CN113796260B - Poria (Wolfiporia cocos) YX1, and culture medium and cultivation method thereof - Google Patents

Abstract

The invention discloses a Poria cocos (Wolfiporacocos) YX1, which is preserved in China center for type culture Collection with the preservation number of CCTCCNO: m2021434. The invention also discloses a stock culture medium and a mother culture medium of the poria cocos (wolfiporicoocs) YX 1. The invention also discloses a cultivation method of the poria cocos (wolfiporicoocs) YX 1. The poria cocos (wolfiporiaccos) YX1 screened by the method has stable economic characters, high yield, high quality and stable genetic genes; and a high-yield and stable-production culture medium of Poria cocos (Wolfiporacocos) YX1 is screened out; and provides a scientific and practical cultivation method.

Description

Poria (Wolfiporia cocos) YX1, and culture medium and cultivation method thereof

Technical Field

The invention relates to the technical field of poria cocos, in particular to poria cocos (Wolfiporia cocos) YX1 and a culture medium and a cultivation method thereof.

Background

The protection of the quality resources of the wild pine rot fungi and the stability of the transmission of genetic information are both original materials to be utilized in the breeding work, including various strains or strains of wild and cultivated species. At present, researches on the pine decay fungi are mostly focused on the aspects of pharmacological functions, processing technology, polysaccharide, metabolism and the like, and the researches on improved varieties, culture media, raw materials, cultivation modes and cultivation time used in the cultivation of the pine decay fungi, particularly on the qualitative resources and the raw material substitution of the pine decay fungi, are lagged. This also becomes a major factor that restricts the sustainable, rapid and healthy development of the pine rot fungus industry in our country. The tuckahoe in the pine material rot fungi has nutrition and nourishing value, can calm the nerves and prolong the life after being eaten frequently, is one of the traditional Chinese medicinal materials in China, and simultaneously, the tuckahoe fungi are also important pine material rot fungi. The yield and sales of the Chinese poria cocos are over 1.7-3 ten thousand tons every year, the Anhui Yuexi poria cocos is sold in the nation, the product is sold far in the sea and outside, the poria cocos industry is rapidly started and developed, and the planting area is continuously enlarged.

At present, from the whole view, because the collection and protection of wild resources are not strong, some precious germ plasm resources can not be saved, the wild poria cocos is almost extinct, the wild poria cocos is cultivated completely by manpower, and the quality and the yield of the poria cocos are not high and unstable at present. The traditional poria cocos cultivation method takes masson pine segments as a main raw material, the dosage of the pine segments is increased rapidly along with the rapid rise and development of the poria cocos industry, a large number of pines are felled, the price of the pine segments is high, and the production cost of poria cocos is high. In addition, the traditional poria cultivation method taking the masson pine wood segment as the main raw material has limitations on the cultivation mode, cultivation time and the like of the poria, is difficult to realize standardized, normalized and large-scale industrial cultivation, and has low poria quality and yield and easy pollution to mixed bacteria. A high-yield and stable-yield improved cultivation seed, a culture medium and a cultivation method thereof are urgently needed to solve the problems of unstable quality of the tuckahoe fungi for a long time, insufficient supply of raw materials and the like.

Disclosure of Invention

Based on the technical problems in the background art, the invention provides a poria cocos (Wolfiporia cocos) YX1, a culture medium and a cultivation method thereof, the poria cocos (Wolfiporia cocos) YX1 is screened out, identification of poria cocos germplasm resources is carried out on the poria cocos, genetic relationship and genetic diversity are analyzed, and the poria cocos (Wolfiporia cocos) YX1 is good in economic character, high in quality and stable in genetic gene; screening culture substitute materials of Poria cocos (Wolfiporia cocos) YX1 by artificial cultivation means, and finally selecting high-yield and stable-yield improved variety culture medium; the method is scientific and practical, changes the traditional cultivation mode, solves the problems that the traditional cultivation takes the masson pine section as the main raw material, the cultivation mode and the cultivation time are limited, and solves the problems that the quality of the tuckahoe fungus is unstable for a long time, the raw material supply is insufficient, and the like.

The invention provides a Poria cocos (Wolfiporia cocos) YX1, which is preserved in China center for type culture Collection with the preservation number of CCTCC NO: m2021434, the China center for type culture Collection, the university of Wuhan, China, the preservation date is 2021, 4 months and 22 days.

The invention also provides a stock culture medium of the poria cocos (Wolfiporia cocos) YX1, which comprises the following raw materials in percentage by weight: the main material is more than or equal to 60 percent, the auxiliary material is 0-38 percent, the cane sugar is 0.5-2 percent, the gypsum is 0.5-1 percent, the natural resin is 0-1 percent, the monopotassium phosphate is 0-0.1 percent, the vitamin B is 0-0.0002 percent, and the gram mould king is 0-0.1 percent, wherein the total weight percentage of all the raw materials is 100 percent, and the main material is a mixture of pine sawdust and at least one of miscellaneous wood chips, pine branches, pine needles and turpentine or pine sawdust.

Preferably, the auxiliary material is at least one of rice bran, wheat bran, corncob and cottonseed hull.

Preferably, the natural resins are: at least one of mastic resin, shellac, rosin resin, and amber.

Preferably, the weight percentage of the main material is more than or equal to 70 percent.

Preferably, the material mixing humidity of the stock culture medium is 50-60%.

The material mixing humidity refers to the water content of the culture medium after water is added into the culture medium and the mixture is uniformly mixed.

The pine tree can be Pinus massoniana, Pinus sylvestris, Pinus densiflora, Pinus sylvestris, Pinus armandii, etc.

The stock culture medium of Poria (Wolfipora cos) YX1 can also be added with vitamin C, mildew-proof phytophthora additive (such as King mould, etc.), and yeast tablet; the Kemeiwang and Kemeisheng tablets are commercially available.

