CN112273390A

CN112273390A - Herbicidal activity of compounds harzianums and producing bacteria thereof

Info

Publication number: CN112273390A
Application number: CN202010622724.5A
Authority: CN
Inventors: 张礼文; 徐玉泉; 王辰; 岳群; 尹淼淼
Original assignee: Biotechnology Research Institute of CAAS
Current assignee: Biotechnology Research Institute of CAAS
Priority date: 2020-06-30
Filing date: 2020-06-30
Publication date: 2021-01-29
Anticipated expiration: 2040-06-30
Also published as: CN112273390B

Abstract

The invention discloses a compound harzianums and producing bacteria thereofTrichoderma brevicompactumAs an application in herbicide, the compound shows herbicidal activity in a biological activity test and has wide application value.

Description

Herbicidal activity of compounds harzianums and producing bacteria thereof

Technical Field

The invention belongs to the field of herbicides, and particularly relates to a compound harzianum and application of a Trichoderma brevicompactum producing strain thereof in herbicide preparations and microbial agents.

Background

Harzianums are tetracyclic sesquiterpene epoxides of similar chemical structure, in which a double bond is formed between C-9 and C-10 and an epoxy group is formed between C-12 and C-13. Such compounds can be classified into four types according to their chemical structure: type A contains H or OH at C-8 position, and no carbonyl, such as T-2 toxin, trichodermin, Harzianum A, etc.; form B contains a carbonyl group at the C-8 position, such as Nivalenol (NIV); type C contains an epoxy group at the C-7, 8 position, such as palmatine; form D has a macrocyclic structure in C-4 and C-15, with forms A and B being more common. More than 200 such compounds have been isolated from nature. The tetracyclic sesquiterpene epoxides are produced by fusarium, Myrothecium, Stachybotrys and other fungi, and are mycotoxins with certain toxicity to plants and animals. Wherein, the toxicity of the A and B is low. Therefore, the tetracyclic sesquiterpene epoxide with higher phytotoxicity and lower animal toxicity has the potential to become a herbicide. For example, Abbas et al (2013) found that T-2tetraol is less cytotoxic and more phytotoxic to mammals [1 ].

Trichoderma umbiliciformis (Trichoderma brevicompactum) was first discovered and named in 2004 by Kraus et al, also called Trichoderma brevicompactum or Trichoderma brevicaulis, belongs to Pleurospora species, Trichoderma, while sexual stage belongs to Hypocrea, a Trichoderma widely present in soil, plants [2 ]. Conidial clusters of trichoderma umbiliciforme are hemispherical, conidia are in a subsphere or oval structure with a smooth surface, the color of the conidia is gray green, yellow green or dark green, and the conidia on the flat plate form typical concentric annular colonies from the center to the periphery. Trichoderma umbiliciformis is a biocontrol strain, has good control effect on various plant pathogenic fungi, and can generate secondary metabolites with biocontrol activity to inhibit the growth of plant pathogenic fungi. Strains such as trichoderma umbilicalis CBS 109720, IBT 40839 and IBT 40840 are all able to metabolise the synthesis of trichodermin (trichodermin), whereas t.cf.brevicompactum ATCC 90237, IBT 40863 and NRRL 3199 are mainly able to metabolise the synthesis of the compound Harzianum a.

Harzianum class compounds belong to class A and are structurally similar to T-2tetraol, where Harzianum A was originally isolated from Trichoderma Harzianum of Trichoderma in 1994 and has antibacterial activity [3], and Harzianum B was isolated from Hypocrea sp.F000527 and has anticancer activity [4 ].

