A biological agent containing *Coccidioidomyces chinensis*, its preparation method and application

Abstract

This invention discloses a biological agent containing *C. praecox*, its preparation method, and its application, relating to the field of biological control technology. The method involves spraying the cut surface of *Solidago canadensis* stalks with the agent containing *C. praecox* MP1 after harvesting. The agent developed using this *C. praecox* strain, combined with harvesting conditions, has been demonstrated in indoor potted plant and field plot control trials to cause necrosis of the entire root system of *Solidago canadensis*, exhibiting highly effective and stable control. This method overcomes the limitations of solid herbicide granules using *Sclerotium sclerotiorum* strain SC64 as the main component, which require soil tilling before application. The method of this invention has a wider range of applications, suitable for almost all terrain and habitat types.

Description

Technical Field

[0001] This invention relates to the field of biological control technology, specifically to a biological agent containing *Cyclocarya paliurus*, its preparation method, and its application. Background Technology

[0002] Canadian goldenrod (Solidago canadensis L.), a plant belonging to the Asteraceae family in the order Asterales, is also known as goldenrod or goldenrod. It is a perennial herb with long rhizomes. The stems are erect and can reach up to 2.5 meters in height. It has become a globally invasive weed. Introduced to my country as an ornamental plant in earlier years, it escaped its native habitat due to the absence of numerous natural enemies and pests that controlled its spread, becoming a highly virulent weed and one of the most serious invasive alien plants today. It has been listed in the National Key Management List of Invasive Alien Species. Currently, it has invaded and spread to many provinces in my country, covering a large area and seriously threatening my country's biodiversity, even potentially leading to the extinction of native species. More importantly, the invasion of Canadian goldenrod has caused direct economic losses to my country's agriculture, forestry, and animal husbandry. It can invade farmland and surrounding areas, causing a sharp decline in crop yield and quality. The economic losses caused by the invasion of Canadian goldenrod into orchards are significant, sometimes even resulting in total crop failure. Currently, the technology for controlling Canadian goldenrod mainly relies on physical and chemical control methods. Canadian goldenrod reproduces sexually and asexually through seeds and rhizomes, exhibiting extremely high reproductive capacity. Furthermore, after invading my country, it has undergone chromosome doubling due to climatic and environmental factors, resulting in exceptional environmental adaptability and resistance. This renders physical control methods such as manual cutting or uprooting, mechanical mowing, and chemical control ineffective, as the rhizomes are not damaged and regenerate quickly. Moreover, physical control is labor-intensive, and chemical control can damage the soil ecosystem and biodiversity.

[0003] Chemical herbicides experienced rapid development in the 20th century due to their affordability, rapid effectiveness, ease of use, and labor-saving benefits, bringing tremendous changes to agricultural production and human life. However, even so, the harm they cause is immense. The unscientific use of large quantities of highly toxic and persistent chemical herbicides has caused irreparable environmental damage. Even glyphosate, once considered a safe and environmentally friendly herbicide, has gradually revealed problems with its impact on human health and effectiveness. On the one hand, chemical herbicide residues in the soil are difficult to degrade, affecting the growth of subsequent crops and the quality of agricultural products; residues in the soil are easily absorbed by crops, causing toxins to accumulate in large quantities in the human body through the food chain, posing a significant threat to human health; soil compaction, water quality deterioration, and reduced soil fertility; on the other hand, they adversely affect local biological communities, impacting biodiversity; increasing weed resistance; and affecting crop photosynthesis and metabolism. Therefore, exploring and finding broad-spectrum, highly efficient, low-toxicity, environmentally friendly, and sustainable green herbicides is key to solving these problems. New environmentally friendly biological herbicides are one important approach to addressing this issue. However, the development of biological herbicides faces many limiting factors, such as limitations imposed by climatic environmental factors, especially humidity, and limitations in production and application technologies. Therefore, developing new application technologies is one of the important steps in promoting the research and industrialization of biological herbicides.

[0004] Developing bio-based herbicides using naturally occurring pathogenic microorganisms of invasive weeds is an important alternative to chemical control. Due to the rich diversity and complexity of pathogenic fungi and microorganisms, and their diverse metabolic pathways, microorganisms provide a wealth of biological resources for modern agriculture and green production. Currently, in China, bio-herbicide granules developed using the *Sclerotium rolfsii* strain SC64, when combined with a double-chain rotary tiller, have shown effective control of *S. glabra* in small-scale trials by simultaneously tilling the soil. However, given that solid granular bio-herbicides require significant investment in soil tillage, and that most *S. glabra* habitats lack the conditions for tillage (i.e., tillage is not feasible), there is a need to develop a control method that is easy to apply, lower in cost, has a wider applicability, and is convenient for mass production. Summary of the Invention

