CN115918438B

CN115918438B - Cultivation method of new black fungus variety

Info

Publication number: CN115918438B
Application number: CN202211627524.4A
Authority: CN
Inventors: 马宝宏; 解修超; 马明元
Original assignee: Shaanxi Sensheng Mushroom Technology Co ltd; Shaanxi University of Technology
Current assignee: Shaanxi Sensheng Mushroom Technology Co ltd; Shaanxi University of Technology
Priority date: 2022-12-16
Filing date: 2022-12-16
Publication date: 2024-05-14
Anticipated expiration: 2042-12-16
Also published as: CN115918438A

Abstract

The application provides a cultivation method of a new black fungus variety, which comprises the following steps: collecting wild black fungus: collecting wild black fungus in natural environments with different altitudes in a target area and sterilizing; culturing wild black fungus strains; primary screening in the growth period; and (3) re-screening: comprises mycelium re-screening and fruiting body re-screening; and (3) carrying out cellulase activity measurement, polysaccharide content measurement and genetic specificity identification on the re-screened wild strain, and finally screening to obtain a new black fungus variety. The cultivation method comprises the following steps: performing tissue separation on the new black fungus variety to prepare mother seeds; inoculating the mother seeds into a stock seed bag for culture to obtain stock seeds; inoculating the stock seeds into a production seed bag for culture to obtain production seeds; and inoculating the production seeds into the log holes and the bag material respectively, and cultivating by adopting two modes of log cultivation and bag material cultivation. The application breeds a new black fungus variety with high bioconversion rate and good product quality, and establishes a matched cultivation method.

Description

Cultivation method of new black fungus variety

Technical Field

The application relates to the technical field of edible fungus breeding and planting, in particular to a cultivation method of a new black fungus variety.

Background

The black fungus is one of main cultivated varieties in the production of edible fungi in China, is a precious colloid fungus for both medicine and food in China, is a health-care food accepted in the world, has soft texture and delicious taste, and has the reputation of meat in vegetables and treasures in fungi. Researches show that the dried black fungus product has high contents of protein, vitamins and iron, and the protein contains various amino acids, especially lysine and leucine, which are most abundant. The Auricularia auricula polysaccharide also has anticoagulant, blood lipid reducing, blood sugar reducing, antithrombotic, antioxidant, cough relieving, phlegm eliminating, and humoral immunity enhancing effects.

The black fungus can be wild or cultivated artificially, and the artificial cultivation of the black fungus also comprises two cultivation methods of using a log cultivation device and a cultivation device. For a long time, vast scientific and technological workers in China do a great deal of work on the aspect of fine variety breeding of black fungus, and a great deal of excellent strains are bred. By 2008, 22 black fungus varieties are approved by national edible fungus varieties.

The Qinba mountain area has proper climate and rich fungus materials, and is suitable for the wide area for the growth of the black fungus, has great development potential, and the black fungus is also a main fungus crop in the Qinba mountain area. However, as the cultivation area of the black fungus in the Qinba mountain area is continuously enlarged, the problems existing in the production are increasingly prominent. The existing black fungus strain has low bioconversion rate and low yield, even has the phenomenon of harvest failure, and seriously damages the benefits of producers. Therefore, the research on wild planting resources is developed, and the breeding of excellent strains aiming at the ecological environment of Qinba mountain areas has important theoretical and practical significance.

Disclosure of Invention

The application provides a cultivation method of a new black fungus variety, which is used for solving the problems mentioned in the background art.

The cultivation method of the new black fungus variety comprises the following steps:

s1, collecting wild black fungus specimens: collecting wild black fungus specimens in natural environments with different altitudes in a target area, sterilizing the surfaces of the wild black fungus specimens with 0.1% mercuric chloride, and cleaning the wild black fungus specimens with sterile water for 3-5 times.

S2, culturing wild black fungus sample strains: the sterilized wild black fungus specimen is subjected to tissue separation under aseptic operation condition, inoculated on a culture dish filled with CPDA culture medium and cultured at 28 ℃.

S3, primary screening in the growth period: observing the growth condition of hyphae, and selecting the strain with vigorous hyphae growth vigor and hyphae growth speed greater than 0.40cm/d as the primary screening wild strain.

S4, re-screening: and (3) carrying out activation and expansion culture on the primary screening wild strain obtained in the step (S3) to prepare a stock seed, inoculating the stock seed into a culture medium which is contained in a polypropylene plastic bag and is subjected to sterilization treatment according to a sterile operation procedure, and respectively carrying out mycelium rescreening and fruiting body rescreening in a bag cultivation mode to obtain the rescreening wild strain.

S5, character identification: and (3) carrying out cellulase activity measurement, polysaccharide content measurement and genetic specificity identification on the re-screened wild strain, and finally screening to obtain a new black fungus variety.

The mycelium re-screening step comprises the following steps: and (5) performing mycelium re-screening according to the mycelium growth condition in the culture medium.

The fruiting body re-screening step comprises the following steps: and (3) observing the fruiting situation of the black fungus in the culture medium, carrying out fruiting body rescreening according to the agronomic characters and the yield, and simultaneously selecting a strain which is middle-early maturing, high in yield, high in bioconversion rate and good in commodity characters as a rescreening wild strain by combining with a mycelium rescreening result.

The commercial properties refer to the fruiting body shape (monolithic or chrysanthemum), color, size, thickness, rotten and worm-eaten ear and smell of the ear.

Agronomic traits are fruiting body morphology, fruiting time and fruiting temperature after fruiting.

Middle-early ripening: according to different accumulation temperatures required in the black fungus after-ripening process, the black fungus can be divided into three varieties of early, medium and late ripening, and when in bag material cultivation, the early ripening variety hypha grows up to full bags quickly or the opening is cut for 4-7 days when the bag is just full; marking the mouth for 15-25 days when the mycelium of the late-maturing variety grows and fills the bag, and discharging the ear; the variety between early and late maturing is called medium-maturing variety.

The re-screened wild strain should satisfy: (1) agronomic traits: the fruiting body form (single piece or chrysanthemum shape) after the fruiting body is in a single piece shape is preferable, the fruiting time is short, the fruiting temperature is low, and the range is wide. (2) commodity traits: the fruiting body is preferably in the form of a single sheet, has black brown color, large and even and thick fungus, has no rotten fungus and worm-eaten fungus, has no peculiar smell, and has peculiar smell of black fungus.

Optionally, in step S1, when the wild black fungus specimen is collected, a natural environment with a target area altitude of more than 800m is selected.

Optionally, step S2 further includes: after the wild black fungus specimen tissue is separated, inoculating each two different strains on a culture dish of the same CPDA culture medium, performing opposite culture at 28 ℃, and screening the wild black fungus strain with antagonism.

Optionally, the formula of the CPDA medium is as follows: 200g of potato, 20g of glucose, 3g of KH ₂PO₄5g,MgSO₄·7H₂ O, 10mg of VB1, 20g of agar powder, 1000ml of water and pH value of 6.5.

Optionally, the cultivation substrate comprises: 78.0% of broadleaf wood dust, 16.0% of wheat bran, 1.0% of sucrose, l.0% of gypsum, 1.0% of lime, 1.5% of bean cake powder, 1.5% of corn flour, 56-62% of water content and 1.1kg of culture medium in each bag.

Optionally, sterilizing the culture medium under normal pressure or high pressure, wherein the temperature of the normal pressure sterilization is 100-110 ℃, and the sterilization time is 10-12h; the high-pressure sterilization temperature is 124-126 ℃, the sterilization time is 3-4h, and the pressure is 0.14-0.15MPa.

The cultivation method of the new black fungus variety screened according to the breeding method comprises the following steps:

(1) Preparing mother seeds: performing tissue separation on the new black fungus variety under the aseptic condition, inoculating the strain to the inclined plane of the mother culture medium, and culturing at constant temperature in a 28 ℃ incubator until mycelium grows to be full of the inclined plane to obtain mother seeds;

(2) Manufacturing an original seed: aseptically inoculating the mother seeds into a stock seed bag, and culturing at constant temperature in a 28 ℃ incubator until mycelia grow full of the stock seed bag to obtain stock seeds;

(3) And (3) manufacturing production seeds: sterile inoculating the stock seeds into a production seed bag, and culturing at constant temperature in a 28 ℃ incubator until mycelia grow full of the production seed bag to obtain production seeds;

(4) Cultivation: the cultivation is carried out by adopting two modes of log cultivation and bag material cultivation, and the production seeds are respectively inoculated into log holes and bag materials for cultivation until the ears are produced.