Reference to the literature

[1]H.K.Abbas,T.Yoshizawa and W.T.Shier,2013.Cytotoxicity and phytotoxicity of trichothecene mycotoxins produced by Fusarium spp,Toxicon,74,68-75.doi:10.1016/j.toxicon.2013.07.026；

[2]G.F.Kraus,I.Druzhinina,W.Gams,J.Bissett,D.Zafari,G.Szakacs,A.Koptchinski,H.Prillinger,R.Zare and C.P.Kubicek,2004.Trichoderma brevicompactum sp nov,Mycologia,96,1059-1073.doi:10.2307/3762089；

[3]D.G.Corley,M.Miller-Wideman and R.C.Durley,1994.Isolation and structure of harzianum A:a new trichothecene from Trichoderma harzianum,J Nat Prod,57,422-425.doi:10.1021/np50105a019；

[4]H.Z.Jin,J.H.Lee,W.D.Zhang,H.B.Lee,Y.S.Hong,Y.H.Kim and J.J.Lee,2007.Harzianums A and B produced by a fungal strain,Hypocrea sp.F000527,and their cytotoxicity against tumor cell lines,J Asian Nat Prod Res,9,203-207.doi:10.1080/10286020500531977。

Disclosure of Invention

Therefore, the invention aims to provide a compound harzianums (compound 1, compound 2 and a mixture thereof) and application of a Trichoderma brevicompactum producing strain thereof as a herbicide. Thus, a herbicidal agent and a microbial agent comprising the above compound or its producing bacterium are also provided.

The invention obtains a fungus strain which can inhibit the germination and growth of dicotyledonous plant pakchoi by screening from 211 strains of soil fungi, and the fungus strain is identified as Trichoderma brevicompactum (Trichoderma brevicompactum) by ITS. The strain is preserved by China general microbiological culture Collection center (CGMCC) at 9.4.2020 (the preservation address is No. 3 of Silu 1. Cheng. in the area of the rising of Beijing), the preservation number is CGMCC No.19618, the strain is classified and named as Trichoderma brevicompactum, and the strain survives after detection. Through large fermentation, sufficient extract is obtained and then separated and purified, and a mixture of compounds Harzianum A (compound 1) and Harzianum B (compound 2) is obtained. The chemical structure is shown as formula (1):

the technical scheme provided by the invention is as follows: use of the compounds harzianum a, harzianum B or mixtures thereof as herbicidal agents.

In the above application, the weeding is carried out by referring to dicotyledonous or monocotyledonous weeds, preferably dicotyledonous weeds of the genus Brassica or monocotyledonous weeds of the genus Echinochloa, more particularly barnyard grass.

The invention also provides a bacterial strain of the compound harzianum A and harzianum B producing bacteria (Trichoderma brevicompactum), wherein the strain preservation number is as follows: CGMCC No. 19618.

Meanwhile, the invention provides a method for producing compounds of harzianum A and harzianum B by using the bacterial strain, which comprises the following steps: the compounds of harzianum A and harzianum B are separated from thalli of the strain by culturing the strain, and are separated and purified by chromatography and high pressure liquid chromatography, and identified by high performance liquid chromatography-mass spectrometry.

The invention also provides an application of the strain and the fermentation product thereof in weeding, wherein weeding refers to dicotyledonous plant or monocotyledonous plant weeds, preferably dicotyledonous plant weeds of Brassica and monocotyledonous plant weeds of Echinochloa, and more particularly barnyard grass.

The application, wherein the fermentation product is a crude extract obtained by PDA solid fermentation, three primary extractions with equal volume of methanol and evaporation drying.

The invention also provides a herbicide which comprises the compounds of harzianum A, harzianum B or a mixture thereof as effective components or fermentation products of the strains, can be dissolved in methanol and diluted into solution by water for use.

The herbicide refers to dicotyledonous or monocotyledonous weeds, preferably dicotyledonous weeds of Brassica and monocotyledonous weeds of Echinochloa, and more particularly barnyard grass.

