A biological alternative control method to suppress the invasion of *Chrysanthemum indicum*

Abstract

This invention discloses a biological alternative control method for suppressing the invasion of *Chrysanthemum indicum*, belonging to the field of biological control technology. The method involves intercropping *Artemisia annua* and *Euphorbia milii* in ecosystems where *Chrysanthemum indicum* has already invaded or is susceptible to invasion, thereby inhibiting the growth and development of *Chrysanthemum indicum* and achieving ecosystem restoration. Experimental results show that *Artemisia annua* and *Euphorbia milii* are capable of playing a key role in ecosystems where *Chrysanthemum indicum* is susceptible to or has already invaded. In the dry spring of northern regions, intercropping *Artemisia annua* and *Euphorbia milii* effectively inhibits the growth of *Chrysanthemum indicum* and allows it to occupy its ecological niche, significantly reducing the plant height, biomass, and number of flower buds. This invention provides a solid foundation for the alternative control of the invasive alien species *Chrysanthemum indicum* and the restoration and sustainable management of damaged ecosystems.

Description

Technical Field

[0001] This invention relates to the field of biocontrol technology, and more particularly to a bio-alternative control method for inhibiting the invasion of *Chrysanthemum indicum*. Background Technology

[0002] Biological invasion is considered one of the main causes of ecosystem degradation and biodiversity loss worldwide. Yellow-topped daisy (Flaveria bidentis), also known as two-toothed yellow daisy, is an annual herbaceous plant belonging to the genus Flavedia in the tribe Flavedia of the family Asteraceae, native to South America. Due to its high seed production, small and light seeds, and ease of dispersal, yellow-topped daisy can rapidly invade farmland, orchards, woodlands, river channels, and embankments, often covering large areas. It is a malignant invasive plant, urgently requiring effective control and management measures.

[0003] Currently, the main methods for controlling yellow chrysanthemum include physical control, chemical control, and biological control. Physical control is one of the most effective methods, but it consumes a lot of manpower and resources, is inefficient, and is not suitable for areas with severe infestation or large areas. Chemical control uses non-selective herbicides such as glyphosate and paraquat, which are effective against yellow chrysanthemum, but they are ineffective against seeds in the soil and cannot eradicate it completely. Furthermore, they pose threats to human health and non-target species. Biological control utilizes natural enemies or pathogenic microorganisms to control yellow chrysanthemum, but it is currently not an effective way to completely eradicate it.

[0004] Alternative control is considered an effective measure to both control the invasion of invasive alien plants and maintain a healthy and stable ecological environment. Some scholars have studied the competitive effects of native plants on invasive plants, finding that mixing native plants with different functional traits can effectively inhibit the growth, development, and spread of invasive plants. For example, using *Amorpha fruticosa*...

[0005] The combined use of plants such as *Amorphafruticosa*, *Hippophae rhamnoides*, *Coronilla varia*, *Poa pravensis*, and *Helianthus tuberosus* can effectively control the germination and growth of the invasive alien plant *Ravengrass*. Dense vegetation formed by *Syngonium podophllum* and *Cuphea hyssopifolia* has also shown initial success in controlling the invasive plant *Macfadyena unguis-cati* (L.) A. Gentry. Currently, researchers have used plants such as *Sorghum succinum*, *Alternanthera philoxeroides*, and *Cosmos bipinnatus* to suppress *Gynostemma pentaphyllum*. While these methods have some control effect, their application is limited. Given the diversity of my country's geographical environment, it is necessary to explore different biological alternatives to effectively control the invasive alien species *Gynostemma pentaphyllum*, while simultaneously promoting the restoration of damaged ecosystems and the optimal utilization of plant resources. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of existing technologies and provide an effective and sustainable method for controlling the invasion of the invasive species *Chrysanthemum indicum* and effectively curbing its spread, thereby achieving ecological restoration and improving plant resources. Specifically, this invention provides a biological alternative control method for inhibiting the invasion of *Chrysanthemum indicum*.