For example, the stock culture medium of the above Poria cocos (Wolfiporia cocos) YX1 may be:

the raw materials comprise the following components in percentage by weight: 60-80% of pine sawdust, 18-37% of wheat bran, 1-2% of cane sugar and 0.5-1% of gypsum, wherein the sum of the weight percentages of the raw materials is 100%;

secondly, the raw materials comprise the following components in percentage by weight: 60-80% of pine sawdust, 18-37% of miscellaneous wood chips, 1-2% of cane sugar and 0.5-1% of gypsum, wherein the sum of the weight percentages of the raw materials is 100%;

thirdly, the raw materials comprise the following components in percentage by weight: 50-60% of pine branches, 10-33% of pine sawdust, 5-15% of rice bran, 5-15% of wheat bran, 3-8% of corncobs, 0.5-1% of sucrose and 0.5-1% of gypsum, wherein the sum of the weight percentages of the raw materials is 100%;

the material comprises the following components by weight percent: 10-20% of pine sawdust, 46-58% of pine needles, 5-15% of rice bran, 5-15% of wheat bran, 5-10% of corncobs, 0.5-1% of cane sugar and 0.5-1% of gypsum, wherein the sum of the weight percentages of the raw materials is 100%;

the raw materials comprise the following components in percentage by weight: 50-78% of pine sawdust, 5-10% of pine needles, 5-10% of pinecones, 5-20% of wheat bran, 5-10% of corncobs, 0.5-1% of cane sugar and 0.5-1% of gypsum, wherein the sum of the weight percentages of the raw materials is 100%;

the raw materials comprise the following components in percentage by weight: 65-78% of pine sawdust, 20-31% of cottonseed hull, 1-2% of cane sugar, 0.5-1% of gypsum, 0.1-1% of rosin resin, 0-0.1% of monopotassium phosphate, 0-0.0002% of vitamin B10 and 0.05-0.1% of Kemeiwang, wherein the sum of the weight percentages of the raw materials is 100%.

The invention also provides a mother culture medium of the poria cocos (Wolfiporia cocos) YX1, which comprises the following raw materials: 1000mL of an aqueous solution having a pH of 5-6 contains: 150 g of potato, 20-30g of glucose, 15-20g of agar powder, 0-1g of monopotassium phosphate and 0-0.5g of magnesium sulfate;

alternatively, 1000mL of an aqueous solution having pH 5-6 contains: 20-30g of glucose, 10-15g of peptone, 15-20g of agar powder, 0-1g of monopotassium phosphate and 0-0.5g of magnesium sulfate.

The water in the mother culture medium can be sterile water or distilled water.

The mother strain is pure mycelium obtained by artificially culturing spore, fruiting body, sclerotium or mycelium in medium, and the culture medium is mother strain culture medium.

Stock refers to mycelium obtained by inoculating the stock onto a stock culture medium.

The invention also provides a cultivation method of the poria cocos (Wolfiporia cocos) YX1, which comprises the following steps:

s1, sequentially culturing Poria (Wolfipora cos) YX1 in a mother culture medium and a stock culture medium to obtain a Poria (Wolfipora cos) YX1 stock;

s2, sleeving a fungus bag filled with Poria cocos (Wolfipora cocos) YX1 stock seeds on one end of a pine section, sealing and fixing the fungus bag on the pine section, sleeving an empty fungus bag on the other end of the pine section, keeping the opening state, adjusting the water content of the pine section to be 40-50%, culturing at 28-32 ℃ for 40-50 days, burying the pine section in a cellar, and growing polyporus;

wherein the stock culture medium is a stock culture medium of Poria (Wolfipora cos) YX1, and the stock culture medium is a stock culture medium of Poria (Wolfipora cos) YX 1.

Preferably, in S2, the empty fungus bag is a fungus bag with both ends open.

Preferably, in S2, the empty fungus bag is extended 7-8cm beyond the pine wood segment end of the empty fungus bag.

Preferably, in S2, the middle position of the pine segment does not cover the fungus sack.

In the above S2, the size of the fungus bag may be selected according to the volume of the pine wood segment, so that one end of the pine wood segment is sleeved with the fungus bag filled with the stock seed and the stock seed culture medium; the other end of the pine section is sleeved with an empty fungus bag with two open ends, one end of the empty fungus bag is fixedly tied to the pine section, the other end of the empty fungus bag is kept in an open state, and the middle position of the pine section is not covered with the fungus bag.

Performing stock culture in the strain bag containing stock culture medium in S1 and S2 until mycelia of Poria (Wolfipora cocos) YX1 grow over the strain bag to obtain Poria (Wolfipora cocos) YX1 stock; then sheathing the fungus bag full of Poria cocos (Wolfipora cocos) YX1 original strain hypha on one end of pine wood segment, and proceeding with the step S2; generally, 350-450g of a bag containing mycelia of an original strain of Poria cocos (Wolfiporia cocos) YX1 is used per 6kg of pine segments.

In S2, the weight of the pine segments is preferably 6kg or more, and the diameter of the pine segments may be 8cm, 9cm, 10cm, 20cm, or the like.

The material of the fungus bag can be polypropylene, polyethylene and the like.

Sterilizing the mother culture medium and the stock culture medium; the fungus bags are sterilized.

