Strain mc-m1, bio-herbicide for preventing and controlling artemisia and application thereof

Abstract

This invention provides a strain MC-M1 for controlling Artemisia weeds, a biological herbicide, and its application, belonging to the field of Artemisia control technology. The gene sequence of strain MC-M1 is shown in SEQ ID NO: 1, and it was isolated from the leaves of naturally infected alfalfa. The fermentation broth of this strain showed a 100% inhibition rate on the germination of Artemisia seeds and an 83.33% disease incidence rate after spraying potted Artemisia. When its fermentation broth was mixed with a 1:1 volume ratio of bensulfuron-methyl solution at a bensulfuron-methyl concentration of 0.1165 g / L, the resulting compound microbial herbicide improved the control effect of Artemisia to 100%, significantly better than the single treatment, achieving synergistic effect. This provides a new strain resource and technical solution for the green control of Artemisia weeds, with good application prospects and ecological value.

Description

Technical Field

[0001] This invention belongs to the field of Artemisia control technology, specifically a strain MC-M1 for controlling Artemisia weeds, a biological herbicide and its application. Background Technology

[0002] Artemisia spp. weeds are a type of noxious weed widely distributed in northern and western my country. Their pollen is a major airborne allergen in spring and autumn, easily triggering respiratory diseases such as allergic rhinitis and bronchial asthma, seriously threatening public health. At the same time, Artemisia plants have strong reproductive capabilities and wide dispersal ranges, competing with crops for water and fertilizer, severely impacting agricultural and livestock production, and have become an ecological and agricultural problem urgently needing to be addressed.

[0003] Most herbicides on the market today are chemical herbicides. Although they are effective in the short term and easy to use, they are prone to causing environmental pollution and herbicide resistance in weeds in the long run. Therefore, developing a green, safe and efficient microbial herbicide that is effective against Artemisia is of great significance and urgent importance for the scientific management of Artemisia and the sustainable development of the ecosystem. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a strain MC-M1 for controlling Artemisia spp. weeds, a biological herbicide, and its application, as detailed below: A biological herbicide for Artemisia species is prepared by mixing fermentation broth of strain MC-M1 with a solution of bensulfuron-methyl at a volume ratio of 1:0.5-2. The gene sequence of strain MC-M1 is shown in SEQ ID NO: 1. Strain MC-M1 is Alternaria alternata, accession number: CGMCC No. 42536; depositary institution: China General Microbiological Culture Collection Center (CGMCC); deposit address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; deposit date: March 25, 2026.

[0005] Preferably, the strain MC-M1 is isolated from the leaves of naturally infected alfalfa plants.

[0006] Preferably, the leaves of the naturally infected alfalfa plant are those with black, brown, or yellowish lesions.

[0007] Preferably, the preparation process of the fermentation broth of strain MC-M1 is as follows: strain MC-M1 is inoculated into liquid PDB medium and cultured in a shaker at 150-210 rpm and 22-28℃ for 6-8 days. After filtering through two layers of gauze to remove mycelia and mycelial cake, the obtained fermentation filtrate is the fermentation broth of strain MC-M1.

[0008] Preferably, the concentration of the phenylsulfuron-methyl is 0.1-0.15 g / L.

[0009] Preferably, the concentration of the phenylsulfuron-methyl is 0.1165 g / L.

[0010] Preferably, the volume ratio of the fermentation broth of strain MC-M1 to the bensulfuron-methyl solution is 1:1.

[0011] Preferably, the Artemisia species is Artemisia argyi.

[0012] On the other hand, the present invention discloses the application of a biological herbicide for Artemisia spp. weeds, wherein the herbicide is applied by spraying it onto the surface of Artemisia spp. weed plants or seeds.

