A biocontrol agent for amaranthus retroflexus in alfalfa field and application thereof

Abstract

This invention belongs to the field of biotechnology, specifically relating to a biocontrol agent for alfalfa fields resistant to the herbicide *Amaranthus retroflexus* and its application. The biocontrol agent contains a suspension of mycelium and / or spores of *Morchella* strain MXBP304 as its active ingredient. The microbial strain is MXBP304, belonging to the genus *Morchella*. Mortierella sp The fungus, accession number MXBP304, was deposited on December 22, 2021, at No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, with accession number CCTCC NO:M 20211660. The concentration was 2.8 × 10⁻⁶. 7 A CFU / mL spore suspension significantly inhibited seed germination of *Amaranthus retroflexus*, with a relative germination rate of 27.45% and a significant decrease in root length of 29.57%. Meanwhile, this treatment had no adverse effects on seed germination, growth, or major nutritional quality of alfalfa, demonstrating excellent selectivity and growth-promoting effects.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a biocontrol agent for herbicide-resistant amaranth in alfalfa fields and its application. Background Technology

[0002] Amaranth rotundifolia ( Amaranthus retroflexus L. (Amaranthus retroflexus) is the first malignant invasive weed listed in the "List of Invasive Alien Species in China" (Third Batch) released by the Ministry of Ecology and Environment of my country, posing a serious threat to agricultural production and the ecological environment. In alfalfa-growing areas, Amaranthus retroflexus is one of the most harmful broadleaf weeds. Due to its tall stature and numerous branches, it fiercely competes with alfalfa for sunlight, water, and soil nutrients, severely affecting the normal growth and development of alfalfa. It often leads to a decrease in alfalfa grassland productivity of more than 30%, and in severe cases, even causes alfalfa planting failure.

[0003] More seriously, in recent years, due to the long-term reliance on chemical herbicides for weed control in alfalfa fields, the problem of herbicide resistance in weeds has become increasingly prominent. Taking Chifeng, Inner Mongolia as an example, the alfalfa planting area in this region is approximately 500,000 mu (about 33,333 hectares), and the *Amaranthus retroflexus* population in the fields has developed varying degrees of resistance to several commonly used herbicides such as glyphosate and imidacloprid. Conventional chemical control methods are no longer effective in controlling its damage, leading to a significant decline in the effectiveness of chemical weed control. This forces production to continuously increase the dosage of herbicides or change the types of herbicides used, which not only significantly increases the cost of control but also exacerbates the risk of soil pollution and excessive pesticide residues in forage products, seriously restricting the green and sustainable development of the alfalfa industry. Therefore, developing safe and efficient ecological control technologies for herbicide-resistant *Amaranthus retroflexus* has become a key issue that urgently needs to be addressed in the development of the alfalfa industry.

[0004] In the current technology, there is a relative scarcity of microbial inhibitory strains targeting major broadleaf weeds in alfalfa fields, and a lack of targeted products and applications. At the same time, research on the inhibition of weed seed germination and seedlings by *Morchella* fungi is still lacking, and the *Morchella* resources enriched in the alfalfa rhizosphere have not been fully developed and utilized. In addition, the optimal culture conditions of *Morchella* in the alfalfa rhizosphere and its inhibitory effect on broadleaf weeds in alfalfa fields have not been systematically explored, resulting in a lack of necessary scientific basis for the industrial application of microbial herbicides. Summary of the Invention

[0005] To address the shortcomings of the existing technology, this invention aims to provide a strain of *Morchella* isolated and screened from the rhizosphere of alfalfa. Mortierella We investigated the taxonomic position and optimal culture conditions of strain MXBP304 (sp.), and applied it to inhibit the growth of herbicide-resistant amaranth in alfalfa fields, providing new strain resources and technical support for the microbial ecological control of weeds in alfalfa fields.

[0006] The herbicide-resistant amaranth strain MXBP304 described in this invention is a member of the genus *Morchella*. Mortierella sp The fungus, with accession number MXBP304, was deposited on December 22, 2021, at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, with accession number CCTCC NO: M 20211660.