[0005] The purpose of this invention is to at least solve one of the technical problems existing in the prior art, and to provide a method for preparing a biological agent containing *Coccidioidomyces chinensis*, comprising the following steps:

[0006] S1: Inoculate *Cyclocarya spp.* MP1 onto PDA medium and incubate in a constant temperature incubator for 5-7 days; take the mycelial cakes from the edge of the vigorously growing colonies; place the mycelial cakes in PDB liquid medium and incubate on a shaker at 30-35℃ and 200-215rpm for 3-5 days. After the formation of mycelial balls, crush the mycelial balls to make a uniform mycelial suspension.

[0007] S2: Prepared by adding 10-15 ml of Tween 80 to every 1 L of mycelial suspension.

[0008] As a preferred embodiment of the present invention, the PDA culture medium comprises the following weight components: 200-215 g potato, 20-25 g glucose, 15-20 g agar, and 1000 ml distilled water. After sterilization at 115-120°C for 20-25 minutes, the medium is cooled for later use.

[0009] As a preferred embodiment of the present invention, the PDB culture medium has the following weight composition: 300-310 grams of potato and 20-25 grams of glucose per liter of culture medium, dissolved in water to 1L, autoclaved at 121℃ for 20-25 minutes, and cooled to 50-55℃ for later use.

[0010] As a preferred embodiment of the present invention, the Macropomina phaseolina MP1, classified as Macropomina phaseolina, was deposited on December 28, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 41077.

[0011] As a preferred embodiment of the present invention, the gene sequence of the *Coccobacillus davidii* MP1 is shown in SEQ ID NO.1.

[0012] The present invention also discloses a biological agent containing *Coccidioidomyces beanus*, which is prepared by the method described above.

[0013] This invention also discloses the application of a biological agent containing *Cyclocarya spp.* in the control of *Solidago canadensis*. After treating *Solidago canadensis* stalks, the agent containing *Cyclocarya spp.* MP1 is sprayed onto the cross-section of the stalk stems.

[0014] As a preferred embodiment of the present invention, the application rate of the microbial agent is 6000-8000 ml / mu.

[0015] As a preferred embodiment of the present invention, the spraying is specifically carried out during or before the flowering period of Canadian goldenrod.

[0016] As a preferred embodiment of the present invention, the spraying is carried out using a high-pressure sprayer.

[0017] The beneficial effects of this invention are:

[0018] (1) The biological agent developed by the present invention using the strain of Coccidioides buergerianum, when combined with mowing, has been shown to cause necrosis of the entire root of Canadian goldenrod in indoor potted plants and field control experiments, and the effect is stable.

[0019] (2) The present invention uses a liquid herbicide containing *Sclerotium praecox*, which can be efficiently sprayed in the field using a high-pressure sprayer, effectively causing the roots of *Solidago canadensis* to rot and die. It overcomes the limitations of solid herbicide granules with *Sclerotium sclerotiorum* strain SC64 as the main component, which require application after tilling the habitat soil. The method of the present invention has a wider range of applications and is applicable to almost all terrain and habitat types.

[0020] (3) This invention realizes the preparation of a suspension of *Cyclocarya benzi* MP1 mycelium using PDB liquid culture medium and the addition of adjuvant Tween 80. The prepared biological herbicide *Cyclocarya benzi* agent significantly improves the control effect, greatly shortens the production time, and improves the production efficiency, laying the foundation for mass production and commercialization.

[0021] (4) This invention develops a novel, green, and sustainable biological herbicide, which, when combined with high-pressure spraying equipment, opens up a new control method for invasive weeds such as Canadian goldenrod. Moreover, it offers excellent control and is environmentally friendly. The herbicide, made primarily from highly efficient *Syngonium spp.*, can be directly sprayed onto the cut surface of the stem after harvesting Canadian goldenrod stalks to achieve excellent control, eliminating the need for tilling the habitat soil, reducing costs, and significantly increasing its applicability. Attached Figure Description

[0022] Figure 1 The images show the growth of Canadian goldenrod under different treatments, with rows 2 and 4 being overhead views of rows 1 and 3.

[0023] Figure 2 The root necrosis manifestations of Canadian goldenrod under different treatments;

[0024] Figure 3 The field control effects of different treatment methods;

[0025] Figure 4 The number of regenerated seedlings of Canadian goldenrod under different treatments, where A, B, and C represent the significance level of the difference between treatments at the P<0.01 level;

[0026] Figure 5 The root characteristics of Canadian goldenrod under different control treatments. Detailed Implementation

[0027] The embodiments of the present invention are described in detail below. These embodiments are exemplary and are only used to explain the present invention, and should not be construed as limiting the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0028] The technical problem addressed by this invention, namely its purpose, is to provide a process for the mass production of a biological herbicide based on highly active microorganisms as the main component, along with corresponding supporting technologies, for the control of Canadian goldenrod. This method is highly efficient, environmentally friendly, pollution-free, safe for crops, and can significantly restore local biodiversity.

