CN115838632A - Phlebopus portentosus endophytic trichoderma fungus and application thereof - Google Patents
Phlebopus portentosus endophytic trichoderma fungus and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of edible fungus cultivation, and particularly relates to a boletus meiloti endophytic trichoderma fungus and application thereof. The preservation number of the provided boletus meiloti endophytic trichoderma fungus is CCTCC NO: M2022944. Also provides application of the trichoderma in preparation of liquid strains and solid strains of the phlebopus portentosus. The trichoderma has obvious promotion effect on the hypha growth of exophytic mycorrhizal fungi bolete liquid culture and solid culture, can shorten the production period of bolete reticuloendothellus sporocarp and improve the yield. In addition, the trichoderma fungus can reduce pollution in the culture of the boletus meiloti, and the use of chemical agents is avoided.
Description
Technical Field
The invention belongs to the technical field of edible fungus cultivation, and particularly relates to a boletus meinetii endophytic trichoderma fungus and application thereof.
Background
Boletus reticulatus (Boletus reticulatus Schaeff.) is a rare exogenous mycorrhizal edible fungus, belongs to Boletales (Boletales), boletaceae (Boletaceae) and Boletus (Boletus), has delicious taste and high nutritional value, and is widely distributed in a plurality of areas of China. The Phlebopus portentosus commonly coexists with the root system of the Pinus plant, and the fruiting body is not easily formed due to the unique symbiotic ecological environment, nutrition mode and differentiation conditions of the fruiting body. Due to the change of ecological environment, the yield of wild Phlebopus metschnikowii in China is sharply reduced, and the development of Phlebopus metschnikowii resources is necessary to protect the diversity of wild edible fungus resources. For more than ten years, researchers at home and abroad carry out the strain preparation and cultivation work of the boletus meiloti, and partial researchers use pure cultures of the boletus meiloti mycelium to inoculate pine root systems, and mushroom fruiting is carried out under proper natural climate conditions when the biomass of the mycelium is accumulated to a certain amount. However, the bolete reticulorum shows slow hypha growth, long fruiting period and low fruiting amount, which is necessarily connected with the fact that the bolete reticulorum hyphae accumulation needs a long time under natural conditions.
In addition, in the semi-artificial cultivation process of the wild mycorrhizal edible fungi, inoculated mycorrhizal edible fungi are easy to rot, producers often use commercial chemical bacteriostatic agents to prevent and control diseases of the edible fungi, potential chemical substance pollution exists, and side effects of the chemical bacteriostatic agents are always puzzled to consumers and managers.
Disclosure of Invention
The invention aims to improve the growth speed of the hyphae of the boletus meiloti and reduce the pollution in the culture process.
In order to solve the technical problem, the invention provides a boletus meiloti endophytic trichoderma fungus with a preservation number of CCTCC NO: M2022944 and a name of trichoderma SF-2. When the strain is cultured on an improved PDA plate culture medium, the hyphae are white, dense and felt-shaped in the initial stage, and the aged hyphae are yellow.
The invention also provides a microbial inoculum which comprises the boletus meiloti endophytic trichoderma fungus SF-2.
The microbial inoculum can comprise bacterial powder of the trichoderma fungus SF-2; the preparation method of the bacterial powder comprises the following steps: liquid culturing the trichoderma fungus for 3-5 days, performing aseptic filtration on the obtained trichoderma fungus culture solution to obtain trichoderma fungus hyphae, washing with sterile water, drying and inactivating at 60 +/-1 ℃ (no growth activity is detected by PDA plate culture), grinding the hyphae into powder, and sieving with a 60-mesh sieve.
The microbial inoculum can also comprise immobilized bacterial powder of the trichoderma fungi SF-2, wherein the immobilized bacterial powder is sodium alginate microsphere immobilized bacterial powder or sodium alginate sponge immobilized bacterial powder.
The preparation method of the sodium alginate microsphere immobilized bacterial powder can comprise the following steps: preparing 2-3% sodium alginate solution, adding 0.25-2g of powder of Trichoderma SF-2 into 20mL of the solution, stirring, slowly adding the mixed solution dropwise into 2-4% CaCl 2 Fixing in the solution for 3-5h, sterile filtering to obtain sodium alginate microsphere immobilized bacteria powder, washing with sterile water, and oven drying at 37 + -1 deg.C.
The preparation method of the sodium alginate sponge immobilized bacterial powder can comprise the following steps: preparing 1-2% sodium alginate solution from skim milk, adding 0.25-2g of powder of Trichoderma SF-2 into 20mL of skim milk, stirring, standing at room temperature for a period of time to discharge air in the mixed solution, freezing at-20 deg.C for 3-5h, and vacuum freeze-drying at-80 deg.C for 24-48h; placing the vacuum freeze-dried sample in 1-3% CaCl 2 Crosslinking for 3-5h in the solution to obtain sodium alginate sponge immobilized bacteria powder, washing with sterile water, and drying at 37 + -1 deg.C.