[0007] This invention is achieved through the following technical solution, specifically providing a biological alternative control method to inhibit the invasion of *Chrysanthemum indicum*. The method involves intercropping *Artemisia annua* and *Euphorbia milii* in ecosystems where *Chrysanthemum indicum* has already invaded or is easily invaded, thereby inhibiting the growth and development of *Chrysanthemum indicum* and achieving ecosystem restoration.

[0008] As a further optimization of the present invention, the Artemisia annua is sown by spot sowing, and the sowing depth of the Artemisia annua seeds is 1cm-3cm.

[0009] As a further optimization of the present invention, the Napier grass is propagated asexually by sowing and planting, specifically by transplanting the stem nodes of the Napier grass at a depth of 2cm-3cm.

[0010] As a further optimization of the present invention, the method of transplanting and planting using the stem nodes of Napier grass includes: selecting mature and healthy stem nodes, with at least one bud on each node, disinfecting the stem nodes and pre-seeding them in March, and transplanting them one month after pre-seeding.

[0011] The pre-seedling process involves placing a disinfected stem node obliquely into the soil, covering it with soil, and watering it thoroughly. The pre-seedling is completed after 7-10 days when it sprouts.

[0012] As a further optimization of the present invention, the planting density of Artemisia annua and Napier grass is 2-4 plants / m². 2 The planting row spacing for Artemisia annua is 30cm-35cm, and the planting row spacing for Napier grass is 30cm-50cm.

[0013] As a further optimization of the present invention, the ratio of the mixed planting of Artemisia annua and Napier grass is (1-2):(1-2).

[0014] As a further optimization of the present invention, the type of ecosystem that the yellow-topped chrysanthemum is prone to invade is wasteland without large vegetation cover, with an average annual herbaceous vegetation cover of 40-70%.

[0015] Compared with the prior art, the present invention has the following technical advantages:

[0016] This invention introduces Artemisia annua and Napier grass as alternative control methods for the invasive species Chrysanthemum indicum. The results show that Artemisia annua and Napier grass are fully capable of playing a key role in ecosystems where Chrysanthemum indicum is susceptible to or has already invaded. In the dry spring of northern regions, mixed planting of Artemisia annua and Napier grass effectively inhibits the growth of Chrysanthemum indicum and allows it to occupy its ecological niche, significantly reducing the plant height, biomass, and number of flower buds. This invention provides a solid foundation for the alternative control of the invasive species Chrysanthemum indicum and for the restoration and sustainable management of its ecosystem.

[0017] Furthermore, this invention has found that intercropping Artemisia annua and Napier grass can effectively inhibit the growth of Napier grass, demonstrating its effectiveness in biological control against Napier grass invasion. In addition, adjusting the planting ratio between the control plants can also increase the biomass of Napier grass to a certain extent, providing a material basis for the high-value and efficient utilization of Napier grass biomass resources. Attached Figure Description

[0018] Figure 1 A schematic diagram of the experimental treatment design provided for this invention (in the figure: A represents Chrysanthemum indicum; B represents Artemisia annua; C represents Napier grass). Detailed Implementation

[0019] The present invention will now be described in further detail. It should be noted that the following specific embodiments are only used to further illustrate the present invention and should not be construed as limiting the scope of protection of the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention based on the above application content.

[0020] This invention provides a biological substitution method for suppressing the invasion of *Chrysanthemum indicum*, specifically employing *Artemisia annua* and / or *Euphorbia milii* as biological substitutes. In this invention, the term "biological substitution method" refers to a method that, by planting one or more plants, inhibits the growth and development of one or more other plants in the same growing environment, thereby allowing the "one or more plants" to replace the "other one or more plants" in the same growing environment. This biological substitution method belongs to a biological control method targeting the replaced plant. In this invention, the replaced plant is *Chrysanthemum indicum*, and the plants used to replace *Chrysanthemum indicum* are *Artemisia annua* and / or *Euphorbia milii*.

