Method for judging resistance of trichoderma to lentinula edodes strain and application thereof

Abstract

The present application belongs to the technical field of Lentinula edodes breeding, and particularly relates to a method for judging the strength of Trichoderma resistance of Lentinula edodes strains and application thereof. The present application provides a method for judging the strength of Trichoderma resistance of Lentinula edodes strains, comprising the following steps: inoculating different to-be-tested Lentinula edodes strains on the surface of natural substrate respectively, avoiding light for 21-23 days, then inoculating Trichoderma blocks, and co-culturing for 10-20 days, and comparing the resistance of the to-be-tested Lentinula edodes strains based on the mycelium density. The present application can truly evaluate the resistance of Lentinula edodes strains to Trichoderma by using the natural substrate similar to the actual production substrate of Lentinula edodes. The method is simple to operate, suitable for batch screening, can effectively prevent poor-resistant Lentinula edodes strains from entering the cultivation bag production link, significantly reduce the time and screening cost, has a good application prospect, and is suitable for Lentinula edodes production and breeding enterprises.

Description

Technical Field

[0001] This invention belongs to the field of shiitake mushroom breeding technology, specifically relating to a method for determining the resistance strength of Trichoderma to shiitake mushroom strains and its application. Background Technology

[0003] In conventional shiitake mushroom hybridization breeding, the process often proceeds directly to large-scale log preparation and cultivation trials. However, the introduction of numerous hybrid progeny strains with undefined Trichoderma resistance into pilot-scale and large-scale cultivation greatly increases the risk of Trichoderma contamination during the mycelial growth and fruiting stages. This not only leads to log spoilage and yield losses but also significantly increases the inefficient input of materials, labor, and space, drastically raising breeding screening costs and shortening the variety selection cycle, thus hindering the efficient breeding of high-quality, stress-resistant shiitake mushroom germplasm. Therefore, establishing accurate and efficient methods for detecting Trichoderma resistance in shiitake mushrooms is of great significance. Summary of the Invention

[0004] The purpose of this invention is to provide a method for determining the resistance of *Trichoderma* to shiitake mushroom strains and its application. The method described in this invention can accurately determine the resistance of *Trichoderma* to shiitake mushroom strains in batches and is applicable to shiitake mushroom production and breeding.

[0005] This invention provides a method for determining the resistance strength of *Trichoderma* to *Lentinula edodes* strains, comprising the following steps: different *Lentinula edodes* strains to be tested are inoculated onto the surface of a natural substrate, cultured in the dark for 21-23 days, and then inoculated with *Trichoderma* mycelium blocks for 10-20 days. The resistance of the *Lentinula edodes* strains to be tested is compared based on the mycelial density of *Lentinula edodes*.

[0006] As a preferred embodiment, the natural substrate comprises wood chips; the moisture content of the natural substrate is 58% to 60%.

[0007] As a preferred embodiment, the Trichoderma includes Trichoderma fulvidraco (Yellow-green Trichoderma). Trichoderma aureoviride ).

[0008] As a preferred option, it also includes setting up a control group for the main shiitake mushroom varieties.

[0009] As a preferred method, the mycelial density of the tested shiitake mushroom strain was significantly weaker than that of the main cultivated shiitake mushroom variety, indicating that the tested shiitake mushroom strain had weak resistance to Trichoderma.

[0010] As a preferred option, the main shiitake mushroom variety is Liaofu No. 4 (0912).

[0011] As a preferred option, after comparing resistance, the following also applies: discarding the tested shiitake mushroom strains whose mycelial density is significantly weaker than that of the main cultivated shiitake mushroom varieties.

[0012] As a preferred embodiment, the natural matrix is ​​filled into a test tube.

[0013] As a preferred embodiment, the temperature for the light-protected culture is 25±1℃; the temperature for the co-culture is 25±1℃.

[0014] This invention also provides the application of the method described above in shiitake mushroom breeding.

