Multi-species cooperative truffle mycorrhizal seedling cultivation method and application

Abstract

The application discloses a multi-species synergistic truffle mycorrhiza seedling cultivation method and application, and belongs to the technical field of truffle cultivation.The application first adopts Mongolian oak and scots pine as the symbiotic hosts of summer truffle, adopts a two-species synergistic mode, carries out seedling cultivation and cultivation on mycorrhizal fungi, achieves the synergistic effect, significantly shortens the mycorrhizal formation time of mycorrhizal seedlings, improves the infection rate of mycorrhizal and the survival rate of mycorrhizal seedlings, greatly promotes the yield of summer truffle mycorrhizal, and provides technical support for artificial cultivation of summer truffle.

Description

Technical Field

[0001] This invention relates to the field of truffle mycorrhizal seedling cultivation and technology, and more specifically to a method and application of multi-tree species synergistic summer truffle mycorrhizal seedling cultivation. Background Technology

[0002] Summer truffle (Tube aestivum Vitt.) belongs to the phylum Ascomycota, order Tubrales, family Tuberaceae, and genus Tube. As a type of truffle, it is mainly produced in Europe and is a popular and delicious ingredient in European kitchens. The mature fruiting body of summer truffle is rich in nutrients and has a distinctive aromatic odor. It lives in symbiosis with its host and is mainly found in the understory of broad-leaved forests in Northern Europe, such as the UK, and North America. In recent years, it has been discovered in the mountainous areas of northern and southwestern my country. Currently, the commercially available truffles in the domestic market are primarily sourced from wild resources. Due to the unique growth characteristics of truffles and improper harvesting methods, natural truffle beds and their ecological environment have been severely damaged, leading to a sharp decline in truffle production, which is far from meeting market demand. Therefore, developing artificial cultivation methods for summer truffles has become increasingly important.

[0003] Currently, the artificial cultivation process for truffles is as follows: seedling cultivation → inoculation of truffle spores at the roots of seedlings to form infected seedlings → transplanting of infected seedlings → management such as root turning and pruning → fruiting bodies begin to form after 5-7 years, and fruiting bodies can continue to develop for 20-30 years. Existing artificial cultivation methods for summer truffle mycorrhizal seedlings mostly use chestnut, red pine, and American pecan as hosts for truffle cultivation. However, these methods often involve a single-species parasitic relationship, which significantly reduces species diversity in forest or woodland ecosystems, leading to a homogenized soil microbial community structure, soil nutrient imbalance, and decreased fertility. Furthermore, a single-species environment easily creates large areas of homogeneity, providing ideal habitats for specific pathogens or pests, increasing the likelihood of disease and pest outbreaks. These problems, to some extent, limit truffle growth, resulting in prolonged mycorrhizal emergence time, low infection rates, low seedling survival rates, and unsatisfactory cultivation results.

[0004] Therefore, how to provide a more effective multi-species synergistic summer truffle mycorrhizal seedling cultivation method is a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0005] In view of this, the present invention provides a method and application for multi-species synergistic cultivation of summer truffle mycorrhizae, using Mongolian oak and Scots pine as symbiotic hosts to cultivate and grow summer truffle mycorrhizae, which significantly improves the seedling and cultivation effect.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A method for multi-species synergistic mycorrhizal cultivation of *Tree Gloyton* seedlings includes the following steps:

[0008] (1) Preparation of summer tuber fruiting body powder: After detoxification, mature summer tuber fruiting bodies are crushed to prepare summer tuber fruiting body powder, which is then stored at low temperature for later use.

[0009] (2) Preparation of liquid culture solution: Add the fruiting body powder of summer truffle and Fusarium oxysporum to the liquid culture medium, place at 22-25℃, shake and culture for 7-10 days to obtain liquid culture solution;

[0010] (3) Inoculation and culture: Place the aseptic seedlings of Mongolian oak and Scots pine in the liquid culture solution obtained in step (2), covering the base of the stem, and soak for 10-20 minutes; plant the soaked Mongolian oak seedlings and Scots pine seedlings in the cultivation substrate in a row-to-row mixed manner of 1-2 rows: 2-3 rows, and pour the remaining liquid culture solution along the roots of the seedlings into the cultivation substrate for culture to obtain Mongolian oak and Scots pine summer truffle mycorrhizal seedlings;

[0011] (4) Transplanting: Transplant the Mongolian oak and Pinus sylvestris var. mongolica mycorrhizal seedlings obtained in step (3) in a row-to-row mixed planting pattern of 1-2 rows: 4-6 rows. After transplanting, manage regularly to prevent the occurrence of diseases and pests. After cultivation, harvest the truffles.

