Lentinus edodes strain FX85 and breeding method and application thereof

Abstract

The invention discloses a shiitake mushroom strain FX85 and a breeding method and application thereof, the shiitake mushroom strain FX85 is preserved in China Center for Type Culture Collection on December 3, 2024, the address is Wuhan University, Wuhan, China, and the preservation number is CCTCC NO: M 20242713. The average yield of fresh mushrooms of the FX85 obtained through breeding is 912 g / stick and is 8.07% higher than that of a control variety of new 808, the polysaccharide content of the strain is 8.75% higher than that of the strain of new 808, the protein content is 21.57% higher than that of the strain of new 808, the stem-cap ratio is 13.77% higher than that of the strain of new 808, the agronomic character and the yield are better than those of the strain of new 808, and the FX85 has popularization value. The novel high-yield shiitake mushroom variety bred by the method can enrich the existing shiitake mushroom germplasm resource library, is beneficial to adjusting the shiitake mushroom planting structure, and improves the shiitake mushroom planting benefit.

Description

Technical Field

[0001] The invention belongs to the technical field of mushrooms, and particularly relates to a shiitake mushroom strain FX85 and a breeding method and application thereof. Background Art

[0002] mushroom( Lentinus edodes (Berk.) sing) belongs to the Basidiomycetes class ( Basidaiomycetes ), Agaricalales ( Agaricales )、Dermacentor spp.( Marasmiaceae )、Lentinus ( Lentinula ), is the second largest edible fungus in the world. It has a wide planting area, is rich in nutrients, tastes delicious, and has high medicinal value and economic benefits. It is a very valuable medicinal and edible fungus.

[0003] At present, there are many known varieties of shiitake mushrooms, such as shiitake 9608, shiitake K5, shiitake 939, shiitake 808, shiitake A6, shiitake 238, shiitake 241-4, shiitake 303, Luyu No. 6 shiitake, Huxiang F2, Shenxiang 1504 and Shenxiang 215. Different varieties of shiitake mushrooms have different shapes and nutritional contents.

[0004] Breeding high-yield, high-nutritional-value shiitake mushroom strains can enrich the shiitake mushroom germplasm resource bank, help adjust the shiitake mushroom industry structure, and improve cultivation benefits.

[0005] It should be noted that the information disclosed in the above background technology section is only used to enhance the understanding of the background of the present disclosure, and therefore may include information that does not constitute the prior art known to ordinary technicians in the field. Summary of the invention

[0006] The invention aims to provide a shiitake mushroom strain FX85 and a breeding method and application thereof. The strain has the characteristics of high yield and large cap-to-handle ratio, can enrich the existing shiitake mushroom germplasm resource bank, help adjust the shiitake mushroom planting structure, and improve the shiitake mushroom planting efficiency.

[0007] In order to achieve the above object, the present invention provides a shiitake mushroom strain ( Lentinus edodes ) FX85, the shiitake mushroom strain FX85 was deposited in the China Center for Type Culture Collection on December 3, 2024, address: Wuhan University, Wuhan, China, with the deposit number CCTCC NO: M 20242713.

[0008] The mycelium of the shiitake mushroom FX85 of the present invention is dense and vigorous, white in color, and the optimum growth temperature is 25°C. The shiitake mushroom FX85 has strong high temperature resistance and impurity resistance, early and uniform color change, darker color change of the mushroom stick, 4 to 6 days earlier than the control new 808, the fruiting body of the shiitake mushroom FX85 is medium-large, hemispherical, and rounder in shape, and the average yield of fresh mushrooms of the strain FX85 is 912 g / stick, which is 8.07% higher than the control variety new 808. The cap is brown, convex in longitudinal section, darker in the middle, and shallower at the edge. The cap of the strain FX85 is 4.55±0.09 cm in diameter, convex, 1.58±0.06 cm in thickness, brown in color, and moderately compact; dark scales are evenly distributed on the edge of the cap, the mushroom meat is white, and has a unique smell of shiitake mushrooms. The gills are off-white, the length of the stipe is 3.38±0.25 cm, the thickness is 1.63±0.08 cm, white, the thickness is equal at the top and bottom, and it is nearly cylindrical. The cap-to-stalk ratio (the ratio of the cap diameter to the stipe diameter) of FX85 is 2.81±0.11, and the cap-to-stalk ratio (the ratio of the cap diameter to the stipe diameter) of Xin808 is 2.47±0.24. The cap-to-stalk ratio of FX85 is 13.77% higher than that of Xin808. In summary, FX85 and Xin808 differ in yield, cap thickness, and stipe length and diameter. The cap-to-stalk ratio of FX85 fruiting bodies is large and the yield is high, which reflects the specificity of the FX85 strain.

