermentation liquid, and then culturing the fermentation liquid;

3) centrifuging the seed fermentation liquid obtained in the step 2), and extracting supernatant to obtain a streptomyces secondary metabolite Nanchangmycin.

Further defined, in step 1) the concentration of Streptomyces sp⁸CFU/mL-10⁶CFU/mL。

Further defined, the seed culture solution of step 1) is cultured for 3 days at 25-28 ℃ and 200-250 rpm.

Further defined, the seed liquid inoculation amount in step 2) is 5% by volume.

Further defined, the culture broth of step 2) is cultured at 25 ℃ to 28 ℃ and 200rpm to 250rpm for 7 days.

The invention also provides application of the streptomyces secondary metabolite Nanchangmycin serving as an effective component of a herbicide.

Further defined, the concentration of the streptomyces secondary metabolite Nanchangmycin in the herbicide is 100 mg/L.

Has the advantages that: the strain provided by the invention has high-efficiency preventing and removing effects on amaranthus retroflexus, green bristlegrass herb, purslane and quinoa, can obviously inhibit the germination of weed seeds and the growth of weed roots, and can generate Nanchangmycin for inhibiting the growth of weeds by Streptomyces sp.NEAU-H4, thereby providing a high-quality material for researching and developing environment-friendly Streptomyces microbial inoculum.

Drawings

FIG. 1 is a diagram of hypha and spore morphology of a strain NEAU-H4 under a scanning electron microscope;

FIG. 2 is a phylogenetic tree of the 16S rRNA sequence of strain NEAU-H4;

FIG. 3 shows the inhibitory effect of an aqueous spore suspension of the strain NEAU-H4 on the root bud growth of weed seeds, where CK is a control, 10⁸Concentration of spores of the strain NEAU-H4, 10⁷Concentration of spores of the strain NEAU-H4, 10⁶The concentration of spores of the strain NEAU-H4;

FIG. 4 shows the effect of fermentation broth of strain NEAU-H4 on the inhibition of weed root bud growth, where CK is control, 10, 50, 100, 1000, 10000, and represents dilution factor of fermentation supernatant treatment solution;

FIG. 5 shows the inhibitory effect of the fermented broth soil treatment of the strain NEAU-H4 on weed growth, wherein panel (a) is Amaranthus retroflexus, panel (b) is Ervatamia viridis, CK is control, 10X, 100X represent dilution factor of the fermented supernatant;

FIG. 6 shows the effect of the fermentation broth seedling treatment of the strain NEAU-H4 on weed growth inhibition, where panel (a) is Amaranthus retroflexus, panel (b) is Cantonella, panel (c) is Portulaca oleracea, panel (d) is Chenopodium, CK is control, 10, 100 represent dilution factor of fermentation supernatant;

FIG. 7 is a structure of Nanchangmycin;

FIG. 8 is a 1H-NMR spectrum of Nanchangmycin dissolved in methanol, with chemical shifts (ppm) on the abscissa and intensity of absorption peaks on the ordinate;

FIG. 9 is a 13C-NMR spectrum of Nanchangmycin in methanol with chemical sites (ppm) on the abscissa and intensity of absorption peaks on the ordinate;

FIG. 10 is a mass spectrum of Nanchangmycin with mass to charge ratio (m/z) on the abscissa and ion intensity on the ordinate;

FIG. 11 is a graph of the inhibitory effect of Nanchangmycin on weed seed germination, wherein panel (a) is a clear water control, panel (b) is a methanol control, panel (c) is an atrazine control, and panel (d) is the compound;

FIG. 12 shows the weed growth inhibition effect of Nanchangmycin soil treatment, where panel (a) is a clear water control, panel (b) is an atrazine control, and panel (c) is the compound;

FIG. 13 shows the effect of Nanchangmycin treatment on the inhibition of weed growth during the seedling stage;

FIG. 14 is a determination of the safety of Nanchangmycin for germination of crop seeds, wherein panel (a) is a clear water control and panel (b) is a compound;

FIG. 15 is a safety test of Nanchangmycin against crop growth.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

The invention provides Streptomyces sp, the strain number is NEAU-H4, the strain is preserved in China type culture Collection (CCTCC for short) 10.8.2020, the preservation site is the preservation center of Wuhan university No. 199 in Wuchang district, Wuhan City, Hubei province, and the preservation number is CCTCC NO. M2020572.

The medium, reagents and the like used in the present invention are commercially available unless otherwise specified.