[0007] The first objective of this invention is to provide a biocontrol agent for alfalfa fields resistant to the herbicide *Amaranthus retroflexus*, wherein the biocontrol agent contains mycelium and / or spore suspension of *Morchella esculenta* strain MXBP304 as the active ingredient.

[0008] Furthermore, the concentration of the spore suspension is 2.8 × 10⁻⁶. 7 CFU / mL.

[0009] The present invention also provides a method for culturing the mycelium of the above-mentioned *Morchella esculenta* strain MXBP304, using GY as the culture medium, pH 6, 28°C, and culturing in complete darkness.

[0010] Furthermore, the formula for the GY culture medium (GY) is: 10 g yeast extract, 20 g glucose, 20 g agar, and 1000 mL deionized water.

[0011] The present invention also includes the application of the biocontrol agent in inhibiting the growth of reverse-branching amaranth in alfalfa fields.

[0012] Furthermore, the *Amaranthus retroflexus* is a herbicide-resistant biotype resistant to glyphosate and / or imidazoline.

[0013] The present invention also includes the application of the biocontrol agent in increasing alfalfa biomass and / or increasing alfalfa crude protein content.

[0014] Furthermore, the application period is after the alfalfa has grown to the 3-4 leaf stage, and the application method is to use a concentration of 2.8 × 10⁻⁶. 7 A CFU / mL spore suspension was evenly sprayed onto the roots and leaves of *Amaranthus retroflexus* seedlings.

[0015] Compared with the prior art, the present invention has the following advantages: New microbial resources were provided: a strain of *Morchella esculenta* with good inhibitory effects against the broadleaf weed *Amaranthus retroflexus* was isolated from alfalfa rhizosphere soil for the first time. Mortierella sp. Strain MXBP304 fills the gap in the application of *Morchella* fungi in the field of microbial control of weeds in alfalfa fields; The cultivation method is simple and efficient: the cultivation conditions of strain MXBP304 were systematically optimized, and the optimal cultivation scheme was determined using GY as the medium, pH 6, 28℃, and complete darkness. Under these conditions, the strain grows rapidly and produces a large amount of sporulation (colon diameter >75mm and sporulation >28×10⁻⁶ at 11 days). 6 (pcs / mL), which facilitates industrial production and large-scale application; Significantly effective and safe for the host: concentration 2.8 × 10⁻⁶ 7 A CFU / mL spore suspension significantly inhibited seed germination (relative germination rate 27.45%) and root length (decreased by 29.58%) in *Amaranthus retroflexus*. Meanwhile, this treatment had no adverse effects on seed germination, growth, or major nutritional quality of alfalfa, demonstrating excellent selectivity and growth-promoting effects. Environmentally friendly: This invention utilizes natural rhizosphere microbial resources, avoiding the resistance, environmental residues and ecological risks caused by chemical herbicides, and meets the requirements of green agriculture and sustainable development. Attached Figure Description

[0016] Figure 1 Morphological characteristics of strain MXBP304 on PDA and GY media; Figure 2 Morphological characteristics of strain MXBP304 in different culture media. Where A is the morphological characteristic diagram of the strain under SDA medium, B is the morphological characteristic diagram of the strain under BS medium, C is the morphological characteristic diagram of the strain under WA medium, D is the morphological characteristic diagram of the strain under MA medium, and E is the morphological characteristic diagram of the strain under GY medium. Figure 3 Figure showing the effect of different culture media on the growth of strain MXBP304; Figure 4 This figure shows the effects of different carbon and nitrogen sources on the growth of strain MXBP304. Figure A shows the effect of different carbon sources on the growth of strain MXBP304, and Figure B shows the effect of different nitrogen sources on the growth of strain MXBP304. Figure 5 The graph shows the effect of different concentrations of spore suspensions on the relative germination rates of alfalfa and amaranth retroflexus. Figure A shows the effect of different concentrations of spore suspension on the relative germination rate of alfalfa, and Figure B shows the effect of different concentrations of spore suspension on the relative germination rate of amaranth. Figure 6 The graph shows the effect of different concentrations of spore suspensions on the root length of alfalfa and amaranth retroflexus. Figure A shows the effect of different concentrations of spore suspension on alfalfa root length, and Figure B shows the effect of different concentrations of spore suspension on alfalfa root length. Figure 7Figure showing the effect of different concentrations of spore suspension on crude protein in alfalfa. Figure 8 Figure showing the effect of different concentrations of spore suspension on alfalfa neutral detergent fiber; Figure 9 The figure shows the effect of different concentrations of spore suspension on alfalfa acid detergent fiber. Detailed Implementation

