Application of novel TeAs compound with herbicidal activity

Abstract

The invention belongs to the field of microbiology and agricultural chemistry, and provides an application of a fermentation metabolite of a huperzia serrata endophytic fungus Alternania alternania FL7 in weeding activity. A series of novel TeAs substances are obtained by separating endophytic fungi FL7 through a plurality of separation means, and the substances are used for an arabidopsis inhibition experiment. The compound disclosed by the invention is remarkable in herbicidal activity, can be used as a broad-spectrum herbicide, and is environment-friendly, low in toxicity and safe to human beings.

Description

Technical Field

[0001] The invention belongs to the fields of microbiology and agricultural chemistry, and in particular relates to the application of a new type of TeAs compound with herbicidal activity. Background Art

[0002] Weeds are considered to be harmful plants and are often unwanted flora in artificial environments (such as green spaces, farmlands, grasslands, and parks) and natural areas (Radhakrishnan R, Alqarawi AA, Abd_Allah EF. Bioherbicides: Current knowledge on weed control mechanism [J]. Ecotoxicology and Environmental Safety, 2018, 158: 131-138). Weeds compete with crops for soil moisture and nutrients, and even sunlight and space (Fang W, Liu F, Wu Z, et al. Plant-Associated Bacteria as Sources for the Development of Bioherbicides [J]. Plants, 2022, 11 (23): 3404). Many weed species also serve as alternative hosts for insect pests or pathogens (Kumar S, Bhowmick MK, Ray P. Weeds as alternate and alternative hosts of crop pests [J]. INDIAN JOURNAL OF WEED SCIENCE, 2021, 53 (1): 14-29). Gharde et al. evaluated the yield and economic losses of 10 major crops in India and estimated that the total economic losses due to weeds alone were approximately US$11 billion (Gharde Y, Singh P K, Dubey RP, et al. Assessment of yield and economic losses in agriculture due to weeds in India [J]. Crop Protection, 2018, 107: 12-18).Between 2007 and 2013, weed disturbance in North American soybeans caused an average yield loss of 52.1%, equivalent to approximately US$17.2 billion per year (Soltani N, Dille JA, Burke IC, et al. Potential Corn Yield Losses from Weeds in North America [J]. Weed Technology, 2016, 30(4): 979-984); in corn, it caused a yield loss of 52%, equivalent to US$28 billion per year (Soltani N, Dille JA, Burke IC, et al. Perspectives on Potential Soybean Yield Losses from Weeds in North America [J]. Weed Technology, 2017, 31(1): 148-154). Weeds in U.S. pastures are estimated to cause losses of more than $2 billion each year due to reduced forage yield and quality, interference with grazing, poisoning animals, increased livestock management and production costs, and reduced land value (Soltani N, Dille JA, Gulden RH, et al. Potential Yield Loss in Dry Bean Crops Due to Weeds in the United States and Canada [J]. Weed Technology, 2018, 32 (3): 342-346). In order to prevent further expansion of crop and economic losses related to weeds, it is urgent to find an efficient means of weed control.

[0003] Currently available weed control methods include manual weeding, tillage, and the use of herbicides. Each technology has its own advantages and disadvantages (Beckie HJ, Ashworth MB, Flower K C. Herbicide Resistance Management: Recent Developments and Trends [J]. Plants, 2019, 8 (6): 161): Manual weeding is time-consuming and expensive, especially for poisonous, thorny and perennial weeds. It is very difficult to deal with cleanly. Tillage is a mechanical weed control method. It effectively controls weeds, but tillage exposes the fertile surface soil to water and wind erosion. Herbicides (mainly chemical herbicides, such as glyphosate, dicamba and 2,4-dichlorophenoxyacetic acid (2,4-D, etc.) can effectively control weeds and are now widely used to inhibit the germination and growth of weeds. However, the long-term use of chemical herbicides will be limited by the emergence of resistant weed populations. At present, 267 weed species (154 dicotyledons and 113 monocotyledons) have been reported to be resistant to 21 of the 31 known herbicide action sites and 165 different herbicides. In addition, weeds can also pollute water and soil, reduce soil fertility, destroy soil structure, pollute water through surface runoff, kill non-target plant crops, and even cause harm to a variety of organisms including humans, such as cancer, Parkinson's disease, etc. (Nez JCJ, Irizarry KB, Cordero HO, et al. GLYPHOSATE POISONING: COMMON WEED KILLER, BUTRARE HEART KILLER[J].Journal of the American College of Cardiology,2021,77(18):2074) diseases. The spread of herbicide-resistant weeds further exacerbates the problem of crop yield reduction. Therefore, it is urgent to establish some new environmentally friendly weed control technologies to address these limitations. The discovery of microbial herbicides has the potential to overcome these limitations.