[0021] The *Artemisia annua* L. selected for this invention, also known as wormwood, is an annual herbaceous plant belonging to the genus *Artemisia* in the family Asteraceae. It is highly branched, hairless, and entirely bright green with a fragrant aroma. The whole plant is used medicinally, possessing properties that clear heat from the blood, reduce fever, and relieve summer heat. It is a major raw material for the antimalarial drug artemisinin and can be used to treat malaria and other ailments. *Artemisia annua* has wide ecological adaptability, is drought-tolerant and tolerant of poor soil, grows rapidly, and exhibits strong interspecific competition. It is commonly found along roadsides, wastelands, and hillsides.

[0022] The Napier grass (Pennisetum hydridum), also known as hybrid Napier grass, selected for this invention, is a perennial triploid grass belonging to the C4 family. It is characterized by high photosynthetic efficiency, rapid growth, high yield, and wide adaptability. In addition, it is rich in nutrients, has a well-developed root system, a long forage supply period, and strong stress resistance. As a fast-growing lignocellulose raw material, Napier grass not only performs well in the energy industry but is also widely used as an environmental remediation material and high-quality forage.

[0023] When using Artemisia annua and / or Napier grass as a biological substitute for Chrysanthemum indicum, the following descriptions are provided regarding the planting method, planting time, planting row spacing, planting depth, planting density, and post-planting maintenance:

[0024] Regarding the planting method, the method of the present invention does not have any particular restrictions on the sowing method of Artemisia annua. For example, row sowing, spot sowing or broadcast sowing can be used. However, considering the comprehensive consideration of more fully inhibiting the growth of Chrysanthemum indicum and facilitating the harvesting of Artemisia annua, spot sowing is preferred. For Napier grass, since its seed germination rate is low, asexual reproduction is adopted, that is, transplanting using stem nodes.

[0025] Regarding planting time, the method of this invention does not impose any particular restrictions on the planting time of Artemisia annua and Napier grass, as long as the climatic conditions are suitable for their emergence and growth. Chrysanthemum 'Yellow Top' seedlings grow slowly; in northern regions, they emerge around late April. Artemisia annua emerges up to 20 days earlier, and generally forms a dominant population before Chrysanthemum 'Yellow Top' emerges, thus inhibiting its growth. Napier grass is usually pre-seeded from February to May, and transplanted about a month after pre-seeding, typically between March and June. For ease of operation, the preferred planting time for both Artemisia annua and Napier grass is early April.

[0026] Regarding the planting spacing, there are no particular limitations in the method of this invention. Considering the need to more fully suppress the growth of Chrysanthemum indicum and facilitate the growth of Artemisia annua and Napier grass, the row spacing for sowing Artemisia annua is preferably 30cm-35cm, and the plant spacing for transplanting Napier grass is generally preferably 30-50cm. For ease of operation, the planting spacing for both Artemisia annua and Napier grass is preferably 30cm.

[0027] Regarding planting depth, if the sowing depth is too shallow, it may not be conducive to the fixation of the roots of Artemisia annua and Napier grass, thus affecting their later growth. The sowing depth of Artemisia annua is generally 1cm-3cm, and the transplanting depth of Napier grass is generally 2cm-3cm.

[0028] Regarding planting density, too low a density may hinder the suppression of *Chrysanthemum indicum*, while too high a density may impede the growth of *Artemisia annua* and *Euphorbia milii*. In the method of this invention, the preferred planting density for *Artemisia annua* and *Euphorbia milii* is 2-4 plants / m². 2 .

[0029] Regarding post-planting maintenance, this invention does not impose any specific limitations; conventional field methods can be used for the maintenance and management of Artemisia annua and Napier grass. Note that human activities unrelated to ecosystem management must be strictly prohibited in the experimental area, especially trampling by humans and livestock, fertilization, or mowing, to avoid adverse effects on plant growth and interspecific competition, and to ensure the reliability of the alternative control experiment results.

