CN106947696B - Black fungus strain - Google Patents

Abstract

The invention discloses a black fungus strain. The black fungus (Auricularia auricula) strain No. 3 belongs to Auricularia, is preserved in China general microbiological culture Collection center (CGMCC), has the preservation address of No. 3 Xilu-Beichen No.1 of the Korean area in Beijing, the preservation date of 2016, 7 and 5 days, and the preservation number of CGMCC No. 12525. The No. 3 strain of the lihe-ear provided by the invention has the quality characteristics of the black fungus, such as single fruiting body, moderate size of the ear, no root, bowl shape or ear shape, gray color of the back of the dry ear, black and glossy ventral surface, large color difference of the front and back surfaces, round edge of the ear, unobvious tendon vein on the back surface, and the like, and the single fruiting rate of the black fungus reaches more than 90 percent, so that the defects of poor resistance of the 'new family' and poor ear shape of '916' are improved, and the strain has good popularization prospect.

Description

Black fungus strain

Technical Field

The invention relates to a black fungus strain.

Background

Auricularia auricula (lex hook) Underw belongs to Basidiomycota, Hymenomycetes, Auriculariales, Auriculariaceae and Auricularia, is a high-colloid Basidiomycetes which is originally artificially cultivated in the world, has the effects of moistening and strengthening, clearing lung-heat and tonifying qi, enriching blood and activating blood and the like, and is popular among people. The Lishui city in Zhejiang province is the main cultivation area of black fungus in south China, and the cultivation scale in recent years is stabilized to about 1.5 hundred million sticks. With the expansion of the cultivation scale of black fungus substitute materials, different standards are generated for the classification of black fungus grades in the market, and characteristics such as blackish brown, small ear, single piece, thick meat and the like become evaluation standards of high-quality ears more and more. In the existing local traditional cultivated varieties, the variety 916 is large in size and low in the proportion of high-quality ears at the early stage; the other main cultivar, new family, has good ear shape and moderate size, but has poor ear-flow resistance, and is mainly used for cut-log cultivation. With the development of the black fungus industry, the problem of insufficient backup varieties becomes increasingly prominent, and the selection of excellent varieties with excellent quality, high yield, good commodity and good market prospect is particularly important.

Disclosure of Invention

The invention aims to solve the technical problems that a black fungus strain used in the existing production is difficult to form a single piece due to small hole emergence, low in yield and poor in quality, and provides the black fungus strain with excellent quality, high yield, good commodity and good market prospect.

The black fungus strain No. 3 belongs to the genus Auricularia, is preserved in the China general microbiological culture Collection center of the culture Collection of microorganisms, has the preservation address of No. 3 Xilu-Beijing, the sunward area, the preservation date of 2016, 7 and 5 days, the preservation number of CGMCC No.12525, and is named Auricularia auricula by classification.

The mycelium of the No. 3 beautiful fungus strain on the PDA culture medium is creeping, the mycelium is dense, the edge is neat, and the branch is obvious. The fruiting body is single-sheet, the single-sheet rate of small-hole ear is up to above 90%, the ear is moderate in size, has no root, is bowl-shaped or ear-shaped, has gray dry ear back, black and glossy abdomen, large difference in color between front and back, round ear edge and unobvious back veins.

The No. 3 Liear is bred by single-double hybridization of the 'New family' and '916' of the black fungus, the number of a hybrid strain is 'X6', the strain enters a cultivation test after being screened, and the strain is 'A18' after being uniformly numbered. The cultivation test shows that the strain has the genetic phenotype characteristics of good earliness, single growth of sporocarp and less back tendon. The strain improves the defects that the parent 'new family' is easy to flow ears and low in yield, and the other parent '916' produces ears to be in a cluster shape, integrates the advantages of the parent 'new family' of precocity, good ear shape and '916' of high yield and good resistance, has excellent comprehensive properties, and is named as 'Liear No. 3'.

