Talaromyces purpureogenus and its application in weeding
By using the metabolites of *Bacillus purpureus* CY-1 as a spraying method, the environmental problems caused by chemical herbicides have been solved, achieving a highly efficient and environmentally friendly weeding effect on broadleaf weeds.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- SICHUAN AGRI UNIV
- Filing Date
- 2023-04-28
- Publication Date
- 2026-07-24
AI Technical Summary
The long-term overuse of existing chemical herbicides has led to herbicide resistance in weeds, environmental pollution, and ecological damage, necessitating a highly efficient and environmentally friendly biological herbicide.
The metabolites of the purple basket-forming bacterium CY-1 were used and sprayed onto the stems and leaves of broadleaf weeds. The fermentation broth had a significant inhibitory effect on broadleaf weeds, including amaranth, lotus, and cocklebur.
It achieves a broad-spectrum inhibition effect on broadleaf weeds, significantly reduces weed growth, and has environmental safety and high-efficiency weed control performance.
Smart Images
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Abstract
Description
Technical Field
[0001] This invention belongs to the fields of bioengineering and weed control technology, specifically relating to a purple basket-forming bacterium and its application in weed control. Background Technology
[0002] Weeds are diverse and widely distributed, causing various forms of harm to farmland. Currently, the application of chemical herbicides remains the most common method for weed control, characterized by high efficiency, rapid action, and low cost. However, long-term overuse has led to many adverse effects, such as herbicide resistance in weeds, damage to soil ecosystems, surface water pollution, and serious harm to humans and livestock. With the increasing demand for modern green agriculture, research into efficient and environmentally friendly biological herbicides is crucial for the development of the agricultural economy.
[0003] Biological herbicides, including those derived from plants, animals, and microorganisms, are characterized by their environmental friendliness, ease of preparation, and excellent efficacy. Among them, microbial herbicides have attracted much attention due to their readily available and abundant material sources and low cost. Microbial herbicides can be classified into live microbial herbicides and herbicides derived from microbial metabolites based on their active ingredients. Compared to live microorganisms, microbial herbicides developed using microbial metabolites have stronger target specificity, higher development success rates, and easier processing characteristics, and have thus become a key research area in microbial herbicides.
[0004] Extensive research has been conducted both domestically and internationally in the field of weed control using microbial metabolites, yielding some results. Currently known herbicidal microbial metabolites include AAL-toxins, clavicin, physcin, anisin, and oxychlortin produced by Alternaria alternata. From existing successful examples, it is clear that utilizing microbial metabolites to develop herbicides is currently a shortcut to developing biological herbicides. Therefore, the collection, isolation, evaluation, and identification of herbicide strains with application potential are of great significance. Summary of the Invention
[0005] The purpose of this invention is to provide a purple basket-producing bacterium and its application in weed control. This purple basket-producing bacterium CY-1 has good application prospects in controlling broadleaf weeds.
[0006] The present invention adopts the following technical solution:
[0007] A purple basket-forming bacterium (Talaromyces purpureogenus), named CY-1, is deposited at the China General Microbiological Culture Collection Center on May 23, 2022, with accession number CGMCC No. 40203.
[0008] The application of the aforementioned purple basket-forming bacterium CY-1 in weed control.
[0009] Furthermore, the metabolites of the purple basket-forming bacterium CY-1 were sprayed onto the stems and leaves of the plant.
[0010] Furthermore, the purple basket-forming bacteria CY-1 was fermented, and the fermentation liquid was sprayed onto the stems and leaves of the plant.
[0011] Furthermore, the aforementioned grasses are broadleaf weeds.
[0012] Furthermore, the aforementioned broadleaf weeds include Amaranthus retroflexus, Alternanthera philoxeroides, Xanthium sibiricum, Chenopodium album, Solanum nigrum, Bidens pilosa, Erigeron tiglium, Sophora flavescens, Pteris vittata, Alfalfa, Amaranthus cuspidatum, Rumex japonicus, Viola yedoensis, Iris tectorum, Abutilon theophrasti, Portulaca oleracea, Leonurus japonicus, Sophora flavescens, Achyranthes bidentata, and Aster tataricus.
