CN117887638B - Tricholoma giganteum growth-promoting microorganism SLTmB strain and application thereof - Google Patents
Tricholoma giganteum growth-promoting microorganism SLTmB strain and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of microorganisms, and particularly discloses a Grifola matsutake growth-promoting microorganism SLTmB strain and application thereof, wherein the strain is GDMCC No:64275 in preservation number, the classification name is Pseudomonastery koreensis, and the strain SLTmB is separated from Grifola matsutake pond soil, can be applied to Grifola matsutake artificial culture, and is suitable for developing a technological innovation of Grifola matsutake ecological growth promotion.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to a microorganism SLTmB strain for promoting growth of tricholoma giganteum and application thereof.
Background
Tricholoma matsutake Tricholoma matsutake (S.Ito & S.Imai) Singer, also known as Tricholoma matsutake, skinning fungus, is a fungus of the genus Tricholoma of the family Tricholomaceae, is listed in "China biological diversity red directory-Large fungus volume" (Redlistof China's Biodiversity-Macrofungi), is a protected precious wild edible fungus, and is widely distributed in Yunnan and has higher economic value. The national geographic mark protection product of the agaricus blazei Murill (DB 5334/T8-2022) is rich in vitamins B1 and B2, contains various amino acids and microelements such as zinc, magnesium and the like necessary for human bodies, can enhance human body immunity, generate essence and qi, regulate qi and resolve phlegm, delay aging, prevent and treat cancer, prevent diabetes and the like, has higher nutritive value, and also has important diet therapy and health care functions, and is popular in the trade of 'the mushroom body is hypertrophic, the meat quality is tender, the fragrance is rich for a long time, the color is good'.
The Shanglira tricholoma matsutake grows in Yunnan pine, mountain pine, ash back oak, huang Bei oak, long spike mountain oak or pine, oak mixed forest at an altitude of 2000-4000 meters, is mycorrhizal fungi which are symbiotic with plant roots, and manual planting cannot be performed at present. In recent years, resources are affected by various factors, and it is difficult to realize sustainable development and utilization. As mycorrhizal fungi symbiotic with plants, the growth mechanism of the tricholoma matsutake under natural conditions is unknown, the pure culture growth is slow, the requirements on environment and biological factors are severe, and the artificial cultivation cannot be realized until now. At present, the research in production is mainly focused on conservation of primordium, mycelium fermentation and the like, and the technology of tricholoma matsutake conservation and propagation promotion still remains in the aspects of forest land management and protection and the like, so that the research on a micro-ecological system is very little, and the breakthrough of a manual intervention technology is difficult to realize.
In the tricholoma matsutake ecological micro system, besides non-biological factors such as soil, temperature, humidity and the like, biological factors such as plants, fungi, bacteria, nematodes and the like form a subtle synergetic community, and the biological factors are mutually beneficial and reciprocal, so that the growth of mycorrhiza of the symbiotic plant is promoted by the fungi, the plants also transmit special nutrition to the fungi, and the fungi are mutually promoted. Research has also shown that natural rhizosphere microorganisms may have a certain promoting effect on the fruiting body formation of large fungi, and the mechanism of action includes various aspects such as improving nutrient absorption of wild fungi, competing ecological niches with pathogenic fungi, improving wild fungi resistance, promoting spore germination and hypha growth.
The invention collects the soil of the fungus pond in natural habitat, separates dominant microorganisms in the soil of the fungus pond, screens the growth-promoting bacteria in a laboratory, combines morphology and genome DNA amplicon sequencing, and identifies the growth-promoting strains screened in situ. The application of the growth promoting bacteria to the tricholoma matsutake is developed in a laboratory, a micro-ecological control technology is developed, a new way is found for constructing an artificial bacterial pond and promoting propagation, and a foundation is laid for the artificial intervention technology for promoting the growth and propagation of the tricholoma matsutake.