Has the beneficial effects that:

1. the invention provides a Poria cocos (Wolfiporia cocos) YX1 with high and stable yield, which enriches germplasm resources of Poria cocos, has stable hereditary character and stable yield, and has the advantages of good Poria quality, regular Poria cocos and high Poria cocos yield; the yield of the poria cocos is improved, the good quality is kept and the stability of the yield can be kept by screening out a proper mother culture medium and a proper stock culture medium; the main materials of the stock culture medium are miscellaneous wood chips, pine branches, pine needles, pinecones and pine wood chips, the raw materials are cheap, and the cultivation cost of the poria cocos can be reduced;

2. the cultivation method that the original seeds are inoculated at one end of the pine section and the empty fungus bags with openings are sleeved at the other end (called the pine section one-end bacterium-sleeved inoculation cultivation method for short) solves the problem that the traditional cultivation mode easily pollutes the mixed fungi, and has the advantages of regular poria, good poria quality, high yield, avoidance of mixed fungus pollution, convenient management and realization of industrial cultivation;

3. in addition, the traditional cultivation mode uses sclerotium inoculation, inoculation is carried out in a mild weather of 4-5 months every year, and the cultivation is carried out in a cellar, so that the inoculation amount is large and the inoculation time is limited; the method uses the fungus bags filled with the original seeds for inoculation, the fungus bags are cultured for a period of time at a proper temperature after inoculation to promote the growth of tuckahoe hyphae, and then the tuckahoe hyphae are put into a cellar for cultivation, the inoculation period is not limited by the climate, the inoculation can be carried out indoors, is suitable for factory unified management, and can be carried out in advance; culturing at proper temperature to promote the growth of Poria cocos hypha, and reducing the inoculation amount; the invention solves the problems of unstable quality of the traditional Chinese medicine tuckahoe fungus for a long time, insufficient raw material supply and the like.

Drawings

FIG. 1 is a photograph showing the culture characteristics of Poria cocos (Wolfiporia cocos) YX1 mycelium, wherein a is the front side of the culture dish, b is the back side of the culture dish, c is 10 times the objective lens, and d is 20 times the objective lens.

Fig. 2 is a genetic phylogenetic analysis diagram of different strains of poria cocos (Wolfiporia cocos) YX1, wherein the various strains are herein labeled poria cocos (Wolfiporia cocos) YX 1.

FIG. 3 is a photograph showing the cultivation process in example 5.

FIG. 4 is a photograph of sclerotium of Poria cocos in example 5.

FIG. 5 is a photograph of sclerotium of Poria cocos in example 6.

FIG. 6 is a drawing showing the cultivation process of example 7, wherein a is a pine wood section with one end inoculated with stock and the fungus sack sealed, and b is a pine wood section with both ends sleeved with fungus sacks.

FIG. 7 is a photograph showing the formation of the polyporus umbellatus of example 5.

FIG. 8 is a photograph showing the formation of the knot in accordance with example 7.

FIG. 9 is a photograph of sclerotium of Poria cocos in example 7.

Detailed Description

The technical solution of the present invention will be described in detail below with reference to specific examples.

Example 1

Screening of Poria cocos (Wolfiporia cocos) YX1

Collecting Monilinia fuscosa sclerotia dormant bodies under rotten pine piles from a pine forest region (30 degrees in northern latitude and 85 degrees in east longitude and 116 degrees and 35 degrees in east China) of village of Guanzhen in Yuexi county of Anqing, Anhui province, taking mycelia, respectively inoculating the mycelia into mother culture media with different formulas in an ultraclean workbench, culturing, and observing the growth of the mycelia of the poria; then, screening good mother seeds, respectively inoculating the mother seeds into stock culture media with different formulas, culturing, observing the growth of poria cocos hyphae, and screening good stock seeds; then, the pure tuckahoe hyphae are obtained by continuously transferring for 2 generations, and the obtained tuckahoe fungus (Wolfiporia cocos) YX1 is preserved in China Center for Type Culture Collection (CCTCC) in Wuhan with the preservation number of CCTCC NO: m2021434.

Example 2

Morphological identification of Poria cocos hyphae was performed on Poria cocos (Wolfiporia cocos) YX1 obtained in example 1.

1. The specific method comprises the following steps: the poria cocos (Wolfiporia cocos) YX1 obtained in example 1 is inoculated into a culture dish filled with a culture medium, the culture dish is cultured for 4-5 days, then the glass slide is obliquely inserted into the culture medium at an angle of 45 degrees, the glass slide is taken out after being cultured for 12h, the glass slide is placed on a cover glass and placed on an optical microscope and an electron microscope, and macroscopic and microscopic growth characteristics such as the formation, color and structure and the existence of separation of the poria cocos hyphae are observed under a field of view of 10 times or 20 times of an objective lens, as shown in figure 1, figure 1 is a picture of the culture characteristic of the poria cocos (Wolfiporia cocos) YX1 mycelium, wherein a is the front surface of the culture dish, b is the back surface of the culture dish, c is the 10 times of the objective lens, and d is the 20 times of the objective lens.

2. As a result: the mycelium of Poria cocos (Wolfipora cos) YX1 comprises mononuclear mycelium and binuclear mycelium, wherein the Poria cocos mycelium is composed of multiple branched mycelium, the mycelium is criss-cross, and penetrates through the substrate densely or grows on the surface of the substrate in an extending manner, the mycelium is white villous and can be seen in a locked joint phenomenon, the mycelium is divided into multiple linear cells by a diaphragm, and the width of the mycelium is 2-5 μm. When the bacterial colony is cultured in a culture dish, the bacterial colony in a concentric ring form is common, the hyphae have strong vitality, grow over the dish for 4-5 days, and fade to be dark brown after being cultured for 28 days.

3. The morphological structural characteristics of tuckahoe show three different morphological characteristics, namely mycelium, sclerotium and fruiting body, at different developmental stages. The poria cocos mycelium is continuously cultured into sclerotium and sporocarp, and macroscopic and microscopic growth characteristics of the poria cocos mycelium are observed.

4. As a result: sclerotium of Poria (Wolfipora cos) YX1, in the shape of sphere or ellipsoid; the sizes are uniform and consistent; is a dormant body with accumulated hypha. The fresh tea is soft and easy to tear, the water content is not more than 30%, the dried tea is hard and not easy to break, and the water content is generally less than 18%; when the sclerotium is covered with soil, the sclerotium is rough in surface, mostly appears as tumor-shaped shrink, and has a layer of peel-shell-shaped exodermis, which is light brown like soil when fresh, and is dark brown after dried, and poria cocos flesh appears white.