[0013] Thirdly, the present invention provides a strain MC-M1 for controlling Artemisia spp. weeds, the gene sequence of which is shown in SEQ ID NO: 1.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. The strain MC-M1 isolated from naturally infected alfalfa plants provides a novel strain resource for the control of Artemisia spp. weeds. Experimental data show that the fermentation broth of MC-M1 can inhibit the germination rate of Artemisia spp. seeds by up to 100%, and the disease incidence rate of potted Artemisia spp. reached 83.33% after 3 days of spraying. It can effectively inhibit Artemisia spp. at both the seed germination and plant growth stages, solving the problem that existing herbicides are not very targeted to Artemisia spp. weeds.

[0015] 2. This invention screened bensulfuron-methyl as the optimal compound chemical herbicide, and the MC-M1 strain showed good compatibility with half-concentration bensulfuron-methyl (0.1165 g / L). Experimental data showed that after the two were compounded at a volume ratio of 1:1, the incidence rate of potted Artemisia argyi reached 100% after 3 days of treatment. The control effect was far superior to the treatment of MC-M1 fermentation broth alone or bensulfuron-methyl alone, realizing the synergistic effect of microorganisms and chemical herbicides, and quickly and thoroughly controlling Artemisia argyi.

[0016] 3. In the technical solution of the present invention, the concentration of halved bensulfuron-methyl (0.1165 g / L) is significantly reduced compared to the concentration of conventional chemical herbicides. This not only retains the advantages of high efficiency and speed of chemical herbicides, but also significantly reduces the amount of chemical agents used, alleviating the environmental pollution and soil residue problems caused by the long-term use of existing chemical herbicides, and also reducing the probability of weed resistance.

[0017] 4. Artemisia species are noxious weeds in northern and western my country. Their pollen is a major airborne allergen and can also affect agricultural and livestock production. The herbicide of this invention is designed for Artemisia species such as Artemisia aquilina and can effectively inhibit their reproduction and spread. This reduces pollen-induced allergic diseases, protects public health, and alleviates the problem of them competing with crops for water and fertilizer, thereby improving the efficiency of agricultural and livestock production. It provides a brand-new technical solution for the scientific management of Artemisia species in northern and western China and has broad application prospects. Attached Figure Description

[0018] Figure 1 These are leaves of alfalfa that have been naturally infected in the field. Figure 2 The images show the colony morphology of the strain in the dish and the strain morphology under a microscope. In this image, A represents the colony morphology and B represents the microstructure of the strain. Figure 3 The MC-M1 phylogenetic tree constructed for ITS; Figure 4 The results of the test on the inhibition of seed germination of Artemisia argyi by strain MC-M1; Figure 5 Results of pathogenicity testing of potted Artemisia annua; Figure 6 The results show the herbicide control efficacy of potted Artemisia annua; where A is the comparison between the CK group and clopyralid, B is the comparison between the CK group and difenosulfuron, and C is the comparison between the CK group and flufenoxuron. Figure 7 The results of the in-plate compatibility test between strain MC-M1 and benzylsulfamethoxam; Figure 8 The results show the efficacy of compound microbial herbicides in potted Artemisia annua; where A is a comparison between group T0 and group T2, B is a comparison between group T0 and group T1, C is a comparison between group T0 and group T3, and D is a comparison between group T0 and group T4. Detailed Implementation

[0019] Example 1 A biological herbicide for Artemisia species is prepared by mixing fermentation broth of strain MC-M1 with a solution of bensulfuron-methyl at a volume ratio of 1:2. The gene sequence of strain MC-M1 is shown in SEQ ID NO: 1.

[0020] Furthermore, the strain MC-M1 is Alternaria alternata.

[0021] Furthermore, the strain MC-M1 was isolated from the leaves of naturally infected alfalfa (Medicago sativa) plants.

[0022] Furthermore, the preparation process of the fermentation broth of strain MC-M1 is as follows: strain MC-M1 is inoculated into liquid PDB medium and cultured in a shaker at 210 rpm and 28°C for 8 days. After filtering through two layers of gauze to remove mycelia and mycelial cake, the obtained fermentation filtrate is the fermentation broth of strain MC-M1.

[0023] Furthermore, the concentration of the phenoxysulfuron is 0.15 g / L.

[0024] Furthermore, the Artemisia species mentioned is Artemisia argyi.

