A Ganoderma lucidum strain resistant to Trichoderma

Abstract

The present invention provides a Ganoderma lucidum strain resistant to Trichoderma disease, which has been deposited with the General Microbiology Center of the China National Committee for the Collection of Microorganisms and is designated CGMCC No. 23845. The Ganoderma lucidum strain has excellent resistance to Trichoderma longibrachiatum, Trichoderma harzianum, and Trichoderma pleuroticola, is suitable for cultivating Ganoderma lucidum fruiting bodies, can avoid Trichoderma disease, and facilitates the production of high-yield, high-quality Ganoderma lucidum fruiting bodies.

Description

Technical Field

[0001] The invention belongs to the biological field, and in particular relates to a Ganoderma lucidum strain resistant to Trichoderma. Background Art

[0002] Ganoderma lucidum is a valuable medicinal fungus with high medicinal value and health benefits. In recent years, with the increase in cultivation years and the expansion of cultivated areas, the incidence of various diseases has increased. In particular, Trichoderma contamination, causing Ganoderma disease, can seriously damage Ganoderma yield and quality, causing significant losses and directly threatening Ganoderma production and industrial development.

[0003] Trichoderma disease can occur throughout the entire Ganoderma cultivation process, from seed and stick production to stick cultivation and production. Numerous factors can contribute to Trichoderma contamination, including incomplete sterilization, non-standard aseptic techniques, bag damage, poor ventilation, and improper cultivation management during stick cultivation. Therefore, to prevent Trichoderma contamination, strict sterilization and standardized operations are essential during Ganoderma cultivation. However, for many mountain farmers who rely on Ganoderma cultivation for their livelihoods, achieving high standards of sterilization and aseptic operation is challenging. Once Trichoderma disease occurs during Ganoderma cultivation, yield and quality decline, severely impacting farmers' profits.

[0004] Therefore, based on the quality of the strain itself, if a Ganoderma strain that can effectively resist Trichoderma contamination can be provided for Ganoderma cultivation, it will have very good economic benefits. Summary of the Invention

[0005] The purpose of the present invention is to provide a Ganoderma lucidum strain resistant to Trichoderma.

[0006] The present invention provides a Ganoderma lucidum strain - ZL18, the origin of which is the Polyporaceae fungus Ganoderma lucidum (Leyss.ex Fr.) Karst., which was deposited on November 8, 2021 in the General Microbiology Center of the China Culture Collection Administration (No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing), with the deposit number CGMCCNO.23845.

[0007] Specifically, the present invention provides a Ganoderma lucidum strain resistant to Trichoderma, which is deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms with a deposit number of CGMCC NO.23845.

[0008] Furthermore, the above-mentioned Trichoderma-resistant Ganoderma strain is a Ganoderma strain resistant to Trichoderma longibrachiatum, Trichoderma harzianum and Trichoderma pleuroticola.

[0009] Furthermore, the above-mentioned Trichoderma-resistant Ganoderma strain is a Ganoderma strain resistant to Trichoderma infection.

[0010] The present invention also provides a ganoderma lucidum fruiting body, which is obtained by culturing and cultivating the above-mentioned ganoderma lucidum strain.

[0011] Furthermore, the cultivation substrate production method of the above-mentioned cultivation includes bag material production or log production, and the cultivation method includes greenhouse cultivation, under-forest cultivation, simulated wild cultivation or factory cultivation.

[0012] The present invention also provides a method for cultivating the above-mentioned Ganoderma lucidum fruiting body, comprising the following steps:

[0013] (1) Making fungus bags;

[0014] (2) inoculating the Ganoderma lucidum strain according to claim 1;

[0015] (3) mycelial culture;

[0016] (4) Cultivation of Zhizhi.

[0017] Furthermore, the formula of the compost in the bag in step (1) is: 60% sawdust, 20% cottonseed hulls, 18% bran, 1% lime and 1% gypsum;

[0018] The conditions for the mycelium culture in step (3) are: temperature 26° C., humidity 60%, and protection from light;

[0019] The conditions for cultivating the Ganoderma lucidum in step (4) are: temperature 26° C., relative humidity 80%, CO2 concentration 500ppm-600ppm, and light 100-200lx.

[0020] The Ganoderma lucidum strain ZL18 of the present invention exhibited excellent resistance to infection by Trichoderma (Trichoderma longibrachiatum, Trichoderma harzianum, and Trichoderma pleuroticola) in both plate confrontation experiments and fungus bag screening experiments. The strain is suitable for cultivating Ganoderma lucidum fruiting bodies, can avoid Trichoderma diseases, and is conducive to obtaining Ganoderma lucidum fruiting bodies with high yield and good quality.

