A pine nematode inhibiting method based on pachyman substitute cultivation

Abstract

The application discloses a pine nematode inhibition method based on pachyman substitute cultivation, and comprises the steps of destroying the survival carrier of pine wood nematode and intermediate host, substitute fermentation directional regulation of microbial environment and pachyman fungus biological control, wherein the substitute fermentation makes the pH of the substrate in the range of 5.0-6.5, and the microbial environment for inhibiting the hatching of pine wood nematode and monochamus alternatus egg, the development of larva and the reproduction of adult is constructed; especially, the pH of the substitute substrate after fermentation is maintained at 5.5-6.5, and the pachyman fungus is combined. Compared with the existing biological control method of diseased wood, the pine nematode inhibition rate can be increased from 60-70% to 100%, and the secondary transmission risk of pine nematode is significantly reduced. While realizing the harmless treatment of diseased wood and completely blocking the transmission of pine nematode, efficient and high-quality cultivation of pachyman is achieved, and the method is suitable for the large-scale treatment of pine wood nematode disease and the industrialized planting of pachyman genuine producing area.

Description

Technical Field

[0001] This invention belongs to the field of forest pest and disease control and biological treatment technology, and more specifically, relates to a method for suppressing pine nematodes based on Poria cocos as a substrate. Background Technology

[0002] Pine wilt disease (Bursaphelenchus xylophilus) is a devastating forest disease caused by the pine wood nematode. The pine wood nematode is a highly pathogenic nematode; each female lays approximately 100 eggs. At a suitable growth temperature (25°C), the eggs hatch into second-instar larvae within 30 hours. The larvae take 3-4 days to develop into adults. At temperatures above 30°C, the nematode completes a generation cycle in just 3 days. Therefore, the pine wood nematode is characterized by its high pathogenicity, rapid spread, and difficulty in control. Furthermore, large numbers of pine wood nematodes and their vector insect, the pine sawyer beetle (Monochamus alternatus), lurk in infected logs, branches, and stumps after felling. If not treated promptly and thoroughly, these nematodes will become a significant source of secondary disease transmission.

[0003] In recent years, biological treatment technologies have gradually attracted attention, among which the method of using Poria cocos to treat pine wilt-infected wood has proven to have certain application value. However, the existing model based on the cultivation of infected logs, although relying on the inhibitory effect of Poria cocos mycelium on pine wilt nematodes to treat infected wood, has some shortcomings in practical applications, such as: (1) Limited effect of pine wilt disease suppression: The existing cultivation techniques for diseased logs lack targeted methods for suppressing pine wilt disease. They rely solely on the inhibitory effect of Poria cocos itself, and the pine wilt disease suppression rate is generally less than 70%.

[0004] (2) High risk of secondary transmission of pine wood nematode: Traditional cultivation of infected logs uses the same treatment as normal pine wood (peeling and leaving the core, stacking and drying), which does not fundamentally destroy the living environment of pine nematodes. Pine wood nematodes are likely to survive inside infected logs, and pine longhorn beetles can lay eggs and reproduce in infected logs, resulting in a high risk of secondary transmission of pests and diseases.

[0005] (3) Poor adaptability to cultivation environment: Soil acidity and alkalinity and microbial community structure are difficult to regulate in log cultivation. The yield of Poria cocos is greatly affected by environmental factors, making it difficult to simultaneously achieve the dual goals of inhibiting pine wilt nematode and achieving stable and high yield of Poria cocos.

[0006] Patent CN121444773A describes a method where infected logs are inoculated with Poria cocos and then buried in pits to block the transmission of pine wilt nematodes through soil isolation. However, the logs must be planted in the pits for at least 15 days before undergoing a period of propagation and killing for at least 60 days. After these two stages, the logs remain in the pits to continue growing Poria cocos. Patent CN117502098A describes inoculating infected logs with Poria cocos or Armillaria mellea spores and then cultivating the mycelium through a film covering to kill pine wilt nematodes and pine sawyer beetles. However, this method has a long treatment cycle, requiring an additional 210-300 days of cultivation after covering with a film and soil to inactivate the pine wilt nematodes and pine sawyer beetles. Therefore, addressing the limitations of existing biotechnology methods for treating infected logs, there is an urgent need to develop an integrated method for treating infected logs and cultivating Poria cocos that offers a short treatment cycle, high inhibition rate of pine wilt nematodes and their intermediate hosts, and superior yield and quality of Poria cocos. Summary of the Invention

[0007] To address the shortcomings of existing methods for suppressing pine wilt nematodes using Poria cocos grown on infected logs, such as low suppression rates, high risk of secondary transmission, and unstable yields, this invention provides a method for suppressing pine wilt nematodes based on Poria cocos cultivation using a substrate substitute. The method aims to destroy the pine wilt nematode and its intermediate host by crushing the substrate, and then use substrate fermentation to regulate the substrate's pH and microbial environment. This ensures the post-fermentation substrate has a pH range of 5.0–6.5, thereby creating a microbial environment that inhibits the hatching of pine wilt nematode eggs and the development of larvae and adults of the pine sawyer beetle. By combining the biological environment with the steps of biocontrol using Poria cocos fungus, it was found that compared with unfermented substrate combined with Poria cocos fungus, this method increased the inhibition rate of pine wilt nematode and pine sawyer beetle from 67.8% and 55.6% to 94%~96% and 85%~93%, respectively, and even reached 100%. It can completely block the transmission path of pine wilt nematode and also promote the growth of Poria cocos. This solves the technical problem that the inhibition rate of pine wilt nematode and pine sawyer beetle using the diseased linden wood cultivation method in practical applications is generally between 60% and 70%, which poses a high risk of secondary transmission.

