Method for improving inoculation rate of desert cistanche

Abstract

The application discloses a method for improving inoculation rate of desert Cistanche, and comprises the following steps: determining host forest planting plot, host forest planting time and host plant of the host forest; digging multiple planting pits with equal interval distribution in the host forest planting plot during the host forest planting time; placing four-winged Atriplex container seedlings in the middle of the planting pit, and digging an inoculation groove in the planting pit around the container outer edge; mixing the soil dug out of the inoculation groove with desert Cistanche seeds after physiological after-ripening, and then filling back; covering the planting pit with soil to the ground level, and watering the planting pit once within 3 days after the soil covering is completed; and maintaining and managing the host forest. The method has the following remarkable effects: not only greatly reducing the seed consumption of Cistanche and improving the seed utilization rate, but also accelerating the planting of the host forest and the subsequent inoculation speed of the desert Cistanche, reducing the requirement for the seedling age of the host plant, and effectively reducing the cultivation cost.

Description

Technical Field

[0001] This invention relates to the field of plant resource development and utilization technology, specifically to a method for increasing the inoculation rate of desert Cistanche deserticola. Background Technology

[0002] *Cistanche deserticola* is a holoparasitic plant belonging to the genus *Cistanche* in the family Orobanchaceae. It parasitizes the roots of other plants, and its fleshy stems are used medicinally. *Cistanche deserticola* is a traditional and precious Chinese medicinal herb, listed as a superior-grade herb in the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica). It possesses extremely high medicinal value, with effects such as tonifying the kidneys and strengthening yang, nourishing blood and moisturizing dryness, and improving complexion and prolonging life. It is known as "desert ginseng." Modern pharmacological studies have shown that *Cistanche deserticola* plays a significant role in hormone regulation, immune regulation, neuroprotection, antioxidation, anti-apoptosis, anti-damage, anti-inflammation, anti-fatigue, and promoting bone formation.

[0003] Due to its weak natural reproductive capacity and the long-term over-harvesting of its fleshy stems, *Cistanche deserticola* has no opportunity for further reproductive development, resulting in a sharp decline in its population and its listing as a national second-class protected plant. To protect the wild resources of *Cistanche deserticola* and its surrounding ecosystem, promote the sustainable development of related traditional Chinese medicine industries, and meet the ever-expanding market demand, it is necessary to vigorously develop large-scale cultivation of *Cistanche deserticola*.

[0004] However, although there is considerable research on the cultivation techniques of Cistanche deserticola, high-quality and stable-yield cultivation techniques are still in the research and development stage, with low inoculation rates and high inoculation costs. Furthermore, currently, the artificial cultivation of desert Cistanche deserticola mostly uses Haloxylon ammodendron as a host, with a smaller portion using Trichosanthes kirilowii, but primarily uses host plants that are two years or older. Establishing host forests requires a long time and results in high cultivation costs. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the purpose of this invention is to provide a method for increasing the inoculation rate of desert Cistanche deserticola. This method can not only significantly reduce the amount of Cistanche deserticola seeds used and improve seed utilization, but also accelerate the establishment of host forests and the subsequent inoculation of desert Cistanche deserticola, reduce the requirements for the age of host plant seedlings, and effectively reduce cultivation costs.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] A method for increasing the inoculation rate of Cistanche deserticola, the key of which includes the following steps:

[0008] Step 1: Determine the host forest establishment site, the host forest establishment time, and the host plants for the host forest, wherein the host plants are container seedlings of Triplophysa quassop at three months of age or older.

[0009] Step 2: During the host forest establishment period, dig multiple planting pits evenly spaced in the host forest establishment plot. The diameter of each planting pit should be at least 20cm larger than the container size of the four-winged quinoa seedling, and the depth should not be less than the height of the container.

[0010] Step 3: Place the four-winged quinoa container seedling in the center of the planting pit, and dig inoculation grooves around the planting pit on the outer edge of the container.

[0011] Step 4: Mix the soil excavated from the inoculation trough with the physiologically matured desert Cistanche deserticola seeds and then fill it back in. The amount of seeds mixed is 0.02g / kg.