[0021] Obviously, based on the above contents of the present invention, according to common technical knowledge and customary means in this field, without departing from the above basic technical ideas of the present invention, other various forms of modifications, replacements or changes can be made.

[0022] The following further describes the above content of the present invention in detail through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the resistance of Ganoderma lucidum strains ZL1, ZL18, and ZL81 10 days after inoculation with Trichoderma.

[0024] Figure 2 This is a comparison chart of the fruiting bodies of the normal group, JY2 group and D14 group in the experimental strain ZL18 and the control variety ZL1. DETAILED DESCRIPTION

[0025] Source of the Trichoderma used in the present invention: Fungus bags and fruiting bodies infected with Trichoderma disease were collected from Ganoderma lucidum production sites. The pathogens were isolated and purified under sterile conditions. Three Trichoderma strains (D14, JY-1, and JY-2) and one purchased Trichoderma strain (Lv) were selected as representative strains based on the texture and color of the colonies on the plates. Molecular biological identification was performed on these strains. The identification results of the four representative Trichoderma used in the present invention are shown in Table 1:

[0026] Table 1 Identification results of Trichoderma strains

[0027]

[0028]

[0029] Example 1: Plate screening of Trichoderma-resistant Ganoderma strains

[0030] Based on the results of previous research on the domestication and cultivation of Ganoderma lucidum strains, the inventors selected 28 Ganoderma lucidum strains with similar growth during the spawning period and low contamination rates (<10%) as screening strains. At the same time, strains ZL212 and ZL253 with medium contamination rates (10-20%) and strains ZL258 and ZL217 with high contamination rates (>20%) were used as controls.

[0031] A plate confrontation experiment was conducted on PDA plates using 32 Ganoderma strains screened by the inventors and four Trichoderma strains. The results of the confrontation were recorded 4, 8, and 12 days after inoculation of each Trichoderma strain. The results are shown in Tables 2-5. "-" indicates that the Ganoderma strain has no resistance to Trichoderma, with Trichoderma hyphae covering those of the hybrid strain; "+" indicates that the Ganoderma strain has some resistance to Trichoderma, with the Ganoderma hyphae growing in confrontation with the Trichoderma hyphae; and "+-" indicates that the Ganoderma strain has weak resistance to Trichoderma, with the Ganoderma hyphae growing in confrontation with the Trichoderma hyphae, and some Trichoderma hyphae can invade the Ganoderma strain's antagonistic line.

[0032] In the standoff experiment against JY-1, only four strains, ZL237, ZL18, ZL5, and ZL266, showed weak resistance on day 4. As the mycelium grew, all strains lost resistance (Table 2). In the standoff experiment against D14, 14 strains showed significant resistance on day 4, 17 showed weak resistance, and only one showed no resistance. On day 8, two strains, ZL247 and ZL265, showed significant resistance, while six strains, ZL81, ZL9, ZL18, ZL251, ZL259, and ZL159, showed weak resistance. On day 12, except for ZL18, which showed weak resistance, all other strains lost resistance (Table 3). In the confrontation experiment against Jy-2, all strains except ZL258 showed significant resistance on day 4. On day 8, 12 strains (ZL81, ZL237, ZL247, ZL9, ZL6, ZL266, ZL265, ZL268, ZL294, ZL231, ZL259, and ZL242) showed significant resistance, while 5 strains showed weak resistance. On day 12, none of the strains showed resistance (Table 4). In the confrontation experiment against Lv, all strains showed significant resistance on day 4. On day 8, 19 strains showed significant resistance, 11 strains showed weak resistance, and only 2 strains showed no resistance. On day 12, 11 strains still showed significant resistance, 14 strains showed weak resistance, and 7 strains showed no resistance (Table 5).

[0033] Table 2 Results of the plate confrontation experiment of Trichoderma strain JY-1

[0034]

[0035]

[0036] Table 3 Trichoderma strain D14 plate confrontation test results

[0037]

[0038]

[0039] Table 4 Results of the plate confrontation experiment of Trichoderma strain JY-2

[0040]

[0041]

[0042] Table 5 Trichoderma strain Lv plate confrontation test results

[0043]

[0044]

[0045] The resistance of all Ganoderma strains to the four Trichoderma strains was ranked in the order: Lv > JY-2 > D14 > JY-1. Nineteen Ganoderma strains were screened and found to be resistant on the plates: ZL247, ZL265, ZL18, ZL259, ZL81, ZL237, ZL266, ZL268, ZL231, ZL2, ZL167, ZL9, ZL246, ZL5, ZL280, ZL252, ZL294, ZL159, and ZL242. Only strain ZL18 showed resistance to Trichoderma longibrachiatum, Trichoderma harzianum, and Trichoderma pleuroticola.