Experiments show that the invention has the following beneficial technical effects:

specifically, the invention evaluates the inhibition effect of the fermentation crude extract of Trichoderma brevicompactum and the mixture of the compounds 1 and 2 on the germination and growth of Brassica chinensis of dicotyledonous plant Brassica. The results show that: the fermentation crude extract of Trichoderma brevicompactum can inhibit the germination of Chinese cabbage seeds by 100% under the dosage of 20-200 μ g/ml; the mixture of the compounds 1 and 2 can inhibit the germination of the Chinese cabbage seeds by 100 percent under the dosage of 2,4,6,8 and 10 mu g/ml; in potting experiments, the mixture of compounds 1, 2 was at 50. mu.g/cm²The spray can inhibit germination and growth of pakchoi. The compound 1, 2 mixture also had inhibitory effect on the monocot Echinochloa crusgalli (L.) Beauv. In addition, the growth inhibitory effect of the mixture of compounds 1 and 2 on mammalian cells was evaluated by in vitro cytotoxic activity assay. The results show that: the mixture of compounds 1 and 2 showed no significant growth Inhibition (IC) on four human tumor cells (A549, HepG2, HeLa and MCF-7) and one Vero cell₅₀>50.0 μ M), indicating that mixtures of compounds 1 and 2 are safe to humans and mammals. Therefore, the compounds 1 and 2 and the Trichoderma brevicompactum producing strain thereof are expected to be developed into green, environment-friendly, efficient and low-toxic herbicide agents and have wide application prospects.

Drawings

FIG. 1 alignment of ITS sequences of tested strains with those of Trichoderma brevicompactum standard strain.

FIG. 2 shows the inhibitory activity of Trichoderma brevicompactum fermentation crude extract on the germination of Brassica rapa.

FIG. 3 liquid chromatogram of crude fermentation extract of Trichoderma brevicompactum at 210 nm. FIG. 4 liquid chromatogram of chloroform fraction at 210 nm.

FIG. 5 is a liquid chromatogram of the mixture of compounds 1-2 separated and purified at 210 nm. .

FIG. 6 inhibitory Activity of Compound 1-2 mixture on the Germination of Brassica rapa.

FIG. 7 inhibitory Activity of Compound 1-2 mixture on growth of Brassica rapa.

FIG. 8 the inhibitory activity of the herbicide 24D on the growth of Brassica rapa.

FIG. 9 inhibition of growth of Brassica rapa by Compounds 1-2 and herbicide 24D in potting experiments (post-emergence application).

FIG. 10 inhibitory Activity of Compounds 1-2 and herbicide 24D on growth of Brassica rapa in potting experiments (Pre-emergence application).

FIG. 11 Effect of Compounds 1-2 and herbicide 24D on the germination percentage of Brassica rapa seeds.

FIG. 12 inhibitory Activity of Compound 1-2 mixtures on Rice Germination and growth.

FIG. 1324D inhibitory Activity on barnyard grass growth.

FIG. 14 inhibition of barnyard grass root and stem growth by compound 1-2 mixture.

Detailed Description

The invention is further illustrated by the following detailed description of specific embodiments, which are not intended to be limiting but are merely exemplary.

Example 1: ITS identification of Trichoderma brevicompactum

1.1 instruments and materials

Strains used in the experiment: the Trichoderma brevicompactum is sourced from the biotechnology institute of Chinese academy of agricultural sciences, and is stored in the laboratory of the inventor. The strain is preserved by China general microbiological culture Collection center (CGMCC) at 9.4.2020 (the preservation address is No. 3 of Silu 1. Cheng. in the area of the rising area in Beijing), the preservation number is CGMCC No.19618, the strain is classified and named as Trichoderma brevicompactum, and the strain survives after detection. The reagents used in the experiment are all domestic analytical pure products. Culture medium: PDA and PDB medium (purchased from BD Difco, USA). The CTAB method is used for DNA extraction, and all reagents are analytically pure.

Other experimental procedures not specifically indicated for the conditions in the examples were carried out according to conventional methods, molecular cloning being carried out according to the conditions described in the Laboratory manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the conditions recommended by the manufacturer.