Weed species and origin: amaranthus retroflexus L seed, Sedum sarmentosum seed, Portulaca oleracea L seed, and Chenopodium album L seed are all purchased at seed stores.

Are purchased at seed stores.

The media and their formulations referred to in the following examples are as follows:

HV medium: humic acid 0.1g, MgSO₄·7H₂O 0.05g，KCl 0.17g，Na₂HPO₄·3H₂O 0.05g，FeSO₄ 0.001g，CaCO₃0.002g, 0.1ml of vitamin complex, 2g of agar and 100ml of sterile water.

GY medium (glucose yeast extract liquid medium): 1g of yeast extract powder, 1g of glucose and MgSO₄ 0.05g，K₂HPO₄0.05g, sterile water 100 ml.

Gao's first agar medium: 20g of soluble starch, 0.5g of NaCl and KNO₃ 1g，FeSO₄.7H 2O 0.01g，K₂HPO₄ 0.5g，MgSO₄.7H₂0.5g of O, 20g of agar and 1000mL of distilled water, and the pH value is 7.2-7.4.

Oat agar medium (ISP 3): boiling herba Avenae Fatuae 20g for 30min, filtering with four layers of gauze, adding tap water to 1000mL, adding KNO₃ 0.2g，MgSO₄.7H 2O 0.2g，K₂HPO₄0.5g of agar and 20g of agar.

Seed culture medium: tryptone17g, soy peptone 3g, NaCl 5g, K₂HPO₄2.5g, 2.5g of glucose and 1000mL of distilled water, and the pH value is 7.2-7.4.

Fermentation medium: 10g of soluble starch, 10g of glucose, 10g of glycerol, 5g of tryptone, 5g of yeast powder and CaCO₃3g, 1000mL of tap water, pH 7.2-7.4.

Example 1 culture and characterization of Streptomyces sp

1. Isolation and cultivation of the Strain

The soil sample is collected from Songshan scenic spot of Shanghai, Dengyan, and the collected soil sample is placed in a dry and ventilated place for two weeks, 5g of the air-dried soil sample is taken and ground into powder, the powder is added into a triangular flask (with glass beads) containing 45ml of sterile water, the vibration is carried out for 30-45 minutes at the rotating speed of 180rpm, the soil is taken and homogenized and diluted by 1000 times, 200 mu l of diluent is absorbed and coated on an HV culture medium plate culture medium, the culture medium plate culture medium is placed at 28 ℃ for culture for 7 days, a single colony is selected and inoculated into a fresh ISP3 plate culture medium, a purified strain is obtained through repeated switching, the purified strain is named as NEAU-H4, and the purified strain is preserved in an ultra-low temperature refrigerator at-80 ℃.

2. Identification of strains

The identification method comprises the following steps: the strain is identified by morphological characteristics, physiological and biochemical experiments, cytochemical components and 16S rRNA sequence analysis.

Morphological characteristics of the thallus: the results are shown in FIG. 1, wherein gram-positive staining was observed on Streptomyces sp.NEAU-H4 aerial hyphae in a spiral shape of spore chain, the surface of the spore was rough, and the growth temperature range was as follows: 10-40 ℃; growth pH range: 5-12; growth NaCl range: the mass fraction is 0-4%.

Physiological and biochemical characteristics: the index results are shown in table 1, the Streptomyces sp.NEAU-H4 starch hydrolysis, Tween hydrolysis, urease production, esculin hydrolysis and gelatin liquefaction experiments are positive, and the peptone and liquefaction, hydrogen sulfide production, cellulose hydrolysis and nitrate utilization experiments of milk are negative;

streptomyces sp.neau-H4 uses carbon sources such as maltose, mannose, rhamnose, galactose, glucose, mannitol, raffinose, inositol, and the like;

streptomyces sp.neau-H4 cannot utilize carbon sources such as sucrose, sorbitol, etc.; a nitrogen source such as glycine, asparagine, serine, glutamine, alanine, tyrosine, proline, glutamic acid, aspartic acid, threonine, arginine, or the like can be used; nitrogen sources such as creatine cannot be utilized.

TABLE 1 physiological and biochemical Properties of NEAU-H4

Note: "+" is positive and "-" is negative.