[0030] The invention will now be described in conjunction with specific alternative prevention and treatment experiments.

[0031] 1. Test materials

[0032] Mature seeds of *Chrysanthemum indicum* and *Artemisia annua* (a type of wild grass) with high density and tall stature, found in their natural growing areas, were collected, purified, and stored at room temperature. Additionally, *Erigeron tigrinum* was selected for an alternative control experiment. Due to the low germination rate of *Erigeron tigrinum* seeds, asexual propagation was employed, using stem segments for planting. Mature, healthy stem segments with at least one bud per segment were selected, disinfected, and pre-seeded. Pre-seeding was carried out in March, with the stem segments placed obliquely in the soil, covered with soil, and watered thoroughly. Germination occurred in approximately 7 to 10 days. Transplanting was carried out about one month after pre-seeding.

[0033] 2. Test site

[0034] The experimental site was an experimental field of the Chinese Academy of Agricultural Sciences, belonging to a wasteland, grassland, and farmland ecosystem with no large vegetation cover. The average annual herbaceous vegetation cover was approximately 40-70%, making it an ecosystem type easily invaded by *Chrysanthemum indicum*. Before conducting the planting experiment, since the experimental field had not yet been invaded by *Chrysanthemum indicum*, only land leveling was required. After land preparation, the experimental field was thoroughly watered to ensure soil moisture.

[0035] 3. Experimental Treatment Design

[0036] Several 1m×1m planting plots were planned in the experimental field. Yellow-top chrysanthemum, Artemisia annua, and Napier grass were used as experimental materials. Three planting patterns were employed: yellow-top chrysanthemum alone, yellow-top chrysanthemum mixed with either Artemisia annua or Napier grass, and yellow-top chrysanthemum mixed with Artemisia annua and Napier grass. The total planting density for each pattern was 6 plants / m². 2 In addition, different mixing ratios were set for the mixed-cropping planting pattern. Specific experimental treatment designs are shown in Table 1 and... Figure 1 As shown. Figure 1 As shown, A represents Chrysanthemum indicum; B represents Artemisia annua; and C represents Napier grass.

[0037] Table 1. Experimental Treatment Design Table

[0038]

[0039] 4. Planting and Post-Planting Management

[0040] The planting of each treatment group was carried out according to the experimental design disclosed in step 3. For *Chrysanthemum indicum* and *Artemisia annua*, the planting method was spot sowing in early April. To ensure even seed distribution, the seeds of *Chrysanthemum indicum* and *Artemisia annua* were mixed with fine river sand at a ratio of 1:10 and evenly sown on the surface soil of the experimental field. A thin layer of soil (1 cm thick) was then used to cover the seeds to facilitate germination and growth. For *Euphorbia milii*, the planting method was based on stem segments. In early April, pre-germinated stem segments were transplanted. The planting holes were dug to a depth of 2 cm, and the stem segments were placed in the holes and then covered with a thin layer of soil to compact the soil and promote stem growth.

[0041] Because the experimental field is located in a northern region where spring is dry and rainfall is scarce, the soil was kept moist by spraying with an electric sprayer every other day for the first two weeks after planting. No fertilizer was applied during the plant's growing season. It is important to strictly prohibit human activities unrelated to ecosystem management in the experimental field area, especially trampling by humans and livestock, fertilization, or mowing, to avoid adverse effects on plant growth and interspecific competition, and to ensure the reliability of the alternative control experiment results. Apart from the above requirements, conventional field management methods can be used for maintenance.

[0042] 5. Determine relevant growth indicators of Chrysanthemum indicum.

[0043] The alternative control trial continued until September of that year. In early September, the plant height, number of flower buds, and biomass of *Chrysanthemum indicum* under different planting patterns were measured.