[0015] Beneficial Effects: This invention provides a method for determining the resistance strength of *Trichoderma* to *Lentinula edodes* strains, comprising the following steps: different *Lentinula edodes* strains to be tested are inoculated onto the surface of a natural substrate and cultured in the dark for 21-23 days, followed by inoculation with *Trichoderma* mycelium blocks and co-cultured for 10-20 days. The resistance of the *Lentinula edodes* strains to be tested is compared based on the mycelial density of *Lentinula edodes*. This invention abandons artificially synthesized substrates and utilizes the similarity between natural substrates and actual *Lentinula edodes* production substrates, which can accurately evaluate the resistance of *Lentinula edodes* strains to *Trichoderma*. Furthermore, the mycelial growth test of *Lentinula edodes* takes a long time (120 days). If strains with low resistance are found in production, the cost loss is high. Using the method of this invention, strains with low resistance can be eliminated, avoiding losses and improving screening efficiency. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0017] Figure 1 The images show the mycelial growth status of the tested shiitake mushroom strains F124, F103, and F104 co-cultured with Trichoderma. Figure 2 The images show the mycelial growth status of the tested shiitake mushroom strains F35 and A85 co-cultured with Trichoderma. Figure 3 The images show the mycelial growth status of the tested shiitake mushroom strains 3272, 1083, and 1583 co-cultured with Trichoderma. Figure 4 The images show the mycelial growth status of the tested shiitake mushroom strains 2550, 3350, 3826, and 4073 co-cultured with Trichoderma. Figure 5 The images show the mycelial growth status of the tested shiitake mushroom strains 4136, 4440, and D22 co-cultured with Trichoderma. Figure 6 The images show the mycelial growth status of the tested shiitake mushroom strains A17, D104, and D130 co-cultured with Trichoderma. Figure 7 The images show the mycelial growth status of the tested shiitake mushroom strains D63 and 2560 co-cultured with Trichoderma. Figure 8 The images show the mycelial growth status of the tested shiitake mushroom strains F124 and D22 and the main cultivated variety 0912. Figure 9The results of plate confrontation of strongly resistant shiitake mushroom strains are shown; where A is F124; B is F103; C is F104; D is F35; and E is A85. Figure 10 The results of plate confrontation of weakly resistant shiitake mushroom strains are shown; where A is A17; B is D104; C is D130; D is D63; and E is D22. Figure 11 This is a schematic diagram of the method for determining the resistance strength of Trichoderma strains in shiitake mushrooms according to the present invention. Detailed Implementation

[0018] This invention provides a method for determining the resistance strength of *Trichoderma* to *Lentinula edodes* strains, comprising the following steps: different *Lentinula edodes* strains to be tested are inoculated onto the surface of a natural substrate, cultured in the dark for 21-23 days, and then inoculated with *Trichoderma* mycelium blocks for 10-20 days. The resistance of the *Lentinula edodes* strains to be tested is compared based on the mycelial density of *Lentinula edodes*.

[0019] Unless otherwise specified, all raw materials described in this invention can be obtained through conventional commercial methods.

[0020] A schematic diagram of the method for determining the resistance strength of Trichoderma strains in shiitake mushrooms according to the present invention is shown below. Figure 11 As shown.

[0021] This invention involves inoculating different shiitake mushroom strains to be tested onto the surface of a natural substrate. The source of the shiitake mushroom strains to be tested is not specifically limited; for example, they can be hybrid strains from the breeding process, progeny of mutagenesis, wild isolates, or existing cultivated varieties. As one embodiment, before inoculating the shiitake mushroom strains to be tested onto the natural substrate surface, the invention further includes: culturing the shiitake mushroom strains on PDA medium and recording the number of days required for mycelium to fully colonize the culture dish; selecting shiitake mushroom strains whose mycelium has fully colonized the culture dish in 11-14 days as the test strains; and discarding shiitake mushroom strains whose mycelium has not fully colonized the culture dish after 14 days. By pre-screening the mycelial growth rate on PDA medium before inoculation into the natural substrate, this invention ensures that the shiitake mushroom strains to be tested have similar and stable growth rates, avoiding misdetection due to individual differences in growth rate.

[0022] After obtaining the shiitake mushroom strain to be tested, the present invention activates the shiitake mushroom strain. The present invention does not impose any particular limitation on the activation method; conventional activation methods in the art can be used.

[0023] In one embodiment, the natural substrate includes sawdust; the moisture content of the natural substrate is 58%~60%, for example, 58%, 59% or 60%. In another embodiment, the sawdust includes oak sawdust. Sawdust is the main raw material for the actual cultivation of shiitake mushrooms. This invention uses sawdust as a natural substrate, which can realistically reproduce the colonization, spread, and competitive, antagonistic, and tolerant growth states of shiitake mycelium against Trichoderma, avoiding the distortion of resistance evaluation caused by substrate differences in artificial culture media systems. This allows the test results to accurately reflect the true Trichoderma resistance level of the strain under actual production conditions, effectively improving the authenticity and accuracy of resistance evaluation.

