CN116267624B

CN116267624B - Lentinus edodes strain L135-1 and application thereof

Info

Publication number: CN116267624B
Application number: CN202211383759.3A
Authority: CN
Inventors: 刘昆; 蒋俊; 路新彦; 宋小亚; 应国华; 吕明亮; 曾凡清
Original assignee: Lishui Academy Of Agricultural And Forestry Sciences
Current assignee: Lishui Academy Of Agricultural And Forestry Sciences
Priority date: 2022-11-07
Filing date: 2022-11-07
Publication date: 2024-03-29
Anticipated expiration: 2042-11-07
Also published as: CN116267624A

Abstract

The invention provides a lentinus edodes strain L135-1 and application thereof in cultivation. The strain is a variant strain of L135 variety, and mycelium grows slowly; the fruiting bodies are small, fruiting times are concentrated, fruiting capacity of head tide is high, picking times can be reduced, and labor cost is reduced; the yield is equivalent to the original L135 variety; classification naming of the strain: the Lenitula edodes is preserved in China general microbiological culture Collection center (CGMCC) under the preservation number of CGMCC No.21088 in the year 2020, 12 and 07.

Description

Lentinus edodes strain L135-1 and application thereof

Technical Field

The invention belongs to the technical field of mushroom breeding, and particularly relates to a mushroom strain L135-1 and application thereof.

Background

Lentinus edodes is the edible fungus type with the largest production amount in China. The total yield of the mushrooms in the whole country in 2020 reaches 1188.21 ten thousand tons and accounts for 34.28 percent of the total yield of the edible mushrooms in the whole country. Lentinus edodes L135 is produced by screening introduced variety from abroad in 2007 by fungus research institute of Sanming city of Fujian province. L135 is low temperature late maturing type, the bacterial age is 160-200 days, the hypha growth temperature is 4-28 ℃, the fruiting temperature is 5-18 ℃, and the optimum temperature is 9-13 ℃. The formation of the mushroom buds requires the stimulation of temperature difference above 6 ℃, the mushroom tide is obvious, and the tide transferring interval is 10-15 days. Fruiting body spread, short stem, thick stem, round and regular mushroom shape, brown color, less scales, cylindrical mycosis, short stem or aseptic stem; the flower mushroom is easy to form, the mushroom quality is compact, the storage is durable, and the flower mushroom is suitable for fresh selling, dehydration and drying. The suitable inoculation time is 2 months-4 months, and the harvesting period is 10 months-4 months next year. The mycelium has poor stress resistance and weak antibacterial capability, is not high-temperature resistant and difficult to overspray, and is suitable for being planted in areas below 32 ℃ or places with high altitude, cool air temperature and easy overspray. The high altitude mountain areas such as Qingyuan county and Jing Ning county in Zhejiang province are widely cultivated by mushroom farmers because of cool summer and large temperature difference in winter and are suitable for the characteristics of L135. After the cultivation for 15 years, the L135 variety is transported, preserved, separated, passaged, bred and cultivated in various forms, and the variety has variation phenomenon. The variant strain is the key for genetic breeding research, particularly provides a material with excellent foundation for the genetic mechanism research of specific characters, and brings a new break for the development of the edible fungus industry.

Disclosure of Invention

The invention provides a lentinus edodes strain L135-1 and application thereof in cultivation. The strain is a variant strain of L135 variety, and mycelium grows slowly; the fruiting bodies are small, fruiting times are concentrated, fruiting capacity of head tide is high, picking times can be reduced, and labor cost is reduced; the yield is equivalent to the original L135 variety.

In a first aspect, the invention provides a Lentinus edodes strain L135-1, designated by the classification: the Lenitula edodes is preserved in China general microbiological culture Collection center (CGMCC) under the preservation number of CGMCC No.21088 in the year 2020, 12 and 07.

In a second aspect, the present invention provides mycelium, protoplasts, sporophores, spores and sticks produced by Lentinus edodes strain L135-1.