[0009] The second object of the present invention is to provide a method for breeding the shiitake mushroom strain FX85, which comprises the following steps: 1) Dilute the high-quality fruiting body spores of Pleurotus eryngii of Shenxiang 215 and Huxiang F2 with sterile water, absorb the spore suspension on the PDA medium plate and spread it evenly, culture it at a constant temperature of 25±2℃, and select the monokaryotic mycelium without lock-like union; wherein the high-quality fruiting body spores have uniform shape and size, smooth surface, no wrinkles and spots, and no deformity and incompleteness; 2) Inoculate the selected monokaryotic mycelia of Shenxiang 215 and Huxiang F2 onto PDA plates, with a spacing of 3 cm between the two monokaryotic mycelia and culture at 25±2°C. When a fusion line is formed between the two colonies, take the mycelia in the middle of the fusion line for microscopic examination and select the binucleate mycelia with lock-like union for culture; 3) After the heterozygotes are transferred to the controlled stock, they are placed in fungus bags for fruiting experiments to screen hybrid strains with good mushroom shape and dense mushrooms; the good mushroom shape means that the mushrooms are uniform in size, without damage, defect or deformity; 4) Carry out fruiting test on the selected hybrid strains, collect the fruiting bodies of Pleurotus ostreatus and the control shiitake mushrooms, and conduct specificity, consistency and stability tests at the same time to screen out the target dominant strains.

[0010] Preferably, in step 1), the spore content in the spore suspension is 10 5 Pieces / mL.

[0011] Preferably, in step 3), the formula of the mushroom bag comprises the following components in percentage by mass: 78% miscellaneous sawdust, 20% bran, 1% white sugar and 1% gypsum.

[0012] Preferably, in step 3), in the fruiting experiment, after inoculating the original seed, the mushroom bag is placed in a dark culture room at 24±2°C and a relative air humidity of 70% for culture; when the mycelium grows all over the bag and irregular ridges appear, the bag is transferred to an outdoor greenhouse, punctured, and pressed to separate the bag, so that the culture medium in the bag is fully in contact with the air; the temperature during the day is maintained at 22-24°C, the scattered light in the greenhouse is increased, ventilation is carried out, a dry-wet alternating environment is created, and temperature and dry-wet difference stimulation is carried out to promote the lodging of aerial mycelium on the surface of the tube, secrete pigments, and form a layer of reddish-brown shiny fungal film. After the color change is completed, the bag is removed and the mushrooms are fruited.

[0013] The third object of the present invention is to provide the fruiting body of the Lentinus edodes strain FX85.

[0014] The fourth object of the present invention is to provide spores of the Lentinus edodes strain FX85.

[0015] The fifth object of the present invention is to provide the mycelium of the Lentinus edodes strain FX85.

[0016] The sixth object of the present invention is to provide a mushroom log of the shiitake mushroom strain FX85.

[0017] The seventh object of the present invention is to provide the application of the shiitake mushroom strain FX85 in food or as a parent in hybrid breeding.

[0018] The shiitake mushroom strain FX85 of the present invention and the breeding method and application thereof have the following advantages: The main color of the fruiting body cap of FX85 bred by the present invention is brown, with white fluffy scales on the edge, and the average fresh mushroom yield of strain FX85 is 912 g / stick, which is 8.07% higher than that of the control variety Xin 808, the polysaccharide content of the strain is 8.75% higher than that of strain Xin 808, the protein content is 21.57% higher, the cap handle ratio is 13.77% higher than that of Xin 808, the agronomic traits and yield are more advantageous than those of strain Xin 808, and it has promotion value. The high-yield new shiitake mushroom variety bred by the present invention can enrich the existing shiitake mushroom germplasm resource bank, help adjust the shiitake mushroom planting structure, and improve the shiitake mushroom planting efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the mycelial growth of FX85 of the present invention.

[0020] Figure 2 These are the bacterial sticks of FX85 of the present invention and the control group.

[0021] Figure 3 This is a morphological diagram of the fruiting body of FX85 of the present invention.

[0022] Figure 4 The sectional views of the FX85 fruiting body of the present invention; (a) cross section; (b) side section.

[0023] Figure 5 This is the phylogenetic tree of FX85 of the present invention.

[0024] Figure 6 This is the ISSR identification diagram of FX85 of the present invention.

[0025] Figure 7 This is the genetic relationship cluster diagram of FX85 of the present invention. DETAILED DESCRIPTION

[0026] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0027] Note: The experimental methods in the following examples are conventional methods unless otherwise specified, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.

