Oudemansiella raphanipes strain with stress resistance and its application

Abstract

The present invention discloses a stress-resistant Oudemansiella raphanipes strain and its application, belonging to the technical field of edible fungi. Aiming at the prominent problems of strain adaptability and stress resistance degradation in the commercial cultivation of Oudemansiella raphanipes strains, especially the problems of high temperature, high humidity and easy outbreak of Trichoderma contamination in summer, high temperature, high humidity, salt-alkali resistance and Trichoderma contamination are used as stress conditions. The collected strains are domesticated under stress conditions on the basis of primary screening, and then the selected dominant strains are bred by protoplast fusion technology. The fusants are regenerated and screened under stress conditions, and finally the dominant resistance is evaluated through physiological tests and fruiting verification, obtaining a stress-resistant Oudemansiella raphanipes strain with strong comprehensive stress resistance, and its preservation number is CGMCC No. 40285, effectively solving the problem that currently restricts the large-scale cultivation of Oudemansiella raphanipes.

Description

Technical Field

[0001] The present invention relates to the technical field of edible fungi, and particularly relates to a stress-resistant Oudemansiella raphanipes strain and its application. Background Art

[0002] Oudemansiella raphanipes, also known as the long-root mushroom, belongs to the genus Oudemansiella of the Physalacriaceae family, Agaricales order, Agaricomycetes class (Zhuliang Y, 2009). The fruiting body of Oudemansiella raphanipes has a grayish-brown to light black-brown cap with a diameter of 1 - 12 cm, a central protrusion, and a slight depression from the center to the edge of the cap. It is sticky when wet. The gills are white to milky white. The stipe is white to black, covered with small black to brown scales on the surface, 2 - 30 cm long, nearly cylindrical. When the epidermis of the stipe is peeled off, the inner flesh is white. The lower part of the stipe is a root-like structure formed by the twisting of hyphae, similar to the rhizome of a plant, so it is called the long-root mushroom.

[0003] Oudemansiella raphanipes is a medium-high temperature edible mushroom, and the optimal fruiting temperature is 23 - 28 °C. It is one of the few edible mushroom varieties suitable for cultivation in early summer and early autumn. It is a rare edible mushroom with the advantages of supplementing the supply of edible mushroom varieties in the market, reducing energy consumption and costs in cultivation, and has high promotion value. However, when cultivated in this temperature range at present, the strains in production show low yield and poor stress resistance, and their excellent traits degenerate year by year. Specifically, the fruiting temperature adaptability of the fruiting body degenerates and the anti-pollution ability decreases. From the original strain property that could normally fruit at 23 °C - 28 °C during commercial cultivation, the adaptable fruiting temperature range gradually shrinks year by year. Especially when cultivated in the original temperature range of 25 °C - 28 °C for normal fruiting, large areas of Trichoderma spp. pollution will occur, indicating a decrease in the anti-microbial ability. The main reason is that most of the strains for artificial cultivation in China at present are wild-domesticated strains. Due to the short cultivation history, they have poor adaptability to artificial cultivation conditions, and the degeneration of wild strains is very obvious. As the disadvantages of cultivated strains in production practice become increasingly prominent year by year, the pollution rate is high during annualized cultivation, especially during summer cultivation, and the pollution rate is so high that it often leads to reduced production or even crop failure. At present, reducing the cultivation temperature is adopted in production to control pollution, but the effect is not significant. This problem has become an outstanding problem restricting the large-scale cultivation of Oudemansiella raphanipes, and there is an urgent need for stress-resistant Oudemansiella raphanipes strains in production. Summary of the Invention

[0004] In view of the above-mentioned prior art, the object of the present invention is to provide a stress-resistant Oudemansiella raphanipes strain and its application. According to the environment with high temperature and high humidity in summer and prone to Trichoderma contamination, high temperature, high humidity and Trichoderma contamination are used as stress conditions, and combined with the resistance to salt and alkali, strains with certain stress resistance are screened out and subjected to stress domestication. The protoplasts of the strains after stress domestication are fused, and after regeneration under conditions and stress screening, several dominant resistances are integrated to obtain an Oudemansiella raphanipes strain with strong comprehensive stress resistance, effectively solving the problem that currently restricts the large-scale cultivation of Oudemansiella raphanipes.