[0013] A biological herbicide comprising the metabolites of the aforementioned bacterium CY-1. This invention provides a bacterium CY-1 whose metabolites are environmentally safe and possess broad-spectrum herbicidal activity. It exhibits significant inhibitory effects on broadleaf weeds such as *Amaranthus retroflexus*, *Alternanthera philoxeroides*, *Xanthium sibiricum*, *Chenopodium album*, *Solanum nigrum*, *Bidens pilosa*, *Erigeron tiglium*, *Sonchus oleraceus*, *Pteris vittata*, alfalfa, *Amaranthus urinaria*, *Rumex japonicus*, *Viola yedoensis*, *Kalimeris indica*, *Abutilon theophrasti*, *Portulaca oleracea*, *Leonurus japonicus*, *Corydalis yanhusuo*, *Achyranthes bidentata*, and *Zanthoxylum bungeanum*, demonstrating excellent herbicidal efficacy. Attached Figure Description
[0014] Figure 1 The morphology of CY-1, a bacterium that produces purple basket-like bacteria, under an optical microscope.
[0015] Figure 2 The morphology of purple basket-producing bacteria CY-1 under a scanning electron microscope.
[0016] Figure 3 Phylogenetic tree diagram of the ITS sequence of CY-1, a bacterium that produces purple basket-like bacteria.
[0017] Figure 4 Phylogenetic tree diagram of the β-tubulin sequence of CY-1, a bacterium that produces purple basket-shaped bacteria. Specific Implementation
[0018] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention; that is, the described embodiments are merely some embodiments of the invention, and not all embodiments.
[0019] Therefore, the following detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.
[0020] Example 1: Isolation, Screening and Identification of Bacterial Strains
[0021] 1. Isolation and purification of bacterial strains
[0022] (1) Using the plate spreading method, 10g of soil samples were collected from the Qinghai-Tibet Plateau and placed in a sterile Erlenmeyer flask. Then, 90mL of sterile water was added to the Erlenmeyer flask.
[0023] (2) Place the Erlenmeyer flask from step (1) on a constant temperature incubator shaker and shake at 120 rpm and 28°C for 30 min until completely mixed to obtain a dilution of 10. -1 The soil dilution was then serially diluted under aseptic conditions to obtain dilutions of 10-10. -2 10 -3 10 -4 10 -5 and 10 -6 Soil dilution solution;
[0024] (3) From dilution of 10 -4 10 -5 and 10 -6 Take 200 μL of the diluted soil solution and place it in the center of the plate. Spread it on the surface of the PDA solid culture medium with a sterile glass rod to ensure even distribution.
[0025] (4) Add 100 mg / L streptomycin sulfate to the PDA solid medium obtained in step (3) (add 10 mg streptomycin sulfate to 100 mL of PDA medium and pour into plates). After culturing in a constant temperature incubator at 25℃ for 7 to 14 days, observe the colony morphology. Based on the size, shape and color of the colony, select a gray-green strain with a diameter of 26 to 28 mm, a low colony and a surface covered with conidia for purification. Purify the fungus by using the single spore isolation method and the hyphal tip cutting isolation method. Finally, transfer the single colony to a test tube and store it in a refrigerator at 4℃. Name the fungus CY-1.
[0026] 2. Identification of fungal strains
[0027] 2.1 Morphological identification of strains
[0028] (1) Morphological characteristics of the strain under an optical microscope
[0029] Colonies were cultured on CYA medium at 25°C for 14 days. A small amount of mycelium was then picked, placed on a glass slide, and examined and photographed using a DM4B upright fluorescence microscope. Figure 1As shown, it can be observed that the mycelium produces conidiophores, which are smooth, branched, and have septate transverse membranes, growing nearly upright on the mycelium; at the tip of the conidiophore grows a conidiophore base and sterigmata, which are closely arranged in a broom-like shape; conidia are strung on the sterigmata. From the typical conidiophore bundles, it can be seen that the CY-1 strain has the morphological characteristics of the genus Penicillium.
[0030] (2) Scanning electron microscopy observation of the strain
[0031] Vigorous colonies grown on CYA medium were fixed with 2.5% glutaraldehyde overnight. On day 2, the cells were washed three times with phosphate buffer, followed by a gradient dehydration with ethanol. The dehydration steps were as follows: dehydration with 30%, 50%, 70%, 85%, and 95% ethanol for 15 min each, then dehydration twice with 100% ethanol for 15 min each time. Centrifugation was performed at 8000 rpm for 5 min at each solvent change. After centrifugation, the supernatant was discarded, and the cells were mixed with the next solvent. This process was repeated. Finally, CO2 critical point drying and gold sputtering were performed sequentially. After processing, scanning electron microscopy was performed for observation. Figure 2 As shown, a clearer three-dimensional structure can be observed than under an optical microscope. The broom-like branches are mainly three-whorls at the top of the same conidiophore base. The top conidiophore is bottle-shaped with a bottle mouth-shaped base, on which conidia are strung. The conidia are spherical with a diameter of 2.7 μm. Thus, it can be seen that the conidiophores of fungus CY-1 have a typical broom-like morphology, and the conidia are in long chains, which have obvious morphological characteristics of the genus Penicillium.