The fruiting body of Poria cocos (Wolfiporia cocos) YX1 is generated on the surface of sclerotium, and also can be seen on aged mycelium, and has the advantages of honeycomb shape, different sizes, no stalk, flat lying, and lignification with thickness of 0.3-1cm and light yellow color. The sporophore pore tubes are densely arranged in a honeycomb shape, the diameter of the pore tubes is 0.5-2 μm, the depth of the pore tubes is 2-3 μm, the pore openings are polygonal and irregular, and the pore openings become tooth-shaped after maturation. The sub-shell layer is planted on the inner wall surface of the pore pipe and is composed of a basilar and has no capsule. The basidiomycetes are rod-shaped, and have a size of 19-22 μm × 5-7 μm, each mature basidiomycetes has 4 peduncles, and each peduncle has 1 basidiomycetes. The spore is oblong or nearly cylindrical, sometimes slightly curved, has a crooked tip, has a size of 6-11 μm × 2.5-4 μm, and is gray.

Example 3

Molecular biological identification was performed on Poria cocos (Wolfiporia cocos) YX1 obtained in example 1.

1. Extracting the genomic total DNA of Poria (Wolfiporia cos) YX 1: placing 10-30mg of mycelium or sclerotium of YX1 of fresh Poria (Wolfipora cos) into a centrifuge tube, adding into a sample preparation system cell crusher, fully grinding into powder to break wall, crushing at 5000rpm for 30s, and repeating for 2-3 times to obtain a broken wall sample; then extracting according to CTAB method or UNIQ-10 column type fungus genome extraction kit (manufacturer: biological engineering (Shanghai) GmbH, No. B511375) to obtain genome total DNA sample, and storing in refrigerator at-20 deg.C;

2. obtaining rDNA sequence, 28S sequence and EF sequence of Poria cocos (Wolfiporia cocci) YX 1: taking the total genomic DNA obtained in the step 1 as a template, then carrying out polymerase chain PCR amplification with a total system of 25 mu l, and utilizing three pairs of primers ITS1 and ITS 4; NL1, NL 4; EF1F and EF1R are used for carrying out tuckahoe sequence analysis, and PCR reaction programs comprise 94 ℃ pre-denaturation for 5min, 94 ℃ denaturation for 30S, 55 ℃ annealing for 45S, 72 ℃ extension for 2min, 30 cycles in total, finally 72 ℃ extension for 10min, and 4 ℃ termination; the PCR target product is detected by agarose gel electrophoresis, and the sequencing of the product is carried out, thus finally obtaining 1652bp rDNA sequence, 583bp 28S sequence and 634bp EF sequence of Poria cocos (Wolfiporia cocos) YX 1.

The nucleotide sequence of rDNA of the Poria cocos (Wolfiporia coccos) YX1 is shown in SEQ ID No.1, the nucleotide sequence of 28S is shown in SEQ ID No.2, and the nucleotide sequence of the transcription elongation factor EF is shown in SEQ ID No. 3.

The components of the PCR amplification system (25. mu.l) are shown in Table 1, and primers ITS1 and ITS 4; NL1, NL 4; the sequences of EF1F and EF1R are shown in Table 2.

TABLE 1 PCR reaction amplification System (25. mu.l)

Reagent	Dosage (mu l)
		10×PCR Buffer	2.5
Bsa	0.5
		dNTP(2.5mM)	2.0
Primer 1 (10. mu. mol/L)	0.5
		Primer 2 (10. mu. mol/L)	0.5
rTaq DNA polymerase (5U. mu.L)	0.2
		Template DNA	1.0

Table 2 primers ITS1, ITS 4; NL1, NL 4; EF1F, EF1R sequences

3. Submitting rDNA sequence, 28S sequence and EF sequence of Poria cocos (Wolfiporia cocos) YX1 in NCBI GenBank, performing BLAST comparison on sequences of similar strains, performing comprehensive comparison by using ClustalW 2.0 software, and finally constructing a phylogenetic tree by adopting an NJ method in software MEGA 6.1. The results are shown in FIG. 2. Fig. 2 is a development analysis diagram of a genetic system of different strains of poria (Wolfiporia cocos) YX1, wherein the respective symbols represent poria (Wolfiporia cocos) YX 1.

As can be seen from fig. 2: the phylogenetic tree NJ clustering method constructed by MEGA6.1 software was used to analyze that Poria cocos (Wolfipora cocos) YX1 is most closely clustered with other strains of 14 Poria cocos strains, and Pachyma hoelen, Wolfipora cocos, Poria cocos and Macrohypoporia cocos are synonyms of Poria cocos. The genetic distance of the three-dimensional vector is relatively close, four homologous and synonym poria cocos ITS sequences are clustered together, and 3 Trichoderma, 1 Fungal sp.P6540 and 1 WolfiPoria cos YX 128S sequences are clustered as the most close sequences. There was also a cluster of 3 Trichoderma koningi and WolfiPoria cos YX1 EF transcriptional elongation factors. The poria ITS sequence, the 28S sequence and the EF sequence are distinguished on a phylogenetic tree, but genetic connection exists among the sequences, and the homology reaches more than 90 percent. It was confirmed that Poria cocos (Wolfiporia cocos) YX1 is a novel strain.

Example 4 screening Medium

When the selected poria cocos (Wolfiporia cocos) YX1 was cultured, a mother culture medium and a stock culture medium of poria cocos (Wolfiporia cocos) YX1 were selected, which could increase the cultivation yield of poria cocos (Wolfiporia cocos) YX 1.