1.2 Experimental methods

After the Trichoderma brevicompactum was inoculated and cultured in 10ml of PDB liquid medium for seven days, the PDB medium was blotted and the mycelia were collected.

DNA extraction: grinding mycelium in liquid nitrogen, placing the mycelium in CTAB lysate, carrying out water bath at 65 ℃ for 30min, extracting DNA extracting solution (phenol/chloroform/isoamyl alcohol 25:24:1) for 2 times, extracting chloroform for 1 time, sucking supernatant, adding equal volume of anhydrous ethanol and one tenth volume of 3mol/L sodium acetate (pH 5.2), precipitating at 20 ℃ for 1 hour, centrifuging at 12000rpm for 10min, discarding supernatant, washing precipitate with 70% ethanol twice, drying, and dissolving with 50 mu L buffer solution.

Amplification of ITS sequences:

the amplification primers are ITS universal primers ITS1 and ITS4, and the primer sequences are as follows:

ITS1:5’-TCCGTAGGTGAACCTGCGG-3’

ITS4:5’-TCCTCCGCTTATTGATATGC-3’

PCR amplification was carried out using PrimeSTAR GXL DNA Polymerase kit from Takara.

1.3 Experimental results and analysis

The ITS sequence obtained by sequencing is searched by using the NCBI Blastn program, the base consistency with the Trichoderma brevicompactum strain 296 is 100 percent, and the unknown strain is identified as Trichoderma brevicompactum.

The sequence alignment of the strain to be tested and Trichoderma brevicompactum strain 296ITS is shown in figure 1.

Example 2: activity assay for Trichoderma brevicompactum fermentation crude extract

2.1 purpose of the experiment

Testing the inhibitory activity of the fermentation crude extract of Trichoderma brevicompactum on the germination of pakchoi

2.2 Experimental methods

After 1L of PDA plate solid is fermented for 14 days, PDA is cut into blocks, methanol with the same volume is used for extraction for three times, the organic phase is decompressed and concentrated (45 ℃ water bath) to obtain a total extract, 5mL of methanol is used for redissolving, the obtained crude extract is fully dissolved by methanol, then the centrifugation and concentration are carried out again for standby, and the methanol is used for dissolving until the concentration is 100 mug/muL.

Adding 50 μ L of water into a 96-well plate, adding 0.2-2.2 μ L of dissolved crude extract, adding 5 seeds (with smooth surface, smooth seeds, no crack, complete phase), and observing growth conditions at room temperature of 25 deg.C.

2.3 Experimental results and analysis

Adding the crude extract of fungus to a final concentration of 0.2mg/mL or more, the Chinese cabbage seed will not germinate.

The inhibitory activity of the crude extract of Trichoderma brevicompactum fermentation on the germination of Brassica rapa is shown in FIG. 2.

EXAMPLE 3 isolation and Structure characterization of Compounds 1-2

3.1 instruments and materials

The solvents chloroform, methanol and ethyl acetate used in the experiment are produced by Beijing chemical reagent factory and are all analytically pure. Chromatographically pure acetonitrile is produced by Fisher Chemical company. The column chromatography silica gel is produced by Qingdao ocean factory (200-300 mesh). The thin-layer silica gel chromatography plate is produced by a cigarette bench yellow affair silica gel development and test factory. BUCHI R-300 rotary evaporator, BUCHI I-300 touch control central control unit, BUCHI V-300 PTFE film vacuum pump, and BUCHI B-300 electric heating constant temperature water bath, all manufactured by BUCHI corporation of Switzerland. The low-temperature cooling liquid circulating pump is produced by Shanghai Xin laboratory instruments and technologies, Inc. The semi-preparative HPLC is Agilent 1260 Infinity II, and the C18 column is Agilent ZORBAX RX-C18(5 μm, 9.4X 250 mm). The HRESI-MS instrument is an Agilent MS 6530Q-TOF type mass spectrometer, and is directly subjected to sample injection and measurement. The HPLC is Agilent 1290 Infinity II, column RRHD Eclipse Plus C18,4.6X100 mm. The NMR spectrum is measured by Bruker AVIII 400M superconducting NMR spectrometer, and is calibrated by the peak of the residual solvent of deuterated dimethyl sulfoxide produced by Cambridge Company (CIL) in America.