[0005] Specifically, the present invention relates to the following technical solutions:

[0006] In the first aspect of the present invention, an Oudemansiella raphanipes strain named Y1 is provided. This strain was deposited at the China General Microbiological Culture Collection Center (abbreviated as CGMCC, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing) on August 26, 2022, and its deposit number is: CGMCC No. 40285.

[0007] The Oudemansiella raphanipes strain Y1 of the present invention has the following characteristics:

[0008] (1) High temperature resistance: After the strain is stressed at 36°C for 24 hours and then restored to normal culture conditions, it still does not lose its activity.

[0009] (2) Salt and alkali tolerance: The strain can grow normally under saline-alkali conditions with a salt content of 0.7% and a pH of 8.5.

[0010] (3) High humidity tolerance: The strain can grow well under conditions with a water content greater than 70%.

[0011] (4) Resistance to Trichoderma contamination: The strain has strong resistance to Trichoderma and still maintains its anti-Trichoderma activity under high temperature and high humidity conditions.

[0012] (5) High yield: High mushroom yield and high proportion of grade I mushrooms.

[0013] The Oudemansiella raphanipes strain (Oudemansiella raphanipes) claimed by the present invention includes the strain corresponding to CGMCC No. 40285; it also includes the offspring produced by the reproduction of the strain and having the same genetic and / or morphological traits as the strain.

[0014] In the second aspect of the present invention, the application of the above-mentioned Oudemansiella raphanipes strain with the deposit number CGMCC No. 40285 as a parent in breeding is provided.

[0015] In the above application, the breeding is for cultivating stress-resistant varieties of the Oudemansiella raphanipes strain.

[0016] In the third aspect of the present invention, there is provided mycelia or spores obtained by culturing the Oudemansiella raphanipes strain with the preservation number of CGMCC No. 40285.

[0017] In the fourth aspect of the present invention, there is provided fruiting bodies obtained by cultivating the Oudemansiella raphanipes strain with the preservation number of CGMCC No. 40285.

[0018] In the fifth aspect of the present invention, there is provided a method for breeding a highly stress-resistant Oudemansiella raphanipes strain, comprising the following steps:

[0019] (1) Domesticate and screen Oudemansiella raphanipes strains resistant to high temperature, screen Oudemansiella raphanipes strains resistant to high humidity, screen Oudemansiella raphanipes strains resistant to Trichoderma, and screen Oudemansiella raphanipes strains resistant to salinity and alkalinity respectively;

[0020] (2) Use the Oudemansiella raphanipes strains screened in step (1) as parents for protoplast fusion, and conduct re-screening for stress resistance on the protoplast fusion strains to breed a highly stress-resistant Oudemansiella raphanipes strain.

[0021] Preferably, in step (1), the method for domesticating and screening Oudemansiella raphanipes strains resistant to high temperature is as follows: conduct cyclic domestication and screening at four temperature levels of (26 °C, 16 h; 30 °C, 8 h), (26 °C, 16 h; 32 °C, 8 h), (26 °C, 16 h; 34 °C, 8 h), and (26 °C, 16 h; 36 °C, 8 h) in sequence. After 16 cycles at each gradient, measure the growth rate of each strain under this temperature condition; finally, screen out the strains that can tolerate 8 h at 36 °C.

[0022] Preferably, in step (2), the re-screening for stress resistance of the protoplast fusion strains specifically includes:

[0023] Transfer the strains regenerated after protoplast fusion and cultured under the condition of (26 °C, 16 h; 32 °C, 8 h) to a new plate, culture them under the condition of (26 °C, 16 h; 34 °C, 8 h), screen out the strains with faster growth rate and better growth vigor, and conduct screening for salinity and alkalinity resistance under the conditions of 0.7% salt content and pH of 8.5; inoculate the strains screened under the salinity and alkalinity resistance conditions with Trichoderma harzianum after high temperature stress, and select the strains with good confrontation performance with Trichoderma harzianum.