[0008] To achieve the above objectives, according to one aspect of the present invention, a method for suppressing pine nematodes based on Poria cocos substrate cultivation is provided, comprising the following steps: (1) Destroy the survival carrier of pine wood nematode and its intermediate host: The infected wood is crushed to destroy the hiding space of pine wood nematode and its intermediate host in the infected wood, and granular infected wood chips are obtained. (2) Directional regulation of the living environment by fermentation of substitute material: The infected sawdust from step (1) is prepared into a substitute material substrate, and the moisture content of the substitute material substrate is adjusted to 50%~55%. The pH value of the substitute material substrate is adjusted by fermentation so that the pH value after fermentation is in the range of 5.0~6.5, thereby constructing a microbial environment that inhibits the hatching of pine wood nematode eggs, the development of larvae and the reproduction of adults. (3) Biocontrol of Poria cocos: The substrate after fermentation in step (2) is divided into cultivation bags. After sterilization, Poria cocos strain is inoculated into the cultivation bags under sterile conditions and cultivated until the Poria cocos mycelium fills the bag. According to the principle of one bag per wood, the exposed Poria cocos mycelium is pressed onto the surface of the peeled diseased wood section to biologically inhibit the pine wood nematode and its intermediate host in the diseased wood section. The wood is then placed in a cellar and Poria cocos is cultivated in the cellar.

[0009] Preferably, in the method, step (2) involves adjusting the pH of the substrate to 5.5-6.5 through fermentation to create a microbial environment that inhibits the hatching of pine wood nematode eggs, larval development, adult reproduction, and promotes the growth of Poria cocos mycelium.

[0010] Preferably, in the method, the fermentation in step (2) is carried out under aerobic fermentation conditions of 28-32℃ and an aeration rate of 0.3~1.0m³ / (h·m³).

[0011] Preferably, in the method, the fermentation time in step (2) is 48~96h.

[0012] Preferably, in the method, the fermentation in step (2) is natural fermentation, and the microorganisms used in the fermentation include Bacillus.

[0013] Preferably, in the method, the substitute substrate comprises, by weight percentage, 55% to 65% sawdust, 15% to 25% corn cobs, and 15% to 25% corn kernels.

[0014] Preferably, in the method, the substrate further includes calcined gypsum; the infected sawdust is made by removing the moldy parts from pine wood nematode-infected wood and crushing it into particles of 0.5-1cm; the corn cob has a particle size of 0.1-1mm, and the corn kernel has a particle size of 0.1-0.2mm.

[0015] Preferably, in the method, the Poria cocos strain inoculated in the cultivation bag in step (3) includes Poria cocos strain WZ-1, whose accession number is CGMCCNO.40108.

[0016] Preferably, in the method, the culture conditions of the Poria cocos strain in step (3) are constant temperature culture at 26℃±2℃ in the dark for 15-20 days.

[0017] Preferably, in step (3) of the method, the cellar planting is carried out according to the principle of one cellar, one bag, and one piece of wood, wherein each cellar contains one cultivation bag full of Poria cocos mycelium and one 1.5~2kg peeled diseased wood section.

[0018] In summary, compared with the prior art, the above-described technical solutions conceived by this invention can achieve the following beneficial effects: This invention provides a method for inhibiting pine wilt nematodes based on Poria cocos substrate cultivation. This method completely destroys the survival and transmission vectors of pine wilt nematodes and pine sawyer beetles through substrate cultivation. It also employs targeted fermentation to regulate the substrate's pH, maintaining a post-fermentation pH range of 5.0-6.5, thus creating a microbial environment that inhibits pine wilt nematode egg hatching, larval development, and adult reproduction. Compared to unfermented substrate cultivation, this method, using a fermented substrate combined with Poria cocos bacteria, can increase the pine wilt nematode inhibition rate from 60%-70% to 94%-100%, while simultaneously increasing the average yield of Poria cocos per pit by 1.3-1.8 times and the triterpenoid content by 1.32-1.45 times. This invention achieves harmless treatment of infected wood, completely blocks the spread of pine wilt nematodes, and enables efficient and high-quality cultivation of Poria cocos. It is applicable to the large-scale treatment of pine wilt-infected wood and the industrialized cultivation of Poria cocos in authentic producing areas. Attached Figure Description

[0019] Figure 1 Flowchart of a process for inhibiting the spread of pine wilt nematode through the cultivation of Poria cocos as a substrate. Detailed Implementation

[0020] 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 embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Furthermore, the technical features involved in the various embodiments of this invention described below can be combined with each other as long as they do not conflict with each other.