Disclosure of Invention
In order to overcome the problems in the background art, the invention adopts an in-situ screening method to screen a Tricholoma matsutake growth-promoting bacterium SLTmB strain from dominant microorganisms separated from the soil of the Tricholoma matsutake pond, and identifies the strain as Pseudomonas Pseudomonastery koreensis of Pseudomonas of Proteus, with the preservation number of GDMCC No: 64275.
More specifically, the invention provides the following technical scheme:
a strain SLTmB of Tricholoma giganteum growth promoting microorganism with the accession number GDMCC No: 64275 and the taxonomic name Pseudomonastery koreensis.
Further, the invention provides a product, which contains the SLTmB strain or SLTmB strain culture solution.
Preferably, the preparation method of the SLTmB strain culture solution comprises the following steps: inoculating SLTmB strain to beef extract peptone liquid culture medium, culturing to obtain bacterial liquid, and aseptically filtering to obtain filtrate.
Preferably, the culture conditions are: shake flask culture at 37deg.C at 150r/min for 24 hr.
Further, the SLTmB strain or the product is used for promoting the growth of mycelium, spore germination or spore growth of the Tricholoma matsutake.
Further, the invention provides a method for culturing the liquid for promoting growth of the tricholoma giganteum, which comprises the following steps:
S1: preparation of SLTmB strain culture solution, wherein the preservation number of the SLTmB strain is GDMCC No: 64275; inoculating SLTmB strain 04 into beef extract peptone liquid culture medium to prepare bacterial liquid with concentration of 6.5X10- 8 CFU/L, and aseptically filtering to obtain strain SLTmB culture liquid;
S2, preparing a fermentation liquid of the Shangri-La tricholoma matsutake, inoculating the Shangri-La tricholoma matsutake strain into an MMN liquid culture medium, and culturing to obtain the fermentation liquid of the Shangri-La tricholoma matsutake with the strain concentration of 0.35 g/L;
s3: adding SLTmB strain culture solution into the fermentation liquid of the Shangri-La tricholoma matsutake according to the volume ratio of 1:10-70, and continuing the liquid fermentation culture of the Shangri-La tricholoma matsutake.
Preferably, in step S3, SLTmB strain culture solution is added into the fermentation broth of the Tricholoma giganteum according to the volume ratio of 1:30, and the fermentation culture of the Tricholoma giganteum is continued.
Further, the invention provides a method for promoting growth and culturing of tricholoma giganteum spores, which comprises the following steps:
(1) Preparation of growth-promoting solid medium: inoculating SLTmB strain 04 into beef extract peptone liquid culture medium to prepare bacterial liquid with concentration of 6.5X10- 8 CFU/L, and aseptically filtering to obtain strain SLTmB culture liquid; the SLTmB-50% of strain culture solution is added into the MMN solid culture medium according to the volume percentage to prepare a growth-promoting solid culture medium, and the preservation number of the SLTmB strain 04 is GDMCC No: 64275;
(2) Preparing a suspension of the spores of the Tricholoma matsutake; coating the suspension on MMN solid culture medium for germination of Tricholoma giganteum spores or culture of spore hyphae.
Preferably, in the step (1), SLTmB strain culture solution is added into MMN solid culture medium according to volume percentage of 20% to prepare the growth-promoting solid culture medium.
The invention has the beneficial effects that:
1. Compared with other isolated dominant bacteria, the Tricholoma matsutake growth promoting strain SLTmB, provided by the invention, has the characteristic of promoting the growth of Tricholoma matsutake hyphae.
2. The SLTmB strain and the tricholoma matsutake are cultivated on a flat plate in a counter manner, so that the effect of promoting the growth of tricholoma matsutake hyphae by the viable cells is shown.
3. The culture solution of SLTmB strain provided by the invention is applied to tricholoma matsutake liquid fermentation culture, and the culture solution of the strain adopts the addition amount of preferably 1:30 volume ratio, so that the liquid culture of tricholoma matsutake hyphae can be effectively promoted.