[0024] Advantages of the present invention:

[0025] (1) The Oudemansiella raphanipes strain Y1 provided by the present invention is obtained by combining artificial stress domestication and protoplast breeding. Compared with conventional Oudemansiella raphanipes varieties, the comprehensive stress resistance (high temperature resistance, high humidity resistance, salinity resistance, and Trichoderma resistance) of the Oudemansiella raphanipes strain Y1 has been greatly improved, effectively solving the problem that the contamination rate of Oudemansiella raphanipes during summer cultivation is as high as to reduce production or even result in a complete crop failure, and it has high popularization and application value in production.

[0026] (2) While having excellent comprehensive stress resistance, especially the strongest Trichoderma resistance under the conditions of 25-28°C, the Oudemansiella raphanipes strain Y1 of the present invention also has good production performance, with relatively high fruiting body yield and a high proportion of grade I mushrooms. Description of the Drawings

[0027] Figure 1 : Results diagram of the confrontation test against Trichoderma.

[0028] Figure 2 : Infection situation after inoculating Trichoderma harzianum after 1 day of stress at 36°C.

[0029] Figure 3 : Fruiting body photos of Y1 and other test strains.

[0030] Figure 4 : Photo of grade I mushrooms of strain Y1.

[0031] Figure 5 : Photo of the fruiting body of strain Y1 in the field. Detailed Embodiments

[0032] It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present application belongs.

[0033] Term Explanation:

[0034] Fruiting body: It is the spore-bearing structure of higher fungi, that is, the fruit body, which is composed of organized mycelium.

[0035] Hypha: A single tubular filament, which is the structural unit of most fungi.

[0036] Mycelium: Many hyphae gather together to form the vegetative body of fungi, that is, the mycelium.

[0037] To enable those skilled in the art to more clearly understand the technical solution of this application, the technical solution of this application will be described in detail below in conjunction with specific embodiments. If the specific test conditions are not specified in the embodiments, they are usually in accordance with conventional conditions or the conditions recommended by reagent companies; for the reagents, consumables, etc. used in the following embodiments, unless otherwise specified, they can all be obtained through commercial channels.

[0038] Example 1: Screening of stress-resistant Oudemansiella raphanipes strains

[0039] 1. Artificial domestication and screening of high-temperature resistant strains

[0040] The similarity matrix diagram and UGMA clustering diagram of 28 collected Oudemansiella raphanipes strains were constructed by SRAP technology for genetic relationship analysis. Based on the analysis, 2 strains with the same name but different strains were removed, and 26 different strains were determined as test materials (Table 1).

[0041] Table 1: Test strains

[0042]

[0043] After the 26 test strains were activated and transferred for cultivation, artificial high-temperature domestication was carried out. Four cyclic temperature gradients were designed for the test and carried out in a full-automatic biochemical incubator: (26°C, 16 h; 30°C, 8 h), (26°C, 16 h; 32°C, 8 h), (26°C, 16 h; 34°C, 8 h), (26°C, 16 h; 36°C, 8 h). Each treatment had 3 replicates. After 16 cycles at each gradient, the growth rate of each strain under this temperature condition was measured, and comprehensive screening was carried out using the growth rate, colony color, and hyphal thickness as indicators. Strains with faster growth rate and good growth were screened.

[0044] After 16 cycles of the test strains under the condition of (26°C, 16 h; 30°C, 8 h), the comparison results of the growth rate and growth trend of the hyphae are shown in Table 2.

[0045] Table 2: High-temperature resistance domestication of strains at 30°C (mm / d)

[0046]

[0047] Note: "+++" indicates strong growth; "++" indicates general growth; "+" indicates weak growth

[0048] The results showed that: for the 26 tested strains at 30°C, the mycelial growth rate decreased compared to that under constant temperature culture at 26°C, and there were also differences in the growth rates among different strains. The color of the mycelia of the 26 strains did not change significantly, and the growth vigor of the mycelia did not change significantly. After the high-temperature acclimation test at 30°C, it was shown that most of the Oudemansiella raphanipes strains could grow at 30°C, but the growth rate was slightly inhibited, and the mycelial color and growth vigor were not significantly affected.

[0049] After the strains were cyclically acclimated 16 times under the condition of (26°C, 16 h; 32°C, 8 h), the measurement results of the growth rate, mycelial growth vigor and mycelial color are shown in Table 3.