1. Screening is carried out on the basis of a basic mother culture medium, a plurality of formulas are obtained and are shown in table 3, and the results are shown in table 3 by carrying out heat preservation culture for 5-7 days at 28 ℃ with the same inoculation amount.

TABLE 3 mother culture Medium formulation and culture results

As can be seen from table 3: when the culture time is the same, the growth speed of hyphae in the culture medium for enriching the mother seeds and the culture medium for peptone glucose is faster than that of the basic culture medium in the culture medium for enriching the mother seeds and the culture medium for peptone glucose; particularly, the enriched mother culture medium and the peptone glucose culture medium of No.1 have the advantages that the growth rate of hyphae reaches more than 76.49 +/-0.43 mm when the culture time is 120h, the culture time of the two culture media is shortened by 24h compared with that of a basic culture medium in a culture dish, and the growth speed is increased by more than 2.1 mm.

2. Taking pine accessories such as pine sawdust and the like as ingredients, screening a stock culture medium of Poria cocos (Wolfiporia cocos) YX1 to obtain 6 groups of formulas shown in Table 4 (100 jin are prepared), preparing the stock culture medium according to each group of formulas, and inoculating and culturing.

The method comprises the following specific steps: mixing the above components, adding water, stirring, respectively pressing stock culture medium, piling for 10-12 hr, spraying water until the material is gripped by fingers but no water is discharged (controlling the humidity of the material for each stock culture medium to be 50-60%), respectively bagging, weighing, sterilizing at 121 deg.C under high temperature and high pressure for more than 6 hr, standing overnight, naturally cooling, and inoculating Poria (Wolfipora coccos) YX1 mother strain in the next morning; then, the temperature was controlled at 28 to 32 ℃ and the culture was carried out until the hyphae were grown over the bags, and the results of the observation of the culture in each group are shown in Table 4.

TABLE 4 stock culture Medium formulation and culture results

Remarking: the natural resin is as follows: at least one of mastic resin, shellac, rosin resin, and amber.

As can be seen from table 4: the shortest culture time of the group 1 is 24 days, the auxiliary material is wheat bran, the growth of hypha is easier to observe in appearance, and the formula of the group 1 is cheaper in price cost; the group 2 only contains pine sawdust as a main material and does not contain auxiliary materials, so that the growth speed of hyphae is slower than that of other groups, but the formula is simple and the cost is low; the main materials of the group 3 are pine sawdust and pine branches, the growth of hyphae is slow, the time for filling the fungus bags is long, and the culture time is 38d at most, but the hyphae grow in the pine branches and are not easy to observe; in groups 4-5, the main materials comprise pine sawdust, pine needles and pinecones, the materials are convenient to obtain and cheap, and the auxiliary materials comprise rice bran, wheat bran and corncobs, so that poria cocos hyphae can grow easily and quickly; group 6 had the most comprehensive and abundant nutrition, the time for filling the fungus bags was 26.6 days, and the average growth rate was 77.26 + -0.28;

therefore, the 6 groups of stock culture media can grow hyphae, the average culture time for the hyphae to overgrow the fungus bags is 30.33d, and the average growth rate is 71.99 +/-0.30 mm; all of 6 groups were used as stock culture medium for Poria (Wolfiporia cocos) YX 1;

when the stock culture medium of Poria (Wolfipora cocos) YX1 is prepared from pine attachment such as pine sawdust, the content of main material cannot be lower than 60%; the pine sawdust can be used as a main material, or a mixture of at least one of miscellaneous sawdust, pine branches, pine needles and pinecones and the pine sawdust can be used as the main material; the content of adjuvant is preferably 0-38%, and the adjuvant can be at least one of testa oryzae, testa Tritici, corn cob, and cottonseed hull. The stock culture medium may also contain vitamin B2, vitamin C, mildew-proof phytophthora additive (such as Clavictoria king, etc.), and yeast tablet.

Example 5 traditional pine wood segment cultivation method

The traditional pine wood section cultivation method of poria cocos (Wolfiporia cocos) YX1 comprises the following steps:

selecting pine wood sections with the diameter of more than or equal to 9cm and the length of about 60cm and with part of envelope removed in months of 11-12, and drying in the sun for later use;

in the next 4-5 months, selecting continuous sunny weather, digging cellars with the length of about 1m, the depth and the width of about 50cm on the prepared greenhouses along the slope, wherein the cellars are spaced by about 20cm, and a plurality of cellars are horizontally and longitudinally spaced (depending on the slope) and a drainage ditch with the width of about 40cm is dug; then, placing a charging barrel of about 25kg in each cellar, commonly called as 'lower cellar' (1 charging barrel means 1 pine segment, the adding amount of the charging barrel is determined according to the size of the pine segment, and generally 3-5 charging barrels are placed in each cellar);

a culture medium of mycelia of a strain of Poria cocos (Wolfiporia cocci) YX1 was obtained as described in S1 of example 7; inoculating a culture medium of Poria cocos (Wolfipora cos) YX1 protospecies hypha to one end of a pine tree section (400 g of the culture medium of Poria cocos (Wolfipora cos) YX1 protospecies hypha is used for every 6kg of the pine tree section as shown in figure 3), covering with 4-6cm thick soil, preserving heat and moisture for a period of time, and forming sclerotium in a cellar, which is commonly called as 'fruiting'; at the moment, cracks of the soil surface are cracked, and the cracks are covered by the soil in time so as to prevent the poria cocos from falling out of the soil surface.

The heat preservation, the moisture preservation and the soil covering can ensure that the hyphae can normally grow and develop under the dark condition, and are beneficial to the formation of sclerotia; the thickness of the covering soil is controlled to be 4-6cm and is not tight or loose. The traditional pine cultivation of tuckahoe takes pine trees as a main source of nutrient substances, and mainly utilizes trunks and stumps of the pine trees to cultivate.