3.2 Experimental methods

The detection method comprises the following steps: and (3) centrifuging the fermentation product at a high speed, and detecting the fermentation product by High Performance Liquid Chromatography (HPLC) and high resolution mass spectrometry (HRMS/MS). The HPLC detection conditions were as follows: performing gradient elution by using methanol-water as a mobile phase, wherein the gradient elution condition is as follows: 0-20min, methanol from 10% → 100%; 20-23min, methanol is 100%; 23-23.01min, methanol from 100% → 10%, 23.01-25min, methanol 10%, flow rate 0.5ml/min, detection wavelength 210 nm. HRMS/MS detection conditions were as follows: negative ion mode, collision energy 25V.

The separation method comprises the following steps: extracting 4L PDA plate solid culture with methanol of the same volume for 3 times, and concentrating organic phase under reduced pressure to obtain total extract (1.8 g). And (3) completely dissolving the total extract by 80% methanol, removing impurities by using n-hexane, dissolving by using 50% methanol, adding equal volume of chloroform to obtain a chloroform layer, and concentrating the organic phase under reduced pressure to obtain a chloroform layer compound. Performing gradient elution preparation by using semi-preparative high performance liquid chromatography, wherein the gradient elution conditions are as follows: 0-30min, methanol from 10% → 95%; 30-45min, 95% → 10% methanol, 45.01-48min, 10% methanol.

3.3 Experimental results and analysis

Separating chloroform layer by semi-preparative high performance liquid chromatography, collecting fraction, and retaining time (min) as follows: peak 1: 14.685, respectively; peak 2: retention time: 16.866, respectively; peak 3: retention time: 25.132, Peak 4: retention time: 31.407, respectively; peak 5: retention time: 34.347, respectively; peak 6: retention time: 31.916, Peak 7: Retention time 33.329, Peak 4 gave a 12.5mg yield of compound.

The structure of the compound 1-2 is identified as shown in the formula (1) by analyzing a high-resolution mass spectrum and one-dimensional and two-dimensional nuclear magnetic resonance spectrums.

Wherein, the compound 1 is reported Harzianum A, the compound 2 is reported Harzianum B, and the two are cis-trans isomers. The structural identification data for compounds 1 and 2 are as follows:

compounds 1 and 2: high resolution mass spectrometry M/z 401.2003[ M + H ]]⁺Calcd.401.1964 with molecular formula C₂₃H₂₈O₆；¹H (400MHz) and¹³the C NMR (100MHz) data are shown in Table 1.

TABLE 1 NMR data for Compounds 1 and 2 (deuterated methanol as test solvent)

The liquid chromatogram of the crude extract of fungi at 210nm is shown in FIG. 3.

The liquid chromatogram of the chloroform fraction at 210nm is shown in FIG. 4.

The liquid chromatogram of the mixture of compounds 1 and 2 after separation and purification at 210nm is shown in FIG. 5.

Example 4: mixture of compound 1 and compound 2 for inhibiting germination of pakchoi seeds

4.1 purpose of the experiment

The mixture of the compounds 1 and 2 after separation and extraction was tested for its inhibitory activity against the germination of pakchoi.

4.2 Experimental methods

After the mixture of the compounds 1 and 2 obtained by separation and extraction was sufficiently dissolved in methanol, it was again concentrated by centrifugation and dissolved in methanol to a concentration of 1. mu.g/. mu.L.

4.3 Experimental results and analysis

When the mixture of the compounds 1 and 2 is added to the final concentration of more than 0.002mg/mL, the seeds of the pakchoi do not sprout.

The inhibitory activity of the mixture of compounds 1 and 2 on the germination of pakchoi is shown in fig. 6.