The application of the boletus edulis endophytic trichoderma fungus SF-2 or any of the inocula in the preparation of liquid strains and solid strains of the boletus edulis also belongs to the protection range of the invention.
The invention also provides a method for preparing the boletus meiloti liquid strain, which comprises the following steps: liquid culture of the boletus meiloti, when the boletus meiloti in the culture solution is in dominant growth, adding the fungus powder of the trichoderma SF-2 or the immobilized fungus powder of the trichoderma SF-2 into the culture solution, and continuously culturing to obtain the boletus meiloti liquid strain.
In the method, 0.25-2g of powder or immobilized powder of Trichoderma SF-2 can be added into 150mL of Boletus Dichotomae culture solution.
In the method, the improved PDA liquid culture medium can be adopted to carry out liquid culture on the boletus omenii; the formula of the culture medium is as follows: weighing peeled potato 200g, cutting into small pieces, adding 1000mL water, heating to boil, maintaining for 5-10min, filtering with gauze while it is hot, removing residue, cooling filtrate to 50-60 deg.C, adding glucose 20g, corn flour 20g, yeast powder 2g, KH, etc 2 PO 4 0.1-0.5g、MgSO 4 1g, vc 0.01-0.05g, add ddH 2 And O, heating to dissolve, and then fixing the volume to 1000mL.
In the above method, the Phlebopus portentosus can be cultured in liquid at 28 deg.C and 160 r/min.
In the method, the powder of the trichoderma SF-2 or the immobilized powder of the trichoderma SF-2 is added into the phlebopus portentosus culture solution, and then the culture can be continued for 5 to 7 days to obtain the phlebopus portentosus liquid strain.
The invention also provides a method for preparing the boletus meiloti solid strain, which comprises the following steps: the method is used for preparing the boletus meiloti liquid strain, the liquid strain is inoculated into a solid culture medium, and the boletus meiloti solid strain is obtained by culture.
The formulation of the solid culture substrate can be: according to the mass percentage, 10-15% of masson pine sawdust, 5-10% of masson pine needle powder, 10-20% of straw powder, 10-20% of cotton seed hulls, 10-15% of cow dung, 10-15% of corncobs, 10-15% of perlite, 1-1.5% of glucose, 1-1.5% of gypsum, 0.01-0.05% of monopotassium phosphate and 0.001-0.005% of Vc, the water content is adjusted to 65-70%, and the pH value is 7.2.
In the method, the liquid strain of the boletus meiloti can be inoculated into a solid culture medium according to the inoculation amount of 1% (v/v), and is cultured in the dark at the temperature of 24 +/-1 ℃ for 19-25 days to obtain the solid strain of the boletus meiloti.
The invention utilizes the low-dose environment-friendly endophytic fungus Trichoderma SF-2 with biocontrol effect and the exomycorrhizal fungus bolete meretrix to co-culture to obtain the excellent bolete meretrix liquid strain and solid strain. Compared with the boletus meiloti strain prepared by a pure culture method, the liquid strain and the solid strain not only contain a proper amount of biocontrol endophytic fungi trichoderma SF-2, but also contain the boletus meiloti in dominant growth, and the trichoderma SF-2 can effectively promote the growth of the boletus meiloti and reduce pollution. If the liquid strain or the solid strain is inoculated to pine forests (such as natural pinus massoniana forest), the synergistic effect can be achieved, and the trichoderma SF-2 can promote hypha growth and fruiting body development of boletus meretrix in the pine forests, so that the production period of the boletus meretrix fruiting body is shortened, and the yield of the boletus meretrix is improved. The method is an efficient semi-artificial cultivation method for the boletus meiloti.
The invention has the following beneficial effects:
(1) The trichoderma SF-2 provided by the invention can obviously improve the hypha growth speed and the extracellular enzyme activity of the boletus meiloti.
Experiments prove that after 0.5g of trichoderma SF-2 powder is added into the culture solution of the boletus meiloti according to the proportion of adding 0.5g of trichoderma SF-2 powder into each 150mL of the culture solution, the dry weight of the hyphae of the boletus meiloti can reach 15.05mg/mL, the activity of laccase can reach 23.61U/mL, the activity of polyphenol oxidase can reach 38.67U/mL, the activity of neutral protease can reach 3.519U/mL, the activity of amylase can reach 0.596U/mL, and the activity of cellulase can reach 0.566U/mL. The bolete meliloti liquid strain is prepared by trichoderma SF-2 induction, and the obtained bolete meliloti liquid strain has good growth vigor, quick spawn running and high hypha biomass, and can be used for cultivation and inoculation after being cultured for 6-7 days. After the liquid strain of the bolete meinetorum is inoculated to the solid culture medium, the hyphae have strong growth vigor and are uniformly distributed in the medium, and the solid culture medium can be overgrown in 19-25 days.
(2) The trichoderma SF-2 provided by the invention has a good biocontrol effect and can reduce pollution.
Experiments prove that after the boletus meliloti liquid strain is inoculated to a solid culture medium, under the condition of not using any chemical agent, when hyphae grow over the solid culture medium, 1-2 bags of solid culture are polluted on average in 10 bags of solid culture.