The black fungus strain of the invention is analyzed by DNA (ISSR) fingerprint polymorphism, and the result is as follows: the 15 primers amplify stable bands for 1-3 strains to be detected, the polymorphism bands are 7-19, the 15 ISSR primers amplify 201 stable bands for 3 strains of black fungus to be detected, wherein the polymorphism bands reach 145, and the polymorphism ratio is 72.14%. The similarity coefficient of the black fungus-Lier No. 3 to be detected and the reference strain black fungus- 'New family' is 62%, the similarity coefficient of the black fungus-Lier No. 3 to be detected and the reference strain black fungus '916' is 47%, and the black fungus-Lier No. 3 to be detected can be presumed to be a new variety different from the two reference strains.

RAPD fingerprint analysis results show that: the similarity coefficient of the black fungus-Lier No. 3 to be detected and the similarity coefficient of the reference strain black fungus- 'New family' is 74%, the similarity coefficient of the reference strain black fungus '916' is 56%, and the black fungus-Lier No. 3 to be detected can also be estimated to be a new variety different from the two reference strains.

The judgment of the difference between the No. 3 fungus and the parent strains 'new family' and '916' by using the antagonistic reaction of the strains shows that hyphae at the junction between the 3 black fungus strains to be tested are all in a groove shape and have obvious antagonistic reaction, and further presumes that the black fungus-No. 3 fungus to be tested is a new variety different from two control strains.

The No. 3 strain of the lihe-ear provided by the invention has the quality characteristics of the black fungus, such as single fruiting body, moderate size of the ear, no root, bowl shape or ear shape, gray color of the back of the dry ear, black and glossy ventral surface, large color difference of the front and back surfaces, round edge of the ear, unobvious tendon vein on the back surface, and the like, and the single fruiting rate of the black fungus reaches more than 90 percent, so that the defects of poor resistance of the 'new family' and poor ear shape of '916' are improved, and the strain has good popularization prospect.

Drawings

FIG. 1 shows ISSR fingerprints of black fungus strain amplification by primers H9, H23, P19, P23 and P26 (from left to right, the serial numbers of strains are 1-3, and the Marker is DL 2000);

FIG. 2 shows ISSR fingerprints of black fungus strain amplification by primers H33, H26, H28, H25 and P11 (from left to right, the serial numbers of strains are 1-3, and the Marker is DL 2000);

FIG. 3 shows ISSR fingerprints of black fungus strain amplification by primers H19, H20, P4, P27 and P20 (from left to right, the serial numbers of strains are 1-3, and the Marker is DL 2000);

FIG. 4 is a cluster diagram of ISSR polymorphisms of strains to be detected in Auricularia auricula;

FIG. 5 shows RAPD fingerprints (from left to right, the serial numbers of strains are 1-3, and the Marker is DL2000) of black fungus strain amplification by primers R2, R4, R5, R13 and R6;

FIG. 6 shows RAPD fingerprints (from left to right, the serial numbers of strains are 1-3, and the Marker is DL2000) of black fungus strain amplification by primers R16, R18, R20, R14 and R7;

FIG. 7 shows RAPD fingerprints of black fungus strain amplification by primers R8 and R9 (from left to right, the serial numbers of the strains are 1-3, and the Marker is DL 2000);

FIG. 8 is a clustering chart of RAPD polymorphisms of strains to be detected of black fungus;

FIG. 9 shows antagonistic reaction patterns of Auricularia auricula No. 3 (left panel) and "New family" (right panel);

FIG. 10 shows antagonistic responses between Auricularia auricula No. 3 (left panel) and "916" (right panel);

FIG. 11 is a graph showing antagonistic reactions of Auricularia auricula "New family" (left panel) and Auricularia auricula "916" (right panel).