[0024] In one embodiment, the natural substrate is filled into test tubes. The present invention does not impose any special limitations on the size of the test tubes; in one specific embodiment, the test tubes are 32 mm × 200 mm. By filling the test tubes with the natural substrate, the mycelial growth rate can be clearly observed.

[0025] In one implementation method, the shiitake mushroom strain to be tested is inoculated at the center of the surface of a natural substrate. If the inoculation site is close to the test tube wall, it will affect the germination of the mycelium and the growth rate of the hyphae. In this invention, inoculating the shiitake mushroom strain to be tested at the center of the surface of the natural substrate is beneficial to the germination of the mycelium and accurately reflects the growth rate of the hyphae.

[0026] In one embodiment, after inoculation, the shiitake mushrooms are cultured in the dark for 21-23 days, for example, 21, 22, or 23 days. In another embodiment, the temperature for this dark culture is 25±1℃. As is known in this invention, compared to molds, the colonization ability of shiitake mycelium is far weaker. If *Trichoderma* and shiitake mushrooms are inoculated simultaneously, the shiitake mushroom mycelium will be infected by *Trichoderma* before germination. Therefore, this invention first inoculates shiitake mushrooms to allow them to grow normally, and then evaluates *Trichoderma* resistance under these conditions. This invention found that under normal circumstances, shiitake mushroom mycelium can grow to half the length of the test tube in about 22 days. At this point, the shiitake mushroom mycelium has fully adapted to the natural substrate, and this position is convenient for subsequent observation. If the time is too short, the shiitake mushroom mycelium may be completely infected by *Trichoderma*, making it impossible to determine the strength of resistance. If the time is too long, it increases the time cost and also makes subsequent observation difficult because the shiitake mushroom mycelium has completely filled the test tube.

[0027] This invention involves co-culturing Trichoderma blocks after 21-23 days of light-protected incubation. As one embodiment, the Trichoderma includes *Trichoderma fulvidraco* (…). Trichoderma aureoviride*Trichoderma luteoliii* is a common Trichoderma species that infects edible fungi, and it can infect the mycelia of edible fungi. This invention uses *Trichoderma luteoliii* as an indicator strain, and the resistance level of *Lentinula edodes* mycelia can be accurately and reliably assessed by observing the growth inhibition or morphological changes of *Trichoderma luteoliii* mycelia under its influence. As one embodiment, the *Trichoderma* block is inoculated at the center of the natural substrate surface, which avoids the test tube wall affecting *Trichoderma* growth.

[0028] In one implementation method, the co-cultivation time is 10-20 days; the co-cultivation temperature is 25±1℃. In a specific embodiment of the present invention, the co-cultivation time can be any value within 10-20 days, such as 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days. For strains with weak resistance to *Trichoderma*, the mycelial density decreases significantly within 10 days of co-cultivation due to cell wall degradation by *Trichoderma* and the absorption of large amounts of intracellular nutrients. Some strains with weak resistance show no significant change in mycelial density in the early stage of co-cultivation (within 10 days), but exhibit a significant decrease in mycelial density after 15-20 days of co-cultivation. The present invention limits the co-cultivation time to 10-20 days, which can accurately reflect the resistance of *Lentinula edodes* strains.