Optionally, the bag material comprises the following components: 77.0% of broadleaf wood dust, 16.0% of wheat bran, 3% of beef extract, 1.0% of sucrose, 0.8% of gypsum, 0.6% of lime, 1.0% of corn meal, 0.3% of mulberry powder, 0.1% of potassium cinnamate, 0.2% of magnesium phosphate and 56-62% of water content.

Optionally, the log cultivation includes:

(1) Preparing log: broad-leaved tree sticks with the length of 100-120cm and the diameter of 8-12cm are selected as the cultivation sections.

(2) Ear field selection and airing rack: the cultivation site is selected in the places with the sun facing leeward, air circulation, water source approaching and irrigation and drainage convenience, and the clean field of the ear site is leveled and sterilized. Meanwhile, the log is piled up in the field in a 'well' -shaped mode, the piled up height is 1.3-1.5m, and the piled up is turned over once in 10-15 days, so that the water content of the log is less than 50%.

(3) Dibbling: opening holes on the surface of the log, wherein the distance between the holes is 7-12cm, the diameter of the holes is 14-16mm, the holes are deep into xylem for 1-2cm, sowing the seeds into the holes, sealing the holes with wood plugs or yellow cement after sowing, and the sowing temperature is above 8 ℃.

(4) Piling up bacteria: discharging the inoculated wood blocks in a well shape to form a log pile, covering the log pile with a plastic film, maintaining the temperature at 18-24 ℃, spraying water to Duan Mudui after 10-14 days, and maintaining the relative air humidity at 85-95%.

(5) Bulk dump drainage: when white fungus film is on the surface of the strain in the hole and white hypha is on the bottom of the hole, raising one end of the log, discharging according to fish scales, turning once in 10-15 days at intervals of 5-6cm, and observing the ear condition in 2-3 months.

(6) Lifting a shed, loading the shed on a frame and harvesting: when 45-50% of the log leaves ears, putting the log by adopting a herringbone frame method or a fish scale type swing method, keeping the relative humidity of air at 85-95%, preferably at 22-35 ℃, and keeping the temperature at 22-30 ℃, so that fruiting bodies grow rapidly; after the earpieces are ripe, picking off the earpieces with roots, immediately airing, spraying water to log after 5-7 days, keeping the relative air humidity to be 65-85%, recovering the growth of hyphae, and forming earbuds again.

Optionally, in the cultivation of the log, each log is inoculated with 0.2-0.24kg of the new black fungus variety.

Optionally, the bag cultivation includes:

(1) Mixing and bagging: the components of the bag material are uniformly stirred according to the proportion, then the bag material is put into a cultivation bag, and high-pressure sterilization is carried out.

(2) Inoculating: the sterilized cultivation bag is cooled to room temperature and then is moved into an inoculation chamber, production seeds are inoculated into bag materials according to aseptic operation, the inoculated production seeds are placed into a culture chamber for culture, the temperature of the culture chamber is kept at 22-26 ℃, the relative humidity of air is kept at 50-60%, hyphae start to grow, and the culture is carried out for 60-65 days.

(3) And (5) punching: after hypha grows fully, the cultivating bags are moved into a cultivating room, holes are uniformly formed on the surface of the cultivating bags, the diameters of the holes are 1-2cm, the distances between the holes are 5-6cm, 10-12 holes are formed in each cultivating bag, the cultivating bags are hung on a bedstead upside down after the holes are formed, the cultivating bags are covered by plastic films, spraying is carried out every day, the relative humidity of air is kept at 65-70%, and the temperature is 15-20 ℃ for 7-10 days, so that the ears can be produced.

(4) Ear-out management: after the ear is removed, the relative humidity of air is kept to be 80-85%, ventilation is enhanced, after 10-15 days, when the ear pieces are flat and the edge is curled inwards, the ear roots are changed from large to small, harvesting is started, and after 5-10 days, ear buds are formed again.

Optionally, in the bag material cultivation, each cultivation bag is inoculated with 5-10g of production seeds of the new black fungus variety.

Optionally, the application performs exemplary cultivation on a new black fungus variety, and comprises the following specific steps: different ecological environments are selected in Qinba mountain areas, the new black fungus variety is used as a test strain, the main cultivated black fungus variety in Qinba mountain areas is used as a comparison variety, and the expansion test is carried out by two modes of log cultivation and bag cultivation.

Alternatively, in the exemplary cultivation, the area with the height of the Qinba mountain area of 400-1300m is selected for cultivation.

The cultivation method of the new black fungus variety provided by the application breeds a new black fungus variety with stable genetic character, strong capability of resisting mixed bacteria infection, high bioconversion rate and good quality, and establishes a matched cultivation method, and has the following beneficial effects:

(1) Collecting wild black fungus specimens in natural environments with different altitudes in Qinba mountain areas, and performing tissue separation, culture, primary screening and secondary screening in a growth period under aseptic operation. During rescreening, the initially screened wild strain is inoculated into a culture medium, and through mycelium rescreening and fruiting body rescreening, the strain which is middle-early maturing, high in yield and high in bioconversion rate and has excellent commodity characters is selected as the rescreened wild strain, cellulase activity, polysaccharide content measurement and genetic specificity identification are carried out on the rescreened wild strain, and finally, a new black fungus variety is obtained through screening.

(2) The mycelium growth conditions of the new variety of the black fungus are explored and optimized, and meanwhile, a cultivation technology system matched with the new variety of the black fungus is established, so that the conclusion is as follows: the new black fungus variety has strong adaptability, and is suitable for both cut-log cultivation and bag material cultivation; the cultivation method is suitable for wide planting range, and is suitable for cultivation in Qinba mountain areas with low altitude (400-700 m), high temperature and high humidity, plain and hilly areas and mountain areas with higher altitude (1300 m). And the yield is high Yu Qinba the current main cultivated variety ear 268 in mountain area.

(3) When the bag material is cultivated, a small amount of mulberry powder and potassium cinnamate are added into the bag material, the addition of the mulberry powder and the potassium cinnamate can play a synergistic effect, the growth of the new black fungus variety is promoted in the mycelium growth period and the growth period of the black fungus sheet after the black fungus is produced, the yield and the bioconversion rate of the new black fungus variety are improved, the period from inoculation to fungus picking of the new black fungus variety can be shortened, the return period of a planter is shortened, the economic benefit is remarkable, and the method is suitable for wide planting and popularization.

(4) The new black fungus variety screened by the application belongs to fungus phylum, basidiomycete, yinyales, auriculariaceae (Auriculariales) and Auricularia (Auricularia) fungi. The bacterial colony is white, the edge is neat and uniform, the mycelium is thick, and the bacterial colony is a medium-temperature type. Is suitable for growth in the acidic environment, and has the highest growth speed when the pH value is 5.0-6.4 and the pH value is 6.2. The mycelium forms fruiting body rapidly in the planting process, 3-4 months earlier than other cultivars, and the fruiting body is monolithic, uniform and thick. The new black fungus variety has the characteristics of stable genetic character, strong capability of resisting mixed bacteria infection, high bioconversion rate, good yield, strong stress resistance and excellent product quality.

Drawings

In order to more clearly illustrate the embodiments of the present application or the technical solutions of the prior art, the following description will briefly explain the drawings used in the embodiments or the description of the prior art, and it is obvious that the drawings in the following description are some embodiments of the present application, and other drawings can be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a flowchart of a method for breeding a new variety of Auricularia auricula-judae provided by an embodiment of the present application;

FIG. 2 shows an electrophoresis spectrum of the nutrition hyphae esterase isozymes of 10 black fungus strains according to an embodiment of the application;

FIG. 3 is a diagram showing esterase isozymes patterns of 10 black fungus strains according to an embodiment of the application; 1 is Sensheng No. 1, 2 is ear 238,3 is ear 268,4 is ear 629,5 is ear 801,6 is ear 913,7 is Qindan 3#,8 is Qindan 4#,9 is Guoxing No. 1, and 10 is Xinke No. 1;

FIG. 4 is a graph of cluster analysis of 10 strains of Auricularia auricula according to one embodiment of the present application;

FIG. 5 is a phylogenetic tree of Sensheng No. 1 bacteria and control varieties according to an embodiment of the present application; 1 is Xinke No. 1,2 is ear 629,4 is ear 238,7 is Qin Dan No. 3, 8 is ear 268,9 is Sensheng No. 1, 10 is ear 801, 11 is Qin Dan No. 4, and 13 is ear 913;

FIG. 6 is an antagonistic experiment diagram of the new variety Sensheng No. 1 of black fungus provided by an embodiment of the application;

FIG. 7 shows the effect of temperature on growth of new variety Sensheng No. 1 of Auricularia according to one embodiment of the present application;

FIG. 8 shows the effect of pH of CPDA medium on growth of new variety Sensheng No. 1 of Auricularia according to one embodiment of the present application;

FIG. 9 is a front morphology of the colony of the new variety of Auricularia, sensheng No. 1;

FIG. 10 is a reverse side morphology of the colony of the new variety of Auricularia, sensheng No. 1;

FIG. 11 is a microscopic view of the hypha of strain No. 1 of the new variety of Auricularia at 40X.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the embodiments of the present application more clear, the technical solutions in the embodiments of the present application will be clearly and completely described below, and it is apparent that the described embodiments are some embodiments of the present application, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are also within the scope of the application.