Detailed Description

Example 1: selecting 'Xinke' and '916' of black fungus planted in current field as hybridization parents, obtaining F1 generation hybrid strains by single hybridization and single-double hybridization technologies, screening strains with vigorous hypha growth, single fruiting body and higher yield than the parents through laboratory hypha growth observation and cultivation tests, collecting the fruiting body, and obtaining 2 generation strains through a tissue separation technology; further carrying out a cultivation test, screening to obtain an excellent strain with stable target agronomic characters, carrying out tissue separation to obtain a hybrid strain number X6, and carrying out the cultivation test after screening, wherein the strain has good earliness, single fruiting body and less back tendon, and has outstanding high and stable yield performance in the cultivation test. And then a stable strain is obtained after single plant screening, the strain improves the defects of easy earring, low yield and cluster generation of the old variety 'Xinke', integrates the advantages of early maturity, good earning, high yield and good resistance of 916 parent neology, and has excellent comprehensive properties, and is named as 'beautiful ear No. 3'.

According to the agricultural industry standard NY/T1730-2009 edible fungus authenticity identification ISSR method of the people's republic of China, the DNA difference comparison between the strain and the parent strain is carried out.

Inspection materials: 3 black fungus strains are provided, wherein the strain to be detected is No. 3 of the fungus, and the reference strain 2 is respectively 'New family' and '916'.

The detection method comprises the following steps: ISSR test primer sequences (Table 1) are synthesized by Ongke biotechnology, Inc., in the main references ISSR fingerprint analysis and SCAR marker of main Auricularia auricula-judae culture strains in China, ISSR fingerprint analysis of wild Auricularia auricula-judae strains in Heilongjiang, and ISSR analysis of Auricularia auricula-judae culture strains.

The mycelium activation, culture, genome DNA extraction, quality detection, PCR amplification and electrophoresis detection of the strain to be detected are carried out according to the ISSR method for identifying the authenticity of the strain of edible fungi of NY/T1730-2009, which is the agricultural industry standard of the people's republic of China.

TABLE 1 ISSR primers for authenticity identification of strains

Results and analysis: ISSR fingerprint of black fungus strain: the method comprises the steps of carrying out ISSR amplification on 3 black fungus strains to be tested by using 49 primers, and selecting 15 primers capable of amplifying stable specific bands from the primers, wherein the 15 primers are H9, H19, H20, H23, H25, H26, H28, H33, P4, P11, P19, P20, P23, P26 and P27. The 15 primers amplify stable bands for 1-3 strains to be detected, the polymorphism bands are 7-19, the 15 ISSR primers amplify 201 stable bands for 3 strains of black fungus to be detected, wherein the polymorphism bands reach 145, and the polymorphism ratio is 72.14% (fig. 1-3).

Clustering analysis: and analyzing the amplification products, counting the stability bands appearing in the test, recording the band as 1, recording the band as 0 when no band exists, and performing cluster analysis by using software NTSYS-pc 2.0.

And detecting a conclusion. The ISSR analysis result shows that: the similarity coefficient between the Auricularia auricula-Li Er No. 3 strain to be detected and the Auricularia auricula-Li Er-Xinke strain is 62%, and the similarity coefficient between the Auricularia auricula-Li Er No. 3 strain to be detected and the Auricularia auricula 916 strain is 47%, and the Auricularia auricula-Li Er No. 3 strain to be detected is presumed to be a new variety different from the two control.

The environmental adaptability and yield results of the No. 3 beautiful ear at different altitudes show that the No. 3 beautiful ear has certain high yield characteristics.

The test strains were: zhe ear 608, 916, Li ear No. 2, Xinke, 842, Zao Hei No.1, 862, Li ear No. 3.

Test site: qingyuan, Chunan, Lishui Bai orchard, Liandu district, Wuyi.

Test arrangement: the strains are manufactured uniformly, and the cultivation management is carried out on each test point according to the local production management mode.

And (3) test results: tests show that the growth speed of the No. 3 hypha of the liriopes is higher, the hypha stick time is shorter than that of other strains by more than 2 days, and the strains grow well.

