Strain of long stem mushroom with high resistance to trichoderma and application thereof

Abstract

The application discloses a long-rooted mushroom strain with high resistance to Trichoderma and application thereof, relates to the technical field of microorganisms, and the long-rooted mushroom strain has a preservation number of CCTCC NO: M 20261245. The long-rooted mushroom strain has outstanding resistance to Trichoderma and excellent agronomic characters, and can be applied to long-rooted mushroom industry cultivation and breeding as high-quality germplasm resources.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and more specifically to a highly resistant Trichoderma longifolia strain and its application, the strain having the preservation number CCTCC NO: M 20261245. Background Technology

[0002] Long-rooted mushrooms ( Oudemansiella raphanipes It is a rare edible and medicinal fungus, belonging to the Basidiomycota ( Basidiomycotina Agaricomycetes ( Hymenomycetes ), Agaricalales ( Agaricales ), Tricholomataceae ( Tricholomataceae Because of its dark brown color and resemblance to termite mushrooms, it is commercially known as "Black-skinned Termite Mushroom" and commonly called "Water Termite Mushroom" in Yunnan. This mushroom is rich in nutrients with significant activity, containing abundant amino acids, vitamins, and polysaccharides. It exhibits excellent pharmacological activity in areas such as anti-tumor activity and enhancing the body's antioxidant capacity. With both market acceptance and development potential, it has become a core variety in industrially cultivated fungi.

[0003] However, during the cultivation of *Pleurotus ostreatus*, its weak resistance to pollution makes it highly susceptible to large-scale contamination. This is mainly because *Pleurotus ostreatus* requires soil covering for cultivation, which contains a large number of miscellaneous microorganisms, leading to frequent mold contamination of the substrate bags during the cultivation period, particularly *Trichoderma*. Trichoderma spp. The pollution accounts for the highest proportion and causes the most damage. During the fruiting period, it faces a complex situation of overlapping damage from mold infection and pests and diseases, which often leads to a sharp decline in the yield and deterioration in quality of long-rooted mushrooms. In extreme cases, it can lead to complete crop failure and seriously restrict the high-quality development of the industry.

[0004] Therefore, cultivating *Trichoderma*-resistant *Pleurotus ostreatus* strains is a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0005] This invention provides a highly resistant Trichoderma longifolia strain and its applications.

[0006] To solve the above-mentioned technical problems, this application adopts the following technical solution:

[0007] A highly resistant *Trichoderma* strain of *Agaricus blazei*, with accession number CCTCC NO: M 20261245, deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, on June 8, 2026, and classified as follows: Oudemansiella raphanipies LS-T.

[0008] As a preferred technical solution, the ITS sequence of the *Pleurotus ostreatus* strain is shown in SEQ ID NO:3.

[0009] Another object of the present invention is to provide the application of the above-mentioned *Pleurotus ostreatus* strain in the cultivation of *Pleurotus ostreatus* and / or in the breeding of germplasm with high resistance to *Trichoderma*.

[0010] Compared with the prior art, the present invention has the following beneficial effects: This invention isolates a wild *Mushroom floribunda* strain with outstanding resistance to *Trichoderma* and excellent agronomic traits, which can be used as a high-quality germplasm resource for cultivation and breeding in the *Mushroom floribunda* industry. Attached Figure Description

[0011] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0012] Figure 1 Phylogenetic tree diagram of *Pleurotus ostreatus* strain CCTCC NO: M 20261245 and other *Pleurotus ostreatus* strains.

[0013] Figure 2 Phenotypic diagram of mushroom strain CCTCC NO: M 20261245 after fruiting.

[0014] Figure 3 The Trichoderma resistance phenotype of *Trichoderma longifolia* strain CCTCC NO: M 20261245 was determined by fixed-position confrontation culture.

[0015] Figure 4 To determine the Trichoderma infection rate of *Pleurotus ostreatus* strain CCTCC NO: M 20261245 in a fixed-position confrontation culture.

[0016] Figure 5 The Trichoderma resistance phenotype of *Trichoderma longifolia* strain CCTCC NO: M 20261245 was determined by constant-day confrontation culture.

[0017] Figure 6 To determine the Trichoderma infection rate of *Pleurotus ostreatus* strain CCTCC NO: M 20261245 during a fixed-day standoff culture. Detailed Implementation

[0018] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. 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.