[0021] Pine wilt disease (also known as pine nematode disease) primarily causes chlorosis and wilting in timber, while the pine nematode (also known as the pine wood nematode) is the direct causative agent. To prevent secondary infection of nematode-infected timber, existing biological treatment methods are mainly based on the cultivation of infected logs. It mainly relies on the inhibitory effect of Poria cocos mycelium on pine wilt nematodes. However, in practical applications, the inhibition rate of pine wilt nematodes is generally only 60%-70%, and the yield of Poria cocos fluctuates greatly due to environmental factors (averaging less than 1.2 kg / cellar), making it difficult to achieve the dual core requirements of harmless treatment of infected wood and stable and high yield of Poria cocos.

[0022] According to the chapter on airborne microorganisms in *Environmental Microbiology and MMBE 305* (Source: *Environmental Microbiology and MMBE 305*; Academic Session 2024-2025, Dr. Samrat Paul, Brainware University, Kolkata), airborne microbiota can be divided into outdoor and indoor microbiota. Among bacteria, *Bacillus* and *Clostridium* are widely found in outdoor air; the most common bacterial genera in indoor air are *Staphylococcus*, *Bacillus*, and *Clostridium*. Airborne microorganisms, including those that are airborne, settle on materials, or are present in various fermentation products through their secretions. *Bacillus* species, such as *Bacillus subtilis*, possess extremely strong resistance to drying and ultraviolet radiation and are among the most common airborne bacteria.

[0023] Studies have shown that the cellulose content of infected wood is almost unaffected, meaning that although the wood turns yellow and becomes brittle, its cellulose content is usually similar to that of healthy wood. Meanwhile, Bacillus species (such as Bacillus subtilis), which are widely distributed in the air, are aerobic bacteria and can secrete cellulase and xylanase, making them the main microorganisms involved in wood fermentation. This invention involves pulverizing pine wood infected with pine wilt disease into granular sawdust, and then combining the sawdust with auxiliary materials such as corn kernels and corn cobs to create a substrate. It cleverly utilizes microorganisms in the air to naturally ferment the substrate under aerobic conditions. The results showed that, compared to the unfermented substrate combined with *Poria cocos*, the fermented substrate combined with *Poria cocos* significantly improved the inhibitory effect on pine wilt disease and pine sawyer beetle in the infected wood. Specifically, after fermentation, the pH value was 5.0–6.5, increasing the inhibition rate of pine wilt disease from 67.8% to 94%–100%, and the inhibition rate of pine sawyer beetle from 55.6% to 85%–100%. In other words, this invention, through substrate fermentation regulation, constructs a microbial environment conducive to the hatching of pine wilt disease eggs, larval development, and adult reproduction of pine sawyer beetles and pine sawyer beetles.

[0024] In particular, by regulating the pH value to 5.5-6.5 through substrate fermentation, the inhibition rate of pine wood nematode increased from 67.8% to 94%-100%, and the inhibition rate of pine sawyer beetle increased from 55.6% to 92%-100%. At the same time, the yield of Poria cocos increased by an average of 1.8 times, and the triterpenoid content increased by 1.45 times. This means that a microbial environment was constructed for the hatching of pine wood nematode and pine sawyer beetle eggs, larval development, adult reproduction, and the promotion of Poria cocos mycelial growth. This achieved the dual goal of effectively inhibiting pine wood nematode and its intermediate host (pine sawyer beetle) while promoting the growth of Poria cocos sclerotia.

[0025] Based on this, the present invention provides a method for suppressing pine nematodes based on Poria cocos substrate cultivation, comprising the following steps: (1) Destroy the survival carrier of pine wood nematode and its intermediate host: The infected wood is crushed to destroy the hiding space of pine wood nematode and its intermediate host in the infected wood, and granular infected wood chips are obtained; for example, the infected wood chips are crushed at the logging site to destroy the hiding space of pine wood nematode and its intermediate host in the infected wood, and 0.5~1cm granular infected wood chips are obtained. Crushing the infected wood at the logging site can avoid secondary transmission of pine wood nematode after the pine sawyer beetle emerges during transportation.

[0026] (2) Directional regulation of microbial environment by fermentation of substitute material: The infected sawdust from step (1) is prepared into a substitute material substrate, and the moisture content of the substitute material substrate is adjusted to 50%~55%. The pH value of the substitute material substrate is adjusted by fermentation so that the pH value after fermentation is in the range of 5.0~6.5, thereby constructing a microbial environment that inhibits the hatching of pine wood nematode eggs, the development of larvae, and the reproduction of adults. (3) Biocontrol of Poria cocos: The substrate after fermentation in step (2) is divided into cultivation bags, sterilized, and then inoculated with Poria cocos strain in a sterile environment until the Poria cocos mycelium fills the bag. Following the principle of one bag per piece of wood, the exposed Poria cocos mycelium is pressed directly onto the surface of the peeled infected wood section to biologically inhibit pine wood nematodes and their intermediate hosts in the infected wood section. The wood section is placed on the side or bottom, with the end facing the groove, and then placed in a cellar to cultivate Poria cocos.