[0012] Preferably, in step (1), the fruiting body is disinfected by soaking in 75% alcohol for 3-5 seconds; the pulverization speed is 8000-19000 rpm and the pulverization time is 10-30 seconds; the storage temperature is 0-4℃.

[0013] Preferably, the liquid culture medium in step (2) has the following composition: glucose 15-25 g / L, maltose 10-13 g / L, peptone 3-4 g / L, KH2PO4 4-5 g / L, MgSO4·7H2O 2-3 g / L and vitamin B1 0.006-0.007 g / L.

[0014] Preferably, the dry weight of mycelium in the liquid culture solution obtained in step (2) is 1-2 g / L; and the content of Fusarium oxysporum in the liquid culture solution is 2 × 10⁻⁶ g / L. 6 -8×10 6 CFU / mL; adjust the pH of the liquid culture to 7.5-8.0.

[0015] Preferably, the shaking in step (2) is performed at a speed of 100-150 rpm, and the culture is carried out in the dark.

[0016] Preferably, the Mongolian oak seedlings in step (3) have a root length of 6-8cm and a stem length of 5-10cm; the Scots pine seedlings have a root length of 5-10cm and a stem length of 10-20cm.

[0017] Preferably, the cultivation substrate in step (3) consists of the following components: 40-50g humus, 30-35g perlite, 15-20g loam and 10-15g limestone particles; the pH of the cultivation substrate is 7.5-8.5.

[0018] Preferably, in step (3), the inoculation amount of Mongolian oak seedlings is 0.2-0.4 ml of liquid culture solution per seedling; the inoculation amount of Scots pine seedlings is 0.2-0.3 ml of liquid culture solution per seedling; the culture temperature is 22-25℃, the humidity of the cultivation substrate is 35-45%, and the light conditions are 1500-4000 Lux.

[0019] Another object of the present invention is to provide the application of the above-mentioned multi-species synergistic summer truffle mycorrhizal seedling cultivation method in summer truffle production.

[0020] As can be seen from the above technical solution, compared with the prior art, the present invention has the following beneficial effects:

[0021] This invention improves the artificial cultivation method of *Truffleia pulcherrima*. Building upon the existing single symbiotic host, it establishes a symbiotic relationship between *Quercus mongolica*, *Pinus sylvestris*, and *Truffleia pulcherrima*. Under this symbiotic relationship, *Quercus mongolica*, *Pinus sylvestris*, and *Truffleia pulcherrima* achieve a relatively stable symbiotic balance, promoting mutual benefit and creating a nutrient complementarity effect. This enhances the ecological stability of the symbiotic environment. Compared to symbiosis between a single tree species and *Truffleia pulcherrima*, this significantly shortens the mycorrhizal formation time, increases the infection rate and survival rate, and greatly improves yield. Furthermore, the *Truffleia pulcherrima* mycorrhizal seedlings prepared using this method exhibit strong stress resistance and can effectively tolerate the adverse effects of temperature and humidity on mycorrhizal growth. Detailed Implementation

[0022] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] The *Fusarium oxysporum* strain used in this embodiment of the invention is a strain already disclosed in the prior art, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 19031.

[0024] Example 1

[0025] A method for multi-species synergistic mycorrhizal cultivation of *Tree Gloyton* seedlings includes the following steps:

[0026] (1) Preparation of summer truffle fruiting body powder: Take mature summer truffle fruiting bodies, soak them in 75% alcohol for 3-5 seconds for disinfection, and then crush them at 8000 rpm for 30 seconds to obtain summer truffle fruiting body powder. Store it at 0-4℃ for later use.