[0012] Step 5: Cover the planting pit with soil until it reaches the ground level. Water the planting pit once within 3 days after covering with soil.

[0013] Step 6: Maintain and manage the host forest.

[0014] Furthermore, in step 1, the site for establishing the host forest should be a flat area with soil consisting of quicksand or sandy loam.

[0015] Furthermore, in step 1, the host forest establishment time is selected from late spring to early autumn, and the minimum temperature is consistently above 0℃ and the average temperature is not lower than 10℃.

[0016] Furthermore, the height of the above-ground part of the four-winged quinoa container seedling is not less than 35cm, and the ends of the newly grown roots are about to grow out of the bottom of the container.

[0017] Furthermore, the container diameter of the container for the four-winged quinoa seedling is not less than 15cm and the height is not less than 18cm.

[0018] Furthermore, the width and depth of the inoculation groove in step 3 are both not less than 10cm.

[0019] Furthermore, in step 5, when watering the planting pits, water should be poured above the inoculation trough where the desert Cistanche deserticola seeds were sown until the soil is thoroughly soaked, and the water should be allowed to seep down at least 2 meters.

[0020] Furthermore, in step 6, during the maintenance and management of the host forest, water is applied three times with a one-month watering cycle after the first watering, and then water is replenished regularly in spring and autumn each year.

[0021] The significant effects of this invention are:

[0022] 1. By transplanting three-month-old or older *Acer tetrawingense* host plants in containers for host forest establishment, the root system is basically not damaged, minimizing the impact of transplantation on the host plant's root system. Therefore, a long root recovery period is not required, which is conducive to the faster adaptation of *Acer tetrawingense* roots and the rapid recovery of normal growth rate. Moreover, the selection of young host plants accelerates the establishment of host forests and the subsequent inoculation of *Cistanche deserticola*.

[0023] 2. Since the root growth of plants is limited within a certain period of time, transplanting the *Cistanche deserticola* host plant in containers can restrict its roots to grow downwards, making the root growth direction predictable and the root density high. This allows for successful inoculation simply by filling the inoculation troughs on the outer edge of the container with soil mixed with *Cistanche deserticola* seeds. This not only significantly reduces the amount of *Cistanche deserticola* seeds used and improves seed utilization, but also limits the main sowing area to the inoculation troughs on the outer edge of the container, reducing the sowing range of *Cistanche deserticola* seeds. Furthermore, restricting the root growth direction increases the density of roots in a fixed direction, increasing the contact opportunities between *Cistanche deserticola* seeds and *Cistanche deserticola* roots, thereby effectively improving the inoculation rate.

[0024] 3. Fill the inoculation trough on the outer edge of the container with soil mixed with the desert Cistanche deserticola seeds. This co-burying method allows the desert Cistanche deserticola seeds to grow within three months while co-burying with the host plant, Trichoderma tetrapanax. The current sowing area and depth of Cistanche deserticola can meet the needs of the seeds to feel the stimulation of the host roots near the Trichoderma tetrapanax, and achieve natural dormancy breaking and germination within three months without affecting the inoculation effect. There is no need to expose the seeds to sunlight in advance to break dormancy. Instead, the seeds break dormancy in the soil and grow towards the host roots while the Trichoderma tetrapanax grows, which greatly improves the efficiency of inoculation.

[0025] 4. An inoculation experiment of *Cistanche deserticola* on 3-month-old *Trichoderma tetragonum* seedlings was conducted. *Trichoderma tetragonum* seedlings of 3 months old could be successfully inoculated with *Cistanche deserticola* after transplanting and growing to half a year old. Since the new roots of the parasitic plant could only grow downwards due to container constraints, the experiment showed that this method of inoculating *Cistanche deserticola* could achieve a 100% inoculation rate, with no significant impact on the growth of the host plant itself. The soil mixing method also saved *Cistanche deserticola* seeds, resulting in more even seed distribution and increasing the probability of contact between the seeds and the root tips of the host plant. This not only improved the efficiency of *Cistanche deserticola* seed utilization and reduced the age requirement for host plant seedlings, but also was simple and easy to implement, effectively reducing cultivation costs. This provides a reference for accelerating the establishment of *Cistanche deserticola* host forests and increasing the inoculation speed, which is conducive to promoting the development of the artificial *Cistanche deserticola* cultivation industry and is a reliable way to improve the efficiency of the *Cistanche deserticola* cultivation industry. Attached Figure Description