[0027] This invention compares the effects of different fermentation processes on the inhibition of pine wood nematode and its intermediate host. The results show that adjusting the pH of the substrate to 5.0-6.5 through fermentation, especially to 5.5-6.5, significantly inhibits the hatching of pine wood nematode eggs, larval development, and adult reproduction. It also promotes the formation of beneficial microbial communities, further enhancing the inhibition effect on pine wood nematode. The inhibition rate of both pine wood nematode and pine sawyer beetle can reach 100%. Further lowering the pH of the substrate inhibits the growth of Poria cocos mycelium. Preferably, the pH of the substrate is maintained at 5.5-6.0. For example, the fermentation in step (2) is carried out naturally under aerobic conditions at 28-32℃ with an aeration rate of 0.3-1.0 m³ / (h·m³) to achieve a substrate pH of 5.5-6.5. The microorganisms utilized in the fermentation include Bacillus.

[0028] In some embodiments, the moldy parts of the infected wood are removed, and the wood is pulverized to 0.5-1 cm particles to obtain infected sawdust, which serves as the main raw material for the substrate. The infected sawdust is then combined with auxiliary materials such as corn cobs and corn kernels in a scientifically proportioned manner to form the substrate. In some embodiments, the substrate comprises, by weight percentage, 55%-65% infected sawdust, 15-25% corn cobs, and 15-25% corn kernels. For example, by weight percentage, the substrate comprises (60±5)% infected sawdust, (20±1)% corn cobs, and 20% corn kernels, providing nutritional support and laying a material foundation for the growth of Poria cocos. In some embodiments, the infected sawdust is obtained by removing the moldy parts of pine wilt-infected wood and pulverizing it to 0.5-1 cm particles; the corn cobs have a particle size of 0.1-1 mm, and the corn kernels have a particle size of 0.1-0.2 mm.

[0029] Preferably, the substrate also includes calcined gypsum, and in some embodiments, the amount of calcined gypsum added is 1%. Calcined gypsum is mainly used to adjust the pH of the Poria cocos substrate, stabilizing the pH value of the substrate at 5.5-6.5 to prevent the substrate from becoming too acidic. At the same time, it can increase the looseness and water retention capacity of the substrate, which helps the formation and growth of Poria cocos sclerotia, making it suitable for large-scale Poria cocos cultivation and production.

[0030] For example, the substrate includes 59% sawdust, 20% corn cob, 20% corn kernels, and 1% by weight of calcined gypsum. The moisture content is adjusted to 50%~55%, the fermentation temperature is set at 28-32℃, the aeration rate is 0.5m³ / (h・m³), the fermentation time is 72h, and the pH value of the substrate is maintained at 5.5-6.5 after fermentation.

[0031] The Poria cocos strain inoculated in the cultivation bags in step (3) includes Poria cocos strain WZ-1, whose preservation number is CGMCC NO.40108. The fermented substrate is packaged into cultivation bags, sterilized, and then inoculated with the Poria cocos strain under aseptic conditions until the bags are fully covered with Poria cocos mycelium, as detailed below: After sterilizing the cultivation bags, inoculate with Poria cocos strain WZ-1 and incubate at 26℃±2℃ in the dark for 15-20 days until the mycelium fills the bag. Place one bag in each cellar, cut a long U-shaped opening in the film along the longitudinal direction, and press the exposed mycelium parallel to the surface of a peeled log (1.5~2kg), with the log on the side or bottom and the end facing the trench. Place the log in the cellar, cover with 8~10cm of soil, and manage in the cellar until the Poria cocos is harvested.

[0032] In some embodiments, the method of using fermented substrate (fermented substrate) for cultivation for 30 days resulted in a 100% inhibition rate of pine wilt nematode, a 100% mortality rate of pine sawyer beetle larvae, a 100% survival rate after inoculation, a 98% formation rate of Poria cocos, an average yield of 1.3767 kg per pit, and a triterpenoid content of 3.2653 mg / g.

[0033] Using traditional infected log cultivation methods for 30 days, 35-42 surviving pine wilt nematodes were still detected per 100g of infected log, with a pine wilt nematode inhibition rate of 65.3%, an inoculation survival rate of 98.7%, a poria cocos formation rate of 96.3%, an average yield of 0.906kg per pit, and a triterpenoid content of 2.83mg / g. Compared to traditional infected log cultivation methods, this method combines a fermented substrate with Poria cocos strains. It not only significantly improved the inhibition rate of pine wilt nematode and its intermediate host (the inhibition rate can reach 100%), and had a better pine wilt nematode inhibition effect, which can avoid the secondary spread of pests and diseases, but also increased the yield of Poria cocos per cellar by 52% and the triterpenoid content by 15.4%.

[0034] The following are examples. Pine wilt disease-infected wood: The pine wilt disease-infected wood treatment plant in Hongshan Town, Yingshan County, Hubei Province, selects Masson pine wood free from mold and insect infestation. After debarking and retaining the core, the wood is cut into sections (35-40cm in length and 8-12cm in diameter), weighing approximately 1-1.5kg. It is then placed in a well-ventilated and dry place to air-dry naturally for 1-2 months until the moisture content of the wood drops to 35%-40% before use. The treatment process complies with the relevant standards for the safe treatment of pine wilt disease-infected wood.