The invention provides trichoderma SF-2, the patent preservation information is as follows:
the preservation name is: trichoderma sp.SF-2
The preservation date is as follows: 22/06/2022
The preservation number is: CCTCC NO: M2022944
The preservation organization: china center for type culture Collection
Address: wuhan, wuhan university.
Drawings
FIG. 1 shows the colony morphology of Trichoderma SF-2 cultured on modified PDA plates.
FIG. 2 shows a phylogenetic tree of Trichoderma SF-2 constructed based on rDNA-ITS sequences.
FIG. 3 shows the results of culturing Trichoderma SF-2 and Phlebopus metschnikoides in a plate-confronted manner. The left side is the colony of Phlebopus meretrix, and the right side is the colony of Trichoderma SF-2.
FIG. 4 shows Trichoderma SF-2 powder.
FIG. 5 shows sodium alginate microsphere immobilized Trichoderma SF-2 powder.
FIG. 6 shows sodium alginate sponge immobilized Trichoderma SF-2 powder.
FIG. 7 shows a liquid strain of Phlebopus meretrix cultured by induction with Trichoderma powder; the CK group is a control group bolete culture solution without trichoderma powder; the group A is a boletus meiloti culture solution added with trichoderma SF-2 bacterial powder; the group B is a phlebopus portentosus culture solution added with sodium alginate microsphere immobilized trichoderma SF-2 strain powder; group C is a Boletus petiolus culture solution added with sodium alginate sponge immobilized trichoderma SF-2 powder.
FIG. 8 shows solid strains of Phlebopus meretrix cultured by induction with Trichoderma powder; the CK group is a solid strain obtained by inoculating a solid matrix to a control group bolete meliloti liquid strain without trichoderma powder and then culturing for 15 days; the treatment group A is a solid strain obtained by inoculating a solid matrix with the liquid strain of the boletus meliloti added with free trichoderma SF-2 powder and then culturing for 15 days.
Detailed Description
The following describes the technical solution of the present invention in detail with reference to the specific embodiments and the accompanying drawings.
The boletus reticuloensis used in the following examples is a strain isolated and preserved in this laboratory. The strain is publicly available from the institute of bioscience and technology of Hubei national university, is used only for repeating the relevant experiments of the present invention, and cannot be used for other purposes. The invention was achieved independently of the strain.
The media used in the examples below:
modified PDA liquid medium: weighing 200g of peeled potatoes, cutting into small pieces, putting into a pot, adding 1000mL of water, heating to boil, maintaining for 5-10min, filtering with 2 layers of gauze while hot, and removing filter residue. Cooling the filtrate to 50-60 deg.C, adding glucose 20g, corn flour 20g, yeast powder 2g, KH 2 PO 4 0.1-0.5g、MgSO 4 1g, vc 0.01-0.05g, add ddH 2 And O, heating to dissolve, fixing the volume to 1000mL, subpackaging in 250mL glass triangular bottles, sterilizing for 20min at 121 ℃ for later use, wherein each bottle is 150mL.
Modified PDA solid medium: 20g of agar powder is added on the basis of the improved PDA liquid culture medium.
The assay methods used in the following examples:
the method for measuring the hypha growth amount of the boletus meinetensis comprises the following steps: putting the sample into a 10mL centrifuge tube, centrifuging at 4 ℃ and 4000r/min for 10min, obtaining a precipitate as hypha after centrifugation, drying the hypha at 60 +/-1 ℃ to constant weight, weighing to obtain the dry weight of the hypha, and measuring the hypha growth amount of the boletus edulis by using the dry weight of the hypha.
The method for measuring the extracellular enzyme activity of the boletus meliloti comprises the following steps: putting the sample into a 10mL centrifuge tube, centrifuging for 10min at 4 ℃ and 9000r/min to obtain a supernatant, namely a crude enzyme solution, and measuring the activity of the extracellular enzyme in the crude enzyme solution.
(1) And (3) laccase activity determination: laccase activity is determined by an o-tolidine method, see the literature (interaction research of ChenGuimei-Chinese pine mycorrhizal associated fungi and ectomycorrhizal fungi [ D ]. Northwest university of agriculture and forestry, 2009). (2) Measurement of Polyphenol oxidase Activity: polyphenol oxidase activity was measured by the catechol method, as described in the literature (Wangweike, lanna, zhouzu, song Guiling, yuanyongong, yan Silun.8 extracellular enzymes have activity changes at different growth stages of Lentinus edodes [ J ]. Zhejiang agricultural science 2014 (4): 498-501). (3) Determination of neutral protease Activity: protease activity was determined by the forskolin phenol method, see literature (petrel, royal vicin. Determination of stichopus japonicus intestinal protease, amylase, lipase and cellulase activity [ J ]. Feed industry, 2012,33 (20): 28-32). (4) And (3) amylase activity determination: amylase activity was measured using DNS colorimetry, see DNS colorimetry in the literature (zhu birch eds. Biochem. Experimental guidelines (2 nd edition) [ M ]. China university of agriculture press, 2020). (5) And (3) cellulase activity determination: the cellulase activity was determined by DNS colorimetry, see DNS colorimetry in the literature (study of change law of extracellular enzyme activity of 8 species of pholiota nameko black umbrella [ D ]. University of agriculture in new jiang, 2018).