Example 1

S1, collecting wild black fungus specimens: collecting wild black fungus specimens in natural environments with different altitudes in Qinba mountain areas, sterilizing the surfaces of the wild black fungus specimens with 0.1% mercuric chloride, and cleaning with sterile water for 3-5 times.

Specifically, 12 wild black fungi grown in natural environments with heights above 800m and different ecological types are selected as specimens in a target area (in this embodiment, qinba mountain areas are taken as an example), because in the Qinba mountain areas, the wild black fungi grown in the environment with higher altitudes have better quality and better taste than the wild black fungi with lower altitudes. Wild black fungus in the original forests of Ningqiang county, chengba county, pingqiang county and Mian County is selected and numbered Lueyang 1#, lueyang 2#, lueyang 3#, chengqian 1#, chengqian 2#, pingqian 1#, pingqian 2#, pingqian 3#, ningqian 1#, ningqian 2#, ningqian 3#, mian County 1#, respectively. Sterilizing the collected wild black fungus specimen, soaking the wild black fungus specimen in 0.1% mercuric chloride for 1-2min, and washing the mercuric chloride remained on the surface of the wild black fungus specimen by using sterile water for 3-5 times to wash away the mercuric chloride remained on the surface of the wild black fungus specimen, thereby playing a role in sterilizing.

Meanwhile, the step S2 further comprises the following steps: after the wild black fungus specimen tissue is separated, inoculating each two different strains on a culture dish of the same CPDA culture medium, performing opposite culture at 28 ℃, and screening the wild black fungus strain with a far relationship (namely antagonism exists).

The antagonism phenomenon between the colonies of two varieties in the opposite culture is observed, and the judgment basis of the antagonism phenomenon is the distinguishing identification antagonism response of edible fungus strains of the agricultural standard NY/T1845-2010. If two black fungus colonies do not generate antagonistic lines but grow together, the two black fungus strains have close relativity and are quite likely to be the same strain, meanwhile, the growth conditions of mycelia are combined and observed, one of the mycelia with poor growth conditions is eliminated, and if the two black fungus colonies do not generate antagonistic lines and the growth conditions of mycelia are close, one of the strains is eliminated randomly. If no elimination is performed, repeated researches are performed, and the workload is increased.

Specifically, the tissue separation method is carried out by a conventional method, and the present application is not described in detail. The wild black fungus strain with the separated tissues is inoculated on a culture dish with the diameter of 90mm, the culture medium is CPDA culture medium, and the culture is carried out at 28 ℃ to enable hyphae to develop and grow. In the counter culture, two different strains were inoculated on a petri dish of the same CPDA medium, the diameter of the petri dish was 90mm, and hyphae grew to form colonies.

Wherein, the formula of the CPDA culture medium is as follows: 200g of potato, 20g of glucose, ₂PO₄5g,MgSO₄·7H₂O 3g,VB₁ mg of KH, 20g of agar powder, 1000ml of water and pH value of 6.5.

Specifically, the growth of hyphae is observed every other day, after 7 days of culture in a culture dish, the diameter of bacterial colonies is recorded, and the strain with white hyphae, vigorous hyphae growth and hyphae growth speed greater than 0.40cm/d is selected as a primary screening wild strain.

The mycelium growth rate was calculated as follows:

Hypha growth rate (cm/d) =colony radius (cm)/growth days (d).

Selecting a wild black fungus strain with a relatively long relationship according to the opposite culture result, and selecting the wild black fungus strain with the relatively long relationship and a fast hypha growth speed as a primary screening wild strain by combining the hypha growth condition in the step S3.

The hypha growth of the different strains was recorded in 3 replicates per 100 treatments, and the results are shown in Table 1.

TABLE 1 growth rate (cm/d) of hyphae of Auricularia strains in CPDA culture medium

Strain	Repeat 1	Repeat 2	Repeat 3	Average value of
					Lueyang 1#	0.54	0.52	0.50	0.52
Lueyang 2#	0.47	0.49	0.47	0.47
					Lueyang 3#	0.41	0.44	0.44	0.43
Dam 1#	0.46	0.48	0.45	0.46
					Dam 2#	0.50	0.46	0.44	0.46
Pad 1 #)	0.50	0.47	0.51	0.49
					Pad 2#	0.43	0.43	0.47	0.44
Pad 3#	0.39	0.38	0.40	0.39
					Ningqiang 1 #)	0.37	0.39	0.40	0.38
Ningqiang 2 #)	0.48	0.49	0.47	0.48
					Ningqiang 3 #)	0.48	0.46	0.45	0.46
Mian County 1#	0.36	0.35	0.35	0.35

The results in Table 1 show that at 28 ℃, the mycelium growth rate of Lueyang # is the fastest and reaches 0.52cm/d, and then the mycelium growth rate of Buddha's 1#, ningqiang 2#, lueyang 2#, retaining dam 1#, retaining dam 2#, and Mian County # is the slowest and is 0.35cm/d. According to the combination of the opposite experiment results, no antagonistic line is generated between the colonies of Lueyang # and Lueyang # and between the colony of the fingerprinting 1# and the colony of the fingerprinting 3#, so that the relatedness between Lueyang # and Lueyang # and between the fingerprinting 1# and the fingerprinting 3# is relatively close, the growth conditions of hyphae are combined, lueyang # and the fingerprinting 3# are eliminated, the Ning1 # and Mian County # are combined, and finally the 8 wild strains of Lueyang # and Lueyang # are screened, the reserved dam 1# and the reserved dam 2# are screened, and the 8 wild strains of Ning2 # and Ningqiang 3# and the fingerprinting 1# and the fingerprinting 2# are screened as initial wild strains.

Specifically, 8 primary screening wild strains are subjected to activation and expansion culture to prepare stock strains, and the activation and expansion culture mode adopts industry conventional means and is not described in detail herein. The cultivation substrate comprises: 78.0% of broadleaf wood dust, 16.0% of wheat bran, 1.0% of sucrose, l.0% of gypsum, 1.0% of lime, 1.5% of bean cake powder, 1.5% of corn flour, 56-62% of water content and 1.1kg of culture medium in each bag. The culture medium meets the technical requirements of safety of culture medium for pollution-free edible fungi for food of NY 5099-2002.

Specifications of polypropylene plastic bag: the specification of the 15cm material bag is resistant to 126 ℃ and accords with the polypropylene plastic bag specified in GB9687-1988 polyethylene molding hygienic Standard for food packaging.

Sterilizing the culture medium: sterilizing at normal pressure or high pressure at 100-110deg.C for 10-12 hr; the high-pressure sterilization temperature is 124-126 ℃, the sterilization time is 3-4h, and the pressure is 0.14-0.15MPa.

S41, re-screening hypha: and (5) performing mycelium re-screening according to the mycelium growth condition in the culture medium.

Specifically, when 8 primary screening wild strains are observed for bag cultivation, mycelium re-screening is carried out on the mycelium in a cultivation substrate, and the strains with high mycelium growth speed, white and dense mycelium, good mycelium growth vigor, short bag filling days when the mycelium grows to be full of bags and large number of lug bases are selected. Each strain was treated in 30 bags, which meets the requirement of large statistical samples, and 3 replicates were used to record the hypha growth of different strains, and the results are shown in Table 2.

TABLE 2 hyphal growth of Auricularia strains on cultivation Medium

Note that: + indicates that hypha grows normally; ++ indicates vigorous hypha growth; ++ indicates hyphae the growth vigor is very vigorous.

S42, re-screening fruiting bodies: and (3) observing the fruiting situation of the black fungus in the culture medium, carrying out fruiting body rescreening according to the agronomic characters and the yield of the black fungus, and simultaneously combining with the mycelium rescreening result, and selecting the strain with high yield, high bioconversion rate and good commodity characters as a rescreening wild strain.

Specifically, the strain comparison test is carried out on the strain of the primary screening wild strain under the conditions of the same volume of material, the same dibbling amount and the same cultivation environment, and the fruiting body shape, the fruiting time, the yield per bag and the bioconversion rate of the strain of the primary screening wild strain are observed, and the results are shown in Table 3.