In a third aspect, the present invention applies the aforementioned Lentinus edodes strain L135-1 to Lentinus edodes breeding; in some embodiments, the application comprises the steps of:

(1) Production of material rod

Filling the cultivation material into a cylindrical bag with a certain specification, tying, preparing a material rod, sterilizing at normal pressure, and keeping for 18 hours when the temperature of the material rod reaches above 98 ℃; after sterilization, opening the door when the temperature of the material rod is reduced to below 80 ℃, moving the material rod to a cooling chamber for cooling while the material rod is hot, and inoculating when the temperature of the material rod is reduced to below 28 ℃;

(2) Inoculating the material rod

Cleaning an inoculation box or an inoculation site before inoculation, sterilizing the surface of the inside, and putting cooled material bars, perforating bars, strains, 75% alcohol, alcohol lamps, aerosol sterilizing agents, bagging and other articles into the inoculation box or the inoculation site; igniting the aerosol disinfectant, sealing the inoculation box, and sterilizing; after the disinfection is finished, the inoculating personnel cleans the hands, and uses 75% alcohol to wipe the hands and the perforating rod; igniting the alcohol lamp, and burning and sterilizing the punching rod or the punching machine; 3 inoculation holes are evenly punched on the material rod, the diameter is about 1.5cm, and the depth is 2 cm-2.5 cm. The strain is plugged into an inoculation hole by hand in blocks, each hole is connected with a bag, and another rod is connected with the bag;

(3) Fungus stick culture

After inoculation, the fungus sticks are stacked in a wall type, the layer height is about 10 layers, and a passageway of 50cm is reserved between each two rows; after the mycelium grows to about 10cm, removing the bagging, puncturing 4-6 holes at the position of 2cm inside the mycelium and 1 cm-1.5 cm deep, performing first deflation, and discharging the fungus sticks to a culture rack; the canopy is covered with a sunshade net, water is sprayed, the sunshade net sags around, and the like, and the environmental condition is controlled between 24 ℃ and 26 ℃; after the hypha grows up the fungus stick, carrying out secondary puncture ventilation, wherein each stick has 30-60 holes; after the color of the fungus sticks is changed, the fungus sticks are deflated for the third time at the temperature of 12-20 ℃ for about 80 fungus sticks before the fungus sticks are removed from the bag. Selecting proper weather for bag removal after exhausting;

(4) Fruiting management

The temperature difference between day and night is pulled to stimulate fungus sticks to fruiting, and the fungus sticks are continuously operated for 7 to 15 days, so that primordia are promoted to occur; after the mushroom buds are formed, water is sprayed in the morning and evening, ventilation is carried out, so that the temperature and the humidity in the mushroom shed are kept stable; picking after the first batch of mushrooms grow to 7-8 minutes of maturity; stopping water spraying after harvesting the first tide mushroom, increasing ventilation to reduce humidity, culturing the mushroom for about 7 days, timely supplementing water according to the water content of the mushroom stick after the hypha at the harvested joint turns white and the hypha is obviously recovered, so that the water content of the mushroom stick reaches 60% -65%, increasing the temperature difference, and stimulating the second tide mushroom to grow; and (5) managing in a reciprocating manner until the harvesting is finished.

In some specific embodiments, the mushroom cultivation formula is 79% of miscellaneous wood dust, 20% of wheat bran, 1% of gypsum, about 55% -60% of water content and natural pH.

In a fourth aspect, the present invention applies the aforementioned Lentinus edodes strain L135-1 to the preparation of Lentinus edodes fruiting bodies and Lentinus edodes mycelia.

Drawings

FIG. 1 is a photograph of a fruiting body of Lentinus edodes strain L135-1.

FIG. 2 is a graph showing the growth rate of hyphae of L135-1 (left panel) and L135 (right panel) in wood chip medium.

FIG. 3 is a photograph of L135-1 fruiting body.

The specific embodiment is as follows:

the invention is further described below in conjunction with the detailed description. It is to be understood that these examples are for illustration of the invention only and are not intended to limit the scope of the invention. Furthermore, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the teachings of the present invention, and such equivalents are intended to fall within the scope of the claims appended hereto.

Example 1 tissue isolation and hypha growth Rate test

The fungus institute of Sanming City of Fujian (national institute of technology, strain 2007005) was used as a starting control strain for developing the strain L135-1 of Lentinus edodes according to the present invention.

(1) Tissue separation

In 2019, fruiting bodies of Lentinus Edodes with smaller fruiting bodies are found in fungus sticks of Lentinus Edodes L135 cultivated normally, the fruiting bodies are collected back to laboratory, sterilized with 75% alcohol surface, and cut into 0.5mm pieces with a scalpel ³ Inoculating large and small tissue blocks onto glucose potato agar medium (PDA) plate, culturing at 25deg.C+ -1deg.C, transferring new strain to new PDA plate for purification and separation after hypha grows, repeating transfer for 3 times, and obtaining final productObtaining the strain L135-1 with smaller fruiting body.