Data processing and analysis methods used in the following examples: experimental data were analyzed for one-way anova using SPSS Statistics 20 software, expressed as mean ± standard deviation.
Unless otherwise specified, the reagents used in the following examples are conventional in the art, and are either commercially available or formulated according to methods conventional in the art, as laboratory grade. Unless otherwise specified, the experimental methods and conditions used in the following examples are all conventional in the art, and reference may be made to relevant experimental manuals, well-known literature, or manufacturer's instructions. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
Example 1 isolation and identification of strains
The inventor of the invention obtains an endophytic fungus on an improved PDA solid culture medium unexpectedly in the process of separating wild Phlebopus metschnikowii. The strain is cultured at 28 ℃ to obtain mycelium. The total DNA of mycelia was extracted using fungal genomic DNA extraction kit (OMEGA, cat # D3471-00) according to the kit instructions. The forward primer ITS1F (5.
The ITS sequence of Trichoderma SF-2 is as follows:
CCTTTTCTAGGGGAACTTCACGAGGGATCATTACCGAGTCCAGCAACTCCCAAACCCAATGTGAACCATACCAAACTGTTGCCTCGGCGAGGGTCACGCCCCGGGTGCGTCGCAGCCCCGGAACCAGGCGCCCGCCGGAGGGACCAACCAAACTCTTTACTGTAGTCCCCTCGCGGACGTTATTTCTTACAGCTCTGAGCAAAAATTCAAATGAATCAAAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCGCCAGTATTCTGGCGGGCATGCCTGTCCGAGCGTCATTTCAACCCTCGAACCCCTCCGGGGGGTCGGCGTTGGGGATCGGGAACCCCTCAGACGGGATCCCGGCCCCGAAATACAGTGGCGGTCTCGCCGCAGCCTCTCCTGCGCAGTAGTTTGCACAACTCGCACCGGGAGCGCGGCGCGTCCACGTCCGTAAAACACCCAACTTCTGAAATGTTGACCTAGGAACAGGTAGAAAAACCCGCTAAACTTACCCATATCAGTAACCGAAGAGAGAATC(SEQ ID NO:1)
trichoderma SF-2 was cultured on modified PDA solid medium, the initial hyphae of the strain was white, dense, felt-like (FIG. 1), yellow after aging, and low in spore yield, similar to the characteristics of the Korean New species Trichoderma sungyi sp. A phylogenetic tree of Trichoderma SF-2 constructed based on the rDNA-ITS sequence is shown in FIG. 2.
The trichoderma SF-2 has been subjected to patent preservation, the preservation organization is China center for type culture Collection, and the preservation number is CCTCC NO: M2022944.
Example 2 culture of Phlebopus metschnikoides in confrontation with Trichoderma SF-2
Using a 6 mm-diameter sterile puncher to punch 1 cake on the edge of an activated colony of the bolete reticuloendothelioides, inoculating the obtained 1 cake in an improved PDA solid culture medium with the diameter of 90mm, placing the obtained product in an incubator at 28 ℃ for dark culture, inoculating 1 cake of trichoderma SF-2 with the diameter of 6mm in the position 30mm away from the colony of the bolete reticuloendothelioides when the colony of the bolete reticuloendothelioides is cultured to 1.5-2cm (after about 8-10 days of culture), continuing to place the obtained product in the incubator at 28 ℃ for dark culture, and observing whether the bolete reticuloendothelioides and the trichoderma fungi grow in a confronting manner.
The results show that: the boletus edulis grows slowly on the improved PDA solid culture medium, and the hyphae are thick and strong. After the trichoderma SF-2 is inoculated, the growth speed of trichoderma hyphae is high, and the hyphae are fine and loose. After the boletus meiloti and the trichoderma SF-2 are co-cultured for 2 days, the boletus meiloti and the trichoderma SF-2 show cross growth, the growth of the boletus meiloti is not influenced by the trichoderma SF-2, and no obvious antagonistic line is formed in the cross growth (figure 3).
Example 3 preparation of liquid strains of Boletus omenii
1. Liquid culture of bacterial species
(1) Liquid culture of boletus meiloti
Selecting boletus omenii with good hypha growth from an improved PDA solid culture medium, punching a fungus cake at the edge of a bacterial colony by using a sterile puncher with the diameter of 6mm, inoculating the fungus cake into 150mL of improved PDA liquid culture medium (3 blocks/bottle and 6 mm/block in diameter), and placing the obtained product at 28 ℃ for shaking culture at 160r/min for 7-10 days to obtain a liquid culture solution of the boletus omenii.