[0050] Table 3: High-temperature acclimation of strains at 32°C (mm / d)

[0051]

[0052] Note: "+++" indicates vigorous growth; "++" indicates normal growth; "+" indicates weak growth

[0053] The results showed that: at 32°C, the mycelial growth rate of most strains decreased significantly compared to that under constant temperature culture at 30°C. The color of the mycelia of some strains changed from pure white to white, and the mycelial growth vigor weakened. At 32°C, the growth rate of each strain was significantly inhibited. Since strain C1 was significantly inhibited by temperature and its growth rate decreased sharply, and the mycelial growth rate of strain T6 was slow, the color of the mycelia changed from pure white to white, and the mycelial growth vigor also became not vigorous. Therefore, strains C1 and T6 were excluded from the acclimation at 34°C.

[0054] After the strains were cyclically acclimated 16 times under the condition of (26°C, 16 h; 34°C, 8 h), the measurement results of the growth rate, mycelial growth vigor and mycelial color are shown in Table 4.

[0055] Table 4: High-temperature acclimation at 34°C (mm / d)

[0056]

[0057] Note: "+++ indicates vigorous growth; "++ indicates normal growth; "+ indicates weak growth

[0058] The results showed that: the high temperature of 34°C had a great impact on the mycelia of Oudemansiella raphanipes. The mycelial growth rate of all strains decreased significantly. Strains S and 2H08 had the fastest growth rate, which was 1.36 mm / d, and the mycelial growth vigor was vigorous, and the mycelial color was white. At 30°C and 32°C, the growth of strains S and 2H08 was relatively fast but not prominent. At 34°C, they could have a relatively high growth rate and relatively vigorous growth potential, indicating that they gradually adapted to the high-temperature environment, developed a certain tolerance to high temperature, and to a certain extent could resist high temperature and repair high-temperature damage.

[0059] After 16 cycles under the conditions of (26°C, 16 h; 36°C, 8 h), the test results of the growth rate, growth potential of the mycelium, and whether there are phenomena such as angular changes or unevenness in the mycelium are shown in Table 5.

[0060] Table 5: High temperature domestication at 36°C (mm / d)

[0061]

[0062]

[0063] Note: "+++" indicates vigorous growth; "++" indicates general growth; "+" indicates weak growth

[0064] The results show that the high temperature of 36°C has a very significant impact on the mycelium of Oudemansiella raphanipes. The mycelium growth rate of all strains has decreased significantly, and the growth rate is less than 1 mm / d. W3 and T4 did not grow, and the growth cycle of the remaining strains also reached 40 days. The high temperature inhibition of all strains is very obvious. The colors of the strains have turned yellow to varying degrees. Some strains have changed from white to lighter white, and some strains have changed to light yellow. The growth potential of the strains gradually weakens, the density of the mycelium changes from thick to sparse, and there are also multiple strains with angular changes, uneven mycelium growth, and serious coral-like bifurcations at the mycelium ends. Because S and 2H08 performed well in the screening tests at 36°C and 34°C, with growth rate and growth potential superior to the other strains, the domesticated S and 2H08 strains were designated as the heat-resistant strains after domestication, renamed Sgw and H08gw, for subsequent test use.

[0065] 2. Screening of high humidity-resistant strains

[0066] All 26 tested strains of Oudemansiella raphanipes were transferred to test tubes with a water content of 67.5%. Strains with fast growth rate and good growth potential were selected for subsequent domestication and screening. The test found that the strains S, T1, C2, W1, 2H01, G, JX, and HP had a relatively fast growth rate. These 8 strains were selected for screening at multiple moisture gradients. Cultivation materials with four different water contents of 67.5%, 70.0%, 72.5%, and 75.0% were loaded into test tubes and cultured in a constant temperature incubator at 26°C for 15 days. Six culture treatments were carried out, and each treatment was repeated 3 times. The water content of 65% was used as the control.

[0067] Three days after inoculation, observe whether the inoculation point germinates and colonizes, measure the mycelium growth rate after culturing for 15 days, count the number of days required to fill the test tube, and select the strains with excellent growth. The results are shown in Table 6.