[0027] (2) Preparation of liquid culture solution: Powdered fruiting bodies of *Ustilago maydis* and *Fusarium oxysporum* were added to a liquid culture medium consisting of 15 g / L glucose, 10 g / L maltose, 3 g / L peptone, 4 g / L KH₂PO₄, 2 g / L MgSO₄·7H₂O, and 0.006 g / L vitamin B1. The medium was incubated at 22-25°C in the dark with shaking at 100 rpm for 10 days to obtain the liquid culture solution. The dry weight of mycelium in the liquid culture solution was determined to be 1.24 g / L, and the content of *Fusarium oxysporum* was 4.32 × 10⁻⁶. 6 CFU / mL; Adjust the pH of the liquid culture to 7.5-8.0;

[0028] (3) Inoculation and culture: Mongolian oak seedlings with roots 7cm long and stems 9cm long and Scots pine seedlings with roots 5cm long and stems 10cm long were placed in the liquid culture solution obtained in step (2), covering the base of the stems, and soaked for 10-20 minutes; the soaked Mongolian oak seedlings and Scots pine seedlings were planted in the cultivation substrate (40g humus, 30g perlite, 15g loam and 10g limestone particles, pH 7.5-8.5) in a 1:3 inter-row mixed planting method, and the remaining liquid culture solution was poured into the cultivation substrate along the roots of the seedlings. The inoculation amount of Mongolian oak seedlings was 0.4ml of liquid culture solution / each seedling, and the inoculation amount of Scots pine seedlings was 0.3ml of liquid culture solution / each seedling. The seedlings were cultured at 22-25℃, 35-45% humidity, and 1500-4000 Lux to obtain Mongolian oak and Scots pine summer truffle mycorrhizal seedlings;

[0029] (4) Transplanting: The Mongolian oak and Scots pine summer truffle mycorrhizal seedlings obtained in step (3) are transplanted in a row-to-row mixed manner of 1 row: 4 rows. After transplanting, they are managed regularly to prevent the occurrence of diseases and pests. Summer truffles are harvested after cultivation.

[0030] Example 2

[0031] A method for multi-species synergistic mycorrhizal cultivation of *Tree Gloyton* seedlings includes the following steps:

[0032] (1) Preparation of summer truffle fruiting body powder: Take mature summer truffle fruiting bodies, soak them in 75% alcohol for 3-5 seconds for disinfection, and then crush them at 19000 rpm for 10 seconds to obtain summer truffle fruiting body powder. Store it at 0-4℃ for later use.

[0033] (2) Preparation of liquid culture medium: Powdered fruiting bodies of *Ustilago maydis* and *Fusarium oxysporum* were added to a liquid culture medium consisting of 25 g / L glucose, 13 g / L maltose, 4 g / L peptone, 5 g / L KH₂PO₄, 3 g / L MgSO₄·7H₂O, and 0.007 g / L vitamin B1. The medium was incubated at 22-25°C in the dark with shaking at 150 rpm for 7 days to obtain the liquid culture medium. The dry weight of mycelium in the liquid culture medium was 1.36 g / L, and the content of *Fusarium oxysporum* was 3.84 × 10⁻⁶. 6 CFU / mL; Adjust the pH of the liquid culture to 7.5-8.0;

[0034] (3) Inoculation and culture: Mongolian oak seedlings with roots of 8cm and stems of 10cm and Scots pine seedlings with roots of 10cm and stems of 20cm were placed in the liquid culture solution obtained in step (2), covering the base of the stems, and soaked for 10-20 minutes; the soaked Mongolian oak seedlings and Scots pine seedlings were planted in the cultivation substrate (50g humus, 35g perlite, 20g loam and 15g limestone particles, pH 7.5-8.5) in a 2:3 interrow mixed planting method, and the remaining liquid culture solution was poured into the cultivation substrate along the roots of the seedlings. The inoculation amount of Mongolian oak seedlings was 0.2ml liquid culture solution / each seedling, and the inoculation amount of Scots pine seedlings was 0.2ml liquid culture solution / each seedling. The seedlings were cultured at 22-25℃, 35-45% humidity, and 1500-4000 Lux to obtain Mongolian oak and Scots pine summer truffle mycorrhizal seedlings;

[0035] (4) Transplanting: The Mongolian oak and Pinus sylvestris var. mongolica mycorrhizal seedlings obtained in step (3) are transplanted in a 2:6 interrow mixed manner. After transplanting, they are managed regularly to prevent the occurrence of diseases and pests. After cultivation, the truffles are harvested.

[0036] Example 3

[0037] A method for multi-species synergistic mycorrhizal cultivation of *Tree Gloyton* seedlings includes the following steps:

[0038] (1) Preparation of summer truffle fruiting body powder: Take mature summer truffle fruiting bodies, soak them in 75% alcohol for 3-5 seconds for disinfection, and then crush them at 10000 rpm for 20 seconds to obtain summer truffle fruiting body powder. Store it at 0-4℃ for later use.