(2) Liquid culture of trichoderma fungi
Selecting trichoderma SF-2 with good hypha growth from an improved PDA solid culture medium, punching a bacterial cake at the edge of a bacterial colony by using a sterile puncher with the diameter of 6mm, inoculating the bacterial cake into 150mL of the improved PDA liquid culture medium (3 blocks/bottle and 6 mm/block), and performing shake culture for 3-5 days at the temperature of 28 ℃ and at the speed of 160r/min to obtain a liquid culture solution of the trichoderma SF-2.
2. Preparation of Trichoderma powder
Filtering the liquid culture solution of Trichoderma SF-2 obtained by culturing for 3-5d with 2 layers of sterile gauze to obtain Trichoderma fungal mycelium, washing the mycelium with sterile water for 2 times, drying and inactivating at 60 + -1 deg.C (no growth activity detected by PDA plate culture), grinding the mycelium into powder, sieving with 60 mesh sieve to obtain Trichoderma SF-2 powder, and sealing for use.
3. Optimization of trichoderma powder adding amount
Treatment group: placing the inactivated trichoderma SF-2 powder in an ultra-clean workbench for ultraviolet disinfection for 30min, adding the powder into a liquid culture solution of cultured 3-5d of the bolete reticulorhizioides (at the moment, the bolete reticulorhizioides is in dominant growth and shows that hypha balls with the size of rice grains appear in the culture solution) at different gradient concentrations (0 g/150mL, 0.25g/150mL, 0.5g/150mL, 1g/150mL, 1.5g/150mL and 2g/150 mL), and then placing the mixture at 28 ℃ for shaking culture at 160 r/min. After adding the trichoderma SF-2 powder, adopting an aseptic operation technology to suck culture solution samples every day, sampling 10mL each time, repeating each treatment for 3 times, continuously sampling for 7d, and determining the dry weight of boletus edulis hyphae in the samples.
Control group (CK group): the rest of the treatment groups were the same without addition of Trichoderma SF-2 powder.
The results are shown in table 1, compared with the CK group without adding trichoderma powder, the weight average of the hyphae dry weight of the boletus meiloti of the treatment group with different amounts of trichoderma powder is obviously increased, and the hyphae dry weight of the treatment group with different amounts of trichoderma powder reaches the maximum value after the trichoderma powder is added at the 6 th day, wherein the hyphae dry weight of the treatment group with 0.5g/150mL trichoderma powder is most obviously enhanced, and the hyphae dry weight reaches the maximum value of 12.89mg/mL.
TABLE 1 influence of different addition amounts of Trichoderma powder on the dry weight of Boletus metschnikoides hyphae
Each value in the table is expressed as the mean (mean) ± Standard Deviation (SD) of three tests, with different letters in the same column indicating significant differences (P < 0.05).
4. Optimization of trichoderma powder adding mode
(1) Preparation of Trichoderma powder
(1) Trichoderma powder: trichoderma SF-2 powder was prepared according to the method described in 2 above.
(2) Preparation of sodium alginate microsphere immobilized trichoderma powder
Preparing 20mL of 2-3% (g/mL) sodium alginate solution, adding 0.5g of trichoderma SF-2 powder, stirring uniformly, and slowly dripping the mixed solution into 100mL of 2-4% (g/mL) CaCl by using a 20-30mL sterile syringe 2 Fixing in the solution for 3-5h, filtering with sterile gauze to obtain sodium alginate microsphere immobilized Trichoderma powder, washing with sterile water for 2 times, oven drying at 37 + -1 deg.C, sealing, and storing at 4 deg.C for use.
(3) Preparation of sodium alginate sponge immobilized trichoderma powder
Preparing 20mL 1-2% (g/mL) sodium alginate solution by using commercially available skim milk, adding 0.5g of trichoderma SF-2 powder, uniformly stirring, standing at room temperature for a period of time to discharge air in the mixed solution, freezing at-20 ℃ for 3-5h, and transferring to-80 ℃ for vacuum freeze drying for 24-48h. The vacuum freeze-dried sample was placed in 100mL 1-3% (g/mL) CaCl 2 Crosslinking in the solution for 3-5h to obtain sodium alginate sponge immobilized trichoderma powder, washing with sterile water for 2 times, drying at 37 +/-1 ℃, and sealing for later use.
(4) Preparation of sodium alginate microspheres
Preparing 20mL of 2-3% (g/mL) sodium alginate solution, not adding trichoderma powder, slowly dripping the sodium alginate solution into 100mL of 2-4% (g/mL) CaCl by using a 20-30mL sterile syringe 2 Fixing in the solution for 3-5 hr, filtering with sterile gauze to obtain sodium alginate microspheres, washing with sterile water for 2 times, oven drying at 37 + -1 deg.C, sealing, and storing at 4 deg.C for use.
(5) Preparation of sodium alginate sponge
Preparing 20mL 1-2% (g/mL) sodium alginate solution by using commercially available skim milk, standing at room temperature for a period of time without adding trichoderma powder to discharge air in the solution, freezing at-20 ℃ for 3-5h, and transferring to-80 ℃ for vacuum freeze drying for 24-48h. The vacuum freeze-dried sample was placed in 100mL of 1-3% (g/mL) CaCl 2 Crosslinking in the solution for 3-5h to obtain sodium alginate sponge, washing with sterile water for 2 times, drying at 37 + -1 deg.C, and sealing for use.