[0068] Table 6: Growth rate of humidity-resistant screened strains

[0069]

[0070] The results showed that: under the condition of the water content of the cultivation material being 70%, the mycelial growth rate of the 8 strains reached the fastest, and with the increase and decrease of the water content, the mycelial growth rate gradually decreased. When the water content of the cultivation material was 65%, the mycelial growth rate of strain 2H01 was the fastest, and with the increase of the water content, the mycelial growth rate gradually decreased. Strain C2 did not grow under the conditions of water contents of 72.5% and 75%. When the water content was 65%, the mycelial growth rate of strain S was the fastest among the 8 strains. However, with the increase of the water content, the characteristic of strain HP preferring high humidity became more obvious, exceeding strain S and reaching the strain with the fastest growth rate. Moreover, with the increase of the water content, the change of the growth rate of strain HP was small, showing its tolerance to high humidity conditions. Therefore, strain HP was selected as the moisture-tolerant strain.

[0071] 3. Screening of Trichoderma-resistant strains

[0072] Ten samples were collected from the contaminated soil blocks and spawn bags at the mushroom fruiting site for isolation, culture and purification. The ITS and tef1 sequences of the 8 obtained strains were sequenced and compared with the sequences in the NCBI database. The results showed that 7 isolates were Trichoderma harzianum and 1 isolate was Trichoderma asperellum. Trichoderma harzianum was selected as the test material for the experiment, and the above-mentioned Oudemansiella raphanipes strains were subjected to a plate confrontation test with Trichoderma harzianum to further screen the Oudemansiella raphanipes strains resistant to Trichoderma harzianum.

[0073] The specific plate confrontation test was as follows:

[0074] A mycelial block of Oudemansiella raphanipes with a diameter of 0.5 cm was inoculated on one side of a petri dish and cultured in the dark at 26 °C in an incubator for 6 d. Then, a mycelial block of Trichoderma harzianum of the same size was inoculated at the corresponding position on the other side of the plate. The two mycelial blocks were separated by 5 cm. After inoculation, it was cultured at 26 °C. There were 3 replicates for each treatment and cultured in the dark for 7 d. Observe the infection of Trichoderma harzianum on the Oudemansiella raphanipes mycelium on the petri dish.

[0075] Figure 1The results of the confrontation test of 21 strains after culturing for 6 days at 26℃ and inoculating Trichoderma at a distance of 5 cm for 7 days. Among them, W1, D1, Q, and Qgw failed to resist the infection of Trichoderma and were infected by Trichoderma across the antagonistic line. The hyphae of multiple strains can form a confrontation with Trichoderma, grow across the antagonistic line, and cover the surface of Trichoderma. Strains C1 and D2 can maintain a confrontation with Trichoderma, but the hyphae of Odetteria oosporea failed to cover the hyphae of Trichoderma. Strain 2H09 has a brown skin, showing the characteristics of strain aging. Strains Sgw, Lgw, and H08gw are strains obtained by high temperature acclimation of strains S, L, and 2H08. In the confrontation test with Trichoderma, they performed better than S, L, and 2H08, which were not acclimated to high temperature. Sgw, Lgw, and H08gw strains covered the entire culture dish at the same culture time, but the color of the hyphae changed to white to light yellow. The F2 strain grew beyond the antagonistic line, and its mycelium was thick and strong, and its growth was good. Therefore, in the Trichoderma confrontation test, the Sgw strain and the F2 strain were selected as the anti-Trichoderma strains.

[0076] 4. Screening of salt-alkali tolerant strains

[0077] (1) Screening of salt-tolerant strains

[0078] Use liquid strains to prepare a salt solution of a certain concentration. After the liquid strain culture medium is prepared, it is promptly placed in a triangular flask, and 10 glass beads are added at the same time to prepare three liquid culture media with concentrations of 0.3%, 0.7%, and 1.1%, respectively. After tightly wrapping and sealing, sterilize at 121°C for 30 minutes, take out and cool to room temperature before inoculating the corresponding strain, and mark the strain name and inoculation date. When the strain is cultured in a shaker in this experiment, the temperature is 26°C, the speed is 200r / min, and the culture time is 10d. Generally, when the culture medium in the bottle is transparent and contains more mycelial balls, and at the same time exudes the unique smell of the fungus, the shake bottle liquid strain culture is completed.