[0044] The methods for determining the plant height, number of flower buds, and biomass of *Chrysanthemum indicum* in each treatment group are as follows:

[0045] (1) Plant height: The plant height of each treatment group of Chrysanthemum in yellow top was measured with a tape measure and the average plant height of each treatment group was calculated. The unit is cm.

[0046] (2) Number of flower buds: The number of flower buds of each treatment group of Chrysanthemum in yellow tops was counted manually, and the average number of flower buds of Chrysanthemum in yellow tops in each treatment group was calculated. The unit is: flower buds.

[0047] (3) The biomass of *Chrysanthemum indicum* was measured as follows: All whole *Chrysanthemum indicum* plants in each treatment group were dug up from the soil, rinsed thoroughly with clean water, and the remaining roots were removed with tweezers. The plants were separated into roots, stems, and leaves, and then blanched at 105℃ for 2 hours, followed by drying at 65℃ for 72 hours. The total biomass was determined as the sum of the biomass of the roots, stems, and leaves of *Chrysanthemum indicum*. Based on the total biomass of *Chrysanthemum indicum* in each treatment group, the average biomass of each treatment group was calculated, in g.

[0048] The results are shown in Table 2.

[0049] Table 2. Measurement of plant height, number of flower buds and biomass of *Chrysanthemum indicum* under different planting patterns.

[0050]

[0051] Note: The numbers in the planting patterns represent different planting densities, and the letters represent different plants.

[0052] As can be seen from Table 2, when Artemisia annua or Napier grass were used alone as biological substitutes for Chrysanthemum morifolium, the average plant height, average number of flower buds, and average biomass of Chrysanthemum morifolium were significantly reduced compared to Chrysanthemum morifolium planted alone. Furthermore, the table shows that the degree of reduction in the average plant height, average number of flower buds, and average biomass of Chrysanthemum morifolium is also somewhat correlated with the mixed planting ratio of Chrysanthemum morifolium with Artemisia annua or Napier grass.

[0053] Table 2 also shows that the intercropping of Artemisia annua and Napier grass is more effective in biologically substituting for Chrysanthemum infestation than using either alone. Artemisia annua, with its rapid growth, drought tolerance, tolerance to poor soil, and earlier emergence than Chrysanthemum infestation areas, exhibits a strong competitive advantage, thus inhibiting the growth of Chrysanthemum seedlings. Napier grass, with its well-developed root system, rapid growth, and strong resistance, possesses a spatial advantage, competing with Chrysanthemum for light and thus suppressing its growth. Furthermore, due to the differences in their growth and development stages, Artemisia annua and Napier grass can continuously emerge from April to September under suitable temperatures, forming a mixed population with different growth stages, occupying the ecological niche of Chrysanthemum infestation. This diversified population structure is particularly effective in suppressing the growth of Chrysanthemum in the later stages. Therefore, the intercropping of Artemisia annua and Napier grass can successfully and effectively control the invasion of the harmful invasive plant Chrysanthemum infestation.

[0054] In addition, Table 2 also shows that there is a certain correlation between different mixed planting ratios of Artemisia annua and Napier grass and the biological substitution effect of Chrysanthemum indicum. The best inhibitory effect on Chrysanthemum indicum can be achieved by adjusting different mixed planting ratios of Artemisia annua and Napier grass.

[0055] To investigate the changes in biomass of Artemisia annua and Napier grass when they were used as biological substitutes for Chrysanthemum indicum, the biomass of Artemisia annua and Napier grass was further measured at the end of the substitution control experiment using the same method as that used for Chrysanthemum indicum. The results are shown in Table 3.

[0056] Table 3. Biomass determination of Artemisia annua and Napier grass under different planting patterns

[0057]

[0058] As shown in Table 3, under the mixed planting pattern of Artemisia annua and Chrysanthemum indicum, increasing the planting density of Artemisia annua has a positive effect on improving its biomass, provided that the total planting density remains unchanged. Similarly, under the mixed planting pattern of Napier grass and Chrysanthemum indicum, increasing the planting density of Napier grass also has a positive effect on improving its biomass, provided that the total planting density remains unchanged.

