Boschniakia rossica symbiotic cultivation method with alnus japonica as host

Abstract

The invention discloses a boschniakia rossica symbiotic cultivation method with alnus japonica as hosts, and belongs to the technical field of boschniakia rossica cultivation The problem that boschniakia rossica symbiotic cultivation is low in survival rate and unstable in yield is solved. The method comprises the following steps: selecting a healthy alnus japonica middle-age forest with the tree age of 10-30 years as a host forest; oblique cuts are cut in the lateral roots of the alnus japonica at the positions 20-40 cm away from the trunk of the alnus japonica; mixing the boschniakia rossica seeds subjected to cold stratification treatment and hormone combined treatment with a biological adhesive to prepare seed paste; coating a root nodule symbiotic nitrogen-fixing actinomycete inoculant at a host root notch, filling the seed paste into the host root notch, covering the host root notch with wet humus soil, and compacting; the humidity of the covered wet humus soil is kept to be 15%-25%, low-concentration compound fertilizer and microbial agents are dressed in May, inoculation points are checked in June and September every year, and inoculation is supplemented if the parasitic rate is lower than 80%; after the boschniakia rossica germinates in the spring of the second year, collecting flower stems.

Description

Technical Field

[0001] This invention belongs to the field of Cistanche deserticola cultivation technology, specifically relating to a symbiotic cultivation method of Cistanche deserticola with Alnus davidii as the host. Background Technology

[0002] *Cistanche deserticola* is a perennial parasitic herb belonging to the genus *Cistanche* in the family Orobanchaceae. The plant grows to a height of 15–35 cm, with a creeping rhizome and usually 2–3 erect, fleshy stems. The mature fruit contains numerous tiny seeds, approximately 0.4–0.7 mm long and 0.2–0.3 mm in diameter. Lacking chlorophyll, *Cistanche deserticola* cannot photosynthesize and relies solely on the host's roots for water and nutrients, making it a holoparasitic plant. It is distributed in the cold, high-altitude regions of Northeast China, typically growing in moist valleys and riverbanks at elevations of 1500–1800 m. It prefers shade, moisture, and cool conditions, growing along riverbanks, streamsides, or under forests of *Alnus davidii*, *Alnus spp.*, and *Alnus spp.* on hillsides. It is an obligate parasite of middle-aged or mature forests of *Alnus davidii* and *Alnus spp.* (betulaceae family), forming a parasitic body at the root tip in the topsoil of the host plant. *Cistanche deserticola* is rarely found in young or over-mature *Alnus* forests. Due to its extremely small seeds and the fact that germination requires contact with the host root tip and stimulation from root secretions, wild *Cistanche deserticola* populations are scarce, making it a Class II protected plant in China. Current near-natural biomimetic cultivation methods often involve clearing humus under *Alnus spp.* forests, sowing *Cistanche deserticola* seeds, and covering them. However, this method results in low seed-to-host root contact rates, low survival rates, and unstable yields. To protect and utilize *Cistanche deserticola* resources, there is an urgent need for a highly efficient artificial cultivation method adapted to the local *Alnus spp.* host. Summary of the Invention

[0003] The problem to be solved by this invention is to provide an efficient method for the symbiotic cultivation of Cistanche deserticola, and to propose a method for the symbiotic cultivation of Cistanche deserticola with Alnus davidii as the host.

[0004] To achieve the above objectives, the present invention provides the following technical solution:

[0005] A method for symbiotic cultivation of Cistanche deserticola with Alnus dahurica as the host includes the following steps:

[0006] S1. Forest site selection: Select healthy middle-aged Alnus dahurica forests with tree ages of 10–30 years as host forests;

[0007] S2. Host pretreatment: In September, collect fallen leaves at a distance of 20–40 cm from the trunk of the Alnus davidii tree, dig up the topsoil, and make oblique cuts on the lateral roots of the Alnus davidii to obtain host root incisions.

[0008] S3. Seed collection and processing: After cold stratification and combined hormone treatment, the seeds of Cistanche deserticola are mixed with a biological binder to make a seed paste.

[0009] S4. Artificial inoculation: Apply Frankia inoculant to the cut surface of the host root nodule symbiotic nitrogen-fixing actinomycete, then fill the cut surface of the host root with seed paste, then cover the cut surface of the host root with moist humus and compact it, and then cover it with the fallen leaves collected in step S2.