4. The culture solution of SLTmB strain 04 disclosed by the invention is applied to germination of tricholoma matsutake spores, can promote germination of tricholoma matsutake spores and growth of spore hyphae, and is added with the optimal volume percentage of bacterial filtrate of 20%.
Drawings
FIG. 1 is a diagram of the test strain of the present invention in opposition to a plate of Tricholoma giganteum, annotated: a is the contrast CK of the Grifola matsutake, b is the contrast of the Grifola matsutake with SLTmB;
FIG. 2 is a morphological map of growth-promoting strain SLTmB04 according to the invention, given by: a is a plate growth chart, b is a colony chart, c is a 40-fold micro chart;
FIG. 3 is a comparative graph of SLTmB strain culture medium of Tricholoma giganteum of the present invention after 15d of spore growth, and is annotated: a is control CK, b is 20% bacterial liquid.
Detailed Description
In order to make the objects, technical solutions and advantageous effects of the present invention more apparent, preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings, so as to facilitate understanding of the skilled person. The experimental methods used in the following examples are all conventional methods unless otherwise specified; reagents, biological materials, etc. used in the examples described below are commercially available unless otherwise specified.
The biological material provided by the invention is a Tricholoma giganteum growth-promoting microorganism SLTmB strain with the preservation name: pseudomonastery koreensisSLTmB04, accession number: the collection of microorganism strains in Guangdong province; preservation address: building 5, guangzhou city martyr, road 100, college 59; preservation time: 2024, 01, 12; accession number GDMCC No: 64275, taxonomic designation: pseudomonastery koreensis the biological material was received by the collection at month 12 of 2024, registered with the book, and upon request, the viability of the biological material was checked by the collection at month 12 of 2024 from this date, and as a result, survived.
Example 1
Step I: tricholoma matsutake culture and separation of dominant bacteria in fungus pond
1) Collection of Shanglira matsutake
The pool town Ji Dicun is established in the main production area of the internal matsutake in Shangri-La city in Diqing and is used as the investigation and sample collection place of the internal matsutake in Shangri-La, which is positioned at 99 o65'39" E,28o '87' N, the altitude is 3.355.2+/-99.6 m, and the hillside faces southwest and the fossa habitat tree species are high mountain matsutake. Selecting wild Tricholoma matsutake pond distributed in a dispersed manner, collecting fresh fruiting body (more than 50 g), packaging into sterile bag, and preserving at low temperature.
2) Tissue isolation and activation of matsutake
Adopts an MMN solid culture medium, and the formula is as follows: 0.025g/L NaCl, 10.0g/L glucose, 2PO4 0.5.5 g/L KH, 3.0g/L malt extract powder, 20g/L vitamin B10.1mg/L,CaCl20.05g/L,(NH4)2HPO40.25g/L,FeCl30.012g/L,MgSO4·7H2O 0.15g/L, agar, and pH5.7+ -0.1, placing the tissue blocks with 0.5cm growing points at the tricholoma matsutake folds on a test tube slant culture medium, recording as "fragrant Tm 05", culturing at constant temperature and dark for 1 month, and transferring to a tube for 2 times.
3) Collecting soil in tricholoma matsutake mushroom pond
Selecting 5 fruiting body development stages of wild Tricholoma matsutake pond, removing surface humus, gravel, etc. by 5-point sampling method, collecting soil with depth of 2cm and surrounding range of 10cm by a small sterilizing shovel to form mixed soil sample 20g, placing into a sampling sealing bag, preserving at 4deg.C, and performing subsequent treatment within 24 h.
4) Separation of symbiotic dominant bacteria in bacteria pond
The bacteria pond soil is separated by dilution and plate streaking method to culture microorganisms. Beef extract peptone medium (BPM solid medium) for bacterial isolation, formulation: beef extract 3 g/L, peptone 10g/L, naCl 5g/L, agar 20g/L, pH 7.2-7.5; the formula of the Bengalia red agar culture medium for fungus separation comprises: peptone 5g/L, glucose 10g/L, KH 2PO41g/L,MgSO4·7H2 O0.5 g/L, agar 20g/L,1/3000 Bengal red solution 100mL/L, natural pH.