[0059] Furthermore, comparing planting patterns where Artemisia annua and Napier grass are intercropped with Chrysanthemum indicum, for example, group 3A1B2C versus group 4A2C, it can be seen that when the planting density of Napier grass is 2 plants / m², the results are more effective. 2 Adjusting the planting density of *Chrysanthemum indicum* and *Artemisia annua* has a positive effect on increasing the biomass of *Euphorbia tirucalli*, while ensuring the growth-inhibiting effect on *Chrysanthemum indicum*. Conversely, in groups 3A2B1C and 4A2B, when the planting density of *Artemisia annua* was 2 plants / m², the results were different. 2 Adjusting the planting density of *Artemisia annua* and *Euphorbia milii* did not significantly change the biomass of *Artemisia annua*, but the 3A2B1C group showed a slightly better inhibitory effect on the growth of *Artemisia annua* than the 3A1B2C group. In summary, the intercropping of *Artemisia annua* and *Euphorbia milii* can effectively inhibit the growth of *Artemisia annua*, demonstrating its application in biological control against its invasion. Furthermore, adjusting the planting density can also increase the biomass of *Euphorbia milii*, providing a material basis for the high-value and efficient utilization of *Euphorbia milii* biomass resources.

[0060] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively 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 modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A biological alternative control method for inhibiting the invasion of *Chrysanthemum indicum*, characterized in that, The method is as follows: by interplanting Artemisia annua and Napier grass in ecosystems where Chrysanthemum 'Yellow Top' has invaded or is easily invaded, the growth and development of Chrysanthemum 'Yellow Top' is inhibited, thereby restoring the ecosystem. Specifically, in the dry spring in the north, interplanting Artemisia annua and Napier grass effectively inhibits the growth of Chrysanthemum 'Yellow Top' and allows it to occupy its ecological niche, resulting in a significant reduction in the plant height, biomass, and number of flower buds of Chrysanthemum 'Yellow Top'. When the ratio of Artemisia annua and Napier grass is 2:1, it can effectively prevent and inhibit the invasion of Artemisia annua while promoting the increase of Napier grass biomass.

2. The biological alternative control method for inhibiting the invasion of *Chrysanthemum indicum* according to claim 1, characterized in that, The Artemisia annua is sown using a spot-seeding method, and the sowing depth of the Artemisia annua seeds is 1cm-3cm.

3. The biological alternative control method for inhibiting the invasion of *Chrysanthemum indicum* according to claim 1, characterized in that, The Napier grass is propagated asexually by sowing and planting, specifically by transplanting stem nodes. The transplanting depth of the Napier grass stem nodes is 2cm-3cm.

4. The biological alternative control method for inhibiting the invasion of *Chrysanthemum indicum* according to claim 3, characterized in that, The method of transplanting and planting using stem nodes of Napier grass includes: selecting mature and healthy stem nodes, with at least one bud on each node, disinfecting the stem nodes and then raising seedlings in March, and transplanting the seedlings one month after raising them. The seedling raising process involves placing the disinfected stem nodes obliquely into the soil, covering them with soil, and watering them thoroughly. Seedlings will sprout in 7-10 days, at which point the seedling raising is complete.

5. The biological alternative control method for inhibiting the invasion of *Chrysanthemum indicum* according to claim 1, characterized in that, The planting row spacing for Artemisia annua is 30cm-35cm, and the planting row spacing for Napier grass is 30cm-50cm.

6. The biological alternative control method for inhibiting the invasion of *Chrysanthemum indicum* according to claim 1, characterized in that, The types of ecosystems that the yellow-topped chrysanthemum is susceptible to invasion are wastelands without large vegetation cover, with an average annual herbaceous vegetation cover of 40-70%.

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