[0029] After co-culturing for 10-20 days, this invention compares the resistance of the tested *Lentinula edodes* strains based on mycelial density. The mycelial density of the tested *Lentinula edodes* strains is positively correlated with their resistance to *Trichoderma*; that is, the higher the mycelial density, the stronger the resistance to *Trichoderma*. As one implementation method, the mycelial density of the tested *Lentinula edodes* strain after dark-weather cultivation is used as a benchmark. The resistance is divided into four levels, I to IV (with resistance decreasing progressively from I to IV, and I representing the highest resistance). The criteria for level I are: if the mycelial density of the tested *Lentinula edodes* does not change significantly during co-cultivation, the strain is considered to have level I resistance to *Trichoderma*. The criteria for level II are: if the mycelial density of the tested *Lentinula edodes* decreases within 1-10 days of co-cultivation, and then gradually recovers and increases in mycelial density within 10-20 days of co-cultivation, the strain is considered to have level II resistance to *Trichoderma*; or, if the mycelial density of the tested *Lentinula edodes* does not change significantly within 1-10 days of co-cultivation, and then decreases in mycelial density within 10-20 days of co-cultivation, the strain is considered to have level II resistance to *Trichoderma*. The criteria for level III are: if the mycelial density of the tested *Lentinula edodes* does not change significantly within 1-10 days of co-cultivation, and then decreases in mycelial density within 10-20 days of co-cultivation, the strain is considered to have level II resistance to *Trichoderma*. Within d, if the mycelial density of the tested shiitake mushroom decreases, and during co-cultivation from day 10 to 20, the mycelial density of the tested shiitake mushroom at the inoculation surface of the test tube is significantly reduced, then the strain is determined to be at level III resistance to Trichoderma. The criteria for level IV are: during co-cultivation, the mycelium of the tested shiitake mushroom is decomposed by Trichoderma, and Trichoderma produces sporulations, then the strain is determined to be at level IV resistance to Trichoderma. In this invention, strains with weak resistance (levels II to IV) are eliminated to avoid entering subsequent breeding procedures; strains with strong resistance (level I) are selected for small-scale and pilot-scale experiments, and fruiting is achieved through bag cultivation. The mycelial growth, susceptibility to contamination, and agronomic traits such as fruiting body morphology and yield are observed.

[0030] As one implementation method, it further includes: setting up a control group for the main cultivated variety of shiitake mushroom; the main cultivated variety mentioned in this invention is a crop variety that has been approved or recognized under certain regional ecological and cultivation conditions, has strong adaptability and excellent performance, and has been widely promoted and dominated by cultivation. In this invention, the local main cultivated variety can be selected according to the area where the shiitake mushroom strain is to be promoted. As one implementation method, the main cultivated variety of shiitake mushroom is Liaofu 4 (0912). As one implementation method, if the density of the shiitake mushroom strain to be tested is significantly weaker than that of the main cultivated variety, it is determined that the shiitake mushroom strain to be tested has weak resistance to Trichoderma, and low-resistance strains are eliminated to avoid entering the subsequent breeding process. In this invention, the main cultivated variety of shiitake mushroom has a resistance level of I to Trichoderma. When the resistance level of the shiitake mushroom strain to Trichoderma is II to IV, it is determined that the shiitake mushroom strain to be tested has weak resistance to Trichoderma, and low-resistance strains are eliminated. Liaofu No. 4 (0912) has rapid mycelial growth and a high rate of high-quality mushrooms, making it one of the main varieties of shiitake mushroom cultivation in my country. Using Liaofu No. 4 (0912) as a positive control can reflect the resistance of the tested shiitake mushroom strain.

[0031] As one implementation method, after comparing resistance, the method also includes discarding test shiitake mushroom strains with significantly weaker mycelial density than the main cultivated shiitake mushroom varieties. In conventional shiitake mushroom hybridization breeding, the selected hybrids are often directly used in large-scale log preparation and cultivation trials. However, shiitake mushroom mycelium growth time is relatively long, about 120 days. If directly used in small-scale and pilot-scale experiments, the time, labor, and material costs required to detect low-resistance strains are high. This invention discards strains with weak resistance, avoiding losses and improving screening efficiency. Subsequently, strains with strong resistance are selected for small-scale and pilot-scale experiments, where fruiting is achieved through bag cultivation. The mycelial growth, susceptibility to contamination, and agronomic traits such as fruiting body morphology and yield are observed.

[0032] This invention also provides the application of the method described above in shiitake mushroom breeding. This invention detects the resistance of shiitake mushroom strains to *Trichoderma* on a natural substrate, observes changes in mycelial density after co-culturing with *Trichoderma*, and screens out and eliminates strains with weak resistance to prevent them from entering subsequent breeding processes. Then, strains with strong resistance are selected for small-scale and pilot-scale trials, where fruiting occurs through bag cultivation. Mycelial growth, susceptibility to contamination, and agronomic traits such as fruiting body morphology and yield are observed. The method described in this invention is simple to operate, suitable for batch screening, and can effectively prevent poorly resistant shiitake mushroom strains from entering the cultivation bag production stage, significantly reducing time and screening costs. It has good application prospects and is suitable for use by shiitake mushroom production and breeding enterprises.