FIG. 3 is a photograph showing the cultivation in example 5.

According to the traditional pine tree section cultivation method, the poria cocos can be introduced after hypha grows on the pine tree section, so that the yield of the poria cocos is improved; the specific steps of guiding the poria cocos can be as follows: after the pine wood section is buried in the cellar for 20-30 days, the soil is dug from the cellar, the lower section (i.e. the end which is not inoculated with the stock) of the pine wood section has white hypha growth, and the smell of tuckahoe is smelled, so that the tuckahoe hypha can be confirmed to have grown in the pine wood section; at the moment, first-generation or second-generation sclerotium (sclerotium size is 4-8cm, is fat, mature, thin and light red in color, white in flesh, much in juice, and not easy to rot and deteriorate) cultivated by the same poria cocos strain is selected, the sclerotium is dug out and then placed in a refrigerator at about 4 ℃ for storage, the outer skin of the sclerotium is peeled off, and the peeled sclerotium is inserted into the crack of pine by using a wooden toothpick at the end of the pine section where the original species is not inoculated and then buried in the soil.

Example 6

The China-nationwide cultivated Poria cocos farmers generally adopt the China academy of sciences microbial culture Collection management center CGMCC 5.78 strain for wide cultivation.

Poria cocos (Wolfiporia cocos)5.78 strain was cultured to obtain a Poria cocos (Wolfiporia cocos)5.78 stock, and then the Poria cocos (Wolfiporia cocos)5.78 stock was inoculated and cultivated according to the procedure of example 5. And comparing the cases of the cultivated polyporus umbellatus of examples 5-6, the results are shown in Table 5 and FIGS. 4-5. FIG. 4 is a photograph of sclerotium of Poria cocos in example 5; FIG. 5 is a photograph of sclerotium of Poria cocos in example 6.

TABLE 5 results of fruiting on traditional pine wood cultivation of examples 5-6

Remarking: 1. the yield rate is the ratio of the dry weight to the fresh weight of the sclerotium of the poria cocos; 2. the poria cocos forming time refers to the time from cellar to the first poria cocos picking, and poria cocos is picked twice in each pine tree section.

As can be seen from table 5 and fig. 4-5, the poria (Wolfiporia cocci) YX1 showed a shorter clotting time by 7 days than the 5.78 species, the poria (Wolfiporia cocci) YX1 showed a clotting rate of 99.76%, the poria (Wolfiporia cocci) YX1 showed an average sclerotic weight of 0.152kg than the 5.78 species, the poria (Wolfiporia cocci) YX1 showed a higher yield by 12.4% than the 5.78 species, the poria (Wolfiporia cocci) YX1 showed a better skin/flesh than the 5.78 species, and the poria (Wolfiporia cocci) YX1 showed a lower sclerotic flesh than the 5.78 species;

the poria cocos obtained by the poria cocos (Wolfiporia cocos) YX1 has short poria cocos hardening time, high poria cocos rate, heavy average sclerotium yield, large individual specification of the poria cocos, low water content, smooth and fine fungus blocks, thin skin and white flesh, firm sclerotium, no sediment, high yield and little skin and much flesh.

Example 7 cultivation method of pine tree section by interplanting bacteria at one end

The method for cultivating Poria cocos (Wolfiporia cocos) YX1 by means of one-end bacterium interplanting comprises the following steps:

s1, preparing a mother culture medium (containing 30g of glucose, 15g of peptone, 20g of agar powder, 1g of monopotassium phosphate and 0.5g of magnesium sulfate in 1000mL of pH 6 aqueous solution), and sterilizing at 121 ℃ and 0.105Mpa for more than 6 hours; then inoculating 2g Poria (Wolfipora cos) YX1 into cooled 10mL mother culture medium, and culturing at 28 deg.C for 5 days to obtain Poria (Wolfipora cos) YX1 mother culture;

preparing a stock culture medium (the raw materials comprise 80 wt% of pine sawdust, 18.5 wt% of wheat bran, 1 wt% of sucrose and 0.5 wt% of gypsum, and the humidity of the mixed material is 50-60%), filling the mixture into a fungus bag, sterilizing the mixture at the high temperature of 121 ℃ for more than 6 hours, and naturally cooling the mixture overnight; inoculating Poria (Wolfipora cos) YX1 mother strain into 400g stock culture medium, and culturing at 28-32 deg.C until the mycelia overgrow the strain bag (about 24-38 days) to obtain Poria (Wolfipora cos) YX1 stock strain;

s2, selecting a pine section with a diameter of more than or equal to 9cm and a length of about 60cm and with partial envelope cut off, sleeving a fungus bag filled with poria cocos (Wolfiporia cocos) YX1 protospecies hyphae in S1 at one end of the pine section (400 g fungus bags filled with poria cocos (Wolfiporia cocos) YX1 protospecies hyphae are used for every 6kg of the pine section), and then tightly binding and sealing the fungus bags on the pine section by using rubber bands (as shown in figure 6 a); then sleeving an empty fungus bag with two open ends at the other end of the pine section, fixing one end of the empty fungus bag on the pine section by using an adhesive tape, keeping the other end of the empty fungus bag in an open state, extending the empty fungus bag out of one end of the pine section by 7-8cm, not covering the fungus bag at the middle position of the pine section (as shown in figure 6b), transferring the empty fungus bag into a culture room with the temperature of 28-32 ℃ and the relative humidity of RH 70-80%, covering newspaper on the surface of the pine section, keeping the water content of the pine section at 40-50%, and culturing 40-day-grown hyphae; then, the pine segments are buried in the cellar to grow the nodulation tuckahoe.

FIG. 6 is a drawing showing the cultivation process of example 7, wherein a is a pine wood section with one end inoculated with stock and the fungus sack sealed, and b is a pine wood section with both ends sleeved with fungus sacks.