[0010] S5. Cultivation and management: Maintain the moisture content of the covered humus soil at 15%–25%. Apply low-concentration compound fertilizer and microbial agents in May of the second year. In summer, provide shade to maintain 15%–30% light intensity under the forest canopy. In June and September each year, check the inoculation sites. If the parasitism rate is below 80%, supplement the inoculation.

[0011] S6. Nurturing and harvesting: In the spring of the second year, after the Cistanche deserticola sprouts, harvest the flower stalks without damaging the host root system, and then fill in the soil with humus.

[0012] Furthermore, the altitude conditions of the selected middle-aged alder forest in step S1 are 1500–1800m, annual precipitation of 300–400mm, and effective accumulated temperature of ≥1800℃.

[0013] Furthermore, in step S1, the selected middle-aged alder forest has a tree diameter of 2–5 cm and a stand canopy closure of 0.4–0.6.

[0014] Furthermore, in step S2, in late autumn or early spring, the root system of the selected alder is examined, and diseased or weak branches and roots are removed.

[0015] Furthermore, in step S2, the angle of the oblique incision is 30-60°, the length is 1-2 cm, the depth is 1 / 3 of the root diameter, the spacing between the incisions is 10-15 cm, and 1-2 incisions are made on each side of the root.

[0016] Furthermore, the specific implementation method of step S3 includes the following steps:

[0017] S3.1. Select the fruit spikes of Cistanche deserticola that matured the previous year, dry them in the shade, and then extract the seeds of Cistanche deserticola;

[0018] S3.2. Mix Cistanche deserticola seeds with sand and stratify them in cold storage for 45–60 days for cold stratification treatment; then soak the seeds in a mixed solution of 0.3% gibberellin and 0.1% polyoxin B for 12 hours, and air dry until semi-dry to obtain pretreated seeds;

[0019] S3.3. Mix the bio-binder, pretreated seeds and peat moss in a mass ratio of 1:1:1 to make a seed paste.

[0020] Furthermore, in step S3, the bio-adhesive is starch glue or chitosan glue.

[0021] Furthermore, the inoculation depth in step S4 is controlled at 5–10 cm.

[0022] Furthermore, in step S5, the low-concentration compound fertilizer is a low-concentration compound fertilizer with an N:P:K mass ratio of 1:1:1 and a total concentration of ≤0.1%, and the microbial agent is Trichoderma harzianum or Bacillus subtilis. The number of topdressings in spring is 2–3 times.

[0023] The beneficial effects of this invention are:

[0024] This invention describes a symbiotic cultivation method for Cistanche deserticola using Alnus davidii as the host. Compared to the traditional method of sowing seeds under Alnus davidii forests, this invention achieves higher parasitism success rates by inoculating Alnus davidii roots with a specific incision and pre-treating the seeds. This allows Cistanche deserticola seeds to directly contact the host cambium. The survival rate of this method can reach over 85%, which is more than 30% higher than natural sowing, and the yield per plant is more than 40% higher. Utilizing the dominant Alnus davidii species and existing forest land in the region, this method achieves integrated forestry and medicinal herb management, protecting the rare Cistanche deserticola resource while increasing economic benefits in the forest area. Attached Figure Description

[0025] Figure 1 This is a schematic diagram illustrating the inoculation process of a symbiotic cultivation method for Cistanche deserticola with Alnus dahurica as the host, as described in this invention.

[0026] Figure 2 This is a schematic diagram illustrating the symbiotic cultivation method of Cistanche deserticola with Alnus davidii as the host, as described in this invention. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention; that is, the described specific embodiments are merely a part of the embodiments of the invention, and not all of them. The components of the specific embodiments of the invention described and shown in the accompanying drawings can generally be arranged and designed in various different configurations, and the invention may also have other embodiments.

[0028] Therefore, the following detailed description of specific embodiments of the invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to illustrate selected specific embodiments of the invention. All other specific embodiments obtained by those skilled in the art based on these specific embodiments without inventive effort are within the scope of protection of this invention.