5 Test tubes with 9ml of sterile water are respectively numbered 10 -1、10-2、10-3、10-4、10-5, 1g of soil sample is weighed and added into the test tube with the number 10 -1, 1ml of the soil sample is sucked by a pipette after shaking and shaking uniformly, and the soil sample is transferred into the test tube with the number 10 -2, shaking and shaking uniformly and sequentially diluted to 10 -5. Bacteria were diluted to 10 -5 and fungi were diluted to 10 -4. The pipette draws 0.1ml of diluted sample solution, adds the diluted sample solution to the surface of the culture medium, uniformly coats the surface of the culture medium by using a coating rod, and repeats 3 plates for each sample solution. Placing the coated culture medium into an incubator for inverted culture, culturing bacteria in a constant temperature incubator at 37 ℃, and culturing fungi in a constant temperature incubator at 28 ℃. After 24 hours of bacterial culture, single colony grows out, and after 3-5 days of fungal culture, single colony grows out. And comparing a control group with a test group by using a test design, further picking up dominant single colony streak pure culture on a gradient plate of a sample, and separating and purifying dominant bacteria.
Results: the experiment successfully realizes the culture and activation of the mycelium of the 'fragrant Tm 05' of the tricholoma matsutake in a laboratory by carrying out tissue separation on the collected wild tricholoma matsutake fruiting body; the collected wild Tricholoma matsutake pond is subjected to dominant bacteria separation, purified and preliminary morphological identification, and the target strain SLTmB is screened, so that a stable test material is provided for screening and researching growth-promoting bacteria in a laboratory.
Step II: screening and identification of growth-promoting bacteria
1. Dominant strain and Tricholoma matsutake mycelium plate counter test
The method comprises the following steps: and (3) adopting an MMN enrichment medium (MMN formula and peptone 0.5%), taking Tricholoma matsutake strain-Tm 05 as an indicator strain, carrying out a growth-promoting test on the dominant strain obtained by the separation in the step (I) by adopting a flat plate counter culture method, and carrying out screening of Tricholoma matsutake mycelium growth-promoting bacteria. And (3) taking mycelium blocks with the size of 5mm 2 and the good 'fragrance Tm 05', inoculating the mycelium blocks to one side of an MMN+ flat plate, and culturing the mycelium blocks in a constant-temperature incubator at the temperature of 22 ℃ for 15d. And (3) inoculating the activated dominant bacteria of 24 h to the 4cm distance from the edge of the mycelium of the Tricholoma matsutake in the treated group, and repeating for 3 times without inoculating the dominant bacteria serving as a control group CK. The culture is continued at a constant temperature of 22 ℃, the colony diameter is measured by a crisscross streak method every 10 d times, the average daily speed (mm/d) of hyphae is obtained, and the growth rate is calculated to determine the influence of dominant bacteria on the growth of the hyphae of the Tricholoma giganteum.
The calculation formula is as follows:
average daily rate of hyphae (mm/d) = [ (colony diameter-5)/(2 ]/number of days of hyphae growth (1))
Growth rate = [ (treated colony growth rate-control colony growth rate)/control colony growth rate ] ×100 (2)
Results: among the dominant strains selected, the results of the opposite culture of the target strain SLTmB04 with the mycelia of Tricholoma matsutake are shown in FIG. 1 and Table 1.
TABLE 1 influence of dominant bacteria of the pool on the growth of Tricholoma giganteum hyphae
Table 1 shows the effect of the dominant bacteria selected in step I on the growth of mycelium of Tricholoma matsutake strain-Tm 05, the growth promoting effect (growth rate) of SLTmB strain is up to 61.23% compared with control CK.
FIG. 1 shows that SLTmB strain is opposite to Tricholoma matsutake "Tm 05" plate, and it is clear from SLTmB strain cultured on Tricholoma matsutake mycelium plate that SLTmB strain exhibits Tricholoma matsutake mycelium growth promoting effect.