The operations and management of digging the cellar, burying the pine wood segments in the cellar, and growing the noduliferous trees after burying the pine wood segments in the cellar in example 7 are the same as those of the conventional pine wood segment cultivation method in example 5.

In example 7, the sclerotium of Poria cocos (Poria cocos) was buried in the cellar and the product was obtained after 3-4 months.

In the above S1, the medium cannot be extinguished and cooled midway during sterilization, so as to ensure thorough sterilization.

In the above-mentioned S1, the inoculation amount of Poria cocos (Wolfiporia cocos) YX1 mother strain in the stock culture medium is not specified; in general, 50-100g of Poria cocos (Wolfiporia cocos) YX1 mother strain is inoculated to 400g of stock culture medium.

In the step S2, a fungus bag is tied and sealed on the pine section by a rubber band at one end of the pine section inoculated with the stock, so that the infection of other mixed fungi in the soil can be prevented; in addition, the problem that the fungus bags are contaminated or separated from the log due to the problems of overlarge humidity and the like caused by the fact that the fungus bags are washed by rainwater on the day of leaving the cellar can be prevented, and new fungus bags need to be replaced in the later period.

In the above S2, a crack, a gap or a new wound is formed at one end of the pine segment inoculated with the stock seed, so that the pine can grow bacteria more easily.

In the above S2, the middle position of the pine wood segment is not covered by the fungus bag, so that the pine wood segment can fully absorb the nutrients and water in the soil, and the surrounding of the pine wood segment can be kept for transporting and exchanging the nutrients with the outside.

In the above S2, the other end of the pine wood section is sleeved with an empty fungus bag with both ends open, one end of the empty fungus bag is fixed on the pine wood section, the other end of the empty fungus bag is kept in an open state, and the empty fungus bag extends out of one end of the pine wood section by 7-8cm, so that the pine wood section can fully absorb nutrients and moisture in soil, the surrounding of the pine wood section is kept in transportation and exchange of nutrients with the outside, and meanwhile, fresh hyphae growing on the cross section can be prevented from polluting sundry fungi, and the hyphae grow white, clean and dense.

In the step S2, before the pine section is placed in the cellar, one end of an empty fungus bag is sleeved, the empty fungus bag which extends out of one end of the pine section by 7-8cm can be tied by a rubber band, when hyphae grow on the cross section of the pine section, the rubber band is loosened, and the tuckahoe is placed in the cellar to grow, so that pollution can be further isolated, the tuckahoe is not easy to be infected with infectious microbes, and the quality of the tuckahoe is good.

In the poria cocos cultivation process, a hole can be drilled on the pine wood section for cultivation, so that the purposes of avoiding the pollution of mixed bacteria and reducing the inoculation amount are achieved; the specific steps of punching cultivation can be as follows: taking a pine section with the length of 60-70cm, punching holes (the punching positions can be that the center of the cross section of the pine section a, the side face of the pine section at the position 2-4cm away from the cross section b, the center of the cross section of the pine section c and the side face of the pine section at the position 2-4cm away from the cross section c) on the pine section, the diameter of each hole is about 4cm, the depth of each hole is 4-6cm, then, the original poria cocos hyphae are plugged immediately, then, hole sealing pastes or wax sealing are pasted, the pine section is cultured for about 60-70 days until the pine section is full of hyphae, the water content of the pine section is adjusted to about 40-50%, and then, the pine section is buried in a cellar to grow polyporus.

The sclerotium conditions of examples 5 and 7 were counted, and the results are shown in Table 6 and FIGS. 7 to 9.

FIG. 7 is a photograph showing the formation of the knot in accordance with example 5; FIG. 8 is a photograph showing the formation of the knot in accordance with example 7; FIG. 9 is a photograph of sclerotium of Poria cocos in example 7.

TABLE 6 sclerotium results of example 5 and example 7

As can be seen from table 6 and fig. 7-9, the sclerotia obtained in example 5 and example 7 are not very different, and the method in example 7 is not prone to contaminating other bacteria, can reduce the stock inoculum size, is convenient for indoor cultivation, is simple and convenient, and avoids being limited by the cultivation time; in addition, in the case of the embodiment 7, the tuckahoe is pure white and does not pollute the mixed bacteria, while in the case of the embodiment 5, the cross section of the pine segment is not protected, and the pine segment is directly buried in the soil, so the tuckahoe is easy to pollute the mixed bacteria.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and equivalent substitutions or changes according to the technical solution and the inventive concept of the present invention should be covered by the scope of the present invention.

Sequence listing

<110> university of teacher's university of Anqing

Anhui Yuelan Pharmaceutical Co., Ltd.

<120> Poria cocos (Wolfiporia cocos) YX1, and culture medium and cultivation method thereof

<130> 2021

<141> 2021-09-14

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1652

<212> DNA

<213> Poria cocos (Wolfiporia cocos)

<400> 1

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ttgggttatc gtgtccggtc ggagtgggtt catggcacac gttgtaacga cacgcgtgtg 180