[0004] Microbial herbicides refer to products derived from organisms or their natural metabolites, which can control weed populations without damaging the environment (Anwar T, Qureshi H. Role of biocontrol agents in weed management – ​​recent developments and trends [J]. Acta Scientiarum Polonorum Hortorum Cultus, 2022, 21 (6): 155-162). Microbial herbicides degrade quickly in the environment, leave little residue, and have little impact on soil, water and ecosystems, which meets the development requirements of green agriculture. In addition, microbial herbicides usually have strong host specificity, are safer for non-target organisms (such as crops), will not damage the ecological balance, and have less health risks to humans and animals. Microbial herbicides can control weeds through a variety of mechanisms (such as direct infection, secretion of toxins, interference with metabolism, etc.), while chemical herbicides usually act through a single target, and long-term use can easily lead to weed resistance. The application of microbial herbicides will not fail due to the increase of weed resistance, thus providing a sustainable solution for long-term weed control. Microbial resources (such as microorganisms in the soil) are widely present in nature, easy to separate and screen, and can be produced through fermentation technology. The resource utilization efficiency is high and the source is sustainable. The application of microbial herbicides is in line with the goal of sustainable agricultural development, reduces the side effects of chemical pesticides (Lei Q, Zhong J, Chen SF, et al. Microbial degradation as a powerful weapon in the removal of sulfonylureaherbicides [J]. Environmental Research, 2023, 235: 116570), reduces the pressure on the environment, and has become a potential alternative to chemical herbicides. Its commercial development has attracted much attention (Radhakrishnan R, Alqarawi AA, Abd_Allah E F. Bioherbicides: Current knowledge on weed control mechanism [J]. Ecotoxicology and Environmental Safety, 2018, 158: 131-138).

[0005] However, there are very few microbial herbicides that have been successfully applied, and the market for microbial herbicides is vast. Summary of the invention

[0006] In view of this, the object of the present invention is to provide an application of a new class of TeAs compounds having herbicidal activity.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] After 30 days of large-scale culture of the endophytic fungus FL7, TeAs compounds 1-9 were isolated using macroporous resin, forward and reverse column chromatography, etc.

[0009] New compounds 3-9 were selected as the TeAs herbicidal active substances for study. Arabidopsis seeds were surface sterilized by soaking in 1% sodium hypochlorite for 10 minutes, then rinsed with sterile water 5 times, and dried with sterile filter paper. The seeds were vernalized at 4°C for 48 hours to break dormancy, and then sown in a six-well plate containing 1 / 2MS solid culture medium. The seeds were cultured at 22°C, 16 hours light / 8 hours dark for 7 days to obtain uniform Arabidopsis seedlings.

[0010] The Arabidopsis in the incubator was taken out for observation and photographing every other day for a total of 6 days. By observing the growth and yellowing state of the leaves, it was found that the Arabidopsis leaves to which the compound was added showed yellowing or withering to varying degrees. However, no similar situation was found in the blank control group. The TeAs-based substances of the present invention can be further synthesized into substances with higher herbicidal activity through structural modification and optimization.

[0011] Compared with the prior art, the present invention has the following beneficial effects:

[0012] The series of new TeAs compounds of the present invention have significant herbicidal activity against Arabidopsis. Substances with better herbicidal activity can be developed around such substances by biological or chemical means. The TeAs substances of the present invention can be used as lead compounds for the synthesis of new pesticides or lay the foundation for the development of new herbicides. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The structures of TeAs compounds 1-9;

[0014] Figure 2 The following are the herbicidal activity results of compounds 3-9. DETAILED DESCRIPTION

[0015] Endophytic fungus FL7 was inoculated on PDA plate medium for activation culture for 7 days (28°C). Mycelium was picked up with a sterilized bamboo stick and expanded into a 250mL conical flask containing 120mL PDB medium for 5 days (28°C, 120rpm). 500 bottles of rice solid medium (80g rice, 120mL tap water) were prepared, 5mL seed solution was inoculated into each bottle, and static culture was carried out at 28°C for 30 days.

[0016] After the cultivation is completed, methanol is used for extraction 11 times, the volume ratio of the culture to methanol is 2:1, each time for 24 hours, and 10.5 kg of methanol extract (containing solvent) is obtained. The methanol extract is dissolved in ultrapure water (1:1, v / v) to form an aqueous suspension, which is extracted 3 times with petroleum ether and ethyl acetate respectively, the volume ratio of the aqueous suspension to petroleum ether is 1:1, and the volume ratio of the aqueous suspension to ethyl acetate is 1:1, to obtain petroleum ether extract and ethyl acetate extract (165 g).

[0017] The ethyl acetate extract (165 g) was subjected to a coarse separation using a macroporous resin, and then gradient eluted using pure water, 20% ethanol, 50% ethanol, 70% ethanol, and 100% ethanol, and was separated into 6 components according to polarity from small to large, namely Fr.1 (23.2 g), Fr.2 (14.6 g), Fr.3 (19.7 g), Fr.4 (32.5 g), Fr.5 (26.7 g), and Fr.6 (21.3 g).

[0018] The extracts of the six components were mixed with 1% DMSO to form an appropriate amount of reagent and then dripped onto Arabidopsis leaves. The analysis results showed that Fr.1 had the highest inhibitory activity on Arabidopsis, which was manifested by large-area yellowing on Arabidopsis leaves and wilting of Arabidopsis leaves. It is speculated that this component contains metabolites with relatively strong herbicidal ability, so this component is separated first.