TABLE 3 agronomic traits and yield comparison of Auricularia strains

In Table 2, the result of re-screening hyphae of 8 primary screened wild strains on a culture medium in a bag cultivation mode shows that the growth speed of the hyphae of Lueyang # 1 strain is the fastest, the average growth speed is 0.52cm/d, the hyphae are thick and white, and the occurrence of ear bases is few. Hypha growth of three strains of Ningqiang 3#, buddhist apron 2# and Ningqiang 2# is slower, average growth speed is 0.40cm/d, 0.41cm/d and 0.44cm/d respectively, and the number of days of filling bags is more than 40 d. And hyphae of the fingered citron # 2 and the Ningqiang # 2 are thicker, white, ear-based and continuous.

The fruit body is better in form of single piece, because the single piece of black fungus has small root, high edible rate, and the chrysanthemum black fungus has good appearance, but large root and low edible proportion. In Table 3, the re-screening results of the fruiting bodies show that the fruiting bodies of the reserved dam 1#, the fingered citron 2# and the Ningqiang 2# are all in a single-piece shape, and meet the breeding target strain. Simultaneously, the strain with shorter ear-out time, high yield and high bioconversion rate is preferentially selected, the Ningqiang No. 2 ear-out time is shortest, and the bioconversion rate is higher, which reaches 76.34%; next is Lueyang #. In addition, in the planting process, besides Lueyang # 1, ningqiang 2# forms fruiting bodies fastest, 3-4 months earlier than other varieties, and the fruiting bodies are single-piece, uniform and thick. And combining the mycelium re-screening results in Table 2, and comprehensively evaluating to obtain 5 re-screened wild strains, namely Lueyang # 1, 1# retaining dam, 2# Ningqiang and 2# fingered citron.

S5, character identification: and (3) carrying out cellulase activity measurement, polysaccharide content measurement and genetic specificity identification on the re-screened wild strain, and finally screening to obtain a new black fungus variety, which is named as Sensheng No. 1.

Cellulase activity determination of S51, 5 re-screened wild strains

Culturing 5 re-screened wild strains according to the conditions in the re-screening of the fruiting bodies, and measuring the cellulase activity of hyphae at 20d of the fungus age. As mycelium grows more actively when the bacterial age is 20d, the measurement of cellulase activity is facilitated. Periodically sampling, wherein the weight of each sampling is 20.0g, adding 40mL of physiological saline, rapidly sealing, extracting enzyme solution in water bath at 40+ -l ℃, filtering, absorbing 3mL of enzyme solution, and measuring cellulase activity at mycelium growth stage by 3, 5-dinitrosalicylic method, wherein the result is shown in Table 4.

Table 45 cellulase Activity of the rescreened wild strains

Strain number	Enzyme activity (U/g)
		Lueyang 1#	3839
Dam 1#	2789
		Dam 2#	4142
Ningqiang 2 #)	4712
		Pad 2#	2523

Cellulase is secreted during the mycelium growth process, and cellulose is hydrolyzed into monosaccharide as energy necessary for mycelium metabolism, which is always accompanied with the growth of black fungus mycelium until the formation of black fungus fruiting body. Therefore, the higher the cellulase activity is, the more favorable the growth of mycelium is. As can be seen from Table 4, the cellulase is strongest in Ningqiang # 2 hyphae and is retained for # 2 times.

S52, polysaccharide content determination of 5 re-screened wild strains

Taking 5 fruiting bodies of the re-screened wild strain, accurately weighing 4.00g after impurity removal, and soaking in a small amount of water for 2h. The excess water was sucked by the filter paper repeatedly, and 50mL of distilled water was added thereto to carry out high-pressure extraction (0.1 MPa,121 ℃ C. Extraction). Filtering to obtain clear liquid, removing impurities by Seveg times of frozen ethanol to precipitate polysaccharide. The OD value was measured at 550nm by anthrone colorimetry, and the reducing sugar content was calculated from the standard curve. The results are shown in Table 5.

Table 55 fruit body polysaccharide content of double-screened wild strains

Strain number	Total sugar content (mg/100 g)
		Lueyang 1#	1545.7
Dam 1#	1330.6
		Dam 2#	1857.3
Ningqiang 2 #)	1729.7
		Pad 2#	1648.9

Polysaccharide substances contained in the black fungus can enhance the immune function of organism cells, so that strains with more polysaccharide content are selected during screening. As shown in Table 5, the polysaccharide content in the fruiting body of the retaining dam 2# is the greatest, the fruiting body of the retaining dam 2# is single-piece after the combination of the screening step, and the yield and the bioconversion rate are obviously higher than those of the retaining dam 2#, so that the combination of the evaluation results finally selects the novel black fungus strain with the retaining dam 2# as the black fungus strain with excellent properties, and the novel black fungus strain is named Sensheng No. 1.

Further, the selected new black fungus variety Sensheng No. 1 is subjected to genetic specificity identification through a protein electrophoresis experiment and DNA molecular fingerprint research.

The names and sources of the respective test strains for genetic specificity identification are shown in Table 6.

TABLE 6 names and sources of test strains

Name of the name	Source(s)
		New family No. 1	Shaanxi Ankang institute of agriculture
Qin sheet 1 #)	Shanxi Ningqiang fungus institute
		Qin sheet 3 #)	Shanxi Ningqiang fungus institute
Qin Shan 4#	Shanxi Ningqiang fungus institute
		Ear 268	Fujian Sanming fungus institute
Ear 801	Shaanxi province edible and medicinal fungus engineering research center
		Ear 913	Shaanxi province edible and medicinal fungus engineering research center
Ear 268	Shaanxi province edible and medicinal fungus engineering research center
		Ear 629	Shaanxi province edible and medicinal fungus engineering research center
Ear 238	Shaanxi province edible and medicinal fungus engineering research center
		Guoxing No. 1	Research institute for black fungus cultivated in Xingzhi of Lueyang country of Shaanxi
Shensheng No. 1	Ningqiang county Wuding Guan Linchang

S53, protein electrophoresis experiment: the specific method refers to an agricultural standard NY/T1097-2006 edible fungus strain authenticity identification esterase isoenzyme electrophoresis method. Liquid culture is carried out by taking domestic part of main cultivated black fungus varieties as comparison varieties, the bred new black fungus variety Sensheng No. 1 is taken as a test strain, the protein electrophoresis test is carried out by preparing nutrition hyphae, and the relationship between the new black fungus variety Sensheng No. 1 and the new black fungus variety is measured.

And (3) performing esterase isoenzyme electrophoresis on the nutrition hyphae of the tested black fungus strains under the electrophoresis condition that the concentration of the separating gel is 12.0% and the concentration of the concentrated gel is 5.0%, so as to obtain an esterase isoenzyme electrophoresis spectrogram of the nutrition hyphae of the 10 black fungus strains shown in figure 2. The control varieties are ear 238, ear 268, ear 629, ear 801, ear 913, qin Shan #, qin Shan #, guoxing No. 1 and Xinke No. 1.

The results in FIG. 2 show that the esterase isoenzyme activities of the 10 tested black fungus strains were all relatively high. There are 36 bands of esterase isoenzyme in the electropherograms, and the enzyme bands of each strain are between 2 and 5, and most between 3 and 5. Wherein, 5 esterase isoenzyme bands appear in ear 629 and ear 801, 4 esterase isoenzyme bands appear in ear 238, ear 913 and Guoxing No. 1, 3 esterase isoenzyme bands appear in Sensheng No. 1, ear 268, qin Shan 3#, xinke No. 1, and Qin Shan 4# is the least, and only 2 isoenzyme bands are present.

According to the difference of enzyme bands in the enzyme spectrum, an esterase isoenzyme pattern diagram of 10 black fungus strains shown in figure 3 is drawn. In fig. 3, 1 is sen No. 1,2 is ear 238,3 is ear 268,4 is ear 629,5 is ear 8016 is ear 913,7 is Qindan 3#,8 is Qindan 4#,9 is Guoxing No. 1, and 10 is Xinke No. 1.

The results in FIG. 3 show that 10 different enzyme spectrum types occur for 10 black fungus strains, without the same enzyme spectrum type, wherein the banding patterns for Sensheng No. 1 and the strains of ear 268, ear 238 and ear 913 are substantially similar. 10 test strains had 2 common enzyme bands at Rf values of 0.615 and 0.510, which are characteristic enzyme bands of the species Auricularia.

Meanwhile, 10 black fungus strain esterase isozymes Rf values shown in table 7 and 10 black fungus strain esterase isozymes enzyme spectrum similarity coefficients (Sc) shown in table 8 are obtained, and 10 black fungus strain cluster analysis diagrams shown in fig. 4 are constructed.