atgccctcac gtaacccagt cgagccctga gtggatcgcg cggaagatgc tgtccggtga 240

gcgaagccaa tcgcgacccg ggccgtcctg ttcggaggca agcagatatc cgttagtgtg 300

aagccgataa ggacccagtc cgatgtgtta agccgaagac gattcggtcg gacctgtgag 360

tcttggaagg tccttcggct tgggtccgcc tctatggccg ccgtcgtggg ggacgacatt 420

ctctcttgaa ctcggaaaac cggacgccct cccattcatg gaggagagag ggtcgtctaa 480

gacccgcttg gattgacctg ttgcaccgtc tacagtcatc ttccgagtgt gtgcaatggg 540

agagaacgaa gcccgcgatt ggggaattcg agatgccctc ccatttattg gggcgtgggg 600

aggtttgtgt actcccagac cagctccgag tcgtgccgcc gtctactact acagaccttt 660

tgtctgagat atctggtcga tgccgtcgag cacgtcacaa gtcattctcg aattcatatc 720

cgtccgtcta tgacaggcgc ggcccactac ggacgtgaga gttcgttaac acgggtcgcg 780

agcgtccgtt aacacgggcg tgagcgtccg ttacggtacc gtctttctcg atagacaaaa 840

gaccctttgg cacccctttc acataccaca cccgtgcacc tatttgccgc ggtgcaagga 900

cccacgttcg gtccttccgc gcgtgtgaag accctctgcc cgcggcgccc ttacaaaccc 960

cataatgtca gaacgttgtc ccgatataac aatgaaagag tttaataaca actctcagcg 1020

acggatctct tggctctcgc atcgatgaag aacgcagcga aatgcgataa gtaatgtgaa 1080

ttgcagaatc cagtgaatca tcgaaccttt gaacgcacct tgcgcccctc ggtattccga 1140

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ctggccccta gggtccgagt tgtaatttgt agaggatgct tttggtgagg tgccgcccga 120

gttccctgga acgggacgcc acagagggtg agagccccgt ctggctggcc accgagcctc 180

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gtcttctaaa gctaaatatt ggccagagac cgatagcgca caagtagagt gatcgaaaga 300

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gcgtcacacc ccgcttggcc tgtctacccc tcctttggca gcaaattttt ctgctgcctc 180

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atccatcgtc ccaacaaaat gcactcattc aatcgcatcg tcttttgact cgatttctct 300

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ctttcaagta tgcgtgggtt cttgacaagc tcaaggccga gcgtgagcgt ggtatcacca 420

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tattcctggc tcttgacacg tcgaaatcat cattctaacg tgccaataca gacgctcccg 540

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Claims (10)

1. Poria cocos (Poria cocos) fungusWolfiporia cocos) YX1, which is characterized in that the preservation number is CCTCC NO: m2021434.

2. The Poria cocos bacterium (F) (Poria cocos bacterium) of claim 1Wolfiporia cocos) The stock culture medium of YX1 is characterized by comprising the following raw materials in percentage by weight: the main material is more than or equal to 60 percent, the auxiliary material is 0-38 percent, the cane sugar is 0.5-2 percent, the gypsum is 0.5-1 percent, the natural resin is 0-1 percent, the monopotassium phosphate is 0-0.1 percent, the vitamin B is 0-0.0002 percent, and the gram mould king is 0-0.1 percent, wherein the total weight percentage of all the raw materials is 100 percent, and the main material is a mixture of pine sawdust and at least one of miscellaneous wood chips, pine branches, pine needles and turpentine or pine sawdust.

3. Poria cocos bacterium (hoelen) according to claim 2Wolfiporia cocos) The stock culture medium of YX1 is characterized in that the auxiliary material is at least one of rice bran, wheat bran, corncob and cottonseed hull.

4. Poria cocos bacterium (hoelen) according to claim 2 or 3Wolfiporia cocos) The stock culture medium of YX1 is characterized in that the natural resin is: at least one of mastic gum resin, shellac, rosin resin, and amber.

5. Poria cocos bacterium (hoelen) according to claim 2 or 3Wolfiporia cocos) The stock culture medium of YX1 is characterized in that the weight percentage of the main material is more than or equal to 70 percent.

6. Poria cocos bacterium (hoelen) according to claim 2 or 3Wolfiporia cocos) The stock culture medium of YX1 is characterized in that the mixing humidity of the stock culture medium is 50-60%.

7. The Poria cocos bacterium (F) (Poria cocos bacterium) of claim 1Wolfiporia cocos) The mother culture medium of YX1 is characterized in that the raw materials are as follows: 1000mL of an aqueous solution having pH =5-6 contains: 150 g of potato, 20-30g of glucose and 15-2 g of agar powder0g, 0-1g of monopotassium phosphate and 0-0.5g of magnesium sulfate;

alternatively, a 1000mL aqueous solution having pH =5-6 contains: 20-30g of glucose, 10-15g of peptone, 15-20g of agar powder, 0-1g of monopotassium phosphate and 0-0.5g of magnesium sulfate.

8. The Poria cocos bacterium (F) (Poria cocos bacterium) of claim 1Wolfiporia cocos) The cultivation method of YX1 is characterized by comprising the following steps:

s1, adding PoriaWolfiporia cocos) YX1 sequentially culturing in mother culture medium and stock culture medium to obtain PoriaWolfiporia cocos) YX1 stock;

s2, adding PoriaWolfiporia cocos) Sleeving a fungus bag of a stock strain YX1 at one end of a pine section, sealing the fungus bag, fixing the fungus bag on the pine section, sleeving a hollow fungus bag at the other end of the pine section, keeping the opening state, adjusting the water content of the pine section to 40-50%, culturing at 28-32 ℃ for 40-50 days, burying the pine section in a cellar, and growing polyporus;

wherein the mother culture medium is the Poria cocos (Schw.) wolf fungus (Poria cocos (Schw.) wolf) of claim 7Wolfiporia cocos) YX1, wherein the stock culture medium is the Poria cocos (Poria cocos) strain of claim 2Wolfiporia cocos) Stock culture medium of YX 1.

9. Poria cocos bacterium (hoelen) according to claim 8Wolfiporia cocos) The cultivation method of YX1 is characterized in that in S2, the empty fungus bag is a fungus bag with two open ends; in S2, the empty fungus bag extends 7-8cm beyond the end of the pine section, where the empty fungus bag is sleeved.

10. Poria cocos bacterium (hoelen) according to claim 8 or 9Wolfiporia cocos) The cultivation method of YX1, wherein the middle position of pine wood segment is not covered with fungus sack in S2.

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