[0019] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0020] Example 1 Wild *Pleurotus ostreatus* fruiting bodies were collected from Jinxiang County, Jining City, Shandong Province. The fruiting bodies were disinfected with 75% ethanol, and residual moisture was blotted dry with sterile absorbent paper. Under aseptic conditions, the fruiting bodies were longitudinally split open, and internal tissue blocks were extracted using sterile forceps and inoculated onto potato dextrose agar (PDA) plates containing a final concentration of 50 μg / L kanamycin. The PDA medium consisted of 200 g potato, 20 g glucose, and 18 g agar powder, diluted to a final volume of 1000 mL with water. After inoculation, the plates were placed in a 25°C incubator in the dark, and mycelial growth was observed periodically.

[0021] After the mycelia had fully grown on the plate, genomic DNA of the strain was extracted using a fungal genomic DNA extraction kit (Nanjing Novizan Biotechnology Co., Ltd., catalog number N808). Using the obtained genomic DNA as a template, PCR amplification was performed using the universal fungal ITS primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3', SEQ ID NO: 1) and ITS4 (5'-TCCTCCGCTTATTGATATGC-3', SEQ ID NO: 2) (SEQ ID NO: 3). The amplified products were detected by 120 V constant voltage agarose gel electrophoresis for 30 min. Positive samples showing a single band were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing identification.

[0022] ITS sequence: , SEQ ID NO:3.

[0023] Blast alignment of the sequencing results and phylogenetic tree construction using ITS sequences from other *Pleurotus ostreatus* strains revealed that this strain was most closely related to strain KX688229.1, with 97.8172% homology. Therefore, we confirmed that we had isolated a *Pleurotus ostreatus* strain. Oudemansiella raphanipes ) strains (see appendix) Figure 1The sample was deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M 20261245. The depositary institution is the China Center for Type Culture Collection, located at Wuhan University, Wuhan, China. The deposit date was June 8, 2026. The classification and naming are as follows: Oudemansiella raphanipies LS-T.

[0024] Example 2 Cultivation and Fruiting Management of Pleurotus ostreatus strain CCTCC NO: M 20261245 0.5 cm × 0.5 cm mushroom cakes (CCTCC NO: M 20261245) were prepared using a punch and inoculated into potato dextrose liquid medium (PDB). The PDB medium consisted of 200 g potato and 20 g glucose, with water added to a final volume of 1000 mL. After inoculation, the culture was placed in a constant temperature shaking incubator at 25 ± 1 °C and 130 r / min for 7 days. The resulting culture was used as a liquid culture medium for later use. The cultivation substrate formula, by weight percentage, is 68% broadleaf tree sawdust, 20% wheat bran, 4% corn flour, 4% cottonseed flour, 1% sucrose, 1% superphosphate, 1% light calcium carbonate, and 1% gypsum. The material-to-water ratio is 1:1.2-1.3, and the initial pH is adjusted to 6.5. The substrate is mechanically stirred evenly, dispensed into polypropylene cultivation bags, and sterilized by high-pressure steam at 112℃-118℃ for 120-150 minutes. After sterilization, the bags are transferred to a cooling room at 18℃-20℃ to cool to below 28℃ for later use. Inoculation is performed in the ozone-sterilized inoculation room. Strict aseptic procedures are followed in the aseptic inoculation workshop, and comprehensive disinfection measures are implemented. The outer polypropylene film of the cultivation bag is cut open with a sterile knife, and liquid spawn is inoculated at 10% of the substrate dry weight. After inoculation, the bags are transferred to the incubation room for mycelial growth. The incubation temperature is controlled at 25±1℃, with good ventilation to provide suitable environmental conditions for mycelial growth. The temperature in the incubation room is recorded daily, the mycelial growth status is observed, and the time to full colonization of the bag is recorded and photographed periodically for subsequent comparative analysis of mycelial growth. Once the mycelium has fully colonized the bag, the post-ripening management stage begins, lasting 30–45 days. The incubation temperature is strictly controlled not to exceed 25℃ to avoid excessively thick mycelial skin caused by high temperatures. During this period, the growth status of the bags is continuously observed. When a distinct dark brown mycelial skin forms on the surface of the bag, it indicates that the mycelium has reached physiological maturity and can enter the fruiting management stage. The incubation time should not be the sole criterion for determining maturity.Before mushroom cultivation, the mushroom shed should be thoroughly cleaned and disinfected. The interior should be rinsed with clean water to remove dust. In humid environments, trichloroisocyanuric acid fumigation should be used at a dosage of 1500 mL per cubic meter. Every 2-3 days, the walls and roof of the shed should be sprayed with carbendazim for disinfection. Before the mushroom bags are placed in the shed, they should be fumigated for 24 hours in a sealed environment with a combination of trichloroisocyanuric acid and chlorothalonil. A disinfection passage should be set up at the entrance of the shed. Personnel entering the shed should undergo full-body disinfection with carbendazim spray and change into clean work clothes and shoes before entering. A 0.5-1 cm layer of quicklime should be spread on the surface of the shelves, serving both sterilization and moisture-proofing purposes. After disinfection, the mushroom bags should be neatly arranged on the shelves, closely adjacent to each other, ready for covering with soil. After secondary mycelium has formed on the surface of the bags, they should be opened. Before opening, the shelves should be disinfected again with carbendazim. Operators should disinfect their hands with 75% ethanol and wear sterile gloves. A sterilized utility knife should be used to evenly cut 3-5 cm × 3-5 cm openings at the top of the mushroom bags. Open the square bags with a 1 cm opening, and after opening, cover the surface of the bags with 2-3 cm of sterilized humus soil. Use loose, fertile, pathogen-free humus soil, which should be sterilized by sun exposure or soaking in a carbendazim solution before use. Keep the soil moist after covering, but avoid waterlogging. During the bud induction stage, provide a diurnal temperature range of 5-8℃, maintain a cultivation temperature of 20-24℃, a relative humidity of 85%-90%, and 200-500 lx diffused light to promote primordia differentiation. After primordia formation, reduce diurnal temperature fluctuations. The optimal temperature for fruiting body growth is 18-23℃, with a relative humidity of 85%-92%. Control the environmental CO2 concentration to 800-1200 ppm, and keep the soil moist but not waterlogged to obtain high-quality fruiting bodies. Observe the fruiting status.