[0035] Corn cob: Purchased from Kaizheng Agricultural Development Co., Ltd., Wudi County, Linyi City, Shandong Province. It is the cob of fresh corn after threshing, free from mold, odor, and impurities. After natural air drying, it is crushed to a particle size of 0.18-0.83mm using a pulverizer, dried in an oven at 105℃ for 2 hours, cooled to room temperature, and then sealed for storage. Its main components are cellulose 39.7%, xylan 31.5%, and lignin 10.8%, which meets the standards for corn cob used as a substrate for edible fungi cultivation and can be used as an auxiliary carbon source substrate for Poria cocos cultivation.

[0036] Corn kernels: Selected from "Shenke Sweet 811" fresh sweet corn kernels cultivated by the Shanghai Academy of Agricultural Sciences, purchased from a local formal agricultural input market. The kernels are plump, undamaged, free from mold and insect infestation, and uniformly mature (harvested at the milk stage 21-25 days after pollination). After removing impurities, rinse with clean water, place in a ventilated place to air dry naturally until the moisture content is 12%-15%, crush into powder (particle size 0.1-0.2mm), seal and store for later use. Rich in soluble sugars, dietary fiber and minerals, it can supplement the nutrients needed for the growth of Poria cocos.

[0037] Calcined gypsum: Purchased from Anhui Tongling Hengyuan Gypsum Products Co., Ltd., in 25kg / bag specifications, industrial grade standard, chemical composition CaSO4·0.5H2O, white powder, odorless, free of obvious impurities, purity ≥95%, density 2.2-2.4g / cm³, Mohs hardness 1.5-2.0, fineness 80 mesh, meeting the gypsum standards for large-scale edible fungi cultivation. Origin: Tongling, Anhui. Before use, spread out in a ventilated and dry place and air-dry naturally for 2-3 days to remove any possible lumps. Then pass through an 80-mesh sieve before use.

[0038] Poria cocos strain: strain number WZ-1 (CN117070377A), jointly bred by Hubei Provincial Hospital of Traditional Chinese Medicine and the Institute of Microbiology, Chinese Academy of Sciences. It was obtained through multispore hybridization and multiple rounds of field screening, using wild Poria cocos strain (898) from Yunnan and the main cultivated strain 5.78 as parents. The taxonomic name of Poria cocos strain WZ-1 is Wolfiporia hoelen, its accession number is CGMCC NO.40108, its accession date is March 1, 2022, and its depository institution is the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing (see CN117070377A for details). This strain is characterized by rapid feeding, quick growth on pine cultivation substrate, early sclerotium formation, a sclerotium formation rate 17.4%-19.4% higher than the main strain 5.78, high yield, with an average yield per cellar 154-1394.75g higher than the main strain 5.78, and dense, white sclerotia with a thin outer skin, regular shape, and high marketability.

[0039] Example 1: Suppression of pine nematodes and pine sawyer beetles based on Poria cocos as a substrate cultivation method (1) Preparation of raw materials Infected wood: In-situ harvested pine wood infected with pine wilt disease. Select pine wood infected with pine wilt disease on-site, remove the moldy parts, and crush it into 0.5-1cm particles to obtain infected sawdust.

[0040] Preparation of substitute substrate: Weigh 60% sawdust, 20% corn cob, 20% corn kernels and 1% calcined gypsum by mass percentage, mix them evenly to make substitute substrate, and add water to adjust the moisture content to 50%~55%.

[0041] (2) Group fermentation Water was added to the substrate to adjust its moisture content to 50%~55%, and it was divided into 4 groups. The unfermented substrate served as the control group, and the other 3 groups were placed in fermentation tanks with different fermentation parameters set for each group. Substrate Group 1: Fermentation temperature was 25℃, aeration rate was 0.3 m³ / (h·m³), fermentation time was 48 h, and the pH value of the substrate after fermentation was 6.8-7.2; Substrate Group 2: Fermentation temperature was 28-32℃, aeration rate was 0.5 m³ / (h·m³), fermentation time was 72 h, and the pH value of the substrate after fermentation was 5.5-6.5; Substrate Group 3: Fermentation temperature was 35℃, aeration rate was 0.7 m³ / (h·m³), fermentation time was 96 h, and the pH value of the substrate after fermentation was 5.0-5.3.

[0042] Substitute group 4 (control group): Substitute group 4 does not ferment and is placed in a sterile environment for 48~96 hours, with a pH value of 7.5.

[0043] The fermentation site for the above-mentioned substrate groups 1-3 was Fengnong Agricultural Cooperative in Yangliuwan Town, Yingshan County.

[0044] (3) Poria cocos cultivation Bagging and sterilization: The fermented substrate from step (2) was packed into cultivation bags (3 kg per bag), the bag opening was sealed, and sterilized at 105°C for 12 hours under normal pressure, and then cooled to room temperature; the substrate of the control group was not fermented, packed into cultivation bags, the bag opening was sealed, and sterilized at 105°C for 12 hours under normal pressure, and then cooled to room temperature.