[0017] Example 1 Morphological observation of strain MXBP304 Cut washed potatoes into small pieces. Boil 200 g of the potato pieces in 1000 mL of deionized water for 30 min, centrifuge at 4000 r / min for 2 min, collect the supernatant, add 20 g of glucose and 20 g of agar, and add deionized water to 1000 mL. Stir thoroughly and autoclave at 121℃ for 20 min to obtain potato glucose medium (PDA). Dissolve 10 g of yeast powder in 500 mL of deionized water, add 20 g of glucose and 20 g of agar, and add deionized water to 1000 mL. Stir thoroughly and autoclave at 121℃ for 20 min to obtain glucose yeast medium (GY).

[0018] The *Morchella* strain MXBP304 was inoculated onto PDA and GY plates, respectively, and incubated at 28°C. After successful colony growth, morphological descriptions were performed according to the *Fungal Identification Handbook*. The results showed that on GY medium, the colonies of strain MXBP304 were white, dense, cottony, and petal-like. See [link to relevant documentation] for colony morphology characteristics on different media. Figure 1 Morphological characteristics of strain MXBP304 on PDA medium (left) and GY medium (right).

[0019] Example 2 Determination of optimal culture conditions for strain MXBP304 1. Effects of different culture media on colony growth and sporulation This embodiment uses the following five culture media for screening: Sabouraud dextrose agar (SDA), bean sprout juice dextrose agar (BS), water agar (WA), modified Martin agar (MA), and glucose yeast agar (GY).

[0020] All culture media were autoclaved at 121℃ for 20 min before use. Mycelial discs with a diameter of 5 mm were inoculated into the center of each of the seven culture media plates and incubated at 28℃, with three replicates for each medium. Colony diameter was measured every 24 hours using the cross-hatching method, continuing until day 11. Simultaneously, sporulation was examined microscopically using a hemocytometer, and colony morphology was recorded. The results are as follows Figure 2 and Figure 3 As shown, Figure 2 In the diagram, A represents the morphological characteristics of the strain under SDA medium. Figure 2 B in the diagram represents the morphological characteristics of the strain in BS medium. Figure 2 C in the diagram represents the morphological characteristics of the strain in WA medium. Figure 2 D in the diagram represents the morphological characteristics of the strain under MA medium. Figure 2 E in the figure represents the morphological characteristics of the strain in GY medium. Figure 3 The figure shows the effect of different culture media on the growth of strain MXBP304. Among the five media, GY medium showed the largest average colony diameter, reaching 75.29 mm on day 11, with dense, white colonies; BS medium showed the smallest average colony diameter, at 52.26 mm, with sparse hyphae. The spore yield on GY medium was 28.23 × 10⁻⁶. 6 The other four culture media produced only a very small number of spores per mL. Therefore, GY was determined to be the optimal culture medium for strain MXBP304.

[0021] 2. Effects of different carbon and nitrogen sources on colony growth and sporulation Based on Czapek's medium (CK, formula: 30 g sucrose, 2.0 g NaNO3, 1.0 g K2HPO4, 0.5 g KCl, 0.5 g MgSO4·7H2O, 0.1 g FeSO4, 20 g agar, 1000 mL deionized water, autoclaved at 121℃ for 20 min), carbon source and nitrogen source substitution experiments were conducted.