2. Identification of growth-promoting Strain species
The method comprises the following steps: the growth promoting strain SLTmB04 obtained by screening the dominant bacteria is subjected to plate culture and then is directly observed in colony morphology. Molecular biological identification of the growth-promoting strain SLTmB04 was also carried out: the test strain was purified and identified by molecular system analysis using 16S rDNA sequence analysis. The genomic DNA of the strain was extracted and stored by silica gel adsorption column method using TSINGKE-plant DNA universal extraction kit (TSINGKE). Bacterial 16SrDNA universal primer pair was used: 27F/5'-AGAGTTTGATCCTGGCTCAG-3', 1492R/5'-GGTTACCTTGTTACGACTT-3'. PCR primer synthesis and amplification product sequencing are completed by the Optimago (Kunming), the obtained sequences are subjected to bidirectional measurement and splicing treatment, BLAST sequence comparison is carried out in NCBI database, and high similarity sequences are downloaded. Multiple sequence alignment was performed using Clustalx, DNA Star, etc. software to generate multiple sequence alignment maps.
Results: as can be seen from FIG. 2, the SLTmB strain isolated and selected grew slowly on beef extract peptone plates (BPM solid medium), colony morphology: transparent, round umbilical-shaped bulges, smooth and moist surface, luster and neat edge. And (3) dyeing and observing: gram-negative bacteria, rod-shaped, sporeless, capsular, aerobic. The morphological structure is substantially similar to that of Pseudomonas Pseudomonastery koreensis of Pseudomonas of Proteus. SLTmB 04A strain 16S rDNA amplified sequence fragment size is 1383 bp, the sequence is shown as SEQ ID No.1, and the similarity between the sequence and Pseudomonastery koreensis sequence with the sequence number OP380017 in NCBI database is 100% after Clustalx comparison. Combining the morphological characteristics of SLTmB strain and the 16S rDNA sequence comparison result, determining SLTmB strain separated from the study as a Tricholoma giganteum growth promoting bacterium, pseudomonas, taxonomic name: pseudomonastery koreensis, deposited under the cantonese Collection of microorganisms and cell cultures, accession number GDMCCNo: 64275, was submitted at 1 and 12 of 2024.
SLTmB04 Strain 16S rDNA amplification sequence (SEQ ID No. 1):
>SLTmB04
ACCGTCCTCCCGAAGGTTAGACTAGCTACTTCTGGTGCAACCCACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGACATTCTGATTCGCGATTACTAGCGATTCCGACTTCACGCAGTCGAGTTGCAGACTGCGATCCGGACTACGATCGGTTTTATGGGATTAGCTCCACCTCGCGGCTTGGCAACCCTCTGTACCGACCATTGTAGCACGTGTGTAGCCCAGGCCGTAAGGGCCATGATGACTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCCTTAGAGTGCCCACCATAACGTGCTGGTAACTAAGGACAAGGGTTGCGCTCGTTACGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCAGCACCTGTCTCAATGTTCCCGAAGGCACCAATCCATCTCTGGAAAGTTCATTGGATGTCAAGGCCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCATTTGAGTTTTAACCTTGCGGCCGTACTCCCCAGGCGGTCAACTTAATGCGTTAGCTGCGCCACTAAGAGCTCAAGGCTCCCAACGGCTAGTTGACATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCACCTCAGTGTCAGTATCAGTCCAGGTGGTCGCCTTCGCCACTGGTGTTCCTTCCTATATCTACGCATTTCACCGCTACACAGGAAATTCCACCACCCTCTACCATACTCTAGCTCGCCAGTTTTGGATGCAGTTCCCAGGTTGAGCCCGGGGATTTCACATTCAACTTAACGAACCACCTACGCGCGCTTTACGCCCAGTAATTCCGATTAACGCTTGCACCCTCTGTATTACCGCGGCTGCTGGCACAGAGTTAGCCGGTGCTTATTCTGTCGGTAACGTCAAAACACTAACGTATTAGGTTAATGCCCTTCCTCCCAACTTAAAGTGCTTTACAATCCGAAGACCTTCTTCACACACGCGGCATGGCTGGATCAGGCTTTCGCCCATTGTCCAATATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGACCGTGTCTCAGTTCCAGTGTGACTGATCATCCTCTCAGACCAGTTACGGATCGTCGCCTTGGTGAGCCATTACCTCACCAACTAGCTAATCCGACCTAGGCTCATCTGATAGCGCAAGGCCCGAAGGTCCCCTGCTTTCTCCCGTAGGACGTATGCGGTATTAGCGTTCCTTTCGAAACGTTGTCCCCCACTACCAGGCAGATTCCTAGGCATTACTCACCCGTCCGCCGCTGAATTCAGGAGCAAGCTCC