[0079] After the mycelium has grown well, filter out the mycelium with filter paper. Number and weigh the empty glass culture dishes, transfer the filtered mycelium to the corresponding plate, and put it into a blast dryer at 80°C for drying. After drying, weigh the plate and calculate the mass of the mycelium. Tests were conducted at three gradients of salt content: 0.3%, 0.7%, and 1.1%, and the strains with faster growth and better growth were selected. The results are shown in Table 7.

[0080] Table 7: Mycelium drying quality of salt-tolerant strain screening test

[0081]

[0082] Table 7 shows the screening results of the salt tolerance tests of 6 strains. The 6 strains include the strains screened out in the high-temperature acclimation test, high-humidity screening test, and Trichoderma-resistant strain screening test, as well as the unscreened strain T3. Under the condition of a salt concentration of 0.3%, the growth of each strain was not significantly inhibited. At this time, the growth of strain S was the most vigorous, and the dry weight of the mycelium was the highest. When the salt concentration increased to 0.7%, the mycelial growth rates of all strains were inhibited to varying degrees. Among them, the change range of the dry weight of the mycelium of strain S was the largest. Therefore, it is considered that S is easily affected by the salt concentration and has poor salt tolerance characteristics; the dry weight of the mycelium of the HP strain was the highest among the 6 strains under this condition. When the salt concentration increased to 1.1%, the growth rate of all strains was slow. Affected by the high osmotic pressure, the mycelium formed a compact mycelial ball around the inoculated fungal block in the conical flask, and the number of small mycelial balls formed was less than that under the low-concentration condition. At a salt concentration of 1.1%, the dry weights of the mycelia of each strain were relatively low. The dry weight of the mycelium of the HP strain was the highest among the 6 strains, and the HP strain also showed good performance at a concentration of 0.7%. Therefore, HP was selected as the salt-tolerant strain.

[0083] (2) Screening of alkali-tolerant strains

[0084] Using the method of liquid spawn culture, it was initially found in the test that the strain Oudemansiella raphanipes could not grow in the liquid medium with a pH of 11.5. Therefore, liquid medium solutions with pH values of 7.5, 9.5, and 10.5 were respectively prepared. After the liquid spawn medium was prepared, it was promptly filled into Erlenmeyer flasks, and at the same time, 10 glass beads were added. After tightly wrapping and sealing, it was sterilized at 121 °C for 30 min. After taking it out and cooling to room temperature, three flasks of the medium were selected to measure the pH. Usually, the pH value would decrease to 7. The pH of these three flasks of the medium was readjusted, and the amount of NaOH added to these three media was averaged. The pH of the remaining media was readjusted, and then the corresponding spawn was inoculated, and the strain name and inoculation date were marked. When culturing the spawn in a shaker in this test, the temperature was 26 °C, the rotation speed was 200 r / min, and the culture time was 10 d. Generally, when the medium in the flask was permeable and contained more mycelial balls, and at the same time emitted the unique smell of this fungus, the shake-flask liquid spawn culture ended.

[0085] After the mycelium grew well, the mycelium was filtered out with filter paper. The empty glass Petri dishes were numbered and weighed, and the filtered mycelium was transferred to the corresponding Petri dishes and placed in a blast dryer at 80 °C for drying. After drying, the Petri dishes were weighed, and the mycelium mass was calculated. Tests were carried out at three gradients of pH values of 7.5, 9.5, and 10.5 respectively, and the strains with faster growth rates and better growth conditions were selected. The results are shown in Table 8.

[0086] Table 8: Mycelial growth rates in the alkali-tolerant strain screening test

[0087]

[0088] Table 8 shows the screening results of the alkali tolerance tests of 6 strains. During the test process, it was found that all strains did not germinate in the liquid medium with a pH of 11.5. Therefore, the pH of the last gradient was set at 10.5. The growth of each strain was the best at a pH of 7.5. When the pH increased to 9.5 and 10.5, the growth of all strains was greatly inhibited. Among them, the best-performing one was Sgw, which was selected as the alkali-tolerant strain.

[0089] 5. Protoplast breeding

[0090] The purpose of protoplast fusion breeding is to combine the advantageous traits of the parents to obtain stable fusion strains with excellent genetic traits of the parents.