[0022] Carbon source experiment: Five different carbon source media were obtained by replacing sucrose in Czapek's medium with equal weights of glucose, lactose, maltose, fructose and soluble starch, respectively, with Czapek's medium (CK) as the control.

[0023] Nitrogen source experiment: Four different nitrogen source media were obtained by replacing NaNO3 in Czapek's medium with equal weights of peptone, beef extract, yeast extract and ammonium sulfate respectively.

[0024] The results showed that ( Figure 4 ), Figure 4 In the figure, A represents the effect of different carbon sources on the growth of strain MXBP304. Figure 4 Figure B shows the effect of different nitrogen sources on the growth of strain MXBP304. The optimal carbon source is soluble starch. After 11 days of culture, the average colony diameter reached 71.66 mm, which was significantly larger than that of CK and other carbon sources. P<0.05). Peptone, beef extract, and yeast were the most suitable nitrogen sources, with average colony diameters reaching 82.70, 82.67, and 82.80 mm, respectively, after 7 days of culture. Ammonium sulfate utilization was relatively weak, with a colony diameter of 76.60 mm. Sporulation was minimal on any carbon or nitrogen source medium based on Czapek's medium, indicating that Czapek's medium is unfavorable for sporulation of MXBP304, and GY medium should be used for sporulation culture.

[0025] 3. Determination of the optimal pH value GY medium was adjusted to pH 4, 5, 6, 7, 8, 9, and 10, respectively, and inoculated with mycelial cakes before being cultured at 28°C. The medium at pH 4 and 10 exhibited poor coagulation and was unsuitable for colony culture. In each treatment ranging from pH 5 to 9, three replicates were performed, and colony diameter and sporulation were measured after 11 days of culture.

[0026] The results are shown in Table 1. Strain MXBP304 could grow and produce sporulation within a pH range of 5–9. The average colony diameter was largest at pH 6 (74.83 mm), and the sporulation yield was highest at 25.67 × 10⁻⁶. 6 The concentration / mL was significantly higher than under other pH conditions. P <0.05). Both excessive acidity and excessive alkalinity inhibited colony growth and sporulation to varying degrees. The optimal pH was determined to be 6.

[0027] Table 1. Effects of different pH values ​​on bacterial growth and sporulation. Note: Different letters indicate significant differences between groups. P< 0.05.

[0028] 4. Determination of the optimal incubation temperature The GY medium inoculated with mycelial cakes was incubated at 15℃, 20℃, 25℃, 28℃, 30℃, 35℃, and 37℃, with three replicates for each treatment. Since the colonies had completely covered the plates by day 14 at different temperatures, data from day 11 were used for comparison.

[0029] The results are shown in Table 2. Mycelial growth was observed within the temperature range of 20–35℃, ceasing below 15℃ and above 37℃. The fastest mycelial growth was observed at 28℃, with an average colony diameter of 77.97 mm and a sporulation yield of 28.23 × 10⁻⁶. 6 The concentration of cells / mL was the highest under all temperature conditions, significantly higher than under other temperature conditions. P <0.05). The optimal culture temperature was determined to be 28℃.

[0030] Table 2. Effects of different temperatures on strain growth and sporulation yield 5. Determination of optimal lighting conditions Two light conditions were set up: complete darkness and alternating light and dark (12 h light / 12 h darkness). After inoculation with mycelium, the culture was carried out at 28°C. Each treatment was replicated in 3 places. On day 11, the colony diameter and sporulation were measured.

[0031] The results are shown in Table 3. Under dark conditions, the growth and sporulation of strain MXBP304 were significantly better than those under alternating light and dark conditions. P <0.05). Under dark conditions, the average colony diameter reached 57.17 mm, and the sporulation yield reached 20.53 × 10⁻⁵. 6 Cells / mL. The optimal light condition was determined to be incubation in complete darkness.