application example 1: application of growth promoting strain SLTmB04 in tricholoma matsutake fermentation culture
1) After activation of the growth-promoting strain SLTmB, beef extract peptone liquid medium (BPM solid medium formula removed agar) was used for shake flask culture: culturing at 37deg.C and rotation speed of 150r/min for 24 hr, and measuring bacterial liquid concentration by ultraviolet spectrophotometer to 6.5X10 8 CFU/L; filtering the bacterial liquid by using sterile gauze, and collecting filtrate by using a 0.22 mu m sterile filter, and marking the filtrate as liquid A;
2) Cutting Tricholoma matsutake 'Tm 05' strain (mycelium block), inoculating into 100ml MMN liquid culture medium (MMN solid culture medium formula removes agar), culturing at 24deg.C (130 r/min) for 10d, transferring 5ml of uniform bacterial liquid into 15 100ml MMN liquid culture medium, fermenting and culturing for 15d, and measuring bacterial liquid concentration by dry weight method to 0.35 g/L at 24deg.C and rotation speed of 180r/min, and recording as B liquid;
3) Adding the growth promoting bacteria filtrate A into the tricholoma matsutake fermentation liquid B according to the volume ratio of 4 groups of 1:10,1:30,1:50 and 1:70, and repeating 3 groups of treatments at 24 ℃ for 30d by using the liquid A as a control CKt; the comparative examples are: the Morchella fermentation broth is transferred into the A-M test group according to the volume ratio of 1:30, the Morchella fermentation broth without the A-liquid is used as a contrast CKm, and the Morchella fermentation culture is carried out for 3 days according to the conventional YPD formula (10 g/L yeast extract, 20g/L peptone, 20g/L glucose) by shaking the bottle, and the parameters are the same as above;
4) Determination of mycelium biomass: the 7 groups of experiments comprise 21 bottles of bacterial liquid co-cultured with the growth promoting bacterial filtrate, centrifuging for 20min at 3000r/min, repeatedly washing mycelium with deionized water, drying at 105 ℃ to constant weight, and weighing by an analytical balance.
Results:
TABLE 2 comparison of SLTmB04 bacterial filtrate and fermentation co-culture of Tricholoma giganteum
Table 2 compares the results of SLTmB fungus filtrate and Tricholoma matsutake 'Tm 05' fermentation co-culture, the growth of Tricholoma matsutake mycelium is promoted by the growth promoting fungus filtrate A and Tricholoma matsutake fermentation broth B at different concentration levels, the growth promoting effect is different, the accumulation trend of the Tricholoma matsutake mycelium biomass is increased from rapid to decrease along with the decrease of the concentration of the filtrate A from high to low, the promotion effect is highest at the level of volume ratio of 1:30, the mycelium biomass is 411.5mg, and exceeds the control (247.7 mg), and the increase rate reaches 66.13%; in the comparative example of co-culture with Morchella of different species, the growth promoting effect of the growth promoting bacteria is not obvious. The result shows that the growth promoting strain SLTmB can be applied to the fermentation culture of the tricholoma matsutake in the form of a bacterial filtrate, and the liquid culture of the tricholoma matsutake hyphae can be effectively promoted by adding the sterile growth promoting bacterial filtrate in a volume ratio of 1:30.