[0032] 2.2 Molecular identification of strains
[0033] (1) After the strain CY-1 obtained by isolation and purification in step 1 was grown in PDA and MEA medium at 25°C for 7 days, the genomic DNA of strain CY-1 was extracted using the Ezup column fungal genomic DNA extraction kit, according to the instructions.
[0034] (2) Using the genomic DNA obtained in step (1) as a template, strain CY-1 was amplified using universal primers ITS1 and ITS4. The primer sequences of ITS1 and ITS4 are shown in Table 1. After amplification, the ITS sequence of strain CY-1 was obtained, with a gene fragment length of 553 bp. Samples with clear bands were selected and sequenced by Shanghai Sangon Biotech Co., Ltd. The ITS sequence of strain CY-1 was uploaded to the NCBI database and compared using BLAST. The most similar sequence was searched and downloaded. Using MEGA 7.0 software, the maximum likelihood method and Tamura-Nei mode were used for 1000 replicates to construct the phylogenetic tree of strain CY-1, as shown in Table 1. Figure 1 As shown, the comparison revealed that strain CY-1 had a sequence similarity of 86% with the fungi in the gene bank, namely Talamoyces purpureogenus (accession number: OM372890), Talamoyces flavus (accession number: HQ443252), and Penicillium funiculosum (accession number: JN676119). Therefore, strain CY-1 can be identified as belonging to the genus Talamoyces or Penicillium.
[0035] (3) To amplify the partial sequence of the β-tubulin gene of strain CY-1, primers bt2a and bt2b were synthesized. The primer sequences of bt2a and bt2b are shown in Table 1. Using the genomic DNA obtained in step (1) as a template, the bt2a and bt2b sequences were used as primers for amplification to obtain the β-tubulin sequence of strain CY-1. The gene fragment length was 439 bp. Samples with clear bands were selected and sequenced by Shanghai Sangon Biotech Co., Ltd. The β-tubulin sequence of strain CY-1 was searched in the NCBI database and the most similar sequence was downloaded. The phylogenetic tree of strain CY-1 was constructed using MEGA 7.0 software, as shown in Table 1. Figure 2 As shown, the comparison revealed that strain CY-1 has a 90% similarity to Talamomyces purpureogenus (accession number: LT559063) in the gene bank. Therefore, it can be concluded that strain CY-1 is Talamomyces purpureogenus.
[0036] Table 1 Amplification Primer Sequences
[0037]
[0038] Example 2: Preparation of Fermentation Products from Strains
[0039] The fermentation products of the strain were prepared by shake-flask fermentation. 100 mL of prepared PD liquid culture medium was placed in a 250 mL Erlenmeyer flask, sealed with a breathable membrane, and sterilized in a high-temperature sterilizer at 121 °C for 30 min. After cooling, strain CY-1 was inoculated in a clean bench, with 6 colonies of 5 mm in diameter placed in each Erlenmeyer flask. The flasks were then incubated in a constant-temperature shaker for 7 days (shake speed 200 r / min, incubation temperature 25–28 °C). The fermentation broth was then obtained by filtration through 6 layers of degreased gauze. The metabolites of the strain were distributed in the fermentation broth.
[0040] Example 3: Preliminary Screening Test of Fermentation Broth for Weed Control
[0041] The experimental site was a vacant lot overgrown with various weeds within the Chongzhou Experimental Base of Sichuan Agricultural University. This area contained broadleaf weeds such as *Amaranthus retroflexus*, *Alternanthera philoxeroides*, and *Xanthium sibiricum*, as well as grasses such as *Setaria viridis* and *Eleusine indica*. The vacant lot was divided into two areas, and two experimental groups were set up: a fermentation liquid group and a water group. The water group served as a blank control. The fermentation liquid and water were sprayed until the leaves were dripping wet, respectively. Each treatment was replicated three times, with each replicate plot measuring 4 m². 2 Randomized block design; visual inspection was used to observe the damage to different weeds at 1, 3, 7 and 15 days after application.