[0045] Inoculation and Culture: Under aseptic conditions, each bag was inoculated with the culture medium of Poria cocos strain WZ-1 (accession number CGMCC NO. 40108) and cultured in a constant temperature and dark incubator at 26±2℃ for 15-20 days until the mycelium filled the bag. The Poria cocos strain WZ-1 is classified as Wolfiporia hoelen, with accession number CGMCC NO. 40108, accession date: March 1, 2022, and deposited at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0046] Cellar cultivation: Dig a cellar along the slope (40-50cm long, 30-45cm wide, and 25-30cm deep). Make a long U-shaped cut in the plastic film along the longitudinal direction of the substrate. Place the exposed mycelium parallel to the surface of the diseased wood section (with the bark and core removed), with the wood section on the side or bottom and the end facing the trench. Place one bag in each cellar, with 50g of fresh Poria cocos sclerotia placed tightly against the wood section, and cover with 8-10cm of soil.

[0047] Management: Keep the soil dry, clean drainage ditches in a timely manner, and cover the soil with soil in small amounts and multiple times when the soil cracks during the sclerotium formation period.

[0048] After 60 days of cultivation, the inhibition rates of pine wilt nematode and pine sawyer beetle, as well as growth indicators of Poria cocos, were measured. The Bellman funnel method was used to isolate and identify pine wilt nematode, as detailed below: Isolation and identification of pine wood nematodes: The Bellman funnel method was used to isolate pine wood nematodes from infected trees. After a certain number of pine wood nematodes were released, they were examined under a dissecting microscope. Alternatively, the separation solution was collected in a centrifuge sedimentation tube, and the pine wood nematodes were collected by natural sedimentation or centrifugation for microscopic examination.

[0049] Culture and identification of pine wood nematodes: Tomato Botrytis cinerea was used for indoor culture. Tomato Botrytis cinerea was inoculated onto potato dextrose agar (PDA) plates and cultured for 4-5 days until the Botrytis cinerea mycelium completely covered the PDA plates. The isolated pine wood nematodes were then inoculated and cultured at 25℃ for another 7 days. The nematode-laden culture medium was then picked out, placed in a funnel, and the nematodes were washed out with sterile water. The number of pine wood nematodes was counted.

[0050] Determination of pine wilt nematode activity: Neutral red dye was used to determine the activity of pine wilt nematodes. Dead nematodes stained light red, while live nematodes did not stain. Using untreated infected wood from the same batch as a reference, the inhibition rate of different substrate groups against pine wilt nematodes was statistically analyzed. Ten measurements were taken for each group, and the average value was calculated to determine the pine wilt nematode inhibition rate.

[0051] Using untreated infected wood from the same batch as a reference, the survival of pine sawyer beetle larvae was detected by the tunnel and debris inspection method. The number of surviving pine sawyer beetle larvae was counted. Ten measurements were taken for each group and the average value was calculated to determine the inhibition rate of pine sawyer beetle larvae.

[0052] The experimental results are as follows: Substitute substrate group 1: The pH value of the substitute substrate (after fermentation) is 6.8~7.2, the inhibition rate of pine wilt nematode is 83.9%, the inhibition rate of pine sawyer beetle is 68.5%, the average yield of Poria cocos per pit is 1.12 kg, and the triterpenoid content is 3.01 mg / g; Substitute substrate group 2: The pH value of the substitute substrate (after fermentation) is 5.5~6.5, the inhibition rate of pine wilt nematode is 94.4%, the inhibition rate of pine sawyer beetle is 92.7%, the average yield per pit is 1.38 kg, and the triterpenoid content is 3.27 mg / g; Substrate Group 3: The substrate (after fermentation) has a pH of 5.0~5.3, and the inhibition rate of pine wilt nematode is 95.6% and the inhibition rate of pine longhorn beetle is 85.2%. However, the growth of Poria cocos mycelium is inhibited. The average yield per pit is 0.98 kg and the triterpenoid content is 2.97 mg / g.

[0053] Substrate Group 4: The substrate was not fermented, and the inhibition rate of pine wilt nematode was 67.8%, the inhibition rate of pine sawyer beetle was 55.6%, the average yield of Poria cocos per pit was 0.76 kg, and the triterpenoid content was 2.25 mg / g.

[0054] The above results indicate that, compared to unfermented substrate, fermented substrate (pH 5.0–6.5) significantly enhances the inhibitory effect on pine wood nematode and pine sawyer beetle. In particular, adjusting the pH of the substrate to 5.5–6.5 through fermentation also significantly promotes the growth of Poria cocos. This method achieves an inhibition rate of over 92% against both pine wood nematode and pine sawyer beetle, while also significantly promoting Poria cocos growth. Compared to cultivation with unfermented substrate, cultivation with fermented substrate increases the yield of Poria cocos by an average of 1.3–1.8 times, and the triterpenoid content by 1.32–1.45 times. Furthermore, the triterpenoids and other substances produced by Poria cocos further inhibit pine wood nematode and pine sawyer beetle, thus enhancing the inhibitory effect of this method on these organisms.

[0055] Example 2 verifies the pine nematode suppression method based on Poria cocos substrate cultivation. (1) Raw material preparation Infected wood: Select the same batch of pine wood nematode-infected wood as in Example 1, remove the moldy parts, and crush it into infected wood chips with a particle size of 0.5-1cm to completely destroy the hiding or living space of pine wood nematodes and their main parasite, the pine sawyer beetle, in the infected wood, thereby destroying the living carriers of pine wood nematodes and pine sawyer beetles.