[0025] Furthermore, the herbicide is sprayed onto the surface of Artemisia plants or seeds.

[0026] Example 2 A biological herbicide for Artemisia species is prepared by mixing fermentation broth of strain MC-M1 with a solution of bensulfuron-methyl at a volume ratio of 1:0.5. The gene sequence of strain MC-M1 is shown in SEQ ID NO: 1.

[0027] Furthermore, the strain MC-M1 is Alternaria alternata.

[0028] Furthermore, the strain MC-M1 was isolated from the leaves of naturally infected alfalfa (Medicago sativa) plants.

[0029] Furthermore, the preparation process of the fermentation broth of strain MC-M1 is as follows: strain MC-M1 is inoculated into liquid PDB medium and cultured in a shaker at 150 rpm and 22°C for 6 days. After filtering through two layers of gauze to remove mycelia and mycelial cake, the obtained fermentation filtrate is the fermentation broth of strain MC-M1.

[0030] Furthermore, the concentration of the phenoxysulfuron is 0.1 g / L.

[0031] Furthermore, the Artemisia species mentioned is Artemisia argyi.

[0032] Furthermore, the herbicide is sprayed onto the surface of Artemisia plants or seeds.

[0033] Example 3 A biological herbicide for Artemisia spp. weeds is prepared by mixing fermentation broth of strain MC-M1 with a solution of bensulfuron-methyl at a volume ratio of 1:1. The gene sequence of strain MC-M1 is shown in SEQ ID NO: 1.

[0034] Furthermore, the strain MC-M1 is Alternaria alternata.

[0035] Furthermore, the strain MC-M1 was isolated from the leaves of naturally infected alfalfa (Medicago sativa) plants.

[0036] Furthermore, the preparation process of the fermentation broth of strain MC-M1 is as follows: strain MC-M1 is inoculated into liquid PDB medium and cultured in a shaker at 180 rpm and 25°C for 7 days. After filtering through two layers of gauze to remove mycelia and mycelial cake, the obtained fermentation filtrate is the fermentation broth of strain MC-M1.

[0037] Furthermore, the concentration of the phenoxysulfuron is 0.1165 g / L.

[0038] Furthermore, the Artemisia species mentioned is Artemisia argyi.

[0039] Furthermore, the herbicide is sprayed onto the surface of Artemisia plants or seeds.

[0040] Experimental Section 1. Materials and Methods 1.1 Experimental Materials 1.1.1 Sample Collection Samples of naturally diseased alfalfa plants were collected at the Fifth Seed Source Base of Mengcao Grass in Tuozuo Banner, Hohhot City (111°19′46.708″E, 40°40′51.614″N). Figure 1 After collection, the samples were sealed in plastic bags, numbered, and stored at 4 ℃ for later use.

[0041] 1.2 Isolation and purification of pathogens Rinse the infected alfalfa leaves thoroughly with sterile water. Using sterile scissors, take several 5mm × 5mm tissue samples from the junction of diseased and healthy tissue on the infected leaves. In a sterile workbench, disinfect the samples sequentially in 3%-5% NaClO solution for 10 min, rinse three times with sterile water, immerse in 70% C2H5OH solution for 30 s, rinse three times with sterile water, dry on sterile filter paper, and then inoculate onto PDA agar plates. Seal the plates and incubate at 28℃ for 5 days. Pick hyphae from the colony edges and inoculate them into PDA agar to purify the strain and number it. The resulting strain is MC-M1. This strain was deposited on March 25, 2026, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 42536.

[0042] 1.3 Strain identification and phylogenetic tree construction 1.3.1 Morphological identification The strain was inoculated onto PDA solid medium and incubated in the dark at 28 ℃ for 5 days. The colony morphology was observed, and the results are as follows: Figure 2As shown in Figure A, strain MC-M1 initially appears as white colonies on PDA, which later turn grayish-black with smooth and neat edges, typical characteristics of fungal colonies.