Example 5: mixture of compound 1 and compound 2 for inhibiting growth of pakchoi

5.1 purpose of the experiment

Test the inhibitory Activity of the mixtures of Compounds 1 and 2 on the growth of Brassica rapa and compare it with the Positive control 24D

5.2 Experimental methods

The mixture of compounds 1 and 2 was dissolved in methanol to prepare solutions at concentrations of 100. mu.g/. mu.L, 10. mu.g/. mu.L, 1. mu.g/. mu.L, 100 ng/. mu.L, 10 ng/. mu.L, 1 ng/. mu.L.

Positive control 24D was dissolved in methanol and prepared into solutions at concentrations of 100. mu.g/. mu.L, 10. mu.g/. mu.L, 1. mu.g/. mu.L, 100 ng/. mu.L, 10 ng/. mu.L, 1 ng/. mu.L.

Preparing water agar culture medium, adding 8g/L agar powder, and sterilizing at 121 deg.C for 20 min. And (5) pouring the plate.

Sterilizing the surfaces of the pakchoi seeds: washing the Chinese cabbage seed with sterile water, soaking in 0.1% sodium hypochlorite solution for 3min, and washing with sterile water for 5-6 times. After washing, the pakchoi seeds are dried.

And (3) dropping the surface-sterilized pakchoi seeds on a poured water agar plate, dropping only one seed each time, wherein the distance between every two seeds is about 1.5cm, then culturing at 25 ℃, and observing the growth condition of the plants in 24h, 16h illumination and 8h darkness every day.

The Chinese cabbage seeds are placed on a 0.8% water agar culture medium, 1 mu L of the prepared mixture solution of the compound 1 and the compound 2 or the positive drug 24D solution is dripped, and a methanol negative control is arranged at the same time.

5.3 Experimental results and analysis:

TABLE 2 growth of the aerial and underground parts of pakchoi when the mixture of compounds 1 and 2 is added

	Aerial parts/mm	Underground part/mm
			control	4.10±0.10a	83.00±1.00a
100μg	0.00b	0.00±0.00e
			10μg	0.00b	2.00±0.00d
1μg	4.10±0.10a	5.67±0.58c
			100ng	4.17±0.15a	66.67±1.53b
10ng	4.20±0.10a	68.00±1.00b
			1ng	4.10±0.10a	81.33±1.53a

TABLE 3 growth of the aerial and underground parts of pakchoi with 24D addition of herbicide

	Aerial parts/mm	Underground part/mm
			control	4.10±0.10a	81.00±1.00a
100μg	3.23±0.25b	0.00±0.00f
			10μg	3.30±0.20b	0.00±0.00f
1μg	4.00±0.10a	26.00±1.00e
			100ng	4.03±0.06a	66.00±1.00d
10ng	4.10±0.17a	68.33±：1.53c
			1ng	4.10±0.10a	70.33±0.58b

The addition amount of the mixture of the compounds 1 and 2 is 1ng-100 mu g, the growth inhibition degree of the Chinese cabbage is stronger along with the increase of the amount of the compounds, the length of the Chinese cabbage root is gradually shortened (p is less than 0.05) when the amount of the compounds is 1ng-1 mu g, no significant difference is found in the growth condition of the overground part, the growth of the Chinese cabbage root can be influenced by the compounds 1 and 2, and the Chinese cabbage seed does not sprout when the addition amount of the compounds 1 and 2 is 100 mu g.

Compared with 24D herbicide, the compounds 1 and 2 have the same capacity of inhibiting the growth of underground parts as 24D under the dosage of less than 100ng, are slightly inferior to 24D when being 1-10 mu g, and inhibit the seed germination when being 100 mu g stronger than 24D.

The inhibitory activity of the mixture of compounds 1 and 2 on the growth of pakchoi is shown in fig. 7.

The inhibitory activity of the herbicide 24D on the growth of pakchoi is shown in FIG. 8.