TABLE 7 10 Auricularia strains esterase isozymes Rf

The results in Table 7 show that the enzyme bands of the esterase isozymes of the 10 black fungus strains have 8 different bands, the mobility of which is between 0.300 bands, and the mobility of which is respectively 0.300, 0.345, 0.405, 0.445, 0.510, 0.530, 0.565 and 0.615.Rf is 0.510 and 0.615, and the tested black fungus strains have bands, which are common esterase enzyme bands of 10 black fungus strains. The I and II enzyme bands are unique to ear 801; the III enzyme band only appears in the spectrograms of the ear 629, the ear 801 and the Xinke No. 1; IV enzyme bands appear in the enzyme spectra of ear 238, ear 629, ear 913 and Guoxing No. 1; VII only appears in the enzyme spectrums of Qin Dan No. 3 and Guoxing No. 1. The difference of the bands of different strains can be used as the basis for distinguishing 10 black fungus strains from each other. From Table 7, the distribution frequencies of the different enzyme bands can be calculated, and the distribution frequencies of the enzyme bands I to VIII are as follows: 10%, 30%, 40%, 100%, 50%, 20%, 100%. The distribution frequency of the V enzyme band and the VIII enzyme band is 100% at the highest, and the distribution frequency of the VI enzyme band is 20%, and the distribution frequency of the I enzyme band and the II enzyme band is 10% at the smallest, which indicates that each strain of the black fungus has certain genetic similarity.

TABLE 8 similarity coefficients of esterase isozymes (Sc) of 10 Auricularia strains

As can be seen from Table 8, the genetic similarity coefficient (Sc) of 10 strains of Auricularia was between 0.375 and 1.000, and the smaller the similarity coefficient between the two strains, the farther the relationship between the two strains was. Wherein, the similarity degree between the ear 801 and the ear 238, the ear 913 and the Guoxing No. 1 is the lowest, and the similarity coefficient is only 0.375; second, the similarity coefficient of ear 801 to Sensheng No. 1 and ear 268 is 0.500, indicating a far relationship. While the similarity coefficient for Send No. 1 and ear 268 is 1.000, and that for Send No. 1 and ear 238, ear 913, qin Shan is 0.875, indicating that they may be from the same species, even closely related variant types.

Esterase isozymes are expressed by genes and the expression of the genes on a molecular level, and the distance of the genetic relationship among strains can be reacted from the degree of difference of the genotypes of the strains. As can be seen from fig. 4, ear 801 and ear 913, ear 238 and strain 1 have a large difference in enzyme spectra, indicating that they are far apart; the enzyme spectra of Sensheng No. 1 and ear 268, ear 238 and ear 913 have certain similarities, which means that Sensheng No. 1 and ear 268, ear 238 and ear 913 may be derived from the same variant type, even more closely related variant types.

S54, DNA molecular fingerprint research: the mycelium genome total DNA is extracted by adopting a modified CTAB method. The common primers ITS1 and ITS4 (ITS 1:5' -TCCGTAGGTGAACCTGC-3; ITS4:5' -TCCTCCGCTTATTGATATGC-3 ') are selected to amplify 5.8S rDNA and ITS1 and ITS2 gene fragments on both sides. And submitting the sequenced sequence to NCBI for Blast retrieval, downloading data with higher homology, and generating a file in Fasta format. The resulting sequences were manually corrected and aligned using Clusta-X software. And clustering by using Mega5 according to an N-J method, selecting 1000 repeated Bootstrap value analysis, and constructing a phylogenetic tree. The phylogenetic tree of Sensheng No. 1 bacteria and the control variety shown in FIG. 5 was obtained. Wherein, 1 is Xinke No. 1,2 is ear 629,4 is ear 238,7 is Qin Dan No. 3, 8 is ear 268,9 is Sensheng No. 1, 10 is ear 801, 11 is Qin Dan No. 4, and 13 is ear 913.

As illustrated in FIG. 5, the novel families 1, qin Shan, qin Shan are concentrated on one branch, indicating that the genetic distances of the 3 strains are relatively close; likewise, sensheng No. 1 and ear 268, ear 238 and ear 913 focused on one large branch, indicating that the genetic distances between two strains of the 4 strains were relatively close; ear 801 is located in a separate branch, indicating that the strain is phylogenetically different from other strains and has a relatively distant relationship. Wherein, the sen 1 and black fungus (NCBI accession number: JN 712676) are clustered together, and the similarity is highest; the novel strain obtained by the application, sensheng No. 1, is a strain of Auricularia auricula (NCBI accession numbers HQ388383 and HQ 388355) is closer to the Auricularia auricula.

S55 antagonism experiment of new variety Sensheng No. 1 of black fungus

The antagonism reaction is applicable to different types of fungi combined with foreign matters, and the antagonism reaction forms of different types are different and can be generally divided into three types (one of morphological characteristics of antagonism reaction specified in NY/T1098-2006 edible fungus variety description technical specifications) of a bulge type (ridgy), a ditch type (chimb) and an isolation type (separate), and the judging basis of antagonism reaction is agricultural standard NY/T1845-2010 edible fungus variety distinguishing identification antagonism reaction. Has antagonistic reaction, and the two are different varieties.

Based on the genetic specificity identification result, antagonism experiments are carried out on Sensheng No. 1, the ear 268, the ear 238 and the ear 913, and the genetic relationship of the new black fungus variety Sensheng No. 1 is further analyzed. The new variety Sensheng No. 1 of the black fungus is used as a test strain, the black fungus 268, the black fungus 238 and the black fungus 913 are used as control strains, and the black fungus is cultured on a CPDA culture medium plate at 28 ℃ to analyze the relationship between the new variety Sensheng No. 1 of the black fungus, and the result is shown in FIG. 6.

FIG. 6 is a graph showing antagonism of the new variety of black fungus, sensheng No. 1, and shows that the hyphae of black fungus, sensheng No. 1, have antagonism with the hyphae of the ears 268, 238 and 913 to different extents, and the antagonism with the ears 238, 268 and 913 is in the form of grooves. The new variety of the black fungus, sensheng No. 1 strain, is different from the current main cultivated variety, and the black fungus, sensheng No. 1 strain is an independent strain with specific genetic genes.

Example 2

Detecting the influence of temperature on the growth of new black fungus variety Sensheng No. 1

Under the aseptic condition, the new black fungus variety Sensheng No. 1 strain is inoculated on a culture dish with a CPDA culture medium and a diameter of 90mm, and the culture dish is placed in a constant temperature incubator for culture. Wherein, the formula of the CPDA culture medium is as follows: 200g of potato, 20g of glucose, ₂PO₄5g,MgSO₄·7H₂O 3g,VB₁ mg of KH, 20g of agar powder, 1000ml of water and pH value of 6.5. The incubator temperature was controlled at 22 ℃, 24 ℃,26 ℃, 28 ℃, 30 ℃, 32 ℃, 34 ℃ and 36 ℃, respectively. After 7d, the growth of the mycelium was observed, and the growth rate of the mycelium was calculated, and 3 parallel experiments were set at each temperature, and the average value was taken, to obtain the results shown in FIG. 7.

FIG. 7 shows the effect of temperature on the growth of new variety Sensheng No. 1 of Auricularia, and FIG. 7 shows that the new variety Sensheng No. 1 of Auricularia is a medium temperature variety, the mycelium growth temperature range is 5-35 ℃, the mycelium growth speed is faster at 28-32 ℃, and the mycelium growth temperature is the optimal temperature at 32 ℃.

Example 3

Detecting influence of pH value of CPDA culture medium on growth of new variety Sensheng No. 1 of black fungus

Under the aseptic condition, the new black fungus variety Sensheng No. 1 strain is inoculated on a culture dish with a culture medium of CPDA and a diameter of 90mm, the culture dish is placed in a constant temperature incubator for culture, and the temperature of the incubator is controlled to be 28 ℃. Wherein, the formula of the CPDA culture medium is as follows: 200g of potato, 20g of glucose, 20g of KH ₂PO₄5g,MgSO₄·7H₂O 3g,VB₁ mg, 20g of agar powder and 1000ml of water, and the pH value of the CPDA culture medium is adjusted to 4.6, 5.0, 5.2, 5.4, 5.6, 5.8, 6.0, 6.2, 6.4 and 6.6 by adding HCL or NaOH solution with the concentration of 0.1 mol/L. After 7d, the growth of hyphae was observed and the growth rate of hyphae was calculated, 3 parallel runs were set at each pH and averaged to give the results shown in FIG. 8.