[0039] (2) Preparation of liquid culture medium: Powdered fruiting bodies of *Ustilago maydis* and *Fusarium oxysporum* were added to a liquid culture medium consisting of 20 g / L glucose, 12 g / L maltose, 3 g / L peptone, 4 g / L KH₂PO₄, 3 g / L MgSO₄·7H₂O, and 0.007 g / L vitamin B1. The medium was incubated at 22-25°C in the dark with shaking at 150 rpm for 10 days to obtain the liquid culture medium. The mycelial dry weight in the liquid culture medium was 1.64 g / L, and the *Fusarium oxysporum* content was 4.52 × 10⁻⁶. 6 CFU / mL; Adjust the pH of the liquid culture to 7.5-8.0;

[0040] (3) Inoculation and culture: Mongolian oak seedlings with a root length of 6cm and a stem length of 8cm and Scots pine seedlings with a root length of 8cm and a stem length of 16cm were placed in the liquid culture solution obtained in step (2), covering the base of the stem, and soaked for 10-20 minutes; the soaked Mongolian oak seedlings and Scots pine seedlings were planted in the cultivation substrate (45g humus, 30g perlite, 15g loam and 12g limestone particles, pH 7.5-8.5) in a 2:3 interrow mixed planting method, and the remaining liquid culture solution was poured into the cultivation substrate along the roots of the seedlings. The inoculation amount of Mongolian oak seedlings was 0.3ml of liquid culture solution / each seedling, and the inoculation amount of Scots pine seedlings was 0.25ml of liquid culture solution / each seedling. The seedlings were cultured at 22-25℃, 35-45% humidity, and 1500-4000 Lux to obtain Mongolian oak and Scots pine summer truffle mycorrhizal seedlings;

[0041] (4) Transplanting: The Mongolian oak and Scots pine summer truffle mycorrhizal seedlings obtained in step (3) are transplanted in a 2:5 interrow mixed manner. After transplanting, they are managed regularly to prevent the occurrence of diseases and pests. Summer truffles are harvested after cultivation.

[0042] Comparative Example 1

[0043] It is basically the same as Example 3, except that:

[0044] In step (2), Fusarium oxysporum is not added during the preparation of the liquid culture solution.

[0045] Comparative Example 2

[0046] It is basically the same as Example 3, except that:

[0047] Step (3) The root length of Mongolian oak is 9cm and the stem length is 12cm; the root length of Scots pine is 4cm and the stem length is 10cm.

[0048] Comparative Example 3

[0049] It is basically the same as Example 3, except that:

[0050] Step (3) The root length of Mongolian oak is 5cm and the stem length is 5cm; the root length of Scots pine is 11cm and the stem length is 22cm.

[0051] Comparative Example 4

[0052] It is basically the same as Example 3, except that:

[0053] Step (3) Mongolian oak seedlings and Scots pine seedlings are planted in the cultivation substrate in a 3:2 inter-row mixed planting pattern.

[0054] Comparative Example 5

[0055] It is basically the same as Example 3, except that:

[0056] Step (4) Transplant the Mongolian oak and Scots pine truffle mycorrhizal seedlings in a 3:3 inter-row mixed planting pattern.

[0057] Comparative Example 6

[0058] It is basically the same as Example 3, except that:

[0059] Step (3) only provides Pinus sylvestris seedlings and liquid culture solution for inoculation and cultivation.

[0060] Comparative Example 7

[0061] It is basically the same as Example 3, except that:

[0062] Step (3) Provide Scots pine seedlings and Korean pine seedlings with liquid culture solution for inoculation and culture. The root length of the Scots pine seedlings is 8cm and the stem length is 16cm. The root length of the Korean pine seedlings is 8cm and the stem length is 16cm.

[0063] To evaluate the cultivation of *Tuber spp.* mycorrhizal seedlings from different groups, continuous sampling and testing of mycorrhizal infection were conducted. The specific results are as follows:

[0064] Table 1. Mycorrhizal infection status of different groups

[0065]

[0066] Results Analysis: As shown in Table 1, the truffle mycorrhizal seedlings of Examples 1-3 of this invention have short formation time, high infection rate and survival rate, and generally higher yield than those of existing technologies. Furthermore, comparing Example 3 with Comparative Examples 6 and 7, Comparative Example 6, using a single tree species (Pinus sylvestris) for mycorrhizal seedling cultivation, showed results inferior to Comparative Example 3 in terms of mycorrhizal formation time, infection rate, survival rate, and yield. Comparative Example 7 replaced the symbiotic tree species, replacing the combination of Mongolian oak and Pinus sylvestris with Korean pine and Pinus sylvestris, but its mycorrhizal formation time, infection rate, survival rate, and yield were still lower than Example 3. This demonstrates that the diversity of tree species and the specific combination of species both affect the seedling cultivation effect of mycorrhizae. The present invention, by selecting Mongolian oak and Pinus sylvestris for combination, can significantly improve the cultivation effect of truffles.