[0029] The technical solution adopted in this invention is: a biological agent prepared using *Cyclocarya paliurus*, its method and application, comprising: spraying a bacterial agent containing *Cyclocarya paliurus* MP1 onto the cross-section of *Solidago canadensis* stalks.

[0030] Macropommina phaseolina (MP1) is a pathogenic fungal strain discovered and isolated from diseased plants of Solidago canadensis found in nature. It is currently deposited at the China General Microbiological Culture Collection Center (CGMCC, No. 3, Courtyard 1, Beichen Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China), with a deposit date of December 28, 2023, and accession number CGMCC No. 41077.

[0031] The gene sequence of Macropomina phaseolina (MP1) strain is shown in SEQ ID NO.1.

[0032] The preparation methods of the microbial agents used in Examples 1 and 2 below are as follows:

[0033] Preparation of S1 mycelial suspension: *Coccidioidomyces chinensis* MP1 was inoculated onto PDA medium and cultured at 30℃ for 5 days. Mycelial discs were collected from the edges of vigorously growing colonies using a 4-6 mm diameter punch. These discs were then placed in PDB liquid medium and cultured on a shaker at 30-35℃ and 200-215 rpm for 5 days. Once mycelial spheres formed, they were crushed to prepare a uniform mycelial suspension.

[0034] Preparation of S2 inoculum: Add 10 ml of Tween 80 to every 1 L of mycelial suspension to prepare a stable Coccidioides kojiana inoculum.

[0035] The weight composition of the above PDA culture medium is as follows: 200 g potato, 20 g glucose, 15 g agar, 1000 ml distilled water. After sterilization at 115°C for 20 minutes, it is cooled and ready for use.

[0036] The weight composition of the above PDB culture medium is as follows: 300 grams of potato and 20 grams of glucose per liter of culture medium, dissolved in water to 1L, autoclaved at 121℃ for 20 minutes, and cooled to about 50℃ for use.

[0037] The spraying is carried out using high-pressure spraying. The specific spraying device is a new type of electric sprayer, which includes an electric pressure pump, a dual-hole nozzle, a spray control switch, and a connecting rod, but is not limited to this.

[0038] Control efforts are carried out during and before the flowering period of Canadian goldenrod. The application rate of the biological herbicide is 6000-8000 ml / acre. It begins to take effect after 10-15 days and shows control effect after 50-60 days.

[0039] The present invention will now be described in detail with reference to specific embodiments.

[0040] Example 1

[0041] A comparison of the control effects of *Solidago canadensis* and *Sclerotium sclerotiorum* on *Solidago canadensis*.

[0042] To compare the control effects of *Sclerotinia spp.* and *Sclerotinia spp.* on the roots of *Solidago canadensis*, a pot experiment was conducted (two roots per pot). Roots from normal growing conditions (with the above-ground stalks removed) were collected from the wild and brought back to the laboratory, then planted in nutrient pots. Three treatments were set up: *Sclerotinia spp.* inoculum sprayed on the stem section (MP1), *Sclerotinia spp.* inoculum sprayed on the stem section (SR1), and a control treatment sprayed on PDB liquid medium (CK). Each pot was sprayed with 2 ml of inoculum. Each treatment was replicated 10 times (pots), and the treatment lasted for 20 days. The control effect was observed after 20 days, and the results are as follows: Figure 1 , 2 As shown.

[0043] from Figure 1 and Figure 2 It can be seen that the mortality rate of the *Solidago canadensis* inoculant MP1 against the roots of *Solidago canadensis* reached 90%, which is significantly higher than that of the *Sclerotium sclerotiorum* inoculant SR1 (65% mortality rate).

[0044] The roots of the three treatments were dug up, and the results showed that the proportion of root rot and necrosis of *Solidago canadensis* treated with *Sclerotium moniliforme* inoculant MP1 was significantly higher than that treated with *Sclerotium moniliforme* inoculant SR1.

[0045] In conclusion, the control effect of *Sclerotium pratensis* MP1 is significantly better than that of *Sclerotium sclerotiorum*.

[0046] Example 2

[0047] Demonstration effect of field control of Coccidioides mongolicum MP1 inoculant.