FIG. 8 shows the effect of pH of the CPDA medium on the growth of the novel variety Sensheng No. 1 of Auricularia, wherein the novel variety of Auricularia is suitable for growth in a slightly acidic environment, the pH is 5.0-6.4, but the growth rate is faster at pH 6.2 as shown in FIG. 8. At pH values below 5.4 or above 6.4, the rate of hypha growth is significantly reduced.

Example 4

In the bag material cultivation mode, the influence of different bag material components on the growth speed, the number of days of filling bags, the thickness of fruiting bodies and the yield of hyphae of new black fungus variety Sensheng No. 1 is detected

The new black fungus strain Sensheng No. 1 is inoculated into the bag material which is contained in the polypropylene plastic bag and is subjected to sterilization treatment.

Experimental example 1: the bag material comprises the following components: 77.4% of broadleaf wood dust, 16.0% of wheat bran, 3% of beef extract, 1.0% of sucrose, 0.8% of gypsum, 0.6% of lime, 1.0% of corn flour, 0.2% of magnesium phosphate and 60% of water content.

Experimental example 2: the bag material comprises the following components: 77.1% of broadleaf wood dust, 16.0% of wheat bran, 3% of beef extract, 1.0% of sucrose, 0.8% of gypsum, 0.6% of lime, 1.0% of corn meal, 0.3% of mulberry powder, 0.2% of magnesium phosphate and 60% of water content.

Experimental example 3: the bag material comprises the following components: 77.3% of broadleaf wood dust, 16.0% of wheat bran, 3% of beef extract, 1.0% of sucrose, 0.8% of gypsum, 0.6% of lime, 1.0% of corn flour, 0.1% of potassium cinnamate, 0.2% of magnesium phosphate and 60% of water content.

Experimental example 4: the bag material comprises the following components: 77% of broadleaf wood dust, 16.0% of wheat bran, 3% of beef extract, 1.0% of sucrose, 0.8% of gypsum, 0.6% of lime, 1.0% of corn meal, 0.3% of mulberry powder, 0.1% of potassium cinnamate, 0.2% of magnesium phosphate and 60% of water content.

Each experimental example was inoculated with 30 bags and repeated 3 times to obtain an average value, and the results shown in table 9 were obtained.

Table 9 agronomic traits and yield comparisons under different sacks of new black fungus variety sen sheng No.1

As can be seen from Table 9, the addition of mulberry powder and potassium cinnamate promoted the growth rate of hyphae and increased the yield of Sensheng No. 1. The broad leaf sawdust, the wheat bran, the corn flour and the sucrose provide carbon sources for growth of Sensheng No. 1, the beef extract is a composite nitrogen source, growth of mycelia is facilitated, meanwhile, acid substances can be generated in the growth process of black fungus mycelia, the pH value in the bag materials can be regulated and stabilized by gypsum and lime, the excessive pH value in the bag materials is prevented, calcium ions in the growth of black fungus are supplemented, and sulfur elements required by the growth of black fungus can be directly supplemented by gypsum. Magnesium phosphate can provide phosphorus and magnesium required for growth of black fungus. The addition of the mulberry powder can improve the growth speed of hypha of Sensheng No. 1 in the bag material, so that the number of days that the hypha fills the bag can be reduced, and the mulberry powder contains rich anthocyanidin, glucose, vitamin C and malic acid, so that the decomposition of starch and fat is facilitated, and the required nutrition is provided for the growth of the hypha. Meanwhile, the potassium cinnamate can be added to accelerate the growth speed of hyphae, but the promotion effect on the hyphae in the growth process is smaller than that of mulberry powder, however, after the auricles are produced, the potassium cinnamate has a larger influence on the diameter of the auricularia auricula, the water required in the growth of the auricularia auricula fruiting body is mainly absorbed from the culture material, the potassium cinnamate has good solubility and is easily dissolved in water, the bag material can also be helped to absorb the water in the air, the proper humidity is provided for the fruiting body growth, the absorption of carbon, nitrogen and trace elements by the fruiting body is promoted, the growth of the fruiting body is further promoted, the black auricula maturation period is reduced, and meanwhile, the potassium ions and carboxyl are present in the bag material, so that trace element potassium ions can be provided for the growth of the fruiting body, the auricularia auricula can also have a larger thickness with the cooperation effect with other nutrient elements, the diameter of the auricularia auricula is larger, the weight of a single auricula is improved, and the yield of Sensheng No. 1 is also remarkably improved.

According to the comparative experiment example 4 and the experiment examples 1 to 3, the addition of the mulberry powder and the potassium cinnamate can play a synergistic effect, the growth of Sensheng No. 1 is promoted in the hypha growth period and the ear piece growth period after the ear is pulled out, the fruiting body is thick and elastic, colloid and semitransparent, the yield and the bioconversion rate of Sensheng No. 1 are improved, the period from inoculation to ear picking of the Sensheng No. 1 can be shortened, the reporting period of a planter is shortened, and the method is suitable for wide planting.

Example 5

Exemplary cultivation: according to the cultivation method, different ecological environment areas are selected in the Qinba mountain area, sensheng No. 1 is used as a test strain, the main cultivated black fungus variety ear 268 in the Qinba mountain area is used as a control variety, and the expansion test is carried out by two modes of log cultivation and bag cultivation.

Specifically, a region with the height of the sea in the Qinba mountain area of 400-1300m is selected for cultivation, sensheng No. 1 is used as a test strain, the main cultivated black fungus variety ear 268 in the Qinba mountain area is used as a comparison variety, two modes of log cultivation and bag material cultivation are adopted in each place, and the same method is adopted for demonstration planting and management, so that the analysis of the obtained data is facilitated. Production tests were conducted on Ningqiang five-temple (elevation about 980 m), mian County-homed river (elevation about 1190 m), jiuzhong Jinxiang Zhongchuan dam village (elevation about 860 m) in Lueyang county, sichuan Qingchun county Yao Du Yangshan village (elevation about 490 m), and Shahejia river town (elevation about 580 m) in western county, respectively. By adopting a comparative design, 500 bags are cultivated by the bag material, 50 frames are cultivated by the section wood, and 50 sections of wood are used as one frame.

During the exemplary cultivation period, not only are the black fungus yields in different areas counted, but also the bacterial contamination rates of different strains in different time periods are counted to identify the disease resistance of the new black fungus variety Sensheng No.1, and the statistical seed hole infection rate and the seed hole current ear rate are investigated to identify the stress resistance of the new black fungus variety Sensheng No. 1.

In the exemplary cultivation, the cultivation method of the new black fungus variety Sensheng No. 1 comprises the following steps:

(1) Preparing mother seeds: tissue separation is carried out on the new black fungus variety under the aseptic condition, the strain is inoculated on the inclined plane of the mother culture medium, and the mother culture medium is placed in a 28 ℃ incubator for constant temperature culture until mycelium grows on the inclined plane, thus obtaining the mother culture.

Mother culture medium: 200g of potato, 20g of glucose, ₂PO₄5g,MgSO₄·7H₂O 3g,VB₁ mg of KH, 20g of agar powder, 1000ml of water and pH6.2. And (5) subpackaging: 200X 20mm tube, 15ml, plug. Sterilizing a mother culture medium: maintaining at 0.01MPa for 30min, sterilizing at 110deg.C, and swinging inclined plane after sterilization.

Wherein, after tissue isolation of Sensheng No. 1 under aseptic conditions, the strain was inoculated on CPDA medium, and after culturing for 7d at 28 ℃, the colony morphology and microstructure of Sensheng No. 1 were observed, and the results shown in FIGS. 9 to 11 were obtained.

FIG. 9 is a front morphology of the colony of the new variety of Auricularia, sensheng No. 1; FIG. 10 is a reverse side morphology of the colony of the new variety of Auricularia, sensheng No. 1; FIG. 11 is a microscopic view of the hypha 40x of the strain of the new variety of Auricularia, sensheng No. 1.

As shown in FIG. 9, the colony of Sensheng No. 1 is white in front and has a thick and strong mycelium, and as shown in FIG. 10, the colony of Sensheng No. 1 is orange in back. FIG. 11 shows that, at 40X, the microscopic structure of hyphae of Sensheng No. 1 is observed: the mycelium has no septum and branches, the diameter of the mycelium is 1.25-5 mu m, and the lock-shaped complex is obvious.

(2) Manufacturing an original seed: and (3) aseptically inoculating the mother seeds into a stock seed bag, and culturing at a constant temperature in a 28 ℃ incubator until mycelia grow full of the stock seed bag to obtain stock seeds.

Bag-in-stock bag material: 78% of wood dust, 20% of rice bran or wheat bran, 1% of sucrose, 1% of gypsum powder and 60% of water.