[0019] First, Fr.1 (19.7 g) was crudely separated using an MCI column. MeOH:H 2 O = 1:60, 1:40, 1:20, 1:10, 1:5, 1:2, 1:1 and 1:0, which were separated into 8 sub-fractions, named Fr.1.1–8. After rotary evaporation and concentration, the separation of the 10 sub-fractions was determined using a TLC plate. The Fr.1.1 (4.2 g) fraction was mixed with silica gel and subjected to silica gel column chromatography using a dry loading method. CH 2 Cl 2 :MeOH=40:1-0:1 stepwise gradient elution, and finally five super subfractions were obtained, named F r.1.1.1-5. Fr.1.1.1 (150 mg) was purified by semi-preparative liquid phase purification (acetonitrile: water, 80:20, v / v) to obtain compound 1 (12.7 mg; t R =7.3min). Then, the components of Fr1-6 were analyzed by GNPS molecular network, and a series of TeAs-type substances 2-9 were obtained by auxiliary separation with the molecular weight of compound 1 as the center point, with the structural formula as shown in Figure 1 shown.

[0020] Determination of herbicidal activity:

[0021] In order to study the herbicidal activity of novel TeAs compounds, the present invention uses the model plant Arabidopsis thaliana as the research object. Firstly, Arabidopsis thaliana seeds are subjected to surface sterilization and germination treatment.

[0022] The specific steps are as follows:

[0023] Arabidopsis seeds were surface sterilized by soaking in 1% sodium hypochlorite for 10 minutes, then rinsed with sterile water 5 times, and dried with sterile filter paper. The seeds were vernalized at 4°C for 48 hours to break dormancy, and then sown in a six-well plate containing 1 / 2MS solid medium. They were cultured at 22°C, 16 hours light / 8 hours dark for 7 days to obtain uniform Arabidopsis seedlings.

[0024] Arabidopsis seedlings with good growth and more than 6 leaves were selected as research objects. The compound was dissolved in 1% DMSO and prepared into a 1mg / ml solution. At the same time, 1% DMSO solution was used as a blank control group, and glyphosate was used as a positive control group. 5μL of each of the experimental group, negative and positive controls was pipetted and dropped on the surface of Arabidopsis leaves, and placed in a light incubator for observation for 6 days.

[0025] Experimental results:

[0026] New compounds 3, 4, 5, 6, 7, 8 and 9 were selected as the TeAs herbicidal active substances for research. The Arabidopsis thaliana in the incubator was taken out for observation and photographed every other day for a total of 6 days. The growth and yellowing state of the leaves were observed. Figure 2 As shown, it was found that the leaves of Arabidopsis thaliana to which the compounds and glyphosate were added had different degrees of wilting. The positive control group to which glyphosate was added had the most severe wilting, while the blank control group had no obvious wilting and yellowing. In the experimental group to which the compounds were added, it was found that the yellowing effect of compound 9 on Arabidopsis thaliana was the most obvious. On the 6th day, the leaf tips of this group of Arabidopsis thaliana had all turned yellow and had a tendency to spread to the rhizomes of Arabidopsis thaliana, which was exactly in line with the expectation of adding compounds to the leaf tips of Arabidopsis thaliana, while compound 7 had obvious lesions with black spots, which fully demonstrated that this type of compound has significant herbicidal activity.

[0027] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of fermentation metabolites of the endophytic fungus Alternaria alternata FL7 of Huperzia serrata in herbicidal activity, characterized in that: The fermentation metabolites are TeAs substances, compounds 1-9, the molecular formulas are C 10 H 16 N2O2、C 13 H 20 N2O4、C 16 H 26 N2O4、C 16 H 26 N2O4、C 17 H 28 N2O4、C 19 H 24 N2O4、C 20 H 26 N2O5、C 22 H 27 N3O4、C 16 H 26 N2O4.

2. Use of the TeAs substance according to claim 1 in the preparation of herbicides.

3. The method according to claim 2, characterized in that The TeAs-based substance is compound 3-9.

4. The method according to claim 2, characterized in that: The weeds were selected from the model plant Arabidopsis thaliana.

5. The method according to claim 2, characterized in that: Arabidopsis seeds were surface sterilized by soaking in 1% sodium hypochlorite for 10 minutes, then rinsed with sterile water 5 times, and dried with sterile filter paper; the seeds were vernalized at 4°C for 48 hours to break dormancy, and then sown in a six-well plate containing 1 / 2MS solid culture medium; they were cultured at 22°C, 16 hours light / 8 hours dark for 7 days to obtain uniform Arabidopsis seedlings.

6. The method according to claim 2, characterized in that Arabidopsis seedlings with good growth and more than 6 leaves were selected as the research objects. The compound was dissolved in 1% DMSO to prepare a 1 mg / ml solution. At the same time, 1% DMSO solution was used as a blank control group and glyphosate was used as a positive control group. 5 μL of each of the experimental group, negative control and positive control were pipetted and dropped on the surface of Arabidopsis leaves, and the leaves were placed in a light incubator for observation for 6 days.

Images