[0048] In June, before flowering, a field demonstration experiment was conducted on the control effect of the *Solidago canadensis* MP1 inoculant formulated in this embodiment on a typical *Solidago canadensis* invasive area (invaded for more than 3 years, with an invasion density of more than 80 plants / square meter) in Honggutan District, Nanchang City. The demonstration experiment included four treatments: spraying *Solidago canadensis* MP1 inoculant after cutting the above-ground straw (8000 ml / mu), spraying with 20% aminopyridine acid (2000 ml / mu) (the most effective chemical control method developed, with a control efficiency of approximately 100%, used as a positive control), cutting only the above-ground straw, and a control treatment (spraying with water). Each treatment had three replicate plots, each 67 square meters. Observations were taken every 15 days after treatment, and the control effect was observed after 60 days by digging up the roots. The control efficacy, the number of newly regenerated seedlings, and the results were examined through photographic observation.

[0049] The results showed that, Figure 3 As shown in Table 1, the growth of *Coccidioidomyces chinensis* was severely inhibited after treatment with inoculum, and the control efficacy of the corrected-line plants was significantly higher than that of the mowing treatment, while the efficacy was not significantly different from that of the chemical treatment (i.e., the control efficacy was very close to that of the chemical treatment). The number of rhizome-regenerated seedlings was significantly lower than that of the mowing treatment, and it showed a decreasing trend over time. Figure 4 As shown), the degree of root necrosis and the condition of regenerated seedlings were observed after 60 days of treatment. The roots of MP1 and chemically treated plants were completely necrotic. The root vigor of the mowing treatment was stronger than that of the CK control, and a large number of buds / seedlings sprouted. Figure 5 (As shown). In summary, the *Syntheticotropha buergeriana* inoculant is highly effective in controlling *Solidago canadensis* in the field, completely killing the stubborn roots and resulting in a very low rate of seedling regeneration.

[0050] Table 1. Control efficacy of each treatment on corrected plants of *Solidago canadensis*.

[0051]

[0052] Without causing conflict, those skilled in the art can freely combine and use the above-mentioned additional technical features.

[0053] The embodiments described above are merely preferred embodiments of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various other corresponding changes and modifications based on the technical solutions and concepts described above, and all such changes and modifications should fall within the protection scope of the claims of the present invention.

Claims

1. A method for preparing a biological agent containing *Coccidioidomyces chinensis*, characterized in that, Includes the following steps: S1: Inoculate *Cyclocarya spp.* MP1 onto PDA medium and incubate in a constant temperature incubator for 5-7 days; take the mycelial cakes from the edge of the vigorously growing colonies; place the mycelial cakes in PDB liquid medium and incubate on a shaker at 30-35℃ and 200-215 rpm for 3-5 days. After the formation of mycelial balls, crush the mycelial balls to make a uniform mycelial suspension. S2: Prepared by adding 10-15 ml of Tween 80 to every 1 L of mycelial suspension; The *Coccidioidomyces chinensis* MP1 strain is classified and named as follows: Macrophomina phaseolina It was deposited on December 28, 2023, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 41077.

2. The method for preparing a biological agent containing *Coccidioidomyces chinensis* according to claim 1, characterized in that, The PDA culture medium consists of the following components by weight: 200-215 g potato, 20-25 g glucose, 15-20 g agar, and 1000 ml distilled water. After sterilization at 115-120°C for 20-25 minutes, the medium is cooled and ready for use.

3. The method for preparing a biological agent containing *Coccidioidomyces chinensis* according to claim 1, characterized in that, The PDB culture medium has the following weight composition: 300-310 grams of potato and 20-25 grams of glucose per liter of medium, dissolved in water to 1L, autoclaved at 121℃ for 20-25 minutes, and cooled to 50-55℃ for later use.

4. The method for preparing a biological agent containing *Coccidioidomyces chinensis* according to claim 1, characterized in that, The gene sequence of *Cyclocarya beanica* MP1 is shown in SEQ ID NO.

1.

5. A biological agent containing *Coccidioidomyces chinensis*, characterized in that, It is prepared by the preparation method described in any one of claims 1-4.

6. The application of the biological agent as described in claim 5 in the prevention of Canadian goldenrod, characterized in that, After harvesting the straw of Canadian goldenrod, a fungal agent containing *Syntropha buergeriana* MP1 was sprayed onto the cut surface of the straw stem.

7. The application as described in claim 6, characterized in that, The application rate of the microbial agent is 6000-8000 ml / mu.

8. The application as described in claim 6, characterized in that, The spraying is specifically carried out during or before the flowering period of Canadian goldenrod.

9. The application as described in claim 6, characterized in that, The spraying was carried out using a high-pressure sprayer.

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