Example 6: potting experiment for inhibiting germination and growth of pakchoi by using compound 1 and compound 2 mixture

6.1 purpose of the experiment

Test of the Activity of the mixture of Compounds 1 and 2 in inhibiting the Germination and growth of Brassica rapa in the soil Environment

6.2 Experimental methods

Compounds 1 and 2 and the positive control 24D were formulated as 10mg/mL solutions.

And (3) filling the uniformly mixed nutrient soil into culture pots, adding a proper amount of clear water into the culture pots to moisten the nutrient soil, and compacting the surfaces of the nutrient soil to make the height of the nutrient soil of each pot consistent. Then 15 seeds of Chinese cabbage are sown in each pot, a layer of dry nutrient soil is lightly scattered on the surface, and 500 mu L of methanol, compound 1-2 solution and 24D solution are respectively sprayed on the surfaces of different culture pots uniformly. The culture conditions are set as 25 ℃, 24 hours a day, 16 hours of light and 8 hours of darkness, and the growth condition of the plants is observed.

6.3 Experimental results and analysis

For the inhibitory activity of the compound 1 and 2 mixtures and the herbicide 24D on the growth of pakchoi (post-emergence application) in potting experiments see figure 9.

For the inhibitory activity of compounds 1 and 2 and herbicide 24D on the growth of pakchoi in potting experiments (pre-emergence application) see figure 10.

The effect of compounds 1 and 2 and herbicide 24D on the germination rate of pakchoi seeds is shown in fig. 11.

Compared with positive control methanol, compounds 1 and 2 and herbicide 24D both inhibited the growth of pakchoi (fig. 9), but 24D did not significantly reduce the germination rate of pakchoi seeds, while compounds 1 and 2 caused a reduction in the germination rate of pakchoi seeds (fig. 10).

Example 7: inhibition of growth of monocot barnyard grass by mixtures of compound 1 and compound 2

7.1 purpose of the experiment

The compounds 1 and 2 mixtures were tested for their inhibitory activity against barnyard grass growth.

7.2 Experimental methods

Compounds 1 and 2 were dissolved in methanol to prepare solutions at concentrations of 100. mu.g/. mu.L, 10. mu.g/. mu.L, 1. mu.g/. mu.L, 100 ng/. mu.L, 10 ng/. mu.L, 1 ng/. mu.L.

Surface disinfection of barnyard grass seeds: the seeds are washed with sterile water, then soaked in 0.1% sodium hypochlorite solution of available chlorine for 3 minutes, and washed with sterile water for 5 to 6 times. After washing, the seeds are air dried.

And (3) dropping barnyard grass seeds with the surfaces sterilized on a poured water agar plate, dropping only one seed each time, wherein the distance between every two seeds is about 1.5cm, culturing at 25 ℃, and observing the growth condition of the plants in 24h each day, 16h of light and 8h of dark.

Barnyard grass seeds are placed on a 0.8% water agar culture medium, and 1 mu L of the prepared compound 1 and 2 solution or the positive drug 24D solution is dripped, and meanwhile, a methanol negative control is arranged.

7.3 Experimental results and analysis:

the addition amount of the mixture of the compounds 1 and 2 is 1ng-100 mu g, the growth inhibition degree of the barnyard grass is stronger along with the increase of the amount of the compounds, the length of the barnyard grass root and stem is gradually shortened (p is less than 0.05) when the amount of the compounds is 0.1-100 mu g, which shows that the compounds 1 and 2 can influence the growth of the barnyard grass, and the barnyard grass seeds do not sprout when the addition amount of the compounds 1 and 2 is 100 mu g.

Compared with the herbicide 24D, the compounds 1 and 2 have slightly lower capability of inhibiting the growth of underground parts than 24D at the dosage of below 100 mu g.

The inhibitory activity of the mixture of compounds 1 and 2 on barnyard grass growth is shown in FIG. 12.

The inhibitory activity of 24D on barnyard grass growth is shown in FIG. 13.