[0028] In the present invention, all features defined in the form of numerical ranges or percentage ranges, such as values, quantities, contents and concentrations, are only for brevity and convenience. Accordingly, the description of numerical ranges or percentage ranges should be deemed to have included and specifically disclosed all possible secondary ranges and individual values ​​within the range (including integers and fractions).

[0029] The features mentioned in the present invention can be combined arbitrarily, as long as there is no contradiction in the combination of these features, all possible combinations should be considered as the scope of this specification. Each feature disclosed in the specification can be replaced by any alternative feature that can provide the same, equal or similar purpose. Therefore, unless otherwise specified, the disclosed features are only general examples of equal or similar features.

[0030] The quantitative tests in the following experimental examples were repeated three times and the results were averaged.

[0031] Experimental Example 1 High-yield shiitake mushroom strain ( Lentinus edodes FX85 Acquisition The strain of the present invention is obtained by single spore hybridization and systematic breeding of Shenxiang 215 and Huxiang F2. Shenxiang 215 is a strain with large and thick mushrooms, firm caps, and excellent commercial properties. It has shown good adaptability and stability in demonstration and promotion in many places. Huxiang F2 is a variety with significantly improved yield, a high proportion of high-quality mushrooms, and strong stress resistance. It has shown excellent economic value and production potential in the application of scientific and technological cooperation. Therefore, the two are determined as parent materials of the present invention.

[0032] The steps of hybrid breeding are as follows: (1) Single spore preparation Select the excellent fruiting bodies of Pleurotus eryngii of Huxiang F2 and Shenxiang 215 for spore collection and dilute with sterile water to 10 5 / mL, 50 μL of spore suspension was pipetted onto a PDA medium plate and spread evenly, and cultured at 25°C for 3-5 days. The newly germinated star-shaped colonies were examined under a microscope, and the monokaryotic mycelia of Huxiang F2 and Shenxiang 215 without lock-like union were selected for culture.

[0033] (2) Mating type identification The monokaryotic mycelia obtained above were hybridized and paired to identify the genotype, and the identified genotype results were reflected by ITS and ISSR.

[0034] (3) Hybridization Under sterile conditions, use a 0.8 cm diameter puncher to take the monokaryotic mycelia of the two parents on PDA, with a 3 cm interval between the culture blocks, and culture at 25°C for 3-5 days. When a fusion line is formed between the two colonies, take the mycelia in the middle of the fusion line for microscopic examination. If a lock-like union is formed, it is a binucleate mycelia, indicating that the hybridization is successful.

[0035] (4) Heterozygous mycelium growth and mushroom production test The heterozygotes were transferred to tubes and the growth of hyphae was observed. The hybrids were used for fruiting tests. The culture medium formula (by mass percentage) was: 78% sawdust, 20% bran, 1% sugar and 1% gypsum. 17 cm × 33 cm spawn bags were used for cultivation. 20 bags of each strain, each bag was about 1 kg, and steam sterilized at 108-110 ℃ for 5 h. After inoculation, the bags were placed on the shelves of the culture room at 24±2 ℃ and 70% relative humidity for dark culture. When the hyphae grew all over the bag and irregular bulges appeared, the bags were transferred to the outdoor greenhouse and pierced. About 30 holes were pierced in each bag, and the bags were properly pressed to separate the bags from the materials so that the culture medium in the bags was fully in contact with the air. The daytime temperature was maintained at around 23°C, scattered light in the greenhouse was increased, ventilation was strengthened, and a dry-wet alternating environment was created. The temperature difference and dry-wet difference were stimulated to promote the lodging of aerial hyphae on the surface of the tube, secrete pigments, and form a layer of reddish-brown shiny fungal film. After the color change was completed, the bags were removed and the mushrooms were fruited. The fruiting test was carried out under the same environment, and the strains with good mushroom shape, dense or relatively dense mushrooms, and high yield were selected to enter the next round of screening, as shown in Table 1.

[0036] Table 1 Preliminary screening results of dominant strains

[0037] Note: New 808 refers to the existing shiitake mushroom variety L808; mushrooms with FX numbers are strains selected in the first round with good mushroom shape, dense or relatively dense mushroom production, and high yield.

[0038] (5) Further screening of dominant strains The mushroom bags and bag making formula of step (4) were used to conduct further fruiting tests on the hybrid strain screened in the previous step, and the fruiting bodies of Pleurotus eryngii were collected for agronomic trait recording, crude polysaccharide and crude protein testing, and the target dominant strain FX85 was further screened out.