(2) Effect detection of different addition modes of trichoderma powder
Set up the following experimental groups：
Treatment group A: the Trichoderma SF-2 powder prepared in (1) above was added to a liquid culture medium of 3-5 days old Boletus metschnikowii in a ratio of 0.5g Trichoderma SF-2 powder per 150mL of the culture medium.
B, treatment group: adding the sodium alginate microsphere immobilized trichoderma powder prepared in the step (2) into a liquid culture solution of cultured boletus metschnikowii for 3-5 days according to the proportion that 0.5g of trichoderma SF-2 powder is added into each 150mL of culture solution.
Treatment group C: adding the sodium alginate sponge immobilized trichoderma powder prepared in the step (3) into a liquid culture solution of cultured boletus metschnikowii for 3-5 days according to the proportion that 0.5g trichoderma SF-2 powder is added into each 150mL culture solution.
B-A control group: the same amount of the sodium alginate microspheres prepared in (4) above as that in the group B was added to the liquid culture medium of cultured boletus omenii for 3-5 days.
C-A control group: the same amount of the sodium alginate sponge prepared in (5) above as that in the C-treated group was added to the liquid culture of 3-5 days-cultured Phlebopus metschnikoides.
CK control group: liquid culture solution of cultured 3-5 days old Boletus metschnikowii without addition of Trichoderma powder and sodium alginate.
The culture solution of the boletus meiloti of each experimental group is subjected to shaking culture at the temperature of 28 ℃ and the speed of 160 r/min. And (2) absorbing a boletus meiloti culture solution sample by adopting an aseptic operation technology every day, sampling 10mL each time, repeating each experimental group for 3 times, continuously sampling for 7d, and measuring the hypha growth quantity (measured by hypha dry weight) and extracellular enzyme activity of boletus meiloti in the culture solution, thereby selecting the most suitable trichoderma powder adding mode for culturing boletus meiloti liquid strains.
Results of the experiment：
Hypha growth amount of boletus edulis：
TABLE 2 influence of different addition modes of Trichoderma powder on the growth of boletus meiloti hyphae
Each value in the table is expressed as the mean (mean) ± Standard Deviation (SD) of three tests, with significant differences (P < 0.05) indicated by different letters in the same column.
As can be seen from Table 2, the growth rates of hyphae of Phlebopus americanus in the groups A, B and C to which Trichodermase:Sub>A powder was added were significantly increased as compared with the groups B-A, C-A and CK to which Trichodermase:Sub>A powder was not added. After trichoderma powder is added for induction culture for 2 days, the growth vigor of the bolete meinetorum hyphae is obviously increased, the dry weight of the hyphae is obviously increased, the weight average of the hyphae reaches the maximum value when reaching the 6 th day, and then the hyphae begin to age along with the extension of the culture time, and the dry weight of the hyphae is slowly reduced. In each treatment group, the hypha of the boletus meiloti of the C treatment group grows best, and the dry weight of the hypha is as high as 15.05mg/mL and is obviously higher than that of other treatment groups.
Extracellular enzyme activity of boletus meliloti：
The Phlebopus metschnikoides can produce extracellular enzymes such as laccase, polyphenol oxidase, neutral protease, amylase, cellulase and the like which decompose complex nutrients. These enzymes play a very important role in their action of re-parasitizing, degrading lignin and combating surrounding microorganisms, and they not only make the boletus meiloti absorb and utilize simple organic substances, but also absorb and utilize complex organic substances such as plant or fungal residues.
TABLE 3 influence of different addition modes of Trichoderma powder on laccase activity of Ramaria mellea culture solution
Each value in the table is expressed as the mean (mean) ± Standard Deviation (SD) of three tests, with significant differences (P < 0.05) indicated by different letters in the same column.
As can be seen from Table 3, the laccase activity in the culture solution of the Phlebopus metschnikoides in the treatment groups A, B and C was significantly higher than that in the control groups B-A, C-A and CK. The laccase activity of the treatment groups A, B and C has the same trend with the CK control group, and reaches the maximum value at the 6 th d of the induction culture by adding the trichoderma powder, and starts to decline at the 7 th d. In each treatment group, the effect of the C treatment group is optimal, and the activity of the Phlebopus metschnikoides laccase reaches the maximum value of 23.61U/mL at the 6 th d of culture and is obviously higher than that of other treatment groups.
TABLE 4 influence of different addition modes of Trichoderma powder on the activity of polyphenol oxidase in the culture solution of Boletus dictyostelium
Each value in the table is expressed as the mean (mean) ± Standard Deviation (SD) of three tests, with significant differences (P < 0.05) indicated by different letters in the same column.