[0032] Table 3. Effects of different light conditions on strain growth and sporulation yield Based on the above results, the optimal culture conditions for strain MXBP304 are: GY medium, pH 6, 28℃, and complete darkness. Under these conditions, mycelial growth is rapid, colonies are dense, and sporulation is high. After 11 days of culture, the colony diameter can reach over 75 mm, and the sporulation yield can reach 28 × 10⁻⁶. 6 More than 1 / mL.

[0033] Example 3 Inhibitory effects of different concentrations of spore suspensions on alfalfa and weeds 1. Preparation of spore suspension Strain MXBP304 was inoculated onto GY medium and cultured in complete darkness at 28°C for 14 days. The cultured mycelium was then transferred to a centrifuge tube, and 10 mL of sterile water was added and shaken to fully disperse the mycelium, yielding a spore suspension. The spore concentration in the stock solution was 2.8 × 10⁻⁶ using a hemocytometer. 7 CFU / mL. The stock solution was diluted with sterile water 10, 100, 500, and 1000 times, respectively, to obtain concentrations of 2.8 × 10⁻⁶ CFU / mL. 6 CFU / mL, 2.8×10 5 CFU / mL, 1.4×10 5 CFU / mL and 2.8×10 4 A spore suspension of CFU / mL.

[0034] 2. Seed germination experiment Two sheets of filter paper of equal size were placed in petri dishes, and 10 mL of the spore suspensions of different concentrations were added to each. After the filter paper had fully absorbed the spores, 35 seeds of alfalfa and amaranth retroflexus were evenly placed on the filter paper, respectively. Water was used as the control group (CK). Each treatment was repeated in triplicate. The petri dishes were placed in a light incubator at 25°C with a photoperiod of 16 h light / 8 h dark. The number of germinating seeds was observed and recorded daily. On day 10, the root length of the seeds in each treatment was measured uniformly using a centimeter ruler.

[0035] result Figure 5 and Figure 6 As shown, Figure 5 Figure A shows the effect of different concentrations of spore suspension on the relative germination rate of alfalfa. Figure 5 Figure B shows the effect of different concentrations of spore suspension on the relative germination rate of Amaranthus retroflexus. Figure 6 Figure A shows the effect of different concentrations of spore suspension on alfalfa root length. Figure 6 Figure B shows the effect of different concentrations of spore suspension on the root length of *Amaranthus retroflexus*. 1.4 × 10⁻⁶ 5 CFU / mL up to 2.8 × 10⁻⁶ 6 A spore suspension at a concentration of CFU / mL significantly inhibited seed germination of *Amaranthus retroflexus*. Specifically, 2.8 × 10⁻⁶ CFU / mL significantly inhibited seed germination. 7 The spore suspension with a concentration of CFU / mL had the lowest germination rate, at 27.45%. P <0.05), but it has no inhibitory effect on alfalfa seed germination.

[0036] Regarding root length, 2.8 × 10 7 Under CFU / mL treatment, the root length of the reverse-branching amaranth decreased from 21.06 cm in the control group (CK) to 14.83 cm, a significant decrease of 29.58% compared to the CK. P <0.05); 2.8×10 6 Under CFU / mL treatment, the root length of *Amaranthus retroflexus* decreased to 17.62 cm, a significant decrease of 16.33% compared to the control (CK). P <0.05). Different concentrations of spore suspensions had no significant effect on alfalfa seed root length.

[0037] 3. Pot Experiment – ​​Effects on Plant Growth and Quality Select plump, uniformly sized alfalfa and amaranth seeds, disinfect them by soaking them thoroughly in a 10% sodium hypochlorite solution for 10 minutes, and rinse repeatedly with distilled water. Break dormancy in the alfalfa and amaranth seeds using conventional methods. Place the treated seeds in petri dishes lined with moistened filter paper, add 10 mL of distilled water to maintain moisture, and after 7 days, select healthy seedlings. Transplant 9 seedlings of each plant type into potting soil. The potting soil is prepared by sifting field soil and mixing native soil, vermiculite, and perlite in a volume ratio of 3:1:1. The soil type is brown soil with a pH of 5.8.