[0026] Figure 1 This is a flowchart of the method of the present invention;

[0027] Figure 2 This is a schematic diagram of the planting pit and inoculation trough.

[0028] The components in the diagram are: 1. Planting pit; 2. Inoculation trough; 3. Container. Detailed Implementation

[0029] The specific embodiments and working principles of the present invention will be further described in detail below with reference to the accompanying drawings.

[0030] like Figure 1 As shown, a method for increasing the inoculation rate of Cistanche deserticola is described, with the following specific steps:

[0031] Step 1: Determine the site for establishing the host forest, the time for establishing the host forest, and the host plants for the host forest, wherein the host plants are selected as three-month-old or older seedlings of Triplophysa tetrawingata in containers.

[0032] *Cistanche deserticola* grows in the arid desert regions of Northwest my country, characterized by arid climates, low rainfall, high evaporation rates, and sparse vegetation. *Atriplex canescens*, a perennial semi-evergreen shrub belonging to the Chenopodiaceae family, possesses numerous excellent characteristics, including drought resistance, cold resistance, salt and alkali tolerance, and tolerance to poor soil conditions. Furthermore, it is palatable and can be used as forage in grasslands, as well as for windbreak and sand fixation and saline-alkali land improvement. Therefore, *Atriplex canescens* has been selected as a host plant in the arid desert regions of Northwest my country.

[0033] The selection of host forest sites is based on the growth habits of Cistanche deserticola. Cistanche deserticola generally grows on fixed or semi-fixed sand dunes. Therefore, the sites selected for host forest establishment are flat in terrain and have quicksand or sandy loam soil.

[0034] Since the four-winged shrub is a quasi-evergreen shrub, the host forest is established in late spring to early autumn, with the minimum temperature consistently above 0℃ and the average temperature not lower than 10℃. The above-ground height of the four-winged shrub container seedlings is not less than 35cm, and the end of the new root system is about to grow out of the bottom of the container 3 to ensure the survival rate.

[0035] The container 3 for the four-winged pheasant seedlings has a diameter of not less than 15cm and a height of not less than 18cm, ensuring that the roots of the three- to six-month-old four-winged pheasant can develop a relatively dense root system, with the ends of the roots just close to the bottom of the container 3, so that they can quickly extend and take root later.

[0036] It should be noted that while using older seedlings can ensure survival and growth efficiency to some extent, older seedlings generally have larger and harder root systems. When inoculating bare seedlings, only most or the main root system is retained, with minimal soil. This results in a longer recovery period after transplanting, preventing the host plant's root system from quickly adapting to the new environment. Consequently, establishing the host forest takes longer and is more costly. Therefore, this embodiment uses three-month-old *Acer tetrawingense* as the host plant and transplants it in containers for host forest establishment. This method minimizes root damage and reduces the impact of transplanting on the host plant's root system. Therefore, a longer root recovery period is not required, allowing the *Acer tetrawingense* root system to adapt more quickly and resume normal growth. Furthermore, using younger seedlings accelerates the establishment of the host forest and the subsequent inoculation with *Cistanche deserticola*.

[0037] Step 2: During the host forest establishment period, dig multiple planting pits 1 evenly spaced in the host forest establishment plot. The diameter of each planting pit 1 is at least 20cm larger than the container 3 of the four-winged quinoa container seedling, and the depth is not less than the height of the container 3.

[0038] Although a circular container 3 was chosen in this example to save costs and facilitate the excavation of the planting pit 1, other shapes of containers 3 with polygonal cross-sections can also be used. When a polygonal container 3 is used, the diameter of the planting pit 1 should be at least 20cm larger than the length of any diagonal of the container 3.