[0039] (6) Stability cultivation test The strain stability cultivation test was carried out using the fungus bag and bag making formula of step (4), and its genetic stability was observed. The purified strain material, namely the target dominant strain FX85, was obtained through the separation of Pleurotus ostreatus tissue.

[0040] Experimental Example 2 Identification of strains 1. Morphological characteristics 1. Mycelium morphological characteristics The mycelium of the strain FX85 of the present invention is dense and vigorous, white in the early stage, and slightly light brown after a period of cultivation. Figure 1 shown.

[0041] 2. Morphological characteristics of mushroom sticks Depend on Figure 2It can be seen that (the mushroom sticks numbered FX in the figure are the mushrooms obtained from the fruiting test in Experimental Example 1) the color of the mushroom sticks is darker than that of the new 808, and the color is dark brown.

[0042] 3. Fruiting body morphology characteristics The fruiting body of Lentinus edodes FX85 is medium to large, hemispherical, and relatively rounded; the cap is brown, darker in the middle and lighter at the edge, and the longitudinal section of the cap is convex; dark scales are evenly distributed on the edge of the cap, the cap diameter is 4.55±0.09 cm, the cap thickness is 1.58±0.06 cm, the flesh is white, and has the unique smell of Lentinus edodes; the gills are off-white, the stem is 3.38±0.25cm long, 1.63±0.08 cm thick, white, equal in thickness from top to bottom, and nearly cylindrical. The fruiting body morphology and its section are shown in Figure 3 , Figure 4 .

[0043] 2. Molecular Biological Characteristics The present invention uses a fungal DNA rapid extraction kit to extract DNA and amplify ITS sequencing of the fruiting body of the strain FX85 obtained by hybridization breeding. The ITS sequence measured by sequencing is SEQ ID NO.1. A phylogenetic tree is constructed based on the identification results, such as Figure 5 shown.

[0044] SEQ ID NO.1 is as follows: .

[0045] In view of the above morphological characteristics and classification and identification research of ITS sequences, it is determined that the strain FX85 of the present invention belongs to the Basidiomycetes ( Basidaiomycetes ), Agaricalales ( Agaricales )、Dermacentor spp.( Marasmiaceae )、Lentinus ( Lentinula ). This strain Lentinus edodes FX85 was deposited in the China Center for Type Culture Collection (CCTCC) on December 3, 2024, located at Wuhan University, Wuhan, China, with the accession number CCTCC NO: M 20242713.

[0046] Experimental Example 3 High-yield shiitake mushroom strains ( Lentinus edodes ) Comparative analysis of basic properties of FX85 and new 808 This experimental example studies the high-yield shiitake mushroom strain ( Lentinus edodes ) FX85 was compared with strain new 808. The basic properties of the strains were determined according to "Agricultural Industry Standard of the People's Republic of China NY / T 2560-2014, Guide for Specificity, Consistency and Stability Testing of New Plant Varieties - Shiitake Mushrooms". The crude polysaccharide content of Shiitake fruiting bodies was determined according to the sulfuric acid-phenol method, and the protein content was determined using the Kjeldahl method.

[0047] The results are shown in Table 2. In comparison, the high-yield shiitake mushroom strains ( Lentinus edodes ) The mycelium growth rate of FX85 is faster; the fruiting body petiole is nearly white, and the main color of the cap is brown. The average fresh mushroom yield of strain FX85 is 912 g / stick, which is 8.07% higher than that of the control variety Xin 808; there are very small nearly white velvety scales distributed on the edge, and the longitudinal section of the cap is convex; the petiole is of equal thickness at the top and bottom, cylindrical, and of moderate length; the crude polysaccharide content exceeds that of the main promoted variety Xin 808 by 8.75%; the crude protein content exceeds that of the main promoted variety Xin 808 by 21.57%; the agronomic traits and yield are more advantageous than those of Xin 808.

[0048] Table 2 Comparison of basic characteristics of FX85 and control strains

[0049] Experimental Example 4 Identification of strain FX85 by interstitial simple sequence repeat markers (ISSR) The strain was activated and cultured at 25℃ for 7 days. The inoculated block was picked and inoculated on PDA medium and cultured at 25℃ for 10 days. The mycelia were ground by liquid nitrogen grinding method and stored at -80℃ for later use. The mycelial DNA was extracted by CTAB method. Amplification was performed on a BioRad C1000 PCR instrument. The reaction system was: 10 μL of 2×T5 Super PCR MIX (Basic), 8 μL of ddH2O, 1 μL of primer (P1: 5′-AGAGAGAGAGAGAGAGT-3′) and 1 μL of template DNA, a total of 20 μL. The amplification annealing temperature was 54 ℃. Bands were counted after electrophoresis separation and imaging. The presence of ISSR bands in the ISSR map was counted as 1, and the absence of ISSR bands was counted as 0. The initial data matrix was constructed and cluster analysis was performed using NTSYSpc2.1 Professional Edition software.