Application example 2: application of growth promoting strain SLTmB04 in germination of tricholoma matsutake spores
1) Collecting tricholoma matsutake spores: taking a fungus cover of a mature fruiting body of the Shanglira tricholoma matsutake, buckling the fungus cover on a cellophane membrane with 20cm multiplied by 20cm at room temperature, keeping away from light and wind, standing for 5 days to obtain a spore print, cutting off 0.5 cm 2 spore print, placing the spore print into a 1.5ml centrifuge tube with a cover, and washing and fixing the volume by using normal saline to obtain spore liquid;
2) Microscopic examination and spore suspension preparation: the spore liquid is sucked by a pipette and dripped into a cell counting plate, the spores are counted under a microscope, the concentration of the spore liquid is quantified, and the spore liquid is properly diluted to the concentration of 2X 10 4/ml (1% streptomycin is added) by using physiological saline;
3) Preparation of growth-promoting solid medium: using the growth promoting strain SLTmB liquid culture and filtering to obtain growth promoting strain culture solution A, adding 5%, 10%, 20% and 50% of the culture solution A into sterilized MMN solid culture medium cooled to 50-60 ℃ according to volume percentage, uniformly mixing, pouring into a plate to prepare 4 concentrations of MMN growth promoting solid culture medium, setting 3 repeats for each concentration, and taking the MMN solid culture medium without A as a Control (CK);
4) Sucking 0.1ml of spore suspension by a pipette, gently and uniformly coating the spore suspension on the surface of the MMN growth-promoting culture medium by a coating rod, and culturing in dark at 23 ℃;15 Counting the germination number of spores after d, and comparing germination rates;
5) And (3) transferring confirmed germinated and grown tricholoma matsutake spore colonies to the same MMN growth promoting solid medium for dark culture at 23 ℃ for 10 days, observing the growth condition of mycelia, measuring the colony diameter by a cross method, and continuously measuring the average daily growth rate (mm/d) of spore mycelia for 5 times.
The calculation formula is as follows:
Average daily rate of spore hyphae (mm/d) =colony diameter +.2/hyphae growth days (3)
Results: the addition of the bacterial filtrate SLTmB of the growth-promoting bacteria SLTmB influences the germination and growth of the Tricholoma giganteum spores, the treatment levels of the bacterial filtrate A with different concentrations are obvious in germination rate difference of the Tricholoma giganteum spores, but not obvious in growth rate difference of the Tricholoma giganteum spores.
TABLE 3 influence of SLTmB04 bacterial liquid on the spore growth of Tricholoma giganteum
The results in table 3 show that as the concentration of the bacterial filtrate increases, the germination rate of the spores of the agaricus blazei murill and the growth rate of the mycelia increase, and the germination rate of the spores of the agaricus blazei murill and the growth rate of the mycelia decrease when the concentration of the bacterial filtrate increases to a level of 50%, so that the agaricus blazei murill is inhibited to a certain extent. With the increase of the concentration of the bacterial filtrate A, the spore germination rate has obvious rising trend, and the growth rate reaches 62.09% at the concentration level of 20% of the bacterial filtrate, and is 46.75; the growth rate of spore hypha is not significantly different from that of the control, and only 11.98% of spore hypha is increased at the concentration level of 20% of the bacterial filtrate. The results show that: SLTmB 04A 04 strain can promote germination and growth of Tricholoma matsutake spores, and is characterized in that the fungus filtrate is added to an MMN culture medium, and when the optimal adding proportion of the fungus filtrate is 20%, the germination rate of the Tricholoma matsutake spores and the growth speed of spore hyphae are optimal.
FIG. 3 shows that germination of Tricholoma matsutake spores is cultivated in SLTmB% of the bacterial filtrate of the growth promoting strain SLTmB. The control and the addition of 20% of the bacterial filtrate A affect germination and growth of Tricholoma matsutake spores, and are significantly different.