[0033] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0034] Example 1 (1) Preparation of culture medium Potato dextrose agar (PDA): Boil 200 g of fresh, peeled potatoes to extract the juice. Filter the juice through 8 layers of gauze, collect the filtrate, and add 20 g glucose, 0.5 g MgSO4, 2.0 g KH2PO4, and 15 g agar powder. Add water to bring the volume to 1 L. Autoclave at 121°C for half an hour. Cool to approximately 60°C, pour into 90 mm petri dishes (approximately 3-5 mm thick), and allow to solidify.

[0035] Wood chip culture medium formula: 100% oak wood chips, moisture content 58%~60%, natural pH.

[0036] (2) Activation of microbial strains Hybrids of *Lentinula edodes* obtained through hybridization during the breeding process were inoculated onto PDA medium, and the number of days required for each hybrid to fully colonize the culture dish was recorded. Hybrid strains of *Lentinula edodes* that colonized the culture dish in 11–14 days were selected as test strains, totaling 20 strains. For ease of explanation, these 20 hybrid strains were numbered, as detailed in Table 1. These 20 test strains were inoculated onto PDA medium, with inoculation blocks of 4 mm in diameter, and cultured in the dark at 25±1℃ for 7–8 days, activating twice to prepare *Lentinula edodes* mycelium blocks with a diameter of 9 mm for later use.

[0037] Trichoderma ylang-ylang ( Trichoderma aureoviride The strain was inoculated onto PDA medium with inoculation blocks of 4 mm in diameter and cultured in the dark at 25±1℃ for 3-4 days. After two activation cycles, Trichoderma blocks with a diameter of 9 mm were prepared for later use. The study of *Trichoderma chloroticum* was published in "Precise Identification and Detection Technology of *Trichoderma* in Contaminated Shiitake Mushroom Substrate," Cao Zijian, Master's Thesis, Hebei University of Engineering, 2023.

[0038] (3) Trichoderma resistance test The sawdust culture medium from step (1) is loaded into large test tubes with a specification of 32 mm × 200 mm, with each test tube containing approximately 60 g of sawdust culture medium. The tubes are sterilized at 121°C for 2.5 h and then cooled for later use.

[0039] Each test tube was inoculated with a 9 mm diameter piece of shiitake mushroom mycelium placed in the center of the substrate surface and incubated at 25±1℃ in the dark for (22±1) days. Then, a 9 mm diameter piece of Trichoderma mycelium was inoculated in the center of the substrate surface and co-cultured at 25±1℃. Mycelial changes were observed and photographed at 10 and 20 days of co-culture. Each tested shiitake mushroom strain was inoculated into 3-5 test tubes. The results are as follows: Figures 1-7 As shown.

[0040] Based on the mycelial density of the tested shiitake mushroom strains after the end of light-protected culture, the resistance of the tested shiitake mushroom strains was divided into four levels, from I to IV. The resistance gradually decreased from level I to level IV, with level I being the highest resistance. The criteria for determining Grade I resistance to Trichoderma are as follows: If the mycelial density of the tested *Lentinula edodes* does not change significantly during co-cultivation, the strain is considered to have Grade I resistance. The criteria for Grade II resistance are: If the mycelial density of the tested *Lentinula edodes* decreases within 1-10 days of co-cultivation, and then gradually recovers and increases in density within 10-20 days of co-cultivation, the strain is considered to have Grade II resistance; or, if the mycelial density of the tested *Lentinula edodes* does not change significantly within 1-10 days of co-cultivation, and then decreases in density within 10-20 days of co-cultivation, the strain is considered to have Grade II resistance. The criteria for Grade III resistance are: If the mycelial density of the tested *Lentinula edodes* decreases within 1-10 days of co-cultivation, and then gradually recovers and increases in density within 10-20 days of co-cultivation, the strain is considered to have Grade II resistance. During period d, if the mycelium of the tested shiitake mushroom at the inoculation surface of the test tube is significantly reduced, the strain is judged to be resistant to Trichoderma as level III; the criteria for level IV are: during co-culture, the mycelium of the tested shiitake mushroom is decomposed by Trichoderma and Trichoderma produces spores, the strain is judged to be resistant to Trichoderma as level IV.