[0050] ISSR electrophoresis pattern Figure 6As shown in the figure, the results show that Xin 808 has 5 bands from top to bottom, and FX85 has 4 bands. There are obvious differences in the distribution, staining depth, and width of bands between FX85 and Xin 808. The first and second bands of FX85 are brighter and wider than those of Xin 808 and Shenxiang 215, and FX85 lacks the third band of Xin 808.

[0051] Draw a genetic relationship cluster diagram for the band data, Figure 7 It can be seen that (the mushroom sticks numbered FX in the figure are the mushrooms obtained from the fruiting test in Experimental Example 1) are divided into two major categories at a genetic similarity coefficient of 0.77, indicating that the genetic differences between the shiitake mushroom strains FX85 and Xin 808 (X808 in the figure) are relatively large.

[0052] In summary, the high-yield new shiitake mushroom variety bred by the present invention can enrich the shiitake mushroom germplasm resource bank, help adjust the shiitake mushroom industry structure, and improve planting efficiency.

[0053] Although the content of the present invention has been described in detail through the above preferred embodiments, it should be appreciated that the above description should not be considered as a limitation of the present invention. After reading the above content, it will be apparent to those skilled in the art that various modifications and substitutions of the present invention will occur. Therefore, the protection scope of the present invention should be limited by the appended claims.

Claims

1. Shiitake mushroom strains ( Lentinus edodes ) FX85, characterized in that, The shiitake mushroom strain FX85 was deposited in the China Center for Type Culture Collection on December 3, 2024, with the address: Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: M20242713.

2. The method for breeding the shiitake mushroom strain FX85 according to claim 1, characterized in that: The method comprises the following steps: 1) Dilute the excellent fruiting body spores of Pleurotus eryngii of Shenxiang 215 and Huxiang F2 with sterile water, pipette the spore suspension onto a PDA medium plate and spread it evenly, culture it at a constant temperature of 25±2℃, and select the monokaryotic mycelium without lock-like union; 2) Inoculate the selected monokaryotic mycelia of Shenxiang 215 and Huxiang F2 onto PDA plates, with a spacing of 3 cm between the two monokaryotic mycelia and culture at 25±2°C. When a fusion line is formed between the two colonies, take the mycelia in the middle of the fusion line for microscopic examination and select the binucleate mycelia with lock-like union for culture; 3) After the heterozygotes are transferred to the original strains, they are placed in fungus bags for fruiting experiments to screen hybrid strains with good mushroom shape and dense mushroom production; 4) Carry out fruiting test on the selected hybrid strains, collect the fruiting bodies of Pleurotus ostreatus and the control shiitake mushrooms, and conduct specificity, consistency and stability tests at the same time to screen out the target dominant strains.

3. The breeding method according to claim 2, characterized in that: In step 1), the spore content in the spore suspension is 10 5 Pieces / mL.

4. The breeding method according to claim 2, characterized in that: In step 3), the formula of the mushroom bag comprises the following components in percentage by mass: 78% of miscellaneous sawdust, 20% of bran, 1% of white sugar and 1% of gypsum.

5. The breeding method according to claim 2, characterized in that: In step 3), in the fruiting experiment, after inoculating the original seed, the fungus bag is placed in a dark culture room at 24±2°C and a relative air humidity of 70% for culture; when the mycelium grows all over the fungus bag and irregular ridges appear, the fungus bag is transferred to an outdoor greenhouse, punctured, and pressed to separate the bag so that the culture medium in the bag is fully in contact with the air; the daytime temperature is maintained at 22~24°C, the scattered light in the greenhouse is increased, ventilation is performed, a dry-wet alternating environment is created, and temperature and dry-wet difference stimulation is performed to promote the lodging of aerial mycelium on the surface of the fungus tube, secrete pigments, and form a layer of reddish-brown shiny fungus film. After the color change is completed, the bag is removed and the mushrooms are fruited.

6. The fruiting body of the Lentinula edodes strain FX85 as claimed in claim 1.

7. The spores of the Lentinula edodes strain FX85 as claimed in claim 1.

8. The mycelium of the Lentinus edodes strain FX85 as claimed in claim 1.

9. A mushroom log of the Lentinus edodes strain FX85 as claimed in claim 1.

10. Use of the Lentinula edodes strain FX85 as claimed in claim 1 in food or as a parent in hybrid breeding.

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