16S rRNA gene amplification and sequence analysis:

inoculating Streptomyces sp.NEAU-H4 into GY liquid medium, culturing at 28 deg.C and 250rpm for 7 days, centrifuging at 12000rpm for 5min, collecting mycelium, washing with sterile water for 2 times, extracting genomic DNA with bacterial genomic DNA extraction kit, amplifying universal primers 27F (SEQ ID NO.1, i.e., 5'-AGAGTTTGATCCTGGCTCAG-3') and 1541R (SEQ ID NO.2, i.e., 5'-AAGGAGGTGATCCAGCC-3') with 16S rRNA using the extracted DNA as template, and assaying for 16S rRNA gene fragment. The 16S rRNA sequence is shown in SEQ ID NO.3, and the sequence is registered with NCBI, and the accession number of Gene bank is MN 885885. Sequence alignment analysis was performed in ezBioCloud by BLAST program, and NEGA 7.0 was used to construct phylogenetic tree of the strain, as shown in FIG. 2, which indicated that the 16S rRNA sequence of the strain and Streptomyces melanospocifens DSM 40318^TThe similarity was 99.7%, and a stable branch was formed therewith.

Experimental example 2. the weeding method in this example was performed according to the following steps:

1. preparing a weed killer:

1) will 10⁸CFU/mL-10⁶Inoculating Streptomyces sp.NEAU-H4 spore to GY liquid to obtain seed liquid, culturing the seed liquid at 25-28 deg.C and 250rpm for 3 days or growing to logarithmic phase;

2) inoculating the seed liquid obtained in the step 1) into a cottonseed fermentation liquid to obtain a seed fermentation liquid, and culturing the fermentation liquid for 7 days at 28 ℃ and 250 rpm;

3) centrifuging the seed fermentation liquor obtained in the step 2), and extracting supernatant to obtain a weed killer;

2. and (3) treating the amaranthus retroflexus, the green bristlegrass herb, the purslane and the quinoa by using the herbicide obtained in the step (1), wherein the treatment time is 3-7 days.

The description experiment verifies the effect of the invention:

measurement of inhibitory Effect of Streptomyces sp.NEAU-H4 spore water suspension on growth of root bud of weed seed

1. Four kinds of weed seeds of Amaranthus retroflexus, herba Setariae viridis, herba Portulacae and fructus Chenopodii are treated by plate seed germination experiment.

2. Preparation of an aqueous spore suspension: the spore concentration of Streptomyces sp.NEAU-H4 strain is measured by plate dilution and coating method, and gradient dilution treatment is carried out to obtain 10⁶CFU/mL、10⁷CFU/mL and 10⁸CFU/mL of spore liquid of the strain.

3. And (3) treating weeds: and selecting plump seeds for disinfection treatment, placing the seeds into an aseptic plate paved with filter paper, adding 3mL of diluted spore suspension into each 25 seeds, and uniformly wetting the filter paper. Sterile water controls and sterile broth dilution treatment controls were also set, each treatment was repeated 3 times. And (4) placing the culture dish into a constant-temperature incubator at 25 ℃ for dark culture, and counting the root length, the bud length and the growth condition of the seeds after 3 days. As a result, as shown in FIG. 3 and tables 2, 3 and 4, it can be seen that the spore concentration of the strain was 10⁶CFU/mL、10⁷CFU/mL and 10⁸The inhibitor has certain inhibiting effect on the growth of the root buds of amaranthus retroflexus, setaria viridis, barnyard grass and chenopodium album seeds at CFU/mL.

TABLE 2 inhibition of the root bud growth of Amaranthus retroflexus seeds by an aqueous spore suspension of the strain NEAU-H4

Spore concentration (CFU/mL)	Root growth inhibition (%)	Bud growth inhibition (%)
			10⁶	94.6	89.7
10⁷	97.4	95.1
			10⁸	99.3	97.6

TABLE 3 inhibition of the growth of the root bud of the seed of Geranium sibiricum by an aqueous spore suspension of the strain NEAU-H4

Spore concentration (CFU/mL)	Root growth inhibition (%)	Bud growth inhibition (%)
			10⁶	62.4	27.1
10⁷	87.6	53.3
			10⁸	98.4	67.9

TABLE 4 inhibition of growth of purslane, seed root bud by aqueous spore suspension of the strain NEAU-H4

Spore concentration (CFU/mL)	Root growth inhibition (%)	Bud growth inhibition (%)
			10⁶	92.3	90.6
10⁷	94.6	93.4
			10⁸	98.4	96.7

TABLE 5 inhibition of the root bud growth of Chenopodium quinoa seeds by aqueous spore suspensions of the strains NEAU-H4

Second, the inhibition effect of Streptomyces sp.NEAU-H4 fermentation liquor on the growth of weed root buds is determined

1. Preparing a seed solution: culturing Streptomyces sp.NEAU-H4 spore with sterile scraping in GY liquid to obtain seed liquid with spore concentration of 10⁸CFU/mL-10⁶CFU/mL

2. Preparing a fermentation liquid: inoculating the seed liquid obtained in the step 1 into the cottonseed fermentation liquid by an inoculation amount of 5% of volume fraction, carrying out shake culture on a shaking table at 200-250rpm for 7 days at 25-28 ℃, and centrifuging the obtained fermentation liquid to obtain a supernatant for later use.