[0043] A small number of bacterial cells were selected and their morphology was observed using a stereomicroscope with a 100× eyepiece. The size of the bacterial cells was measured, and the results are as follows: Figure 2 As shown in B, no conidia were observed under a microscope in the strain. The mycelium was slender, erect, and had obvious branching.

[0044] 1.3.2 Identification using molecular biological methods The extraction and content determination of genomic DNA (SEQ ID NO: 1) of strain MC-M1 (CGMCC No. 42536), as well as the PCR amplification and sequencing of the gene ITS, were performed by Beijing Ruiboxingke Biotechnology Co., Ltd. Primers used for amplifying the fungal ITS region were provided by Beijing Ruiboxingke Biotechnology Co., Ltd., including forward primer ITS1 (SEQ ID NO: 2) and reverse primer ITS4 (SEQ ID NO: 3). These primers specifically amplified the ITS1-5.8S-ITS2 region of fungal ribosomal DNA. DNA sequences with high homology to the MC-M1 sequence were retrieved and downloaded using NCBIBLAST, and the sequences were assembled. A phylogenetic tree was constructed using the neighbor-joining method with MEGA11 software, and the bootstrap test was repeated 1000 times to determine the taxonomic position of the strain. The results showed that ( Figure 3 The active strain MC-M1 belongs to Alternaria alternifolia and Alternaria alternata The ITS DNA sequence of EF06 showed high consistency. Based on molecular biological identification and morphological characteristics, strain MC-M1 was preliminarily identified as Alternaria alternifolia (…). Alternaria alternata ).

[0045] 1.4 Seed germination inhibition test of Artemisia annua The seed germination method was used. Thirty plump Artemisia argyi seeds were selected and placed on filter paper in a petri dish, with three replicates for each treatment. Three treatment groups were set up: sterile water treatment as a blank control (CK), Artemisia argyi seeds treated with 5 ml of PDB medium (PDB group), and Artemisia argyi seeds treated with 5 ml of MC-M1 strain fermentation broth (MC-M1 group). The seeds were cultured in petri dishes with humidity control. The control group used sterile water to moisten the filter paper. The germination inhibition rate was calculated after seven days.

[0046] Germination inhibition rate = [(germination rate of control group - germination rate of treatment group) ÷ germination rate of control group] × 100%; Germination experiment results as follows Figure 4 On day 7, the CK and PDB groups showed a 0% inhibition rate of seed germination in Artemisia argyi, while the MC-M1 group showed a 100% inhibition rate of seed germination in Artemisia argyi, demonstrating a significant germination inhibition effect.

[0047] 1.5 Pathogenicity determination of potted Artemisia annua The strain was inoculated into liquid PDB medium and cultured in a shaker at 180 rpm and 25°C for 7 days. The fermentation broth was filtered through two layers of gauze to obtain the fermentation filtrate for later use. Artemisia seedlings were transplanted into pots and cultured in a greenhouse at 25±1°C for 30 days until the plants grew normally.

[0048] The bacterial inoculum was sprayed onto potted Artemisia annua (MC-M1 group) using a spray bottle. Artemisia annua treated with PDB medium served as a blank control group (CK group). The inoculum volume was 25 mL / pot. After inoculation, the pots were placed in a light incubator with the following conditions: 25℃, 16h light, 20℃, 8h darkness. Each treatment was repeated 6 times. Disease incidence was observed after inoculation, and the severity of disease was assessed after 3 days to calculate the incidence rate.

[0049] Incidence rate (%) = Number of infected plants ÷ Total number of plants × 100; The results are as follows Figure 5 As shown, the seedlings of Artemisia argyi in the CK group grew well, while the seedlings of Artemisia argyi in the MC-M1 group showed obvious symptoms of disease such as leaf wilting and necrosis. According to statistics, the disease incidence rate of Artemisia argyi in the MC-M1 group reached 83.33% on the third day, while that in the CK group was 0%. This indicates that the strain MC-M1 has a significant pathogenic effect on Artemisia argyi and is suitable for the biological control of Artemisia argyi.