[0029] To further understand the invention's content, features, and effects, the following specific embodiments are provided, along with accompanying drawings. Figure 1 and attached Figure 2 Detailed explanation is as follows:

[0030] Example 1:

[0031] A method for symbiotic cultivation of Cistanche deserticola with Alnus dahurica as the host includes the following steps:

[0032] S1. Forest site selection: Select healthy middle-aged Alnus dahurica forests with tree ages of 10–30 years as host forests;

[0033] Furthermore, the altitude conditions of the selected Alnus davidii middle-aged forest in step S1 are 1500–1800m, annual precipitation of 300–400mm, and effective accumulated temperature ≥1800℃.

[0034] Furthermore, in step S1, the selected middle-aged alder forest has a tree diameter of 2–5 cm and a stand canopy closure of 0.4–0.6.

[0035] S2. Host pretreatment: In September, collect fallen leaves at a distance of 20–40 cm from the trunk of the Alnus davidii tree, dig up the topsoil, and make oblique cuts on the lateral roots of the Alnus davidii to obtain host root incisions.

[0036] Furthermore, in step S2, in late autumn or early spring, the root system of the selected alder is examined, and diseased or weak branches and roots are removed.

[0037] Furthermore, in step S2, the angle of the oblique incision is 30°, the length is 1–2 cm, the depth is 1 / 3 of the root diameter, the spacing between the incisions is 10–15 cm, and 1–2 incisions are made on each side of the root.

[0038] S3. Seed collection and processing: After cold stratification and combined hormone treatment, the seeds of Cistanche deserticola are mixed with a biological binder to make a seed paste.

[0039] Furthermore, the specific implementation method of step S3 includes the following steps:

[0040] S3.1. Select the fruit spikes of Cistanche deserticola that matured the previous year, dry them in the shade, and then extract the seeds of Cistanche deserticola;

[0041] S3.2. Mix Cistanche deserticola seeds with sand and stratify them in cold storage for 45–60 days for cold stratification treatment; then soak the seeds in a mixed solution of 0.3% gibberellin and 0.1% polyoxin B for 12 hours, and air dry until semi-dry to obtain pretreated seeds;

[0042] S3.3. Mix the bio-binder, pretreated seeds and peat moss in a mass ratio of 1:1:1 to make a seed paste.

[0043] Furthermore, in step S3, the bio-adhesive is starch glue;

[0044] S4. Artificial inoculation: Apply Frankia inoculant to the cut surface of the host root nodule symbiotic nitrogen-fixing actinomycete, then fill the cut surface of the host root with seed paste, then cover the cut surface of the host root with moist humus and compact it, and then cover it with the fallen leaves collected in step S2.

[0045] Furthermore, the inoculation depth in step S4 is controlled at 5–10 cm.

[0046] S5. Cultivation and management: Maintain the moisture content of the covered humus soil at 15%–25%. Apply low-concentration compound fertilizer and microbial agents in May of the second year. In summer, provide shade to maintain 15%–30% light intensity under the forest canopy. In June and September each year, check the inoculation sites. If the parasitism rate is below 80%, supplement the inoculation.

[0047] Furthermore, in step S5, the low-concentration compound fertilizer is a low-concentration compound fertilizer with an N:P:K mass ratio of 1:1:1 and a total concentration of ≤0.1%, and the microbial agent is Trichoderma harzianum. The number of times it is applied in spring is 2–3.

[0048] S6. Nurturing and harvesting: In the spring of the second year, after the Cistanche deserticola sprouts, harvest the flower stalks without damaging the host root system, and then fill in the soil with humus.

[0049] Fifty middle-aged *Alnus spp.* trees at an altitude of 1600 m and a canopy closure of 0.5 were selected in the Amur Forestry Bureau of the Greater Khingan Mountains. Root inoculation was performed according to the method described in this embodiment, with approximately 100 mg of *Cistanche deserticola* seed paste inoculated into each root. A survey conducted in May of the following year showed a parasitism survival rate of 86%, with an average of 2–3 *Cistanche deserticola* flower stalks germinating per tree. The dried yield was 41% higher than the control (broadcasting method).