(2) Sawdust culture medium hypha growth rate test

Picking about 0.25cm on the inclined plane ² Transferring the bacterial blocks with the size to a PDA flat-plate culture medium, culturing at the constant temperature of 25+/-1 ℃ in a dark place, and when the diameter of a bacterial colony reaches 4 cm-6 cm, punching by using a puncher with the caliber of 3mm to prepare the inoculating blocks so as to ensure that the bacterial ages and volumes of all the inoculating blocks are consistent. Inoculating the prepared inoculating blocks of different strains onto wood chip culture medium (mixed wood chip 78%, wheat bran 20%, sugar 1%, gypsum 1%, material-water ratio 1:1.2-1.3, pH nature, adding wet weight culture medium 45g per tube), culturing at constant temperature of 25+ -1deg.C, and repeating 5 times per strain. When the colony had reached 2cm in diameter, a line was drawn at the edge of the colony as a hypha growth initiation line (L1). All test replicates were removed at the same time each day, streaked at the forefront of hyphae growth, designated as L2, L3 … …, and the daily growth rate recorded. And drawing a termination line (Ln) at the front edge of the bacterial colony until the strain growing fastest is about to grow into a large test tube, measuring the distance between the start line and the termination line by using a ruler, and dividing the distance by the culture days (d), thus obtaining the average growth speed of the hyphae. Hypha average growth rate= (Ln-L2)/d.

TABLE 1 hyphal growth rates (unit: mm) of L135-1 and L135 in wood chip medium

Strain	Test tube 1	Test tube 2	Test tube 3	Test tube 4	Test tube 5	Average value of
							L135-1	5.13	5.12	5.10	5.38	5.13	5.17±0.12
L135 is normal	7.15	6.86	7.06	6.81	7.26	7.03±0.19

Table 1 and FIG. 2 show that the mycelium production speed of the lentinus edodes strain 135-1 in the wood chip culture medium is obviously slower than that of the L135 normal strain.

EXAMPLE 2 Lentinus edodes strain L135-1 variability test L135

(1) DNA extraction and detection

Inoculating mycelium of L135-1 and normal L135 strain into a 90mm culture dish containing 20mLPDA culture medium, spreading sterilized cellophane glass paper on the surface of the culture medium, and culturing at 25+ -1deg.C. After the mycelia are full, a proper amount of mycelia are collected and ground in liquid nitrogen, and DNA is extracted by using a CTAB method. Analysis of DNA degradation, RNA contamination Using agarose gel electrophoresis, and detection of OD of DNA by Nanodrop _260/280 Ratio, qubit accurately quantifies DNA concentration. Wherein the OD value is between 1.8 and 2.0, and the total amount of DNA samples is more than 1.5ug is used for carrying out the resequencing analysis.

(2) Genome resequencing and sequence analysis

The test was conducted by Beijing Nobela source technology Co.

The passing DNA samples were randomly broken into fragments of 350bp in length by a covarias breaker. The warehouse was built using TruSeq Library Construction Kit. The DNA fragment is subjected to the steps of end repair, ployA tail addition, sequencing joint addition, purification, PCR amplification and the like to complete the preparation of the whole library. The constructed library was sequenced by the HiSeq PE150 platform of Illumina. The Raw image Data file obtained by sequencing is converted into an original sequencing sequence (Raw Data) through base recognition analysis. And obtaining effective data (clean data) by sequentially carrying out sequence quality distribution, sequencing error rate distribution and sequencing data filtering on the sequencing data. Clean data was aligned to the reference genome using BWA software (parameters: mem-t 4-k 32-M). The comparison was subjected to SAMTOOLS software to remove duplicates (parameters: rmdup). The detection of individual SNPs is carried out by adopting SAMTOOLS (mp-m 2-F0.002-d 1000), and the SNP is filtered by taking the reads support number of the SNP as the standard not lower than 4 and the mass value of the NP not lower than 20. Small fragment insertions and deletions (InDel) of less than 50bp in length were detected using SAMTOOLs software (mpileup-m 2-F0.002-d 1000).

Results:

(1) Genome resequencing quality and comparison to a reference genome

The L135-1 strain was re-sequenced with the normal L135 strain using the Illumina platform. The measured raw data are respectively 1,249,076,700bp and 1,443,209,100bp,clean data and respectively 1,245,050,100bp and 1,438,529,400bp,clean data, the effective rate is 99.68 percent and 99.68 percent, the base of Q30 mass in clean data reaches 94.62 percent and 94.36 percent, the base content of GC base of two strains is 43.77 percent to 43.97 percent, and the quality of sample library construction sequencing reaches the resequencing standard.