[0046] Example 2: Screening of fungus bags for Trichoderma-resistant Ganoderma lucidum strains

[0047] According to the PDA plate screening results, strains with strong, medium and weak resistance to Trichoderma were selected, and strains with medium and weak resistance to Trichoderma were used as controls. When the mycelium grew to 1 / 3 of the bag, isolated Trichoderma was inoculated at the other end of the bag to screen the Trichoderma-resistant strains.

[0048] A formal experiment of bag screening was carried out, with a total of 23 Ganoderma lucidum test strains, including 19 resistant strains screened from plates, 2 moderately resistant control strains (ZL212, ZL253) and 2 weakly resistant control strains (ZL217 and ZL258).

[0049] Antagonism between the hyphae of 23 Ganoderma strains and four Trichoderma species was observed. Resistance was recorded and analyzed 10, 20, and 30 days after inoculation with Trichoderma. The results are shown in Tables 6-9. "-" indicates that the Ganoderma strain has no resistance to Trichoderma, with the Trichoderma hyphae covering those of the hybrid strain. "+" indicates that the Ganoderma strain has some resistance to Trichoderma, with the hyphae of the Ganoderma strain and the Trichoderma hyphae growing opposite each other, forming an antagonistic line.

[0050] The infectivity of the four Trichoderma strains was essentially the same as on the plates: JY-1 > JY-2 = D14 > Lv. However, some strains showed different antagonistic behavior against Trichoderma in the bag experiment compared to the plate experiment. Some strains showed resistance on the plates but not in the bags, while others showed the opposite behavior. Figure 1 The resistance of some Ganoderma strains to Trichoderma strain JY-2 10 days after inoculation with Trichoderma is shown. It can be seen that obvious antagonistic lines can be observed in the fungus bags of the three Ganoderma strains ZL1, ZL18 and ZL81, and all three strains are resistant to Trichoderma strain JY-2.

[0051] Table 6 Trichoderma strain JY-1 bag material screening test results

[0052]

[0053]

[0054] Table 7 Trichoderma strain D14 bag material screening experimental results

[0055]

[0056] Table 8 Trichoderma strain JY-2 bag material screening test results

[0057]

[0058] Table 9 Trichoderma strain Lv bag material screening experimental results

[0059]

[0060]

[0061] Through a comprehensive analysis of the resistance performance of 23 strains in four Trichoderma species, two resistant strains (ZL247 and ZL18) were finally screened out. They showed resistance when infected by all four Trichoderma species; the weakly resistant control strain ZL258 had no resistance when infected by all four Trichoderma species and was suitable for use as a non-resistant control strain in subsequent variety comparison experiments.

[0062] Example 3: Comparison of new strains of Ganoderma lucidum resistant to Trichoderma

[0063] 1. Experimental methods

[0064] 1.1 Test strains

[0065] Four Ganoderma strains were tested: two resistant strains, ZL247 and ZL18, identified in previous screening, one non-resistant control strain, ZL258, and one control, "Yao Lingzhi No. 2" (Sichuan Pharmaceutical 2011 003, ZL1). The Trichoderma strains tested were D14 and JY-2, both Trichoderma harzianum strains that demonstrated comparable infectivity in previous screening experiments.

[0066] 1.2 Culture medium

[0067] Potato dextrose agar (PDA): potato extract powder 3 g, glucose 20 g, agar 15 g, distilled water 1000 mL, natural pH.

[0068] 1.3 Mushroom bag production

[0069] Bag material formula: 60% sawdust, 20% cottonseed hulls, 18% bran, 1% lime, and 1% gypsum. Pre-wet the sawdust and cottonseed hulls with clean water for 24 hours. Mix the various culture media according to the formula, add water, and stir thoroughly to a moisture content of approximately 60%. Use 17cm x 33cm x 0.05cm polypropylene bags, packing 1000g of wet material per bag. Autoclave at 121°C for 4 hours, then cool to room temperature before inoculation.

[0070] 1.4 Variety comparison test

[0071] Each of the four strains was divided into three groups: a normal group (not inoculated with Trichoderma), a D14 group (inoculated with Trichoderma strain D14), and a JY-2 group (inoculated with Trichoderma strain JY-2). Each group contained 10 bags, with 30 bags for each strain, for a total of 120 bags. Three identically sized blocks of Ganoderma mycelium were punched out on a fresh Ganoderma mycelium plate using a sterile 5mm punch and inserted into one end of the bag. Mycelium culture was performed in a dark environment at 26°C and 60% humidity. When the Ganoderma mycelium in the D14 and JY-2 groups reached 1 / 3 of the bag, three identically sized blocks of fresh Trichoderma mycelium were inoculated into the other end of the bag. The normal group was not inoculated with Trichoderma. Culture was continued until the bags were full of mycelium, the sealing film was opened, and the bags were uniformly placed in an artificial climate incubator for culturing. All culturing conditions were identical, with a temperature of 26°C, a relative humidity of 80%, a CO2 concentration of 500ppm-600ppm, and a light intensity of 100-200lx.