[0025] The results showed that this strain exhibited uniform fruiting, high fruiting rate, strong resistance to contamination by other microorganisms throughout the cultivation period, and good fruiting body growth. Specifically, the cap was initially hemispherical, gradually flattening as it grew, reaching a diameter of 3–8 cm upon maturity, with a smooth, glossy, and uniformly dark brown surface; the stipe was cylindrical, brownish-brown, with fine longitudinal striations on the surface, reaching a length of 8–15 cm, and its base significantly elongated to form a rhizoid-like structure that penetrated deep into the casing soil; the flesh was white, crisp, and tender, demonstrating excellent overall commercial qualities (see appendix). Figure 2 ).

[0026] Example 3 To illustrate the characteristics of the *Trichoderma* strain CCTCC NO: M 20261245 of this invention, existing strains were used as controls to determine its resistance to *Trichoderma*.

[0027] 1. Determination of the resistance of *Trichoderma* to *Pleurotus ostreatus* To verify the resistance of *Trichoderma repens* strain CCTCC NO: M 20261245 to *Trichoderma*, preserved strains ACCC 52243 and ACCC 51848 of *Trichoderma repens* were selected as control strains. Confrontational culture experiments between *Trichoderma repens* and *Trichoderma* were conducted using both fixed-location and fixed-day confrontational culture methods, respectively, to compare the differences in resistance to *Trichoderma* between strain CCTCC NO: M 20261245 and the control strains.

[0028] (1) Fixed-position confrontation culture: Using a 0.5 cm sterile punch, mycelial blocks were taken from the areas with abundant mycelium of each *Trichoderma harzianum* strain, ensuring that the mycelial blocks were of uniform size. The prepared *Trichoderma harzianum* mycelial blocks were inoculated onto one side of a new PDA medium plate (0.5 cm from the edge of the plate). After inoculation, the petri dishes were sealed and placed in a 25℃ constant temperature incubator for culture. During the culture period, the growth status of *Trichoderma harzianum* mycelium was observed. When the mycelium grew to 5.5 cm from the edge of the plate, a *Trichoderma harzianum* mycelial block of the same size (taking *Trichoderma harzianum* LV1 as an example) was inoculated at the corresponding position on the other side of the plate (0.5 cm from the edge). The growth rate of *Trichoderma harzianum* mycelium was measured and recorded daily, and three replicates were set up for each *Trichoderma harzianum* strain.