The inhibition of barnyard grass root and stem growth by the compound 1 and 2 mixtures is shown in FIG. 14.

Example 8: cytotoxic Activity of Compound 1 and Compound 2 mixtures

8.1 instruments and materials

The microplate reader is a Genois microplate reader (Tecan GENios, Swizerland); four human-derived tumor cell lines, namely a human lung cancer A549 cell, a human hepatoma HepG2 cell, a human cervical cancer HeLa cell, a human breast cancer MCF-7 cell and an African green monkey kidney epidermis Vero cell, are purchased from Kunming animal institute of Chinese academy of sciences; fetal bovine serum FBS, RPMI1640 medium, DMEM medium purchased from Gibco, USA; 3- (4, 5-Dimethylthiazol-2) -2, 5-diphenyltetrazolium bromide salt (MTT) was purchased from Amresco, USA.

8.2 Experimental methods

Human lung cancer A549 cells, human liver cancer HepG2 cells, human cervical cancer HeLa cells, human breast cancer MCF-7 cells and African green monkey kidney epidermis Vero cells in logarithmic growth phase are taken and added into a 96-well plate, and each well contains about 5000 cells. Adding compounds 1 and 2 with different concentrations, using adriamycin as a positive control, using a cell hole without a sample as a control group, arranging 4 parallel holes in each group, placing the group in a carbon dioxide incubator at 37 ℃ for culturing for 72 hours, adding 20 mu L of MTT (5mg/mL) solution 4 hours before the experiment is ended, culturing for 4 hours, removing the culture solution, adding 150 mu L of DMSO, and detecting the OD value of each hole under the wavelength of 570nm on an enzyme-linked detector after the crystal is dissolved. The growth inhibition rate was determined according to the following formula, and the median Inhibitory Concentration (IC) was determined by SPSS (17.0) software₅₀Value). The results are shown in Table 2, in which IC₅₀Expressed as Mean ± s.e.m.

8.3 Experimental results and analysis

As can be seen from the results in table 4, compounds 1 and 2 showed no significant cytotoxicity to mammalian cells.

TABLE 4 tumor cell growth inhibitory Activity (IC) of Compounds 1 and 2₅₀,μM)

Claims

1. Use of the compounds harzianum a, harzianum B or mixtures thereof as herbicidal agents.

2. Use according to claim 1, characterized in that the weeding is against dicotyledonous or monocotyledonous weeds, preferably dicotyledonous weedsBrassicaGenus, monocotEchinochloaWeeds of the genus, more particularly barnyard grass.

3. A compound harzianum A and harzianum B producing bacterium (A)Trichoderma brevicompactum) The strain has the following strain preservation number: CGMCC No. 19618.

4. A method for producing compounds of Harzianum A and Harzianum B by using the strain of claim 3, wherein the compounds of Harzianum A and Harzianum B are separated from thalli of the strain by culturing the strain, and the compounds are identified by chromatography, high pressure liquid chromatography, separation and purification and high performance liquid chromatography-mass spectrometry.

5. Use of the strain and the fermentation product thereof according to claim 3 for weeding against dicotyledonous or monocotyledonous weeds, preferably dicotyledonous weedsBrassicaGenus, monocotEchinochloaWeeds of the genus, more particularly barnyard grass.

6. The use as claimed in claim 5, wherein the fermentation product is a crude extract obtained by solid fermentation of PDA, primary extraction with equal volume of methanol, evaporation and drying.

7. A herbicide, which comprises as an active ingredient a compound of Harzianum A, Harzianum B or a mixture thereof, or a fermentation product of the strain of claim 3, dissolved in methanol and diluted with water to use as a solution.

8. Herbicide formulation according to claim 7, characterized in that the herbicide formulation is a dicotyledonous or monocotyledonous weed, preferably a dicotyledonous plantBrassicaGenus, monocotEchinochloaWeeds of the genus, more particularly barnyard grass.