As can be seen from Table 4, the polyphenol oxidase activity in the culture broth of the processed Boletus metschnikowii was significantly higher than that in the B-A control group, C-A control group and CK control group. The change trend of polyphenol oxidase activity of the A treatment group, the B treatment group and the CK control group is the same, the change trend reaches the maximum at the 6 th d and starts to decline at the 7 th d, while the polyphenol oxidase activity of the C treatment group is in the strong rising trend at the 1 st to the 7 th d, the 7 th d is as high as 38.67U/mL, and the change trend starts to decline at the 8 th d. The polyphenol oxidase activity of the group C was significantly higher than that of the groups a and B.
TABLE 5 influence of different addition modes of Trichoderma powder on the activity of neutral protease in the culture solution of Boletus dictyostelium
Each value in the table is expressed as the mean (mean) ± Standard Deviation (SD) of three tests, with different letters in the same column indicating significant differences (P < 0.05).
As can be seen from Table 5, after the Phlebopus meiloti was induced by Trichoderma powder, the activity of the neutral protease in the culture broth changed significantly. After the induction treatment of trichoderma powder, the activity of neutral protease in the culture solution is rapidly improved from 1 st to 4 th, the activity of neutral protease is kept in a high stable state from 4 th to 6 th, and the activity of neutral protease starts to be reduced from 7 th. Compared with the trichoderma different addition mode groups, the neutral protease activity of the culture solution in the C treatment group in the whole culture process is obviously higher than that of the A treatment group and the B treatment group, and is as high as 3.519U/mL in the 6 d.
TABLE 6 influence of different addition modes of Trichoderma powder on the amylase activity of the culture solution of Boletus dictyostelium
Each value in the table is expressed as the mean (mean) ± Standard Deviation (SD) of three tests, with significant differences (P < 0.05) indicated by different letters in the same column.
As can be seen from Table 6, in the culture of H.reticuloendothelial liquid from No. 1 to No. 4d, the amylase activities of the Trichoderma powder-induced group and the CK control group were increased to different degrees, and the amylase activities of the induced group were higher than that of the CK control group. Amylase activity peaked during culture 4d and then began to decline slowly. The amylase activity of the broth in treatment C was significantly higher throughout the culture than in treatment A and treatment B, up to 0.596U/mL in 4 d.
TABLE 7 influence of different addition modes of Trichoderma powder on cellulase activity of culture solution of Boletus omeiensis
Each value in the table is expressed as the mean (mean) ± Standard Deviation (SD) of three tests, with significant differences (P < 0.05) indicated by different letters in the same column.
As can be seen from Table 7, the cellulase activities of the induction-treated group and the CK control group showed the same tendency of change during the whole culture. The cellulase activity in the culture solution of 1-3d is rapidly increased, the cellulase activity reaches the maximum value in the culture solution of 3d, then the cellulase activity starts to slowly decrease, and the cellulase activity slightly increases back in the culture solution of 5d, but the whole cellulase activity is in a descending trend. The cellulose activity of the broth in the C-treated group was significantly higher throughout the culture than in the a-and B-treated groups, up to 0.566U/mL at 3 d.
Example 4 preparation of solid Strain of Phlebopus metschnikoides
The solid culture medium is prepared by a conventional method, and the formula is as follows: according to the mass percentage, 10-15% of masson pine sawdust, 5-10% of masson pine needle powder, 10-20% of straw powder, 10-20% of cotton seed hulls, 10-15% of cow dung, 10-15% of corncobs, 10-15% of perlite, 1-1.5% of glucose, 1-1.5% of gypsum, 0.01-0.05% of monopotassium phosphate and 0.001-0.005% of Vc, the water content is adjusted to 65-70%, and the pH value is 7.2.
The liquid culture of Phlebopus metschnikoides cultured in section 4 of example 3, treatment group A, treatment group B, treatment group C, control group B-A, control group C-A and control group CK, was inoculated to the prepared solid culture medium at an inoculum size of 1% (v/v) (i.e., the volume ratio of the culture fluid of Phlebopus metschnikoides to the solid culture medium was 1: 100), and was cultured in ase:Sub>A dark environment at 24. + -. 1 ℃ until hyphae overgrown with the solid culture medium. Observing the growth vigor of hyphae of each group of boletus meliloti, inoculating 10 bags of solid culture medium into each group, repeating the experiment for 3 times, and thus selecting the most suitable trichoderma powder adding mode for culturing the boletus meliloti solid strains.
The results are shown in table 8, when the boletus meliloti solid strain is prepared by using the solid culture medium mainly containing the masson pine sawdust and the masson pine needle powder, the trichoderma SF-2 strain powder can obviously promote the growth of the boletus meliloti in the solid culture medium, and the mycelia are thick and strong and have less pollution. Wherein the hyphae of the boletus meiloti of the treatment group A have stronger growth vigor and fastest growth, the solid culture medium is full of hyphae for about 19 days, and about 1 bag of hyphae in 10 bags is polluted on average; the hypha growth vigor of the boletus meiloti in the treatment group B is strong, about 25 days of hypha growth is full of the solid culture medium, and about 2 bags of hypha growth in 10 bags are polluted on average; the hyphae of the boletus meiloti in the treatment group C grow most strongly, the solid culture medium grows over about 22 days, and about 2 bags are polluted in 10 bags on average. hyphase:Sub>A growth of the B-A control group, the C-A control group and the CK control group which are not added with the trichodermase:Sub>A SF-2 bacterial powder is weak, the hyphase:Sub>A can grow full of the solid matrix after about 36-37 days, and 3 bags in 10 bags are polluted on average.