TABLE 2 resequencing quality of two strains

Strain	Original data (bp)	Valid data (bp)	Effective rate (%)	Q30(％)	GC base content (%)
						L135-1	1,249,076,700	1,245,050,100	99.68	94.62	43.77
L135 is normal	1,443,209,100	1,438,529,400	99.68	94.36	43.97

(2) Comparison and analysis of resequencing results

Sequence alignment analysis was performed using the NBRC 111202 strain sequence published in 2017 by the Biotechnology research center (Iwate Biotechnology Research Center) of Yanshou county, japan as a reference genome. By comparison, the yield ratios of the L135-1 strain and the normal L135 strain are 92.89% and 92.81%, respectively, and the average sequencing depth is above 26X. The 1X coverage is 86.78% and 86.88%, the 4X coverage is 85.00% and 85.36% respectively, and the comparison result is normal, so that the method can be used for subsequent mutation detection and related analysis.

TABLE 3 comparison of two strains

TABLE 4 specific SNP/InDel sites of the L135-1 Strain

EXAMPLE 3 cultivation of Lentinus Edodes L135-1 Strain

And (3) carrying out high-canopy shelf cultivation on the obtained L135-1 and L135 normal strains in Jing Ning county of Lishui City of Zhejiang province.

(1) Production of material rod

The mushroom cultivation formula comprises 79% of miscellaneous wood chips, 20% of wheat bran, 1% of gypsum, about 55% -60% of water content and natural pH. 1.8kg of the cultivation material is put into a 15 cm multiplied by 55 cm tubular bag, and the tubular bag is tied up to prepare a material rod. Each strain 108 bars were sterilized at normal pressure, and when the bar temperature reached 98℃or higher, the timing was started and maintained for 18 hours. After sterilization, opening the door when the temperature of the material rod is reduced to below 80 ℃, moving the material rod to a cooling chamber for cooling while the material rod is hot, and inoculating when the temperature of the material rod is reduced to below 28 ℃.

(2) Inoculating the material rod

Cleaning an inoculation box or an inoculation site before inoculation, sterilizing the surface of the inside, and putting cooled material bars, perforating bars, strains, 75% alcohol, alcohol lamps, aerosol sterilizing agents, bagging and other articles into the inoculation box or the inoculation site; igniting the aerosol disinfectant, sealing the inoculation box, and sterilizing; after the disinfection is finished, the inoculating personnel cleans the hands, and uses 75% alcohol to wipe the hands and the perforating rod; igniting the alcohol lamp, and burning and sterilizing the punching rod or the punching machine; 3 inoculation holes are evenly punched on the material rod, the diameter is about 1.5cm, and the depth is 2 cm-2.5 cm. The strain is plugged into the inoculating hole by hand in blocks, each hole is connected with a bag, and another rod is connected.

(3) Fungus stick culture

After inoculation, the fungus sticks are stacked in a wall type, the layer height is about 10 layers, and a passageway of 50cm is reserved between each two rows. After the mycelium grows to about 10cm, removing the bagging, puncturing 4-6 holes at the position of 2cm inside the mycelium and 1 cm-1.5 cm deep, performing first deflation, and discharging the fungus sticks to a culture rack. Each strain was randomly placed in 3 cells with 36 bars per cell. The environmental conditions are controlled between 24 ℃ and 26 ℃ by adopting measures such as capping sunshade net on the shed roof, spraying water, sagging sunshade net around and the like. After the hypha grows up the fungus stick, the second puncture ventilation is carried out, and 30 to 60 holes are formed in each stick. After the color of the fungus sticks is changed, the fungus sticks are deflated for the third time at the temperature of 12-20 ℃ for about 80 fungus sticks before the fungus sticks are removed from the bag. And (5) selecting proper weather for bag removal after exhausting.

(4) Fruiting management

The temperature difference between day and night is pulled to stimulate fungus sticks to produce mushrooms, and the fungus sticks are continuously operated for 7 to 15 days, so that primordia are promoted to occur. After the mushroom buds are formed, water is sprayed in the morning and evening, ventilation is carried out, and the temperature and the humidity in the mushroom shed are kept stable. The first batch of mushrooms are picked after being grown to 7-8 minutes of maturity. After the first tide mushroom is picked, stopping spraying water, increasing ventilation and reducing humidity, culturing for about 7 days, whitening hypha at the joint to be picked, and after the hypha is obviously recovered, timely supplementing water according to the water content of the fungus stick, so that the water content of the fungus stick reaches 60% -65%, and increasing the temperature difference to stimulate the second tide mushroom to grow. And (5) managing in a reciprocating manner until the harvesting is finished.

(5) Statistics

Picking fruiting bodies of 7 minutes of mature fungus caps, and recording fresh weight yield of different cells of each strain. Each strain randomly selects 100 fruiting bodies, and calculates fruiting body morphological data such as single mushroom weight, diameter of the fungus cover, thickness of the fungus cover, length of the fungus handle, upper diameter of the fungus handle, middle diameter of the fungus handle and the like of each fruiting body.