[0050] 1.6 Determination of herbicide control efficacy in potted Artemisia annua Prepare the chemical herbicides concentrations according to the minimum dosage specified in the instructions. Select the following chemical herbicides: difenosulfuron, flufenoxuron, and clopyralid, with concentrations of 0.233 g / L, 1.25 g / L, and 0.332 g / L, respectively.

[0051] Herbicides were sprayed onto potted Artemisia annua using a sprayer. Artemisia annua culture medium sprayed on this medium served as a blank control (CK). The inoculation amount was 25 mL / pot. After inoculation, the pots were placed in a light incubator under the following conditions: 25℃ for 16 hours of light, followed by 20℃ for 8 hours of darkness. Each treatment was repeated six times. The mortality rate of Artemisia annua after herbicide inoculation was observed. The severity of disease was assessed after 3 days, the incidence rate was calculated, and the optimal herbicide was selected. Results are as follows: Figure 6As shown (A is the comparison between the CK group and clopyralid, B is the comparison between the CK group and difenosulfuron, and C is the comparison between the CK group and flumetsulam), compared with the CK group, the application of chemical herbicides on the 3rd day showed a certain degree of inhibition on Artemisia annua seedlings, with symptoms such as leaf curling, wilting, and malformation appearing. Statistical analysis showed that the incidence rates of clopyralid, difenosulfuron, and flumetsulam were 100%, 100%, and 50%, respectively, with difenosulfuron showing the strongest disease severity, causing the entire plant to wither. The mortality rate was also the highest with difenosulfuron, reaching 83.33%. Therefore, difenosulfuron was selected for subsequent compound experiments.

[0052] 1.7 In-plate compatibility test of strain MC-M1 with bensulfuron-methyl The selected strain MC-M1 was cultured on solid PDA medium. A 6mm diameter mycelial disc, cultured for 5 days, was placed in the center of a PDA plate. 150μL of a 0.1165g / L difenosulfuron-methyl herbicide solution was added to Oxford cups located 3cm to either side of the mycelial disc. Each treatment was repeated three times. The results are as follows: Figure 7 As shown, no transparent inhibition zone appeared around the Oxford cup, indicating that at a concentration of 0.1165 g / L, bensulfuron-methyl had no growth inhibitory effect on strain MC-M1, and the two showed good in-plate compatibility. Therefore, bensulfuron-methyl was chosen for subsequent compound experiments. It should be noted that this experiment was based on a pre-experiment in pots (…). Figure 6 The optimal chemical herbicide selected was tested.

[0053] 1.8 Determination of the efficacy of potted Artemisia annua compound microbial herbicide Following herbicide screening tests, bensulfuron-methyl was selected as the subsequent compound herbicide due to its superior control effect on Artemisia annua. The concentration of the chemical herbicide was halved from the concentration in Section 1.6 (0.233 g / L), set at 0.1165 g / L for later use. The preparation method for the bacterial strain fermentation broth was the same as in Section 1.5. The bacterial strain fermentation broth and bensulfuron-methyl were mixed at a 1:1 volume ratio.

[0054] Set up potted plant experimental treatment groups: T0: Blank control group; T1: Fermentation broth of MC-M1 strain was treated separately; T2: Benzylsulfuron-methyl treatment alone; T3: MC-M1 strain fermentation broth + bensulfuron-methyl compound treatment (1:1). T4: Treatment with a mixture of 1 / 2 concentration MC-M1 strain fermentation broth and bensulfuron-methyl.

[0055] Each treatment was repeated six times. Weed disease incidence was observed after inoculation with the bacterial solution. The severity of disease was assessed 3 days later, and the pathogenicity rate was calculated. Results are as follows: Figure 8As shown (A is the comparison between T0 group and T2 group, B is the comparison between T0 group and T1 group, C is the comparison between T0 group and T3 group, and D is the comparison between T0 group and T4 group), on the 3rd day, the seedlings of Artemisia argyi in the treatment groups were inhibited to a certain extent compared with the blank control seedlings. The incidence rates of the treatment groups were 66.67%, 83.33%, 100%, and 50%, respectively.