[0038] Potted plants were cultivated in an artificial climate chamber with a light condition of 16 h light (25℃) / 8 h darkness (20℃) and a light intensity of 150 μmol·m⁻¹. - ²·s - ¹. When the plants reach the 3-4 leaf stage, spray 20 mL of spore suspension of different concentrations evenly around the roots and on the leaves of the seedlings every 10 days, using water as the control (CK). Each treatment was replicated four times. Alfalfa was harvested uniformly when it reached the initial flowering stage, and various indicators were measured. The measurement methods are as follows: (1) Crude protein content: The dried plant was ground and passed through a 60-mesh sieve. 10 mg of powder was placed in tin foil and the total nitrogen content was determined using an elemental analyzer (Vario EL cube). Crude protein content = total nitrogen content × 6.25.

[0039] (2) Content of neutral detergent fiber and acid detergent fiber: 10 mg of plant powder that has been ground through a 60-mesh sieve was used to determine the content using an automated fiber analyzer (ANKOM A2000i).

[0040] The test results are as follows: (1) Effect of crude protein content: at 2.8x10 5 CFU / mL and 2.8x10 4 Under CFU / mL treatment, the crude protein content of alfalfa was 18.01% and 17.45%, respectively, which were significantly increased by 9.16% and 5.24% compared with the control (CK). P <0.05). 2.8x10 7 , is 2.8x10 6 and 2.8x10 4 CFU / mL concentration treatment had no significant effect on the crude protein content of alfalfa. Results are shown below. Figure 7 .

[0041] (2) Effect on fiber content: Different concentrations of spore suspension had no significant effect on the content of neutral detergent fiber (NDF) and acid detergent fiber (ADF) in alfalfa. See results below. Figure 8 , Figure 9 .

[0042] Based on the results of the pot experiment, 2.8 × 10 7 The optimal concentration for this invention is a spore suspension at CFU / mL. This concentration significantly inhibits seed germination and root length of *Amaranthus retroflexus*, while having no significant adverse effects on seed germination rate, root length, NDF, and ADF of alfalfa, and can significantly increase the crude protein content of alfalfa at certain concentrations.

Claims

1. A biocontrol agent for alfalfa fields resistant to herbicide-resistant reverse-branching amaranth, characterized in that: The biocontrol agent contains mycelium and / or spore suspension of the *Morchella spp.* strain MXBP304 as the active ingredient, and the preservation number of the *Morchella spp.* strain MXBP304 is CCTCC NO: M 20211660.

2. The biocontrol agent as described in claim 1, characterized in that: The concentration of the spore suspension was 2.8 × 10⁻⁶. 7 CFU / mL.

3. The biocontrol agent as described in claim 1, characterized in that: The mycelial culture conditions for the *Morchella esculenta* strain MXBP304 were as follows: GY medium, pH 6, 28°C, and culture in complete darkness.

4. The biocontrol agent as described in claim 3, characterized in that: The formula for the GY culture medium is: 10 g yeast extract, 20 g glucose, 20 g agar, and 1000 mL deionized water.

5. The application of the biocontrol agent as described in claim 1 in inhibiting the growth of amaranth retrograde in alfalfa fields.

6. The application as described in claim 5, characterized in that: The aforementioned *Amaranthus retroflexus* is a herbicide-resistant biotype resistant to glyphosate and / or imidazoline.

7. The application of the biocontrol agent as described in claim 1 in increasing the crude protein content of alfalfa.

8. The application as described in claim 7, characterized in that: The application period is after the alfalfa has grown to the 3-4 leaf stage, and the application method is to use a concentration of 2.8 × 10⁻⁶. 7 A CFU / mL spore suspension was evenly sprayed onto the roots and leaves of *Amaranthus retroflexus* seedlings.

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