[0039] Step 3: Place the four-winged gooseberry container seedling in the center of planting pit 1, and dig inoculation grooves 2 around the outer edge of planting pit 1, as shown. Figure 2 As shown, the width and depth of the inoculation groove 2 are both not less than 10cm;

[0040] Since the root growth of plants is limited within a certain period of time, transplanting the *Cistanche deserticola* host plant with container 3 can restrict its roots to grow downwards first, making the root growth direction predictable and the root density high. By limiting the main sowing area of ​​the seeds to the inoculation trough 2 on the outer edge of container 3, the sowing range of *Cistanche deserticola* seeds is reduced. In addition, by restricting the root growth direction, the root density in a fixed direction can be increased, increasing the contact opportunity between *Cistanche deserticola* seeds and *Cistanche deserticola* roots, thereby effectively improving the inoculation rate.

[0041] Step 4: Mix the soil excavated from inoculation trough 2 with the physiologically matured desert Cistanche deserticola seeds and then fill it back in. The amount of seeds mixed is 0.02g / kg.

[0042] The soil-mixing method for inoculating desert Cistanche deserticola seeds only requires filling the inoculation trough 2 on the outer edge of container 3 with soil mixed with desert Cistanche deserticola seeds to successfully inoculate. This method not only greatly reduces the amount of Cistanche deserticola seeds used and improves seed utilization, resulting in more uniform seed distribution, but also increases the probability of contact between the seeds and the root tips of the host plant, which helps to improve the inoculation rate of desert Cistanche deserticola seeds.

[0043] In addition, soil mixed with Cistanche deserticola seeds is filled back into the inoculation trough 2 on the outer edge of container 3. Through co-burying, the seeds of Cistanche deserticola can be sown in an area and at a depth that allows them to feel the stimulation of the host roots near the roots of the host plant, Trichoderma tetrapanax, during the co-burying period. This enables them to naturally break dormancy and germinate without affecting the inoculation effect. There is no need to expose them to sunlight in advance to break dormancy. Instead, they break dormancy in the soil and grow towards the host roots while Trichoderma tetrapanax grows, which greatly improves the efficiency of inoculation.

[0044] Step 5: Cover the planting pit 1 with soil until it is level with the ground. After covering the soil, water the planting pit 1 once within 3 days. When watering, water until the soil is thoroughly soaked. The amount of water used per plant is about 20kg. It is necessary to ensure that the water seeps down to at least 2m. The specific amount of water used should be adjusted according to the soil moisture.

[0045] Step 6: Maintain and manage the host forest. During the maintenance and management process, after the first irrigation, irrigate 3 times with a 1-month irrigation cycle, and then replenish water regularly in spring and autumn each year.

[0046] Specific examples:

[0047] Experiments will be conducted in the semi-arid region of Northwest China (Yuzhong County, Gansu Province) in 2023.

[0048] a) Select sandy loam soil;

[0049] b) The host forest should be established in early May;

[0050] c) Select healthy three-month-old container seedlings of Triplophysa grahami, with an above-ground height of ≥35cm, a container diameter of 15cm, and a container height of 18cm.

[0051] d) Dig a planting pit 20cm larger than the diameter of the container and the same depth as the container. Dig an inoculation trench with a width and depth of 10cm from the planting pit on the outer edge of the container. Mix the soil in the inoculation trench with the physiologically matured Cistanche deserticola seeds and then fill it back in. The amount of seeds mixed is 0.02g / kg.

[0052] e) Cover with soil until level with the ground;

[0053] f) Water the area once within 3 days after vaccination;

[0054] g) The maintenance and management of the host forest involves an irrigation cycle of one month, and successful inoculation of Cistanche deserticola is achieved after three irrigations.

[0055] Statistical analysis revealed a 100% success rate in inoculating *Cistanche deserticola* onto each *Acer tetrawingense* plant, meaning that *Cistanche deserticola* parasitized every single plant. Additionally, a control group was added to determine the role of containers in increasing the inoculation rate. In this case, the containers were removed before planting *Acer tetrawingense*, while all other steps remained the same; the inoculation rate was only 70%.