[0042] (1) During each survey, the five-point sampling method was used to observe and record the degree of damage caused by various weeds, as shown in Table 2;
[0043] The severity of pesticide damage is graded according to the percentage of the entire plant affected, and the grading criteria are as follows:
[0044] Level 0: The plant is not damaged;
[0045] Level 1: The affected part accounts for less than 10% of the entire plant;
[0046] Level 2: The affected part accounts for less than 30% of the entire plant;
[0047] Level 3: The affected part accounts for less than 50% of the entire plant;
[0048] Level 4: The affected parts account for less than 70% of the entire plant;
[0049] Level 5: The affected parts account for more than 85% of the entire plant;
[0050] Table 2. Damage to different weeds after treatment with fermentation broth.
[0051]
[0052]
[0053] As shown in Table 2, one day after spraying the fermentation liquid, broadleaf weeds basically began to show symptoms of phytotoxicity, while grass weeds showed no obvious changes. This indicates that the fermentation liquid of the strain has good activity against broadleaf weeds but poor activity against grass weeds.
[0054] (2) Using the five-point sampling method, dominant weeds *Amaranthus retroflexus* and *Alternanthera philoxeroides* were sampled, and the fresh weight of the aboveground parts was measured. The fresh weight control efficacy was calculated, and the results are shown in Table 3. The formula for calculating the fresh weight control efficacy is as follows:
[0055]
[0056] Table 3. Control efficacy of fermentation broth treatment on the fresh weight of Amaranthus buergerianum and Nelumbo nucifera.
[0057]
[0058] As shown in Table 3, the fresh weight of *Amaranthus retroflexus* and *Alternanthera philoxeroides* sprayed with the fermentation liquid of strain CY-1 gradually decreased over time. On day 15, the control efficacy against *Amaranthus retroflexus* reached 72.46%, and the control efficacy against *Alternanthera philoxeroides* was 57.11%. This indicates that the fermentation liquid of strain CY-1 has a certain control effect on *Amaranthus retroflexus* and *Alternanthera philoxeroides*, and the control effect on *Amaranthus retroflexus* is better.
[0059] Example 4: Pot experiment to determine the herbicidal activity of fermentation broth
[0060] Seeds of different weeds, after germination, were sown in 10cm diameter flowerpots, with about 10 seeds per pot, and covered with 1-3cm of soil. The plant materials were cultured in a greenhouse under normal conditions. When the plants reached the 3-5 leaf stage, foliar spraying was performed. The original fermentation liquid and fermentation liquids of different dilution ratios were sprayed onto the test plants, with water treatment as the control group. The herbicidal activity was initially determined. After 7 days, the above-ground parts were weighed to calculate the fresh weight inhibition rate. The results are shown in Table 4.
[0061] Table 4. Herbicidal activity of fermentation broth against weeds
[0062]
[0063] Table 4 shows that when the undiluted fermentation broth was used to treat weeds, the weeds were basically wilted. The fresh weight inhibition rate against 10 weeds, including cocklebur and amaranth, was higher than 80%, demonstrating broad-spectrum herbicidal activity. However, the activity against four weeds, including barnyard grass and sedge, was poor. When the fermentation broth was diluted 5 times and 10 times, compared with other weeds, it showed a more significant inhibitory effect on cocklebur. These experimental results indicate that the undiluted fermentation broth of strain CY-1 has good herbicidal activity against broadleaf weeds, effectively inhibiting weed growth and causing them to wither and even die.
Claims
1. A purple basket-forming bacterium ( Talaromyces purpureogenus ), characterized in that, The purple basket-shaped bacterium was named CY-1 and deposited at the China General Microbiological Culture Collection Center on May 23, 2022, with accession number CGMCC No. 40203.
2. The application of the purple basket-producing bacterium CY-1 as described in claim 1 in weed control; wherein the weeds are: Amaranthus retroflexus, Alternanthera philoxeroides, Xanthium sibiricum, Chenopodium album, Solanum nigrum, Bidens pilosa, Erigeron tiglium, Sophora flavescens, Pteris vittata, Alfalfa, Amaranthus cuspidatum, Rumex japonicus, Viola yedoensis, Iris tectorum, Abutilon theophrasti, Portulaca oleracea, Leonurus japonicus, Sophora flavescens, Achyranthes bidentata, and Aster tataricus.
3. The application of the purple basket-producing bacterium CY-1 in weed control according to claim 2, characterized in that, The fermentation broth of the purple basket-shaped fungus CY-1 was sprayed onto the stems and leaves of the plant.
4. The application of the purple basket-producing bacterium CY-1 in weed control according to claim 3, characterized in that, Ferment CY-1, a bacterium that produces purple baskets, and spray the fermentation liquid onto the stems and leaves of the plants.
5. A biological herbicide, characterized in that, The fermentation broth includes the purple basket-producing bacterium CY-1 as described in claim 1.