Table 2 statistical units of the germination speed of the test strains: mm/d

Bacterial strains	Qingyuan food	Lishuibao garden	Lotus city district	Chunan	Wuyi (Wuyi)	Average spawn running speed
							Zhe Er 608	3.55	3.96	3.70	3.27	3.50	3.60
916	3.57	3.96	4.11	3.54	3.30	3.70
							Lier No. 2	2.88	3.34	3.50	3.08	3.00	3.16
New science	3.16	3.64	3.57	3.20	3.00	3.31
							842	3.31	3.93	3.94	3.38	3.40	3.59
Morning black No.1	3.05	4.16	4.81	3.37	3.50	3.78
							862	3.22	4.12	4.07	3.20	3.70	3.66
Lier No. 3	3.27	4.48	3.59	3.57	4.00	3.78

The cultivation finds that the formation time of the No. 3 earbud of the liriope esculenta is earlier than that of other strains and is more than 5 days earlier than that of 916 and the new family; the strong earbud forming capability can promote earbuds to seal the puncture openings as early as possible, reduce the infection probability of mixed bacteria and effectively reduce the pollution rate of the bacteria sticks after the stock arrangement.

Yield: the cultivation test result shows that the yield of the liriope 3 is higher than that of other strains. Through analysis of variance and statistical analysis of yield, the treatment room, the site, the variety and the site of each test point have very obvious differences; no. 3 of the Liear strain has no significant yield difference with the strain 842, but has significant yield difference with other 6 strains.

Table 3 statistical units of test strain yield results: g/rod

TABLE 4 analysis of variance of the yield of the tested strains

Source of variation	Degree of freedom	Sum of squares	Mean square	F	F0.05	F0.01
							Block of medicine	2	56.84	28.42	1.57	3.11	4.89
Treatment room	39	25466.55	652.99	35.98**	1.56	1.87
							Location of a site	4	12502.92	3125.73	172.24**	2.49	3.57
Variety of (IV) C	7	4816.44	688.06	37.91**	2.13	2.88
							Location x variety	28	8147.19	290.97	16.03**	1.62	1.98
Error of the measurement	78	1415.51	18.15
							Total variation	119	26938.9

Note: and analyzing the data by adopting Stst variance analysis software, and taking one bit after decimal point in statistical analysis.

TABLE 5 statistical analysis of the yield of the test strains

Variety of (IV) C	Average yield (g/bar)	Difference of 5%	1% difference
				Lier No. 3	63.8	a	A
842	62.0	a	AB
				916	58.8	b	BC
Morning black No.1	58.1	bc	BCD
				New science	55.2	cd	CD
Zhe Er 608	55.2	cd	CD
				862	53.9	d	D
Lier No. 2	41.6	e	E

The No. 3 beautiful fungus shows stronger environmental adaptability at different altitudes, and shows excellent strain characteristics even in severe weather of high temperature and rainy days in autumn and winter of 2015; from inoculation of the fungus stick to first ear harvesting, about 78-89 days are needed for beautiful ear No. 3, and the earliness of the strain is good; the earbud is formed quickly, 2-3 tides can be collected in the year, the total yield of autumn and winter ears is more than 80%, and the proportion of high-quality ears is high; the ear slices are single and good in commodity; the cultivation process can be finished after harvesting 1-2 tides in the early period of the ear, the production period is effectively shortened, the time and the management cost are saved, rainy weather in spring can be avoided, and the product quality and the economic benefit are greatly improved on the whole.

Example 2: the RAPD method is used for comparing the DNA difference of the strain and the parent strain.

The detection basis is as follows: extracting strain DNA and determining an optimal primer according to the agricultural industry standard NY/T1743-2009 method for identifying the authenticity of edible fungus strains RAPD.

Inspection materials: 3 black fungus strains are provided, wherein the strain to be detected is No. 3 of the fungus, and the reference strain 2 is respectively 'New family' and '916'.

The detection method comprises the following steps: the experimental primer sequence (Table 6) was synthesized by Ongko Biotechnology Ltd according to the authentication method for edible fungus strains RAPD by NY/T1743-2009 (RAPD method).

The mycelium activation, culture, genome DNA extraction, quality detection, PCR amplification and electrophoresis detection of the strain to be detected are carried out according to the RAPD method for identifying the authenticity of the strain of edible fungi of NY/T1743-2009, which is the agricultural industry standard of the people's republic of China.