[0056] In summary, this invention isolated and screened a fungal strain MC-M1 from naturally infected alfalfa plants, which exhibits highly effective control of Artemisia arenaria. The study investigated its control effect on potted Artemisia arenaria, providing a new strain and technical solution for the green control of Artemisia arenaria. The results are as follows: The MC-M1 strain isolated in this invention exhibits significant pathogenicity and infectivity to detached alfalfa leaves. Morphological and molecular biological identification confirmed that this strain is *Alternaria alternata*. Alternaria alternata The fermentation broth inhibited the germination rate of Artemisia annua seeds by 100%, and the incidence rate of potted Artemisia annua reached 83.33% after 3 days of spraying, showing significant pathogenicity. Among three chemical herbicides, bensulfuron-methyl, flusulfanilamide, and clopyralid, bensulfuron-methyl was selected as the optimal compound herbicide. The MC-M1 strain showed good compatibility with half-concentration bensulfuron-methyl (0.1165 g / L), and the combination of the two at a 1:1 volume ratio showed the best control effect on potted Artemisia annua, with the incidence rate reaching 100% after 3 days of treatment, achieving synergistic effect. The incidence rates of Artemisia annua in the treatment groups of MC-M1 strain fermentation broth alone, bensulfuron-methyl alone, and 1 / 2 concentration MC-M1 strain fermentation broth combined with bensulfuron-methyl were 66.67%, 83.33%, and 50%, respectively, while the incidence rate in the blank control group was 0%.

[0057] The *Alternaria alternata* MC-M1 strain isolated and identified in this invention exhibits highly effective seed germination inhibition and potted plant pathogenicity reduction against *Artemisia arenaria*. Its fermentation broth shows good compatibility with a 50% concentration of fenpropathrin, and when combined with it at a 1:1 volume ratio, it achieves 100% control of potted *Artemisia arenaria*, demonstrating synergistic effects. This compound microbial herbicide combines the environmental friendliness of microbial herbicides with the high efficiency and speed of chemical herbicides, while significantly reducing the concentration of chemical herbicides used. It provides a new strain resource and green control technology solution for the scientific management of *Artemisia* weeds in northern and western my country, showing promising application prospects and ecological value.

Claims

1. A biological herbicide for Artemisia species weeds, characterized in that, It was prepared by mixing fermentation broth of strain MC-M1 with bensulfuron-methyl solution at a volume ratio of 1:0.5-2. The gene sequence of strain MC-M1 is shown in SEQ ID NO:

1. Strain MC-M1 is Alternaria alternata, with accession number CGMCC No. 42536.

2. The Artemisia bioherbicide as described in claim 1, characterized in that, The strain MC-M1 was isolated from the leaves of naturally infected alfalfa plants.

3. The Artemisia bioherbicide as described in claim 1, characterized in that, The preparation process of the fermentation broth of strain MC-M1 is as follows: strain MC-M1 is inoculated into liquid PDB medium and cultured in a shaker at 150-210 rpm and 22-28℃ for 6-8 days. After filtering to remove mycelia and mycelial cake, the filtrate obtained is the fermentation broth of strain MC-M1.

4. The Artemisia bioherbicide as described in claim 1, characterized in that, The concentration of the phenylsulfuron-methyl is 0.1-0.15 g / L.

5. The Artemisia bioherbicide as described in claim 4, characterized in that, The concentration of the phenylsulfuron-methyl is 0.1165 g / L.

6. The Artemisia bioherbicide as described in claim 1, characterized in that, The volume ratio of the fermentation broth of strain MC-M1 to the difenosulfuron solution was 1:

1.

7. The Artemisia bioherbicide as described in claim 1, characterized in that, The Artemisia species mentioned is Artemisia argyi.

8. The application of the Artemisia bioherbicide as described in any one of claims 1-7, characterized in that, The herbicide is applied by spraying it onto the surface of Artemisia plants or seeds.

9. A strain MC-M1 for controlling Artemisia weeds, characterized in that, The gene sequence of strain MC-M1 is shown in SEQ ID NO: 1.

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