3. And (3) treating weeds: spreading the sterilized circular filter paper sheet in sterile culture dish, selecting and sterilizing plump weed seeds (Amaranthus retroflexus, herba Setariae viridis, Echinochloa crusgalli and Chenopodium album), cleaning with sterile water, and placing on filter paper with 25 seeds per dish. The fermented supernatant was diluted 10, 50, 100, 1000, 10000 times, 3mL of the treatment solution was added to each dish to wet the filter paper evenly, and each concentration was repeated 3 times. The culture dish is placed in a constant temperature incubator at 25 ℃ for dark culture, and the inhibition of the growth of the root and the bud of the seed is counted after 3 days. The results are shown in fig. 4, the strain fermentation supernatant has a relatively obvious inhibition effect on the growth of 3 weed root buds, wherein the inhibition rate on the amaranthus retroflexus root buds is more than 95%; the inhibition rate of the cibotium root reaches 95 percent, and the inhibition rate of the bud reaches 90 percent; the inhibition rate of the barnyard grass roots reaches over 90 percent, and the inhibition rate of the barnyard grass buds reaches 75 percent.

Thirdly, the inhibition situation of Streptomyces sp.NEAU-H4 fermentation liquor soil treatment on weed growth is determined

1. Preparing a seed solution: and (5) obtaining the seed liquid by referring to the method for preparing the seed liquid in the step two.

2. Preparing a fermentation liquid: and (5) obtaining the fermentation liquor by referring to the method for preparing the fermentation liquor in the step two.

3. And (3) treating weeds: weighing soil, loading into a plastic flowerpot, watering, and uniformly spreading weed seeds (Amaranthus retroflexus L., herba Setariae viridis, Echinochloa crusgalli and Chenopodium album L.) to be tested in the plastic flowerpot, wherein each pot contains 25 seeds and a thin layer of soil is coated on the seeds. The fermented supernatant is diluted by 10 times and 100 times respectively and sprayed into flowerpots, and the control group is sprayed with clean water with the same amount. The pots were placed outdoors at 25-30 ℃ for natural growth, and each treatment was repeated 3 times. As shown in FIG. 5, the results showed that the soil treated with 10 times the amount of the fermented supernatant had a relatively good effect of controlling both weeds; when the solution is diluted by 100 times and treated with soil, the inhibitory effect on Amaranthus retroflexus is higher than that of herba Ervatamiae. The experimental result shows that the growth of weeds can be effectively inhibited when the fermentation supernatant with higher concentration is subjected to soil treatment, the results show that the germination inhibition rates of the setaria retroflexa seeds when the fermentation broth is diluted by 10 times reach 90% and 85% respectively, and the germination inhibition rates of the two weed seeds when the fermentation broth is diluted by 100 times are 65% and 50% respectively.

Fourthly, the inhibition capability of the strain Streptomyces sp.NEAU-H4 fermentation liquor in the seedling stage to the growth of weeds is measured

3. And (3) treating weeds: weighing soil, loading into plastic flowerpot, watering, uniformly spreading the seeds (25 seeds per pot) of weed to be tested (Amaranthus retroflexus, herba Setariae viridis, herba Portulacae, and fructus Chenopodii Tribuli), covering with a thin layer of soil, and naturally growing at 25-30 deg.C outdoors. After the weeds grow to 3 leaves, the fermentation supernatant liquid diluted by 10 times and 100 times is uniformly sprayed on the leaves of the weeds, the control group is sprayed with the same amount of clear water, and each treatment is repeated for 3 times. After 5 days of cultivation, the growth of weeds, the degree of withered and dead leaves were observed, and the degree of morbidity was rated as follows. Level 0: the weed leaves do not have any disease spots and wilting; level 1: sporadic scabs appear on the weed leaves; and 2, stage: 1/3-2/3 of the weed's leaves withered and dead; and 3, level: 2/3 above weed leaves wither and die; 4, level: all the weed leaves wither and die. The results are shown in fig. 6, when the fermentation liquor is diluted by 10 times, the fresh weight inhibition of the amaranthus retroflexus, the purslane and the quinoa is higher than 90%, the fresh weight inhibition rate of the green bristlegrass is higher than 85%, the leaves of the first 3 weeds above 2/3 wither and die, the leaves of the green bristlegrass are higher than 1/3 wither and die, and the plant dwarfing is serious; when the fermentation liquor is diluted by 100 times, the inhibition rate of the fresh weight of the purslane is still higher than 90%, the inhibition rate of the fresh weight of the other three weeds is higher than 70%, the disease grades of the amaranthus retroflexus and the purslane are still 3 grades, the leaves of the weeds are withered and twisted, and disease spots appear when the weeds are seriously dwarfed.