[0041] The resistance grading results of the tested *Lentinula edodes* strains are shown in Table 1. The experimental results indicate that *Lentinula edodes* strains with strong *Trichoderma* resistance maintained a relatively stable mycelial density on the natural substrate after 10-20 days of co-cultivation with *Trichoderma*. For example, strains F124, F104, F35, F103, and A85 showed almost no change in mycelial density during 10-20 days of confrontation with *Trichoderma*, and their resistance to *Trichoderma* was classified as Grade I (…). Figures 1-2 In contrast, strains with weaker resistance to *Trichoderma* showed a significant decrease in mycelial density within 10 days of co-cultivation due to cell wall degradation and excessive absorption of intracellular nutrients. Furthermore, some strains with weaker resistance did not show significant changes in mycelial density in the initial stage of co-cultivation (within 10 days), but exhibited a significant decrease in mycelial density after 15-20 days. For example, the tested *Lentinula edodes* strains 3826, 2550, and 4073 showed a decrease in mycelial density after 10 days of confrontation culture; however, with continued culture, the mycelium occupied its ecological niche, gradually recovered growth, and the mycelial density increased, classifying their resistance to *Trichoderma* as level II. Similarly, the tested *Lentinula edodes* strains D104, D130, 2560, and D63 showed no significant change in mycelial density after 10 days of confrontation culture; however, with continued culture, the mycelial density decreased, classifying their resistance to *Trichoderma* as level II. Shiitake mushroom strains 1083, 1583, 4136, 4440, and 3350 were cultured in confrontation for 10 days. The mycelial density of *L. shiitake* decreased. After continued culture, although mycelial growth continued, the mycelial density in the middle of the test tube decreased, classifying their resistance to *Trichoderma* as level III. Shiitake mushroom strains D22, A17, and 3272 showed mycelial decomposition by *Trichoderma*, with *Trichoderma* producing sporulations, classifying their resistance to *Trichoderma* as level IV. Figures 3-7 ).

[0042] Table 1. Grading results of tested shiitake mushroom strains

[0043] (4) Set up a control group of the main cultivated varieties. The commonly used shiitake mushroom variety Liaofu 4 (0912) was selected as a strong resistance control; the Trichoderma resistance test was performed in the same way as in step (3).

[0044] The strong-resistant hybrid F124 and weak-resistant hybrid D22 selected in step (3) were used as experimental materials and compared with the mycelial density of the main cultivated variety. The results are as follows: Figure 8 As shown.

[0045] The results showed that the mycelial density of the weakly resistant hybrid D22 was lower than that of the main cultivated variety, indicating weak resistance to Trichoderma. This demonstrates that this method can be used to screen for weakly resistant strains, avoiding their entry into subsequent breeding processes and saving time, manpower, and raw material costs.

[0046] Verification Example Fruiting experiments were conducted on the 20 shiitake mushroom strains to be tested in Example 1 to verify the strength of Trichoderma resistance in the relevant strains.

[0047] (1) Mycelium growth: The optimal temperature for the mycelium is 20~25℃, the relative humidity of the atmosphere in the greenhouse is <60%, and the carbon dioxide is <2000 ppm, with the optimum being <1600 ppm. Cultivate in the dark, ventilate regularly, and ensure sufficient oxygen.

[0048] (2) Color change: After piercing the large holes, enter the color change management, give it a temperature of 18~21℃, increase the diffused light, and the relative humidity of the atmosphere to about 65%.

[0049] (3) Mushroom cultivation: After the mushroom logs mature, they should be placed on the shelves for mushroom cultivation in a timely manner. Calculate the moisture content. If the moisture content is below 58%, add water to bring it up to 65-70%, with 68-70% being optimal.

[0050] (4) Shelf management: Keep the temperature inside the shed at 15℃~25℃ for 5~7 days before removing the bags, and not lower than 15℃ at night. Keep the temperature of the mushroom logs at 10℃~25℃ for each batch of mushrooms.

[0051] (5) Moisture management: Control the temperature of the substrate sticks to 18~22℃. Keep the surface of the substrate sticks moist and prevent them from drying out.