Producing a bag-in-bag material: 86.5% of hard wood dust, 10% of wheat bran, 2% of bean cake, 1% of gypsum, 0.5% of quicklime and 60-65% of water.

(41) The log cultivation comprises:

(411) Preparing log: broad-leaved tree sticks with the length of 100-120cm and the diameter of 8-12cm are selected as the cultivation sections.

(412) Ear field selection and airing rack: the cultivation site is selected in the places with the sun facing leeward, air circulation, water source approaching and irrigation and drainage convenience, and the clean site on the ground of the ear site is leveled and the carbendazim is sprayed for sterilization treatment. Meanwhile, the log is piled up in a 'well' -shaped place, and the piled up height is 1.3-1.5m, and the piled up is turned over once in 10-15 days, so that the water content of the log is less than 50%.

(413) Dibbling: opening holes on the surface of the log, wherein the hole distance is 7-12cm, the diameter of the holes is 14-16mm, the holes are deep into xylem for 1-2cm, sowing black fungus strains into the holes, sowing 0.2-0.24kg of the new black fungus strain on each log, sealing the tree with a wood plug or yellow cement after sowing, sowing at the temperature of above 8 ℃, and inoculating according to the temperature of each region.

(414) Piling up bacteria: discharging the inoculated wood blocks in a well shape to form a log pile, covering the log pile with a plastic film, maintaining the temperature at 18-24 ℃, spraying water to Duan Mudui after 10-14 days, and maintaining the relative air humidity at 85-95%.

(415) Bulk dump drainage: when white fungus film is on the surface of the strain in the hole and white hypha is on the bottom of the hole, one end of the log is lifted by using a cross beam or masonry, the log is discharged according to fish scales, the space between the log is 5-6cm, the log is turned once in 10-15 days, and the ear condition is observed in 2-3 months.

(416) Lifting a shed, loading the shed on a frame and harvesting: when 45-50% of the log leaves ears, the log is put by a herringbone frame method or a fish scale type swing method, the relative humidity of air is kept at 85-95%, the temperature is kept at 22-35 ℃, preferably, the temperature is kept at 22-30 ℃, and fruiting bodies grow rapidly. After the earpieces are ripe, picking off the earpieces with roots and immediately airing, spraying water to log after 5-7 days, keeping the relative humidity of air to be 65-85%, recovering the growth of hyphae, forming earbuds again, and harvesting once every 10-20 days.

The temperature in winter is low, fruiting bodies stop growing, mycelium also enters dormancy, the auricularia auricula is concentrated and is piled at a clean and dry place according to a 'groined' shape, the temperature in spring in the next year rises to more than 5 ℃, auricularia auricula is sent out, and then scattered piling and lifting are carried out, and water spraying management is carried out.

(42) The bag cultivation comprises:

(421) Mixing and bagging: uniformly stirring the bag materials according to a proportion, then filling the bag materials into a cultivation bag, and sterilizing at the temperature of 124 ℃ for 3 hours under the pressure of 0.14MPa.

The bag material comprises the following components: 77% of broadleaf wood dust, 16.0% of wheat bran, 3% of beef extract, 1.0% of sucrose, 0.8% of gypsum, 0.6% of lime, 1.0% of corn meal, 0.3% of mulberry powder, 0.1% of potassium cinnamate, 0.2% of magnesium phosphate and 60% of water content.

(422) Inoculating: cooling the sterilized cultivation bags to room temperature, transferring into an inoculation chamber, inoculating the production seeds of the new black fungus variety into the bag materials according to aseptic operation, placing the inoculated production seeds into a culture chamber for culture, keeping the temperature of the culture chamber at 22-26 ℃ and the relative air humidity at 50-60%, enabling hyphae to start growing, and culturing for 60-65 days, wherein each cultivation bag is inoculated with 5-10g of the production seeds of the new black fungus variety.

(423) And (5) punching: after hypha grows fully, the cultivating bags are moved into a cultivating room, holes are uniformly formed on the surface of the cultivating bags, the diameters of the holes are 1-2cm, the distances between the holes are 5-6cm, 10-12 holes are formed in each cultivating bag, the cultivating bags are hung on a bedstead upside down after the holes are formed, the cultivating bags are covered by plastic films, spraying is carried out every day, the relative humidity of air is kept at 65-70%, and the temperature is 15-20 ℃ for 7-10 days, so that the ears can be produced.

(424) Ear-out management: after the ear is removed, the relative humidity of air is kept to be 80-85%, ventilation is enhanced, after 10-15 days, when the ear pieces are flat and the edge is curled inwards, the ear roots are changed from large to small, harvesting is started, and after 5-10 days, ear buds are formed again.

Experimental example 5: the results of statistics on the black fungus yields obtained by different cultivation methods in different regions of Sensheng No.1 and ear 268 are shown in Table 10 and Table 11.

Table 10 Auricularia Sensheng No. 1 and Auricularia 268-stage wood cultivation test yield

The results in Table 10 show that the average yield of Sensheng No. 1 strain in the log cultivation production test is 11.226kg dry weight/frame, which is improved by 49.82% compared with the control variety, and the yield is obviously increased.

Table 11 black fungus Sensheng No. 1 and black fungus 268 bag cultivation test yield

The results in Table 11 show that the average fresh ear yield per 100kg of dry material produced by Sensheng No.1 bag cultivation is 83.344kg, which is 16.53% higher than that of the control variety ear 268, and the obvious yield increase phenomenon is shown. The Sensheng No.1 has the advantages of high bioconversion rate and high yield.

Experimental example 6: disease resistance and stress resistance detection of new black fungus variety Sensheng No. 1

The antibacterial infection of the black fungus sen Shengsheng No. 1 strain and the control strain fungus 268 was observed by examining and counting the bacterial contamination rates of different strains in different time periods, and the results are shown in Table 12.

Table 12 statistics of antibacterial infections of Auricularia Sensheng No. 1 strain and Auricularia 268

As can be seen from Table 12, the time for the Auricularia to infect the infectious microbe No. 1 is later than that of the control variety Auricularia 268, the pollution rate is obviously lower than that of the control variety Auricularia 268, and is only 5.3%, and the pollution rate of the control variety Auricularia 268 is 16.7%. According to the application, the Sensheng No. 1 strain with excellent properties is screened from 12 wild black fungus strains, and is obtained from a Ningqiang five Ding Guan forest farm with an altitude of 1300m, and through exemplary cultivation, the Sensheng No. 1 log and bag material cultivation are selected, so that the method has strong adaptability and wide suitable planting range, and is suitable for cultivation in Qinba mountain areas at low altitudes (400-700 m), high-temperature and high-humidity plain and hilly areas and mountain areas with higher altitudes (1300 m).

Experimental example 7: quantitative analysis was used to investigate and count the rate of infection of the auricularia auricula seeds and the rate of auricularia auricula current (rotten) seeds in different areas, and the results are shown in Table 13.

Table 13 strain stress resistance trait investigation statistics

As can be seen from table 13, the seed cavity infection rate of the new black fungus variety sen 1 is extremely significantly lower than that of the control strain fungus 268; from the point of view of the seed cavity current ear rate, the new variety of the black fungus, sensheng No. 1 strain, is also obviously lower than that of the control strain ear 268. The stress resistance of Sensheng No. 1 is obviously better than that of the main cultivated variety ear 268 in Qinba mountain area at present.

Through carrying out exemplary cultivation for 3 years on the new black fungus variety Sensheng No.1 in different altitude environment areas of Qinba mountain areas, the yield, disease resistance and stress resistance of the new black fungus variety Sensheng No.1 are superior to those of the main cultivated variety fungus 268, meanwhile, the thickness (0.10 cm-0.14 cm) and the diameter (5 cm-10 cm) of the black fungus sheet of the new black fungus variety Sensheng No.1 in 3 years are kept stable, no yield reduction condition exists, and the increase of the current fungus, the thin fungus, the worm-eating fungus and the rotten fungus does not exist, so that the new black fungus variety Sensheng No.1 has the advantages of stable genetic character, strong resistance to mixed bacteria infection, good yield and strong stress resistance.