Fifthly, determination of inhibition ability of Nanchangmycin on weed growth

1. Ability of strain Streptomyces sp.NEAU-H4 to produce Nanchangmycin

(1) Preparing a seed solution: and (5) obtaining the seed liquid by referring to the method for preparing the seed liquid in the step two.

(2) Preparing a fermentation liquid: and (5) obtaining the fermentation liquor by referring to the method for preparing the fermentation liquor in the step two.

(3) Obtaining Nanchangmycin: and (3) centrifuging the fermentation liquor obtained in the step (2), removing thalli, extracting the supernatant for 3 times by using equal volume of ethyl acetate, concentrating the extract, and sequentially performing silica gel column chromatography, gel chromatography and HPLC analysis to obtain the compound. The compound is Nanchangmycin which is determined by 13C-NMR, 1H-NMR and MS spectrum analysis, the structural formula is shown in figure 7, 1H-NMR and 13C-NMR are shown in figures 8, 9 and 10.

Determination of inhibition ability of Nanchangmycin on root and bud growth of weed seeds

Preparation of herbicidal Compounds: the herbicidal active compound Nanchangmycin obtained in the step (1) is dissolved by 1mL of methanol and prepared into a liquid medicine (100mg/L) with the lowest inhibitory concentration for later use.

And (3) treating weeds: spreading sterilized filter paper in a sterile plate, sterilizing weed seeds (herba Setariae viridis, Amaranthus retroflexus, Chenopodium album and herba Portulacae), drying surface water with absorbent paper, dispersing into the plate, adding 25 seeds per plate with 3mL of medicinal liquid, setting clear water control and methanol water solution control with the same concentration, and repeating each treatment for 3 times with commercial herbicide atrazine as control agent. And (5) placing the plate in a constant-temperature incubator at 25 ℃ for dark culture, observing the result after 3 days, and counting the germination rate and the rhizome growth condition. The results are shown in fig. 11, the compound has better inhibitory activity on the germination of 4 weed seeds, the methanol aqueous solution treatment group does not inhibit the germination of weed seeds and the growth of root buds compared with the clear water control, and the compound can better inhibit the growth of the root of setaria canicola in the setaria plate experiment compared with the control medicament atrazine.

Determination of the ability of Nanchangmycin to inhibit the growth of weeds

Preparation of herbicidal Compounds: referring to the method for preparing the herbicidal compound in step 2, the herbicidal compound is obtained.

And (3) treating weeds: uniformly spraying 10ml (100mg/L) of diluted compound liquid medicine with the lowest inhibitory concentration on the soil surface, arranging a control group sprayed with 10ml of clear water and methanol aqueous solution with the same concentration, and simultaneously spraying 10ml of commercial herbicide atrazine as a control agent. Each treatment was repeated 3 times; and (3) seedling stage treatment: when the weeds (herba Setariae viridis, Amaranthus retroflexus, Chenopodium album and herba Portulacae) grow to 3-5 leaf stage, the compound liquid medicine is filled in a spray pot and sprayed on the leaf surface, a clear water control and a methanol water solution control with the same concentration are set, and a commercial herbicide atrazine is used as a control agent. Each treatment was repeated 3 times. And observing the result after 3 days, and counting the wilt degree and the disease degree grade of the weeds. The results of the soil treatment are shown in fig. 12, and the experimental results show that the compound can effectively inhibit the growth of 3 kinds of weeds after the soil sealing treatment, compared with the commercial medicament atrazine, the compound has slightly lower sealing effect on the setaria viridis, and the sealing effect on the amaranthus retroflexus and the barnyard grass is basically consistent with that of the atrazine in a control group.