[0029] The results of the culture using the fixed-position confrontation method are attached. Figure 3 As shown: After infection with *Trichoderma harzianum* LV1, the mycelia of strain CCTCC NO: M20261245 remained white and dense, without yellowing, shrinkage, or other signs of aging and degradation. A clear and straight antagonistic line was formed between the strain and the LV1 colony, indicating significant antagonism and the strongest resistance. After co-culturing strain ACCC 52243 with LV1, *Pleurotus ostreatus* mycelia showed obvious aging and degradation, suggesting that both strains experienced a stress response upon LV1 infection, resulting in weaker resistance. Strain ACCC51848 showed almost no antagonistic ability against LV1; after co-culturing, LV1 mycelia could cover ACCC 51848 colonies, indicating that this strain was not resistant to LV1. Plates of each strain not inoculated with LV1 were used as blank controls to statistically analyze the infection rate of *Trichoderma harzianum* strains using the fixed inoculation site method. The results showed that strain CCTCC NO: M 20261245 had the lowest infection rate of Trichoderma, at 10.80±0.63%; strains ACCC 52243 and ACCC 51848 had higher infection rates, at 23±2.34% and 15.63±2.9%, respectively (see Appendix). Figure 4 ).

[0030] (2) Fixed-day cultivation: Using a 0.5 cm sterile punch, mycelial blocks were taken from the areas with abundant mycelium of each *Trichoderma harzianum* strain, ensuring that the mycelial blocks were of uniform size. The prepared *Trichoderma harzianum* mycelial blocks were inoculated onto one side of a new PDA medium plate (0.5 cm from the edge of the plate). After inoculation, the petri dishes were sealed and placed in a 25°C constant temperature incubator for cultivation. After the mycelium had grown for 6 days, *Trichoderma harzianum* mycelial blocks of the same size (taking *Trichoderma harzianum* LV1 as an example) were inoculated at the corresponding position on the other side of the plate (0.5 cm from the other edge of the plate).

[0031] The results of the fixed-day confrontation culture are attached. Figure 5 As shown: CCTCC NO: M 20261245 and LV1 form a clear antagonistic line, with CCTCC NO: M 20261245 exhibiting stronger antagonism and more pronounced resistance. The colonies of ACCC 52243 and ACCC51848 were completely covered by LV1 hyphae after 4 days of co-culture, indicating that neither strain showed significant resistance to LV1. Using plates of each strain without LV1 inoculation as blank controls, the infection rate of *Trichoderma* on different *Pleurotus ostreatus* strains was statistically analyzed using the fixed-day method. The results showed that CCTCC NO: M 20261245 had the lowest infection rate from *Trichoderma*, at 17.61 ± 0.98%; ACCC 52243 and ACCC 51848 had higher infection rates, reaching 85 ± 3.56% and 89.61 ± 3.36%, respectively (see Appendix). Figure 6 ).

[0032] 2. Comparison of fruiting contamination rates among different *Pleurotus ostreatus* strains Given that Trichoderma is the main source of contamination in the production of Mushroom floribunda, the fruiting was carried out according to the "Cultivation and Fruiting Management of Mushroom floribunda CCTCCNO: M 20261245 strain" method in Example 2. The contamination rate of four different Mushroom floribunda strains during the mycelial growth period and the fruiting period was counted, and highly resistant strains were screened out.

[0033] The statistical results are shown in Table 1. Throughout the entire cultivation and production period, including the mycelial growth period of the substrate bag and the fruiting period after opening the bag and covering the soil, strain CCTCC NO: M 20261245 showed a low contamination rate of Trichoderma and other strains, and had stronger resistance than the other three strains.

[0034] Table 1

[0035] 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.

[0036] 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 highly resistant Trichoderma longifolia strain, characterized in that, The preservation number of the *Pleurotus ostreatus* strain is CCTCC NO: M20261245.

2. The *Trichoderma longifolia* strain with high resistance to *Trichoderma* according to claim 1, characterized in that, The ITS sequence of the *Pleurotus ostreatus* strain is shown in SEQ ID NO:

3.

3. The application of the *Pleurotus ostreatus* strain according to claim 1 or 2 in the cultivation of *Pleurotus ostreatus* and / or the breeding of highly resistant *Trichoderma* germplasm.

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