[0052] In the fruiting experiment, when Trichoderma infection occurred, green spots initially appeared on the substrate surface. These spots expanded until a large number of green conidia formed on the entire substrate surface [Study on the physiological mechanism of Trichoderma infection caused by high temperature in Pleurotus ostreatus cultivation. Qiu Zhiheng. Doctoral dissertation, Chinese Academy of Agricultural Sciences, 2018]. The phenotype of Trichoderma infection on the substrate logs was observed to determine the resistance of the hybrids to Trichoderma. If there was no Trichoderma contamination, the resistance was considered strong; if there was Trichoderma contamination, the resistance was considered weak. The results are shown in Table 2. The fruiting experiment confirmed that five shiitake mushroom strains, F124, F103, F104, F35, and A85, showed strong resistance to Trichoderma, with no Trichoderma contamination on their substrate logs. The remaining 15 shiitake mushroom strains showed weak resistance to Trichoderma, consistent with the results of the judgment method in Example 1.

[0053] Table 2. Fruiting Experiment Verification of the Tested Shiitake Mushroom Strains

[0054] Comparative Example 1 Referring to [Yue Yuanyuan, et al. Analysis of genetic diversity of wild lung-shaped Pleurotus ostreatus in my country based on whole-genome SNP loci and phenotypic characteristics. Biotechnology Bulletin, 2025, 41(03): 1-12.], potato dextrose agar medium (PDA medium) was prepared and denoted as artificial synthetic medium.

[0055] Five strongly resistant strains (F124, F103, F104, F35, and A85) and five weakly resistant strains (A17, D104, D130, D63, and D22) were selected for a Trichoderma confrontation culture experiment on synthetic culture medium plates at a temperature of 25±1℃. Shiitake mushroom spawn was inoculated first, and after 6 days of culture, Trichoderma spawn was inoculated. The culture was continued for a total of 30 days, and photographs were taken afterward. For specific methods, please refer to [Evaluation of Trichoderma Resistance in Shiitake Mushroom Germplasm Populations and Study on the LeTLP1-Mediated Molecular Mechanism of Resistance to Trichoderma Dark Green, Ma Xiaolong, Doctoral Dissertation, Huazhong Agricultural University, 2023]. The results are as follows: Figures 9-10 As shown in the figure. The results indicate that the use of artificially synthesized culture media could not predict differences in Trichoderma resistance among shiitake mushroom strains.

[0056] In summary, this invention screens and eliminates strains with weak resistance by detecting the resistance of shiitake mushroom strains to Trichoderma on a natural substrate and observing changes in mycelial density after co-culturing with Trichoderma. The method described in this invention is simple to operate, suitable for batch screening, and can effectively prevent poorly resistant shiitake mushroom strains from entering the cultivation bag production stage, significantly reducing time and screening costs. It has good application prospects and is suitable for use by shiitake mushroom production and breeding enterprises.

[0057] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for determining the resistance strength of *Trichoderma* strains in *Lentinula edodes*, characterized in that, Includes the following steps: Different shiitake mushroom strains to be tested were inoculated on the surface of a natural substrate and cultured in the dark for 21-23 days. Then, Trichoderma mycelium blocks were inoculated and cultured together for 10-20 days. The resistance of the tested shiitake mushroom strains was compared based on the mycelial density of shiitake mushrooms.

2. The method according to claim 1, characterized in that, The natural substrate includes wood chips; the moisture content of the natural substrate is 58% to 60%.

3. The method according to claim 1, characterized in that, The Trichoderma includes Trichoderma chloroticum ( Trichoderma aureoviride ).

4. The method according to claim 1, characterized in that, Also includes: A control group was set up for the main shiitake mushroom varieties.

5. The method according to claim 4, characterized in that, The mycelial density of the tested shiitake mushroom strain was significantly weaker than that of the main cultivated shiitake mushroom variety, indicating that the tested shiitake mushroom strain had weak resistance to Trichoderma.

6. The method according to claim 4 or 5, characterized in that, The main shiitake mushroom variety mentioned is Liaofu No. 4 (0912).

7. The method according to claim 4 or 5, characterized in that, After comparing resistance, the test included discarding shiitake mushroom strains whose mycelial density was significantly weaker than that of the main cultivated shiitake mushroom varieties.

8. The method according to claim 1, characterized in that, The natural matrix was filled into a test tube.

9. The method according to claim 1, characterized in that, The temperature for the light-protected culture is 25±1℃; the temperature for the co-culture is 25±1℃.

10. The application of the method according to any one of claims 1 to 9 in shiitake mushroom breeding.

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