[0067] Furthermore, a comparison between Example 3 and Comparative Example 1 shows that the addition of Fusarium oxysporum can affect the mycorrhizal formation time, infection rate, survival rate, and yield. The reason for this may be that Fusarium oxysporum can secrete some enzymes, such as cellulase and ligninase, and release some antibiotic antibacterial substances, as well as some phenolic and sugar substances, which to a certain extent promote the colonization of truffles and the growth of mycelium.

[0068] Furthermore, a comparison of Example 3 with Comparative Examples 2 and 3 shows that during the synergistic cultivation and propagation of summer truffle mycorrhizal fungi by Mongolian oak and Scots pine, the root length and stem length of Mongolian oak seedlings and Scots pine seedlings must be within a specific range to achieve the technical effect of the present invention. When they are outside the range of the present invention, the growth competition and growth promotion effects of Mongolian oak seedlings and Scots pine seedlings cannot be effectively balanced, resulting in the synergistic effect not being maximized, or even producing antagonistic effects.

[0069] The comparison between Example 3 and Comparative Examples 4 and 5 shows that different hybridization methods affect the final mycorrhizal seedling cultivation and planting effect. When the growth and competition between Mongolian oak and Scots pine reach a relatively stable balance, the different root distribution depths and nutrient absorption methods of different tree species form a nutrient complementarity effect, which improves ecological stability. Conversely, if the hybridization method cannot achieve a stable balance between the growth and competition between Mongolian oak and Scots pine, it will affect the truffle cultivation effect.

[0070] To verify the symbiotic effect of truffle mycorrhizae with Mongolian oak and Scots pine, this invention conducted stress resistance experiments on mycorrhizal seedlings cultivated in different groups of Example 3 and Comparative Examples 6 and 7. The experimental process is as follows:

[0071] Different groups of mycorrhizal seedlings were transplanted at 15℃, 25℃, and 35℃. Specifically, the growth rate of truffle mycelium, fruiting body formation, and yield were measured at the mature stage (5-7 years after transplanting). The experimental results are shown in Table 2.

[0072] Table 2 Temperature tolerance of different groups of mycorrhizal seedlings

[0073]

[0074]

[0075] Results Analysis: As shown in Table 2, when different groups of mycorrhizal seedlings were transplanted into different environments, temperature changes had different effects on the mycorrhizal growth of the seedlings. Through comparison, it can be seen that the mycorrhizal seedlings obtained in Example 3 of this invention have better stress resistance than those in Comparative Examples 6 and 7. The fluctuation range of mycorrhizal growth rate and fruiting body quantity affected by temperature is smaller. This indicates that the mycorrhizal seedlings of summer truffles co-cultured by Mongolian oak and Scots pine in this invention can significantly improve the stress resistance of mycorrhizal seedlings compared with Scots pine alone or the combination of Scots pine and Korean pine.

[0076] Meanwhile, the symbiotic effect of truffle mycorrhizae with Mongolian oak and Scots pine was verified. The present invention conducted stress resistance experiments on mycorrhizal seedlings obtained from different groups in Example 3 and Comparative Examples 6 and 7. The experimental process is as follows:

[0077] Mycorrhizal seedlings from different groups were transplanted at soil moisture levels of 20%, 50%, and 80%. Specifically, the growth rate of truffle mycelium, fruiting body formation, and yield were measured at the mature stage (5-7 years after transplanting). The experimental results are shown in Table 3.

[0078] Table 3 Humidity tolerance of different groups of mycorrhizal seedlings

[0079]

[0080]

[0081] Results Analysis: As shown in Table 3, when different groups of mycorrhizal seedlings were transplanted into different environments, the changes in substrate humidity had different effects on the mycorrhizal growth of the seedlings. Through comparison, it can be seen that the mycorrhizal seedlings obtained in Example 3 of this invention have better stress resistance than those in Comparative Examples 6 and 7. The fluctuation range of mycorrhizal growth rate and fruiting body quantity affected by humidity is smaller. This indicates that the mycorrhizal seedlings of summer truffles co-cultured by Mongolian oak and Scots pine in this invention can significantly improve the stress resistance of mycorrhizal seedlings compared with Scots pine alone or the combination of Scots pine and Korean pine.