TABLE 8 influence of different addition modes of Trichoderma powder on the growth of solid Phlebopus meliloti strains
Note: "+ +++" indicates strong hyphal growth, "+ ++" indicates strong hyphal growth, and "+" indicates weak hyphal growth. Each value in the table is expressed as the mean (mean) ± Standard Deviation (SD) of three experimental tests, with significant differences (P < 0.05) indicated by different letters in the same column.
The above embodiments are only some, not all embodiments of the present invention. The above examples are only used for explaining and illustrating the technical solution of the present invention, and are not used for limiting the protection scope of the present invention. Any modification or variation of the above-described embodiments within the technical scope of the present disclosure by those skilled in the art should be covered by the protection scope of the present disclosure.
Claims (10)
1. A boletus meiloti endophytic trichoderma fungus with a preservation number of CCTCC NO: M2022944.
2. An agent comprising the boletus edulis endophytic trichoderma fungus of claim 1.
3. The inoculant according to claim 2, wherein the inoculant comprises a flour of the trichoderma fungus; the preparation method of the bacterial powder comprises the following steps: culturing the trichoderma fungus in a liquid for 3-5 days, performing aseptic filtration on the trichoderma fungus culture solution to obtain trichoderma fungus hyphae, washing the trichoderma fungus hyphae with aseptic water, drying and inactivating the trichoderma fungus hyphae at 60 +/-1 ℃, grinding the hyphae into powder, and sieving the powder with a 60-mesh sieve.
4. The microbial inoculum of claim 2, comprising immobilized fungal powder of the trichoderma fungus; the immobilized bacteria powder is sodium alginate microsphere immobilized bacteria powder or sodium alginate sponge immobilized bacteria powder.
5. The microbial preparation according to claim 4, wherein the microbial preparation is
The preparation method of the sodium alginate microsphere immobilized bacterial powder comprises the following steps: preparing 2-3% sodium alginate solution, adding 0.25-2g of the bacterial powder of claim 3 into 20mL of the sodium alginate solution, stirring, and slowly dripping the mixed solution into 2-4% CaCl 2 Fixing in the solution for 3-5h, sterile filtering to obtain sodium alginate microsphere immobilized bacteria powder, washing with sterile water, and oven drying at 37 + -1 deg.C; and/or
The preparation method of the sodium alginate sponge immobilized bacteria powder comprises the following steps: preparing 1-2% sodium alginate solution by using skim milk, adding 0.25-2g of the bacterial powder in claim 3 into every 20mL of the sodium alginate solution, uniformly stirring, standing at room temperature for a period of time to discharge air in the mixed solution, freezing at-20 ℃ for 3-5h, and then vacuum freeze-drying at-80 ℃ for 24-48h; placing the vacuum freeze-dried sample in 1-3% CaCl 2 Crosslinking for 3-5h in the solution to obtain sodium alginate sponge immobilized bacteria powder, washing with sterile water, and drying at 37 + -1 deg.C.
6. Use of the boletus meiloti endophytic trichoderma fungus of claim 1 or the microbial inoculum of any one of claims 2-5 in the preparation of liquid and solid strains of boletus meiloti.
7. A method for preparing boletus omenii liquid strains comprises the following steps: liquid culture of Phlebopus metschnikowii, when Phlebopus metschnikowii is in dominant growth in the culture solution, adding the bacterium powder described in claim 3 or the immobilized bacterium powder described in claim 4 or 5 into the culture solution, and continuously culturing to obtain Phlebopus metschnikowii liquid strains.
8. The method according to claim 7, wherein 0.25-2g of the fungal powder according to claim 3 or the immobilized fungal powder according to claim 4 or 5 is added per 150mL of the culture medium.
9. The method of claim 7, wherein the Phlebopus omelanuginosus is cultured in liquid using a modified PDA liquid medium; the formula of the culture medium is as follows: weighing peeled potato 200g, cutting into small pieces, adding 1000mL water, heating to boil, maintaining for 5-10min, filtering with gauze while it is hot, removing residue, cooling filtrate to 50-60 deg.C, adding glucose 20g, corn flour 20g, yeast powder 2g, KH, etc 2 PO 4 0.1-0.5g、MgSO 4 1g, vc 0.01-0.05g, add ddH 2 And O, heating to dissolve, and then fixing the volume to 1000mL.
10. A method for preparing boletus edulis solid strains comprises the following steps: preparing a liquid strain of boletus omenii by using the method of any one of claims 7 to 9, inoculating the liquid strain into a solid culture medium, and culturing to obtain the solid strain of boletus omenii.
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