[0091] The strains obtained by acclimating Oudemansiella raphanipes to high temperature, the strains screened in the high humidity test, the strains screened in the salt tolerance test, the strains screened in the alkali tolerance test, and the strains screened in the Trichoderma resistance test were respectively inoculated into the medium, cultured for 6d - 10d, the mycelia were extracted, enzyme solution was added to prepare protoplasts, the protoplasts were inactivated and fused, and after protoplast fusion and regeneration under the conditions of (26°C, 16h; 32°C, 8h), 20 strains, namely Y1 - Y10 and Z1 - Z10, were screened out. The 20 strains were transferred to a new plate and cultured under the conditions of (26°C, 16h; 34°C, 8h). It was found that the strains Y1, Y4, Y7, Z1, and Z9 grew faster and had better growth under these conditions. The five protoplast fusion and regeneration strains of Y1, Y4, Y7, Z1, and Z9 were subjected to subsequent screening.

[0092] The high-temperature-resistant strains regenerated under high-temperature conditions were screened under saline-alkali conditions. A liquid medium solution with a salt content of 0.7% and a pH of 8.5 was prepared. After the liquid strain medium was prepared, it was promptly filled into a triangular flask, and at the same time, 10 glass beads were added. After tightly wrapping and sealing, it was sterilized at 121°C for 30 min. After taking it out and cooling to room temperature, the pH of the liquid medium was readjusted, and then the corresponding strain was inoculated, and the strain name and inoculation date were marked. When culturing the strains in a shaker in this test, the temperature was 26°C, the rotation speed was 200 r / min, and the culture time was about 10d. Generally, when the medium in the flask was permeable and contained more mycelial balls, and at the same time emitted the unique smell of this fungus, the shake-flask liquid strain culture was completed.

[0093] After the mycelia grew well, the mycelia were filtered out with filter paper. The empty glass culture dishes were numbered and weighed, and the filtered mycelia were transferred to the corresponding petri dishes and dried in a blast dryer at 80°C. After drying, the petri dishes were weighed, the mycelia mass was calculated, and the strains with faster growth rate and better growth were selected as the strains with high-temperature resistance and certain saline-alkali tolerance characteristics.

[0094] Table 9 shows the screening results of the salt and alkali tolerance screening tests for strains Y1, Y4, Y7, Z1, and Z9. The mycelial growth of strain Y7 was poor, and the dried mass of the mycelium in the liquid medium was low. Therefore, strain Y7 was excluded, and strains Y1, Y4, Z1, and Z9 were retained.

[0095] Table 9: Salt and Alkali Screening Test

[0096]

[0097] Using the initial strain S as a control, the infection situation of Oudemansiella raphanipes strains that were regenerated under high-temperature conditions and screened under salt and alkali tolerance conditions after being inoculated with Trichoderma harzianum under high-temperature stress was compared. The Oudemansiella raphanipes strains were inoculated on one side of the culture dish and cultured in the dark at 26°C in an incubator for 5 days, and then cultured at 36°C for 1 day. After that, the same-sized Trichoderma harzianum strain was inoculated at the corresponding position on the other side of the plate, and after inoculation, it was cultured at 26°C. Each treatment had 3 replicates and was cultured in the dark for 5 days. The infection situation of Trichoderma harzianum on the Oudemansiella raphanipes strains on the culture dish was observed. The strains that showed good confrontation with Trichoderma harzianum were selected as strains with high-temperature tolerance, certain salt and alkali tolerance characteristics, and certain resistance to Trichoderma harzianum, that is, the resistant strains screened in this experiment.

[0098] Figure 2 Shows the infection situation of the initial strain S and the strains selected by protoplast breeding after being inoculated with Trichoderma harzianum after 1 day of stress at 36°C. As Figure 2 shown, for the strain S that was not acclimated to high temperature and the strain selected by protoplast breeding after being acclimated to various adversities, after 1 day of high-temperature stress at 36°C, strain S failed to form an obvious confrontation with Trichoderma harzianum and was almost completely covered by the mycelium of Trichoderma harzianum. However, after the selected strain was subjected to high-temperature stress, it maintained its own activity, and when the temperature returned to the optimal culture temperature of 26°C, a high-temperature inhibition line was formed and the mycelium gradually extended outward. From this experiment, it can be seen that the strains that have been acclimated to adversity and screened, and then through protoplast fusion and regeneration experiments under certain conditions, the obtained resistant strains can still maintain a certain level of activity after high-temperature stress and form an effective confrontation with Trichoderma harzianum.