The compound spraying experiment result in the seedling stage is shown in fig. 13, when the weeds grow to 3-5 leaf stages, the compound and the contrast agent are sprayed, the weeds sprayed with the contrast agent all wither and die after 7 days, the leaves sprayed with the compound above Amaranthus retroflexus 2/3 wither and die, the leaves twist is serious, and the dwarfing is obvious; all leaves of the herba Ervatamiae and herba Portulacae sprayed with the compound wither and die; the stems and leaves of the chenopodium album sprayed with the compound are seriously twisted, a large number of disease spots appear, and more than 2/3 leaves are rotten and dead.

The results show that the herbicidal active compound of the strain NEAU-H4 can effectively inhibit the germination and growth of weed seeds in soil sealing treatment and seedling stage spraying treatment experiments, and the source of the herbicidal active compound is a biological source, thereby providing a certain theoretical basis for the research of green biopesticides.

Determination of safety of Nanchangmycin to crops

Effects on crop seed germination: selecting tomato, rape, lettuce, wheat and corn as crop seeds, sucking the water of the disinfected seeds, putting the seeds into a flat dish paved with sterile filter paper, adding 3mL of compound liquid medicine with the lowest inhibitory concentration into each dish of 25 seeds, adding an equal amount of clear water into a control group, and repeating the treatment for 3 times. After 5d, counting the germination and root bud growth conditions of the crop seeds; effect on crop growth: putting the sterilized crop seeds (rape, caraway, lettuce, soybean and wheat) into a dish paved with sterile filter paper, adding a proper amount of clear water into the dish, and uniformly wetting the filter paper. And (3) placing the plate in a constant-temperature incubator at 25 ℃ for accelerating germination, and keeping the seeds for later use after the seeds are exposed to white. The equal amount of soil is filled in a plastic flowerpot with the diameter of 15cm, the flowerpot is watered thoroughly, the crop seeds after germination acceleration are evenly placed in the flowerpot, a layer of thin soil is covered on the flowerpot, and the flowerpot is placed outdoors at the temperature of 25-30 ℃ for natural growth. When the crops grow to 3-4 leaf stage, the diluted compound liquid medicine is filled in a spray pot and uniformly sprayed on the crop leaves, clear water is set for comparison, and each treatment is repeated for 3 times. And (5) counting the results after 3-5 days, and observing the withering and death degree of the leaves. The results of the seed germination experiments are shown in fig. 14, the compound has no inhibition on the germination of tomato, lettuce, rape and corn seeds, has a certain inhibition effect on the growth of wheat seed buds, and has no inhibition effect on the growth of wheat seed roots. The compound is proved to be suitable for pre-emergence weeding of part of crops. The results of the safe seedling spraying experiment of the compound crops are shown in fig. 15, the compound has obvious inhibition effect on rape and cucumber, and the inhibition effect is manifested by leaf distortion, plant dwarfing and yellowing, and withering and death of rape leaves above 1/3; the compound has no obvious inhibition effect on coriander corns, and proves that the compound is suitable for weeding after seedling of part of crops.

Claims

1. The Streptomyces is characterized in that the strain number of the Streptomyces sp is NEAU-H4, and the Streptomyces sp is preserved in China center for type culture Collection (CCTCC for short) with the preservation number of CCTCC NO. M2020572.

2. A secondary metabolite Nanchangmycin of Streptomyces, wherein the secondary metabolite Nanchangmycin is produced by the Streptomyces according to claim 1.

3. The method for preparing the streptomyces secondary metabolite Nanchangmycin as claimed in claim 2, wherein the method comprises the following steps:

4. The process according to claim 4, wherein the concentration of spores of Streptomyces sp⁸CFU/mL-10⁶CFU/mL。

5. The method according to claim 4, wherein the seed solution of step 1) is cultured at 25-28 ℃ and 200-250rpm for 3 days.

6. The method according to claim 4, wherein the amount of the seed liquid inoculated in the step 2) is 5% by volume.

7. The method according to claim 4, wherein the culture broth of step 2) is cultured at 25 ℃ to 28 ℃ and 200rpm to 250rpm for 7 days.

8. Use of the streptomyces secondary metabolite Nanchangmycin of claim 2 as an active ingredient of a herbicide.

9. The use according to claim 8, characterized in that the minimum concentration of the streptomyces secondary metabolite Nanchangmycin in the herbicide is 100 mg/L.