[0056] (2) Fermentation was used to construct a microbial environment that inhibits pine wood nematode and pine sawyer beetle and promotes the growth of Poria cocos. Preparation of substitute substrate: Weigh 59% sawdust, 20% corn cob, 20% corn kernels, and 1% calcined gypsum by mass percentage, add water and mix evenly to prepare substitute substrate.

[0057] Substrate fermentation: Add an appropriate amount of water to the substrate to adjust its moisture content to 50%~55%, put it into a fermentation tank, control the fermentation temperature at 30℃ and the aeration rate at 0.5m³ / (h·m³), ferment for 72 hours, and stir once a day during the period. After the fermentation is completed, the pH value of the substrate is stable at 5.5~6.5.

[0058] (3) Cultivation and Management Bagging and sterilization: Pack the fermented substrate into cultivation bags (3kg per bag), tie the bag tightly, sterilize at 105℃ under normal pressure for 12 hours, and cool to room temperature.

[0059] Inoculation and Culture: Under aseptic conditions, each bag was inoculated with the Poria cocos strain WZ-1 culture medium and cultured in a constant temperature and dark incubator at 26℃±2℃ for 15-20 days until the mycelium filled the bag. The Poria cocos strain WZ-1 (preservation number CGMCCNO.40108) was selected as the culture medium.

[0060] Cellar cultivation: Cut a long U-shaped slit in the film along the longitudinal direction of the substrate. Place the exposed mycelium parallel to the surface of the peeled diseased wood section, with the wood section on the side or bottom and the end facing the trench. Place it in the cellar. Place one bag in each cellar, with 50g of fresh Poria cocos sclerotia placed close to the wood section (1.5~2kg), and cover with 8~10cm of soil.

[0061] Management: Keep the soil dry, clean drainage ditches in a timely manner, and cover the soil with soil in small amounts and multiple times when the soil cracks during the sclerotium formation period.

[0062] Thirty days after cultivation, the survival of pine wilt nematodes in the infected logs was detected using the Bellman funnel method. No surviving larvae were found, indicating a 100% inactivation rate. The survival of pine sawyer beetle larvae in the infected logs was also detected using the tunnel and sawdust inspection method. No surviving larvae were found, indicating a 100% inactivation rate and preventing secondary transmission of pine wilt nematodes.

[0063] Six months after planting, the survival rate of the inoculated Poria cocos was 100%, the formation rate was 98%, the average yield per cellar was 1.3767 kg, and the triterpenoid content was 3.2653 mg / g. The quality of the Poria cocos met the requirements of the Pharmacopoeia of the People's Republic of China (2025 edition). Example 3: Quantitative Validation

[0064] This embodiment is based on the cultivation of Poria cocos using a substrate from March 25 to August 20, 2024, with the substrate fermentation site being Fengnong Agricultural Cooperative in Yangliuwan Town, Yingshan County.

[0065] (1) Raw material preparation Infected wood: Select pine wood infected with pine wilt disease, remove the moldy parts, and crush it into 0.5-1cm particles to obtain infected sawdust.

[0066] Preparation of substitute substrate: Weigh out 59% sawdust, 20% corn cob, 20% corn kernels and 1% calcined gypsum by mass percentage, mix evenly to make 1000 kg of substitute substrate.

[0067] (2) Substrate fermentation Add an appropriate amount of water to the substrate to adjust its moisture content to 50%~55%, control the fermentation temperature to 28~32℃ and the aeration rate to 0.5m³ / (h·m³), ferment for 72 hours, and stir once a day during the period. After the fermentation is completed, the pH value of the substrate is stable at 5.5~6.5.

[0068] (3) Cultivation and Management Bagging and sterilization: Pack the fermented substrate into cultivation bags (3kg per bag), tie the bag tightly, sterilize at 105℃ and normal pressure for 12 hours, and cool to room temperature.

[0069] Inoculation and culture: Poria cocos strain WZ-1 (preservation number CGMCCNO.40108) was selected as the culture strain. Under aseptic conditions, each bag was inoculated with the Poria cocos strain WZ-1 culture strain and placed in a constant temperature and light-proof culture room at 26℃±2℃ for 15-20 days until the mycelium filled the bag.

[0070] Cellar cultivation: Select large-scale cultivation bases in Hubei's authentic producing areas and carry out cellar cultivation according to the steps in Example 2. A total of 200 cellars were planted and managed uniformly.

[0071] Poria cocos was harvested 4-6 months after cultivation, and the inhibition rate of pine wilt nematode, the mortality rate of pine sawyer beetle larvae, and the growth indicators of Poria cocos were tested. In this example, the cultivation period was from March 25th to August 20th, 2024, and the specific test results are as follows: The pine wilt nematode inhibition rate was 99.7%, and the mortality rate of pine sawyer beetle larvae was 98.2%, with no disease or pest transmission observed. The survival rate of Poria cocos inoculation was 99.5%, the formation rate was 97.8%, the average yield per pit was 1.32 kg, and the highest yield per pit was 1.83 kg. The triterpenoid content was ≥3.27 mg / g, and the quality of the Poria cocos met the requirements of the Pharmacopoeia of the People's Republic of China (2025 edition). Compared with traditional large-scale cultivation of infected logs, raw material costs were reduced by 30%, labor costs by 40%, and overall profits increased by 65%, making it suitable for large-scale production needs in authentic producing areas.