[0050] Example 2:

[0051] A method for symbiotic cultivation of Cistanche deserticola with Alnus dahurica as the host includes the following steps:

[0052] S1. Forest site selection: Select healthy middle-aged Alnus dahurica forests with tree ages of 10–30 years as host forests;

[0053] Furthermore, the altitude conditions of the selected middle-aged alder forest in step S1 are 1500–1800m, annual precipitation of 300–400mm, and effective accumulated temperature of ≥1800℃.

[0054] Furthermore, in step S1, the selected middle-aged alder forest has a tree diameter of 2–5 cm and a stand canopy closure of 0.4–0.6.

[0055] S2. Host pretreatment: In September, collect fallen leaves at a distance of 20–40 cm from the trunk of the Alnus davidii tree, dig up the topsoil, and make oblique cuts on the lateral roots of the Alnus davidii to obtain host root incisions.

[0056] Furthermore, in step S2, in late autumn or early spring, the root system of the selected alder is examined, and diseased or weak branches and roots are removed.

[0057] Furthermore, in step S2, the angle of the oblique incision is 60°, the length is 1–2 cm, the depth is 1 / 3 of the root diameter, the spacing between the incisions is 10–15 cm, and 1–2 incisions are made on each side of the root.

[0058] Furthermore, use a sterilized knife to make a slanted cut (1–2 cm long) on ​​the lateral roots of the alder. The cut should be close to the root tip and the depth should be about 1 / 3 of the root diameter.

[0059] S3. Seed collection and processing: After cold stratification and combined hormone treatment, the seeds of Cistanche deserticola are mixed with a biological binder to make a seed paste.

[0060] Furthermore, the specific implementation method of step S3 includes the following steps:

[0061] S3.1. Select the fruit spikes of Cistanche deserticola that matured the previous year, dry them in the shade, and then extract the seeds of Cistanche deserticola;

[0062] S3.2. Mix Cistanche deserticola seeds into sand and stratify them for 45–60 days for cold stratification treatment; then soak the seeds in a mixed solution of 0.3% gibberellin and 0.1% polyoxin B for 12 hours, and air dry until semi-dry to obtain pretreated seeds;

[0063] S3.3. Mix the bio-binder, pretreated seeds and peat moss in a mass ratio of 1:1:1 to make a seed paste.

[0064] Furthermore, the bio-adhesive in step S3 is chitosan glue;

[0065] S4. Artificial inoculation: Apply Frankia inoculant to the cut surface of the host root nodule symbiotic nitrogen-fixing actinomycete, then fill the cut surface of the host root with seed paste, then cover the cut surface of the host root with moist humus and compact it, and then cover it with the fallen leaves collected in step S2.

[0066] Furthermore, the inoculation depth in step S4 is controlled at 5–10 cm.

[0067] S5. Cultivation and management: Maintain the moisture content of the covered humus soil at 15%–25%. Apply low-concentration compound fertilizer and microbial agents in May of the second year. In summer, provide shade to maintain 15%–30% light intensity under the forest canopy. In June and September each year, check the inoculation sites. If the parasitism rate is below 80%, supplement the inoculation.

[0068] Furthermore, in step S5, the low-concentration compound fertilizer is a low-concentration compound fertilizer with an N:P:K mass ratio of 1:1:1 and a total concentration of ≤0.1%, and the microbial agent is Bacillus subtilis. The number of topdressings in spring is 2–3 times.

[0069] S6. Nurturing and harvesting: In the spring of the second year, after the Cistanche deserticola sprouts, harvest the flower stalks without damaging the host root system, and then fill in the soil with humus.

[0070] One hundred Alnus spp. trees were inoculated under the forest canopy in the Jiagedaqi area of ​​the Greater Khingan Mountains according to the method described in this embodiment. During cultivation, the soil moisture content was maintained at 18%–22%, and microbial inoculants were applied three times within the first year. In the second year, the parasitism rate reached 88%, with an average yield of 0.35 kg per tree, representing a 45% increase compared to the biomimetic broadcasting method.

[0071] It should be noted that relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0072] Although this application has been described above with reference to specific embodiments, various modifications can be made and components can be replaced with equivalents without departing from the scope of this application. In particular, as long as there is no structural conflict, the features in the specific embodiments disclosed in this application can be combined with each other in any way. The lack of an exhaustive description of these combinations in this specification is merely for the sake of brevity and resource conservation. Therefore, this application is not limited to the specific embodiments disclosed herein, but includes all technical solutions falling within the scope of the claims.