In summary, the invention uses the in-situ screening method to screen the Tricholoma giganteum SLTmB strain from dominant microorganisms isolated from the soil of the wild Tricholoma matsutake pond, combines morphology and genome 16S rDNA sequencing, and identifies the Pseudomonas Pseudomonastery koreensis. The application of the growth promoting bacteria SLTmB04 to the co-culture of the tricholoma giganteum is developed in a laboratory, and the result shows that the SLTmB strain has the function of promoting the growth of the tricholoma giganteum by the living colony on the tricholoma giganteum mycelium plate; SLTmB04 strain shows the addition amount of 1:30 volume ratio of the fungus filtrate on fermentation culture of Tricholoma matsutake, and can effectively promote liquid culture of Tricholoma matsutake hyphae; SLTmB04 strain shows that when the optimal adding proportion of the bacterial filtrate is 20% on the culture of Tricholoma matsutake spores, the germination rate of Tricholoma matsutake spores and the growth speed of spore hyphae are optimal. The tricholoma giganteum growth promoting bacteria and the growth promoting characteristics thereof provided by the invention can be applied to the artificial culture of tricholoma giganteum, find a new way for an artificial bacteria pond propagation promoting technology and a microecology control technology, and lay a foundation for the artificial intervention of tricholoma giganteum growth promoting and propagation promoting; meanwhile, functional microorganism resources are enriched, and strain resources and application technology are accumulated for microecological construction of agriculture and forestry crops.
Finally, it is noted that the above-mentioned preferred embodiments illustrate rather than limit the invention, and that, although the invention has been described in detail with reference to the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention as defined by the appended claims.
Claims (5)
1. A strain SLTmB of Tricholoma giganteum growth promoting microorganism SLTmB is characterized in that the strain has a preservation number of GDMCC No: 64275 and a classification name of Pseudomonastery koreensis.
2. The method for culturing the liquid for promoting growth of the tricholoma giganteum is characterized by comprising the following steps of:
S1: preparation of SLTmB strain culture solution, wherein the preservation number of the SLTmB strain is GDMCC No: 64275; inoculating SLTmB strain 04 into beef extract peptone liquid culture medium to prepare bacterial liquid with concentration of 6.5X10- 8 CFU/L, and aseptically filtering to obtain strain SLTmB culture liquid;
S2, preparing a fermentation liquid of the Shangri-La tricholoma matsutake, inoculating the Shangri-La tricholoma matsutake strain into an MMN liquid culture medium, and culturing to obtain the fermentation liquid of the Shangri-La tricholoma matsutake with the strain concentration of 0.35 g/L;
s3: adding SLTmB strain culture solution into the fermentation liquid of the Shangri-La tricholoma matsutake according to the volume ratio of 1:10-70, and continuing the liquid fermentation culture of the Shangri-La tricholoma matsutake.
3. The method according to claim 2, wherein in step S3, SLTmB strain culture is added to the fermentation broth of the hon-shimeji mushroom at a volume ratio of 1:30, and the liquid fermentation culture of the hon-shimeji mushroom is continued.
4. The method for promoting growth and culture of the tricholoma matsutake spores is characterized by comprising the following steps of:
(1) Preparation of growth-promoting solid medium: inoculating SLTmB strain 04 into beef extract peptone liquid culture medium to prepare bacterial liquid with concentration of 6.5X10- 8 CFU/L, and aseptically filtering to obtain strain SLTmB culture liquid; the SLTmB-20% of strain culture solution is added into the MMN solid culture medium according to the volume percentage to prepare a growth-promoting solid culture medium, and the preservation number of the SLTmB strain 04 is GDMCC No: 64275;
(2) Preparing a suspension of the spores of the Tricholoma matsutake; coating the suspension on MMN solid culture medium for germination of Tricholoma giganteum spores or culture of spore hyphae.
5. The method according to claim 4, wherein in the step (1), SLTmB% by volume of the strain culture solution is added to the MMN solid medium to prepare the growth-promoting solid medium.
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