The new variety Sensheng No. 1 mycelium of the black fungus bred by the method has the advantages of faster growth, white and thick mycelium, black fruiting body and single-piece cluster, and belongs to middle-early maturing varieties. From inoculation to harvest, 90-115 d are needed, and the diameter of each ear piece is 5cm-10cm. Meanwhile, the black fungus Sensheng No. 1 obtained by exemplary cultivation is sent to a product quality supervision and inspection institute in Hanzhong, and inspection methods and judgment conditions are carried out according to national standards GB/T6192-2008 black fungus, and the black fungus new variety Sensheng No. 1 fruiting body fungus bred by the method has black brown front, luster and dark gray back; no fist ear, thin ear, lost ear, worm-eaten ear and rotten ear; has special smell of Auricularia auricula, and no odor; the lugs are complete and cannot pass through a sieve with the diameter of 3 cm; the dry-wet ratio is 1:14; the thickness of the ear piece is 0.10cm-0.14cm; impurity 0.2%, which is less than or equal to 0.3%; crude protein 9.6%, which meets the standard of 7.0% or more; total sugar (calculated by converted sugar) is 41.7 percent, which accords with the standard not less than 22 percent; crude fat 1.26 percent meets the standard of more than or equal to 0.40 percent. The novel black fungus variety Sensheng No. 1 seed is preserved by adopting a liquid paraffin closed inclined plane or a wheat seed preparation method, and is preserved at the temperature of 4 ℃.

Finally, it should be noted that the above embodiments are merely illustrative of the technical solution of the present application, and not limiting thereof; although the application has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art will appreciate that; the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the application.

Claims

1. A cultivation method of a new variety of black fungus is characterized in that,

The breeding of the new black fungus variety comprises the following steps:

s1, collecting wild black fungus specimens: collecting wild black fungus specimens in natural environments of different altitudes of a target area, sterilizing the surfaces of the wild black fungus specimens with 0.1% mercuric chloride, and then washing with sterile water for 3-5 times;

S2, culturing wild black fungus sample strains: performing tissue separation on the sterilized wild black fungus specimen under the aseptic operation condition, inoculating the wild black fungus specimen on a culture dish filled with a CPDA culture medium, and culturing at 28 ℃; the formula of the CPDA culture medium is as follows: 200g of potato, 20g,KH2PO4 5g,MgSO4.7H2O 3g of glucose, 10mg of VB1, 20g of agar powder and 1000ml of water, adding HCL or NaOH solution with the concentration of 0.1mol/L, and regulating the pH value of the CPDA culture medium to be 5.0-6.4;

s3, primary screening in the growth period: observing the growth condition of hyphae, and selecting a strain with vigorous hyphae growth vigor and hyphae growth speed of more than 0.40cm/d as a primary screening wild strain;

S4, re-screening: activating and performing enlarged culture on the primary screening wild strain obtained in the step S3 to prepare a stock seed, inoculating the stock seed into a cultivation substrate which is contained in a polypropylene plastic bag and is subjected to sterilization treatment according to a sterile operation procedure, and respectively performing mycelium rescreening and fruiting body rescreening in a bag cultivation mode to obtain a rescreened wild strain;

S5, character identification: performing cellulase activity measurement, polysaccharide content measurement and genetic specificity identification on the re-screened wild strain, and finally screening to obtain a new black fungus variety;

the mycelium re-screening step comprises the following steps: performing mycelium re-screening according to the mycelium growth condition in the culture medium;

the step of re-screening the fruiting bodies comprises the following steps: observing the fruiting body re-screening of the black fungus in the culture medium according to the agronomic characters and the yield of the black fungus, and simultaneously combining the mycelium re-screening result to select a re-screened wild strain;

The cultivation method comprises the following steps:

(1) Preparing mother seeds: performing tissue separation on the new black fungus variety under the aseptic condition, inoculating a strain obtained by tissue separation to the inclined plane of a mother culture medium, and culturing at a constant temperature in a 28 ℃ incubator until hyphae grow to fill the inclined plane to obtain a mother culture;

(2) Manufacturing an original seed: aseptically inoculating the stock seeds into stock seed bags, and culturing at constant temperature in a 28 ℃ incubator until mycelia grow full of the stock seed bags to obtain stock seeds;

(3) And (3) manufacturing production seeds: aseptically inoculating the stock seeds into a production seed bag, and culturing at constant temperature in a 28 ℃ incubator until mycelia grow full of the production seed bag to obtain production seeds;

(4) Cultivation: cultivating by two modes of log cultivation and bag material cultivation, and respectively inoculating the production seeds into log holes and bag materials for cultivation until ears are produced;

The bag material comprises the following components: 77.0% of broadleaf wood dust, 16.0% of wheat bran, 3% of beef extract, 1.0% of sucrose, 0.8% of gypsum, 0.6% of lime, 1.0% of corn meal, 0.3% of mulberry powder, 0.1% of potassium cinnamate, 0.2% of magnesium phosphate and 56-62% of water content.

2. The method according to claim 1, wherein in the step S1, a natural environment having a target area altitude of 800m or more is selected when the wild black fungus specimen is collected.

3. The method for cultivating a new variety of black fungus according to claim 1, wherein step S2 further comprises: after the wild black fungus specimen tissue is separated, inoculating each two different strains into the same culture dish, and carrying out opposite culture at 28 ℃ to screen out the wild black fungus strain with antagonism.

4. The method for cultivating a new variety of black fungus according to claim 1, wherein the cultivation substrate comprises: 78.0% of broadleaf wood dust, 16.0% of wheat bran, 1.0% of sucrose, l.0% of gypsum, 1.0% of lime, 1.5% of bean cake powder, 1.5% of corn flour, 56-62% of water content and 1.1kg of culture medium in each bag.

5. The method for cultivating a new variety of black fungus according to claim 1, wherein the log cultivation comprises:

(1) Preparing log: broad-leaved tree sticks with the length of 100-120cm and the diameter of 8-12cm are selected as cultivation log;

(2) Ear field selection and airing rack: the ear field is selected in a place which is leeward and sunny, air circulation, water source approaching and irrigation and drainage are convenient, and the surface of the ear field is leveled and sterilized; simultaneously stacking the log in a 'well' -shaped place, wherein the stacking height is 1.3-1.5m, and the stacking is turned once in 10-15 days, so that the water content of the log is less than 50%;

(3) Dibbling: opening holes on the surface of the log, wherein the hole distance is 7-12cm, the diameter of the holes is 14-16mm, the holes are deep into xylem for 1-2cm, dibbling the production seeds into the holes, sealing the holes by using wood plugs or yellow cement after dibbling, and the dibbling temperature is above 8 ℃;

(4) Piling up bacteria: discharging the inoculated log according to a well shape to form a log stack, covering the log stack with a plastic film, keeping the temperature at 18-24 ℃, spraying water to the log stack after 10-14 days, and keeping the relative air humidity at 85-95%;

(5) Bulk dump drainage: when white fungus films are arranged on the surfaces of the strains in the holes and white hyphae are arranged at the bottoms of the holes, raising one end of the log, discharging the log according to fish scales, turning the log once every 5-6cm for 10-15 days, and observing the ear situation after 2-3 months;

(6) Lifting a shed, loading the shed on a frame and harvesting: when 45-50% of the log leaves ears, putting the log by adopting a herringbone frame method or a fish scale type swing method, keeping the relative air humidity at 85-95% and the temperature at 22-35 ℃, and enabling fruiting bodies to grow rapidly; after the earpieces are ripe, picking off the earpieces with roots, immediately airing, spraying water to log after 5-7 days, keeping the relative air humidity to be 65-85%, recovering the growth of hyphae, and forming earbuds again.

6. The method according to claim 5, wherein each of said cut wood is inoculated with 0.2-0.24kg of said production seed.

7. The cultivation method of a new variety of black fungus according to claim 1, wherein the bag cultivation comprises:

(1) Mixing and bagging: uniformly stirring all the components of the bag material in proportion, then filling the bag material into a cultivation bag, and carrying out high-pressure sterilization;

(2) Inoculating: cooling the sterilized cultivation bag to room temperature, transferring into an inoculation chamber, inoculating the production seeds into a bag material according to aseptic operation, placing into a culture chamber for culture after inoculation, keeping the temperature of the culture chamber at 22-26 ℃ and the relative air humidity at 50-60%, enabling hyphae to start growing, and culturing for 60-65 days;

(3) And (5) punching: after hypha grows up to grow up to cultivate the bag, move the bag into the cultivation room, evenly open holes on the surface of the bag, the diameter of the holes is 1-2cm, the distance between holes is 5-6cm, 10-12 holes are opened in each bag, hang the bag upside down on the bed frame after opening holes, cover with plastic film, spray every day, keep the relative humidity of air at 65-70%, the temperature is 15-20 ℃, can come out ears 7-10 days;

(4) Post-aural management: after the ear is removed, the relative humidity of air is kept to be 80-85%, ventilation is enhanced, after 10-15 days, when the ear pieces are flat and the edge is curled inwards, the ear roots are changed from large to small, harvesting is started, and after 5-10 days, ear buds are formed again.

8. The method according to claim 7, wherein each of said cultivation bags is inoculated with 5-10g of said production seed in said bag cultivation.