[0056] Therefore, the above experiment on inoculating *Cistanche deserticola* on 3-month-old *Trichoderma tetragonum* seedlings shows that *Trichoderma tetragonum* seedlings can be successfully inoculated with *Cistanche deserticola* after transplanting and growing to half a year old. Since the new roots of the parasitic plant are limited by the container, they can only grow downwards first. The experiment shows that this method can achieve a 100% inoculation rate for *Cistanche deserticola*, and the host plant's own growth is not significantly affected. The soil mixing method also saves *Cistanche deserticola* seeds, making the seed distribution more even and increasing the probability of contact between the seeds and the root tips of the host plant. This not only improves the efficiency of *Cistanche deserticola* seed utilization and reduces the age requirement for host plant seedlings, but is also simple and easy to implement, effectively reducing cultivation costs. This provides a reference for accelerating the establishment of *Cistanche deserticola* host forests and increasing the inoculation speed, which is conducive to promoting the development of the artificial *Cistanche deserticola* planting industry and is a reliable way to improve the efficiency of the *Cistanche deserticola* planting industry.

[0057] The technical solution provided by this invention has been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of this invention. The descriptions of the embodiments above are only for the purpose of helping to understand the method and core ideas of this invention. It should be noted that those skilled in the art can make several improvements and modifications to this invention without departing from the principles of this invention, and these improvements and modifications also fall within the protection scope of the claims of this invention.

Claims

1. A method of increasing the inoculation rate of Cistanche deserticola, characterized in that, It comprises the following steps: Step 1, determining the host forest planting plot, host forest planting time and host plants of the host forest, wherein the host plants are three-month-old container seedlings of Aeluropus irpex; Step 2, during the host forest planting time, a plurality of planting holes are dug in the host forest planting plot at equal intervals, and the diameter of each planting hole is at least 20 cm larger than the container size of the Aeluropus irpex container seedlings, and the depth is not less than the height of the container; Step 3, the Aeluropus irpex container seedlings are placed in the center of the planting hole, and an inoculation groove is dug around the planting hole outside the container; Step 4, the soil dug out in the inoculation groove is mixed with the desert Cistanche seeds after physiological after-ripening, and then filled back, so that the desert Cistanche seeds can be buried with the host Aeluropus irpex root system during the co-burial period, and the sowing area and depth of the desert Cistanche seeds can meet the requirement that the seeds can be stimulated by the host root near the Aeluropus irpex root system to naturally break dormancy and germinate, and the seed mixing amount is 0.02 g / kg; Step 5, cover the planting hole to the ground level, and water the planting hole once within 3 days after covering the soil; Step 6, maintaining and managing the host forest; The height of the aboveground part of the Aeluropus irpex container seedlings is not less than 35 cm, and the tip of the new root system will grow out of the bottom of the container; The container diameter of the Aeluropus irpex container seedlings is not less than 15 cm, and the height is not less than 18 cm; The width and depth of the inoculation groove in step 3 are both not less than 10 cm.

2. The method of increasing the seeding rate of C. ferax of claim 1, wherein: The host forest planting plot in step 1 is selected from a plot with flat terrain and soil of drifting sand or sandy soil.

3. The method of increasing the seeding rate of C. ferax of claim 1, wherein: The host forest planting time in step 1 is selected from late spring to early autumn, and the minimum temperature is continuously higher than 0℃, and the average temperature is not less than 10℃.

4. The method of increasing the seed set of C. ferax of claim 1, wherein: In step 5, when watering the planting hole, water is poured above the inoculation groove where the desert Cistanche seeds are sown until it is soaked through, and the water is ensured to infiltrate at least 2 m.

5. The method of increasing the seed set of C. ferax of claim 1, wherein: In step 6, during the process of maintaining and managing the host forest, water is poured 3 times with a watering cycle of 1 month after the first watering, and then fixed water is poured in spring and autumn every year.

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