TABLE 6 RAPD primers for authenticity identification of strains

Species to be examined	Primer name	Primer sequences
			Black fungus	R1	AATCGGGCTG
Black fungus	R2	ACTTCGCCAC
			Black fungus	R3	AGGGGTCTTG
Black fungus	R4	AGTCAGCCAC
			Black fungus	R5	CAATCGCCGT
Black fungus	R6	CAGGCCCTTC
			Black fungus	R7	CCCATATCCC
Black fungus	R8	CCGATATCCC
			Black fungus	R9	CCGCATCTAC
Black fungus	R10	GAAACGGGTG
			Black fungus	R11	GACCGCTTGT
Black fungus	R12	GACGGATCAG
			Black fungus	R13	GGGAATTCGG
Black fungus	R14	GGGTAACGCC
			Black fungus	R15	GGTGATCAGG
Black fungus	R16	GTGACGTAGG
			Black fungus	R17	GTGAGGCGTC
Black fungus	R18	GTGATCGCAG
			Black fungus	R19	GTTGCCAGCC
Black fungus	R20	TCGGCGATAG
			Black fungus	R21	TGAGTGGGTG
Black fungus	R22	TGCCGAGCTG

Results and analysis: RAPD fingerprint of black fungus strain. RAPD amplification is carried out on 3 black fungus strains by utilizing 22 primers, and 12 primers capable of amplifying stable specific bands are selected from the primers, namely R2, R4, R5, R6, R7, R8, R9, R13, R14, R16, R18 and R20. The 12 primer pairs of the black fungus strains 1-3 amplify stable bands, the polymorphism bands are 6-16, the 12 RAPD primer pairs of the 3 black fungus strains 1-3 amplify 133 stable bands together, wherein the polymorphism bands reach 57, and the polymorphism ratio is 57.14% (fig. 5-7).

Clustering analysis: and analyzing the amplification products, counting the stability bands appearing in the test, recording the band as 1, recording the band as 0 when no band exists, and performing cluster analysis by using software NTSYS-pc 2.0.

And (4) detection conclusion: RAPD fingerprint analysis results show that: the similarity coefficient of the Auricularia auricula-Li Er No. 3 strain to be detected and the Auricularia auricula-Li Er-Xinke strain to be detected is 74%, the similarity coefficient of the Auricularia auricula 916 of the reference strain is 56%, and the new variety of the Auricularia auricula-Li Er No. 3 strain to be detected is different from the two reference strains (figure 8).

Example 3: and performing DNA difference comparison between the strain and the parent strain by an antagonistic reaction method.

The detection basis is as follows: inoculating, culturing and observing the sample to be detected according to the agricultural industry standard NY/T1845-2010 edible fungus strain differential identification antagonistic reaction of the people's republic of China.

Inspection materials: 3 black fungus strains are provided, wherein the strain to be detected is No. 3 of the fungus, and the reference strain 2 is respectively 'New family' and '916'.

The detection method comprises the following steps: and (3) according to strict aseptic operation, respectively punching the edges of activated bacterial colonies of the strains with a punch with the diameter of 5mm to be used as inoculation blocks, wherein the distance between the two inoculation blocks is 30mm, the two inoculation blocks are respectively arranged at a position 15mm away from the central point of a PDA (personal digital assistant) plate, and hyphae face upwards. Culturing at 25 deg.C in ventilation and dark until the inoculated mycelium is contacted, and culturing in natural light for 5-7 days.

Results and analysis: the hyphal reaction at the interface between the two strains on the surface of the culture was observed under light or natural light, and the results are shown in the figure (FIGS. 9-11).

And (4) detection conclusion: the detection result shows that hyphae at the junction between the 3 tested black fungus strains are all in a groove shape and have obvious antagonistic reaction, and the new variety of the black fungus-Liou No. 3 strain to be detected is different from the two control strains.

Claims (1)

1. A black fungus (Auricularia auricula) strain No. 3 belongs to Auricularia, is preserved in China general microbiological culture Collection center (CGMCC), has a preservation address of No. 3 Xilu-Beichen No.1 of the Korean area in Beijing, a preservation date of 2016, 7 and 5 days, and a preservation number of CGMCC No. 12525.

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