Claims

1. A method for symbiotic cultivation of Cistanche deserticola with Alnus davidii as the host, characterized in that, Includes the following steps: S1. Forest site selection: Select healthy middle-aged Alnus dahurica forests with tree ages of 10–30 years as host forests; S2. Host pretreatment: In September, collect fallen leaves at a distance of 20–40 cm from the trunk of the Alnus davidii tree, dig up the topsoil, and make oblique cuts on the lateral roots of the Alnus davidii to obtain host root incisions. S3. Seed collection and processing: After cold stratification and combined hormone treatment, the seeds of Cistanche deserticola are mixed with a biological binder to make a seed paste. S4. Artificial inoculation: Apply Frankia inoculant to the cut surface of the host root nodule symbiotic nitrogen-fixing actinomycete, then fill the cut surface of the host root with seed paste, then cover the cut surface of the host root with moist humus and compact it, and then cover it with the fallen leaves collected in step S2. S5. Cultivation and management: Maintain the moisture content of the covered humus soil at 15%–25%. Apply low-concentration compound fertilizer and microbial agents in May of the second year. In summer, provide shade to maintain 15%–30% light intensity under the forest canopy. In June and September each year, check the inoculation sites. If the parasitism rate is below 80%, supplement the inoculation. S6. Nurturing and harvesting: In the spring of the second year, after the Cistanche deserticola sprouts, harvest the flower stalks without damaging the host root system, and then fill in the soil with humus.

2. The method for symbiotic cultivation of Cistanche deserticola with Alnus davidii as the host, as described in claim 1, is characterized in that... The altitude conditions of the selected Alnus davidii middle-aged forest in step S1 are 1500–1800m, annual precipitation of 300–400mm, and effective accumulated temperature ≥1800℃.

3. The method for symbiotic cultivation of Cistanche deserticola with Alnus davidii as the host, as described in claim 2, is characterized in that... The selected middle-aged Alnus davidii forest in step S1 has a tree diameter of 2–5 cm and a stand canopy closure of 0.4–0.

6.

4. The method for symbiotic cultivation of Cistanche deserticola with Alnus davidii as the host, as described in claim 3, is characterized in that... In step S2, in late autumn or early spring, the root system of the selected alder is examined, and diseased or weak branches and roots are removed.

5. The method for symbiotic cultivation of Cistanche deserticola with Alnus davidii as the host, as described in claim 4, is characterized in that... In step S2, the angle of the oblique incision is 30-60°, the length is 1-2 cm, the depth is 1 / 3 of the root diameter, the spacing between the incisions is 10-15 cm, and 1-2 incisions are made on each side of the root.

6. The method for symbiotic cultivation of Cistanche deserticola with Alnus davidii as the host, as described in claim 5, is characterized in that... The specific implementation method of step S3 includes the following steps: S3.

1. Select the fruit spikes of Cistanche deserticola that matured the previous year, dry them in the shade, and then extract the seeds of Cistanche deserticola; S3.

2. Mix Cistanche deserticola seeds with sand and stratify them in cold storage for 45–60 days for cold stratification treatment; then soak the seeds in a mixed solution of 0.3% gibberellin and 0.1% polyoxin B for 12 hours, and air dry until semi-dry to obtain pretreated seeds; S3.

3. Mix the bio-binder, pretreated seeds and peat moss in a mass ratio of 1:1:1 to make a seed paste.

7. A method for symbiotic cultivation of Cistanche deserticola with Alnus davidii as the host, as described in claim 6, is characterized in that... In step S3, the bio-adhesive is starch glue or chitosan glue.

8. A method for symbiotic cultivation of Cistanche deserticola with Alnus davidii as the host, as described in claim 7, is characterized in that... The inoculation depth in step S4 is controlled at 5–10 cm.

9. A method for symbiotic cultivation of Cistanche deserticola with Alnus davidii as the host, as described in claim 8, characterized in that, In step S5, the low-concentration compound fertilizer is a low-concentration compound fertilizer with an N:P:K mass ratio of 1:1:1 and a total concentration of ≤0.1%. The microbial agent is Trichoderma harzianum or Bacillus subtilis. The number of top dressings in spring is 2–3.

Images