[0072] Comparative Example 1: Traditional Cultivation of Diseased Logs (1) Treatment of infected wood Select pine wood infected with wilt disease from the same batch as in Example 1, remove the moldy parts, cut into sections (tail diameter ≥ 6cm, weight 7kg), peel off the bark and leave the sinew (width 4-6cm), and sun-dry for 40 days.

[0073] (2) Log cultivation The same Poria cocos strain WZ-1 as in Example 1 was selected for cultivation, and the infected log cultivation method was used, as detailed below: Digging the cellar: Dig the cellar along the slope (specifications are the same as in Example 1), loosen the soil at the bottom of the cellar, place the barkless logs with the "retaining the core" parts close together, and fill with sand.

[0074] Inoculation: Graft 400g of spawn onto every 7kg of infected wood segments using the grafting method, and cover with 8-10cm of soil; 20 days later, replant 50-100g of young Poria cocos sclerotia and cover with soil a second time.

[0075] Management: The management method is the same as that of the material group in Example 1.

[0076] Thirty days after cultivation, the Bellman funnel method was used to detect surviving pine wilt nematodes in the infected logs. The results showed that 35-42 surviving pine wilt nematodes were still detected in every 100g of infected logs, and the pine wilt nematode inhibition rate was only 65.3%.

[0077] Thirty days after cultivation, the surviving pine sawyer beetles in the infected wood sections were detected by the tunnel and debris inspection method. The results showed that 15%-20% of the larvae in the infected wood sections were still alive and developing, and the mortality rate of pine sawyer beetle larvae was only 42.7%.

[0078] Using the traditional diseased log cultivation method, the survival rate of Poria cocos inoculation was 98.7%, the formation rate was 96.3%, the average yield per pit was 0.906 kg, and the triterpenoid content was 2.83 mg / g.

[0079] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for suppressing pine nematodes based on Poria cocos as a substrate, characterized in that, Includes the following steps: (1) Destroy the survival carrier of pine wood nematode and its intermediate host: The infected wood is crushed to destroy the hiding space of pine wood nematode and its intermediate host in the infected wood, and granular infected wood chips are obtained. (2) Directional regulation of the living environment by fermentation of substitute material: The infected sawdust from step (1) is prepared into a substitute material substrate, and the moisture content of the substitute material substrate is adjusted to 50%~55%. The pH value of the substitute material substrate is adjusted by fermentation so that the pH value after fermentation is in the range of 5.0~6.5, thereby constructing a microbial environment that inhibits the hatching of pine wood nematode eggs, the development of larvae and the reproduction of adults. (3) Biocontrol of Poria cocos: The substrate after fermentation in step (2) is divided into cultivation bags. After sterilization, Poria cocos strain is inoculated into the cultivation bags under sterile conditions and cultivated until the Poria cocos mycelium fills the bag. According to the principle of one bag per wood, the exposed Poria cocos mycelium is pressed onto the surface of the peeled diseased wood section to biologically inhibit the pine wood nematode and its intermediate host in the diseased wood section. The wood is then placed in a cellar and Poria cocos is cultivated in the cellar.

2. The method as described in claim 1, characterized in that, Step (2) The pH of the substrate is adjusted to 5.5~6.5 by fermentation to create a microbial environment that inhibits the hatching of pine wood nematode eggs, larval development, adult reproduction and promotes the growth of Poria cocos mycelium.

3. The method as described in claim 2, characterized in that, The fermentation in step (2) is carried out under aerobic conditions at 28-32℃ and an aeration rate of 0.3~1.0m³ / (h·m³).

4. The method as described in claim 3, characterized in that, The fermentation time in step (2) is 48~96h.

5. The method as described in claim 4, characterized in that, The fermentation described in step (2) is natural fermentation, and the microorganisms used in the fermentation include Bacillus.

6. The method as described in claim 5, characterized in that, The substrate, by weight percentage, comprises 55% to 65% sawdust, 15% to 25% corn cobs, and 15% to 25% corn kernels.

7. The method as described in claim 6, characterized in that, The substrate also includes calcined gypsum; the infected sawdust is made by removing the moldy parts from pine wood nematode-infected wood and crushing it into 0.5-1cm particles; the corn cob has a particle size of 0.1-1mm, and the corn kernel has a particle size of 0.1-0.2mm.

8. The method according to any one of claims 1 to 7, characterized in that, The Poria cocos strain inoculated in the cultivation bag in step (3) includes Poria cocos strain WZ-1, whose accession number is CGMCCNO.40108.

9. The method as described in claim 8, characterized in that, The culture conditions for the Poria cocos strain mentioned in step (3) are constant temperature culture at 26℃±2℃ in the dark for 15-20 days.

10. The method as described in claim 9, characterized in that, In step (3), the cellar planting is carried out according to the principle of one cellar, one bag, and one piece of wood. Each cellar contains one cultivation bag full of Poria cocos mycelium and one 1.5-2 kg peeled diseased wood section.

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