[0072] 1.5 Sample collection and determination

[0073] When the fruiting bodies of Ganoderma lucidum reach maturity (the white or milky white growth ring at the edge of the cap disappears, the cap thickens from thin, the color changes from light to dark brown, and the cap becomes hard), they should be harvested and dried promptly. After harvesting, the yield, size and other characteristics of the dried fruiting bodies, as well as the polysaccharide and triterpenoid contents, should be determined according to the methods in the Chinese Pharmacopoeia (2020 Edition, Part I).

[0074] Calculate the loss rates (%) of the yield, direct, and thickness for each group. The formula is: Loss rate % = (Normal group - Trichoderma group) / Normal group × 100%.

[0075] 2. Experimental results

[0076] All the normal groups and Trichoderma groups of the experimental strain ZL18 and the control variety ZL1 produced Ganoderma lucidum normally, and a variety comparison between the two could be carried out. Among the 3 experimental groups of the experimental strain ZL247, 2 Trichoderma groups produced Ganoderma lucidum normally, but only 2 bags in the normal group produced Ganoderma lucidum, and the fruiting rate was too low to carry out a variety comparison with the control variety ZL1, indicating that although the experimental strain ZL247 showed resistance to Trichoderma in the screening experiment, the effect of further cultivating Ganoderma lucidum fruiting bodies was not good. All the bags in the 2 Trichoderma groups of the non-resistant control ZL258, JY2 and D14, were infected by Trichoderma, and the Trichoderma hyphae grew to the top of the Ganoderma lucidum hyphae inoculation end, and no Ganoderma lucidum fruited in the normal group, so a variety comparison with the control variety ZL1 could not be carried out.

[0077] All the fruiting bodies of the experimental strain ZL18 and the control variety ZL1 were successfully harvested, and experimental data such as yield were measured. As Figure 2 shown, it can be clearly seen that the fruiting bodies of the JY2 and D14 Trichoderma groups of the 2 Ganoderma lucidum strains were significantly smaller than those of the normal groups, and the JY2 group < D14 group. Table 10 details the yield, cap diameter, radius, and thickness data of the Ganoderma lucidum fruiting bodies produced by all groups of the experimental strain and the control variety, as well as the loss rates of the 2 Trichoderma groups compared with the normal groups. It can be seen that the loss rates of the yield, diameter, and thickness of the 2 Trichoderma groups of the experimental strain ZL18 were all smaller than those of the control variety ZL1, proving that ZL18 has stronger resistance to Trichoderma than ZL1, and it is a Ganoderma lucidum strain with strong resistance to Trichoderma.

[0078] Table 10 Results of variety comparison experiment

[0079]

[0080]

[0081] In summary, the present invention provides a Ganoderma lucidum strain ZL18 that shows obvious resistance to Trichoderma infection, is suitable for cultivating Ganoderma lucidum fruiting bodies, can avoid Trichoderma diseases, and is beneficial to obtaining Ganoderma lucidum fruiting bodies with high yield and good quality.

Claims

1. A Trichoderma-resistant Ganoderma lucidum strain, characterized in that: It is deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms with the deposit number CGMCCNO.23845.

2. A Ganoderma lucidum fruiting body, characterized in that: The ganoderma lucidum is cultured and cultivated by the ganoderma lucidum strain described in claim 1.

3. The Ganoderma lucidum fruiting body according to claim 2, wherein The cultivation substrate production mode includes bag material production or log production, and the cultivation method includes greenhouse cultivation, under-forest cultivation, simulated wild cultivation or factory cultivation.

4. The method for cultivating Ganoderma lucidum fruiting bodies according to claim 2, characterized in that: The steps include: (1) Making fungus bags; (2) inoculating the Ganoderma lucidum strain according to claim 1; (3) mycelial culture; (4) Cultivation of Zhizhi.

5. The cultivation method according to claim 4, wherein The formula of the compost in the bag in step (1) is: 60% sawdust, 20% cottonseed hulls, 18% bran, 1% lime and 1% gypsum; The conditions for the mycelium culture in step (3) are: temperature 26° C., humidity 60%, and protection from light; The conditions for cultivating the Ganoderma lucidum in step (4) are: temperature 26° C., relative humidity 80%, CO2 concentration 500ppm-600ppm, and light 100-200lx.

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