[0099] The above strains were subjected to a fruiting test, and the results are as Figure 3 shown. The results show that: compared with other test strains, Y1 fruited early and had strong anti-pollution ability.

[0100] Taking into account the salt and alkali tolerance, high-temperature tolerance, Trichoderma resistance performance, and fruiting performance of the strains, the present invention selects the protoplast fusion strain Y1 for biological preservation.

[0101] The preservation information of strain Y1 is as follows:

[0102] Biological material (strain) referred to: Y1;

[0103] Classification name: Oudemansiella raphanipes; it can also be called Oudemansiella raphanipes (black-skinned Termitomyces albuminosus).

[0104] Depositary institution: General Microbiology Center, China Committee for Culture Collection of Microorganisms

[0105] Abbreviation of the depositary institution: CGMCC

[0106] Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0107] Deposit date: August 26, 2022.

[0108] Registration number in the deposit center: CGMCC No. 40285.

[0109] Example 2: Mushroom fruiting experiment

[0110] The resistant strain Y1 screened by high-temperature domestication (Sgw, H08gw), high-humidity resistance screening (HP), saline-alkali resistance screening (Sgw, HP), Trichoderma confrontation screening (F2, Sgw), and protoplast breeding was subjected to a cultivation experiment.

[0111] In June 2022, soil-covered cultivation was carried out in a winter-warm greenhouse. Mushroom fruiting started on July 1 and ended in mid-August. During the fruiting period, the air temperature was 23°C to 30°C, the ground temperature was 25°C to 26°C, the CO 2 concentration was relatively stable, usually at 25 - 29 ppm, the light intensity was 100 - 200 Lux, and the environmental humidity during fruiting was 80% - 90%. The quality of the fruiting mushrooms was statistically analyzed, and the contamination situation of Trichoderma was also statistically analyzed.

[0112] The grading standards for Oudemansiella raphanipes are shown in Table 10.

[0113] Table 10: Grading standards for Oudemansiella raphanipes

[0114]

[0115] The contamination situation of Trichoderma was calculated based on the contamination rate of the mushroom sticks:

[0116] Mushroom stick contamination rate (%) = (Contaminated mushroom sticks in the soil / Total mushroom sticks in the soil) × 100%

[0117] The photo of the first-grade mushrooms of the resistant strain Y1 is as Figure 4 shown; the photo of the field-grown mushrooms of the resistant strain Y1 is as Figure 5 shown. The specific results of the mushroom fruiting experiment are shown in Table 11.

[0118] Table 11: Oudemansiella raphanipes Mushroom Fruiting and Yield Statistics

[0119]

[0120] Note: The fruiting yield in the table is the total yield of 9 mushroom bags, with each bag weighing about 500g.

[0121] In summary, the comprehensive stress resistance (high temperature tolerance, high humidity tolerance, salinity tolerance, and Trichoderma resistance) of the Oudemansiella raphanipes strain Y1 of the present invention has been greatly improved, effectively solving the problem of high contamination rate during summer cultivation of Oudemansiella raphanipes, which may lead to reduced yields or even crop failures, and has high popularization and application value in production.

[0122] The above are only the preferred embodiments of the present application and are not intended to limit the present application. For those skilled in the art, various changes and modifications can be made to the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. An Oudemansiella raphanipes ( Oudemansiella raphanipes ) Y1, with a preservation number of CGMCC No. 40285.

2. Use of Oudemansiella raphanipes Y1 as claimed in claim 1 as a parent in breeding.

3. The use according to claim 2, wherein the breeding is for cultivating stress-resistant varieties of Oudemansiella raphanipes.

4. Fruiting bodies obtained by cultivating Oudemansiella raphanipes Y1 as claimed in claim 1.

5. Mycelia and / or spores obtained by culturing Oudemansiella raphanipes Y1 as claimed in claim 1.

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