TABLE 5.2 cultivation yield data for the strains

TABLE 62 sub-body morphology data for strains

As can be seen from Table 5, the cultivation yield of L135-1 at 12 months was significantly higher than that of the L135 normal strain. And the average yield of each rod of the two is equivalent, and the statistical difference is not obvious. The results show that compared with the L135 normal strain, the L135-1 strain has concentrated fruiting and fruiting times and strong fruiting capacity of head tide, can reduce picking times and labor cost.

In addition, referring to FIG. 2, the hyphae growth rates of L135-1 and L135 in wood chip medium are exemplified, the mycelium growth rate of the L135-1 strain on the left side of the graph is significantly slower than that of the L135 normal strain on the right side, and the difference is obvious. As can be seen from the data in Table 6, FIG. 1 and FIG. 3, the weight, thickness, length and upper diameter of the stem of the fruiting body of strain L135-1 are smaller than those of the normal strain L135, which proves that the fruiting body and the fruiting body of Lentinus Edodes produced by strain L135-1 are obviously different from those of the normal strain L135.

Claims

1. A lentinula edodes strain L135-1, characterized in that the classification of the strain is named:Lentinula edodesthe strain is preserved in China general microbiological culture Collection center (CGMCC) in the year 2020, 12 and 07, and the preservation number is CGMCC No.21088.

2. Mycelium produced by a Lentinus edodes strain L135-1 as claimed in claim 1.

3. A protoplast produced by shiitake strain L135-1 as claimed in claim 1.

4. Fruiting body produced by Lentinus edodes strain L135-1 according to claim 1.

5. A spore produced by the lentinus edodes strain L135-1 as claimed in claim 1.

6. A stick produced by a lentinus edodes strain L135-1 as claimed in claim 1.

7. Use of a lentinula edodes strain L135-1 as claimed in claim 1 in lentinula edodes breeding.

8. Use of a lentinula edodes strain L135-1 as claimed in claim 1 for the preparation of lentinula edodes fruiting bodies, lentinula edodes mycelia.

9. The application according to claim 7, wherein the application comprises the steps of:

(1) Production of material rod

Filling the cultivation material of the strain in claim 1 into a cylinder bag with a certain specification, tying, preparing a material rod, sterilizing at normal pressure, and starting timing and keeping for 18 hours when the temperature of the material rod reaches more than 98 ℃; after sterilization, opening the door when the temperature of the material rod is reduced to below 80 ℃, moving the material rod to a cooling chamber for cooling while the material rod is hot, and inoculating when the temperature of the material rod is reduced to below 28 ℃;

(2) Inoculating the material rod

Cleaning an inoculation box or an inoculation site before inoculation, sterilizing the surface of the inside, and putting cooled material bars, perforating bars, strains, 75% alcohol, alcohol lamps, aerosol sterilizing agents and bagging objects into the inoculation box or the inoculation site; igniting the aerosol disinfectant, sealing the inoculation box, and sterilizing; after the disinfection is finished, the inoculating personnel cleans the hands, and uses 75% alcohol to wipe the hands and the perforating rod; igniting the alcohol lamp, and burning and sterilizing the punching rod or the punching machine; uniformly punching 3 inoculation holes on the material rod, wherein the diameter is about 1.5cm, and the depth is 2 cm-2.5 cm; the strain is plugged into an inoculation hole by hand in blocks, each hole is connected with a bag, and another rod is connected with the bag;

(3) Fungus stick culture

After inoculation, the fungus sticks are stacked in a wall type, the layer height is about 10 layers, and a passageway of 50cm is reserved between each two rows; after the mycelium grows to about 10cm, removing the bagging, puncturing 4-6 holes at the position of 2cm inside the mycelium and 1 cm-1.5 cm deep, performing first deflation, and discharging the fungus sticks to a culture rack; adopting the measures of capping sunshade net on the shed roof, spraying water and sagging sunshade net around, and controlling the environmental condition between 24 ℃ and 26 ℃; after the hypha grows up the fungus stick, carrying out secondary puncture ventilation, wherein each stick has 30-60 holes; after color change, the fungus sticks are subjected to third deflation at the temperature of 12-20 ℃ for about 80 fungus sticks, wherein the period is half month before bag removal; selecting proper weather for bag removal after exhausting;

(4) Fruiting management

10. The use according to claim 9, wherein the mushroom cultivation formulation comprises 79% of wood chips, 20% of wheat bran, 1% of gypsum, a water content of 55% to 60% and a natural pH.