[0082] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0083] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for multi-species synergistic cultivation of *Tree Truffle* mycorrhizal seedlings, characterized in that, Includes the following steps: (1) Preparation of summer tuber fruiting body powder: After detoxification, mature summer tuber fruiting bodies are crushed to prepare summer tuber fruiting body powder, which is then stored at low temperature for later use. (2) Preparation of liquid culture solution: Add the fruiting body powder of summer truffle and Fusarium oxysporum to the liquid culture medium, place at 22-25℃, shake and culture for 7-10 days to obtain liquid culture solution; (3) Inoculation and culture: Place the aseptic seedlings of Mongolian oak and Scots pine in the liquid culture solution obtained in step (2), covering the base of the stem, and soak for 10-20 minutes; plant the soaked Mongolian oak seedlings and Scots pine seedlings in the cultivation substrate in a row-to-row mixed manner of 1-2 rows: 2-3 rows, and pour the remaining liquid culture solution along the roots of the seedlings into the cultivation substrate for culture to obtain Mongolian oak and Scots pine summer truffle mycorrhizal seedlings; (4) Transplanting: Transplant the Mongolian oak and Pinus sylvestris var. mongolica mycorrhizal seedlings obtained in step (3) in a row-to-row mixed planting pattern of 1-2 rows: 4-6 rows. After transplanting, manage regularly to prevent the occurrence of diseases and pests. After cultivation, harvest the truffles.

2. The multi-species synergistic summer truffle mycorrhizal seedling cultivation method according to claim 1, characterized in that, In step (1), the fruiting bodies are disinfected by soaking in 75% alcohol for 3-5 seconds; the pulverization speed is 8000-19000 rpm and the pulverization time is 10-30 seconds; the storage temperature is 0-4℃.

3. The multi-species synergistic summer truffle mycorrhizal seedling cultivation method according to claim 1, characterized in that, The liquid culture medium in step (2) consists of the following components: glucose 15-25 g / L, maltose 10-13 g / L, peptone 3-4 g / L, KH2PO4 4-5 g / L, MgSO4·7H2O 2-3 g / L, and vitamin B1. 0.006-0.007 g / L.

4. The multi-species synergistic summer truffle mycorrhizal seedling cultivation method according to claim 1, characterized in that, The mycelial dry weight in the liquid culture solution obtained in step (2) is 1-2 g / L; the content of Fusarium oxysporum in the liquid culture solution is 2 × 10⁻⁶. 6 -8×10 6 CFU / mL; adjust the pH of the liquid culture to 7.5-8.

0.

5. The multi-species synergistic summer truffle mycorrhizal seedling cultivation method according to claim 1, characterized in that, The shaking in step (2) is performed at a speed of 100-150 rpm, and the culture is carried out in the dark.

6. The multi-species synergistic summer truffle mycorrhizal seedling cultivation method according to claim 1, characterized in that, The Mongolian oak seedlings in step (3) have roots that are 6-8 cm long and stems that are 5-10 cm long; the Scots pine seedlings have roots that are 5-10 cm long and stems that are 10-20 cm long.

7. The multi-species synergistic summer truffle mycorrhizal seedling cultivation method according to claim 1, characterized in that, The cultivation substrate in step (3) consists of the following components: 40-50g humus, 30-35g perlite, 15-20g loam and 10-15g limestone particles; the pH of the cultivation substrate is 7.5-8.

5.

8. The multi-species synergistic summer truffle mycorrhizal seedling cultivation method according to claim 1, characterized in that, In step (3), the inoculation amount of Mongolian oak seedlings is 0.2-0.4 ml of liquid culture solution per seedling; the inoculation amount of Scots pine seedlings is 0.2-0.3 ml of liquid culture solution per seedling; the culture temperature is 22-25℃, the humidity of the cultivation substrate is 35-45%, and the light conditions are 1500-4000 Lux.

9. The application of the multi-species synergistic mycorrhizal seedling cultivation method of *Tree Truffle* according to any one of claims 1-8 in the production of *Tree Truffle*.

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