CN115838632B - Trichoderma in Phlebopus portentosus and application thereof - Google Patents
Trichoderma in Phlebopus portentosus 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 trichoderma in Phlebopus portentosus and application thereof. The provided trichoderma atroviride endophytic fungi have a preservation number of CCTCC NO: M2022944. Also provides application of the trichoderma fungus in preparing liquid strain and solid strain of the boletus reticulatus. The trichoderma fungus has obvious promotion effect on the growth of hyphae of the liquid culture and the solid culture of the exomycorrhizal fungi Phlebopus portentosus, can shorten the production period of the Phlebopus portentosus fruiting body, and improves the yield. In addition, the trichoderma fungus can reduce pollution in the culture of the Phlebopus portentosus, and avoid the use of chemical agents.
Description
Technical Field
The invention belongs to the technical field of edible fungus cultivation, and particularly relates to trichoderma in Phlebopus portentosus and application thereof.
Background
The Phlebopus portentosus (Boletus reticulatus Schaeff.) is a rare exogenous mycorrhizal edible fungus, belongs to the order of Boletales (Boletales), the family of Boletaceae (Boletaceae) and the genus of Boletus (Boletus), has delicious taste and high nutritional value, and is widely distributed in a plurality of areas in China. The Phlebopus portentosus is often symbiotic with the root system of the Pinus plant, and is not easy to form fruiting bodies due to the unique symbiotic ecological environment, nutrition mode and fruiting body differentiation conditions. Because of the change of ecological environment, the yield of the wild Phlebopus portentosus in China is drastically reduced, and in order to protect the diversity of wild edible fungus resources, the Phlebopus portentosus resources are necessary to be developed. For more than ten years, researchers at home and abroad have developed the strain preparation and cultivation work of the Phlebopus portentosus, and part of the researchers utilize pure cultures of the Phlebopus portentosus mycelium to inoculate pine root systems, and fruiting is carried out under proper natural climate conditions after the biomass of the mycelium is accumulated to a certain amount. But the Phlebopus portentosus is characterized by slow hypha growth, long fruiting period and low fruiting amount, which is necessarily related to the long time required for hypha accumulation of Phlebopus portentosus under natural conditions.
In addition, in the semi-artificial cultivation process of wild mycorrhizal edible fungi, inoculated mycorrhizal edible fungi strains are easy to spoil, and manufacturers often utilize commercial chemical bacteriostats to prevent and treat edible fungi diseases, so that potential chemical pollution exists, and side effects of the edible fungi are always plagued by consumers and managers.
Disclosure of Invention
The invention aims to solve the technical problems of improving the hypha growth speed of the Phlebopus portentosus and reducing the pollution in the culture process.
In order to solve the technical problems, the invention provides a trichoderma reesei endophytic fungus of the Phlebopus portentosus, which has a preservation number of CCTCC NO: M2022944 and is named trichoderma SF-2. When the strain is cultured on the modified PDA plate culture medium, the mycelium is white, dense and felt-like at the initial stage, and the aged mycelium is yellow.
The invention also provides a microbial inoculum which comprises the trichoderma reesei SF-2 endophytic fungus of the Phlebopus portentosus.
The microbial inoculum can comprise microbial powder of the trichoderma fungi SF-2; the preparation method of the bacterial powder comprises the following steps: culturing the Trichoderma fungus in liquid for 3-5 days, aseptically filtering the obtained Trichoderma fungus culture solution to obtain Trichoderma fungus mycelia, washing with sterile water, oven drying at 60+ -1deg.C for inactivation (detected by PDA plate culture, without growth activity), grinding the mycelia into powder, and sieving with 60 mesh sieve.
The microbial inoculum can also comprise immobilized microbial powder of the trichoderma fungi SF-2, wherein the immobilized microbial powder is sodium alginate microsphere immobilized microbial powder or sodium alginate sponge immobilized microbial 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 the trichoderma fungus SF-2 bacterial powder into 20mL, stirring uniformly, slowly dripping the mixed solution into 2-4% CaCl 2 solution, fixing for 3-5h, performing sterile filtration to obtain sodium alginate microsphere immobilized bacterial powder, washing with sterile water, and drying at 37+/-1 ℃.
The preparation method of the sodium alginate sponge immobilized bacterial powder can comprise the following steps: preparing 1-2% sodium alginate solution by using skimmed milk, adding 0.25-2g of the Trichoderma fungus SF-2 powder into 20mL, stirring, mixing, standing at room temperature for a period of time to discharge air in the mixed solution, freezing at-20deg.C for 3-5h, and vacuum freeze-drying at-80deg.C for 24-48h; and (3) placing the vacuum freeze-dried sample into 1-3% CaCl 2 solution for crosslinking for 3-5 hours to obtain sodium alginate sponge immobilized bacteria powder, washing with sterile water, and drying at 37+/-1 ℃.
The application of the trichoderma reesei SF-2 or any one of the microbial inoculum in preparing the liquid strain and the solid strain of the boletus reesei belongs to the protection scope of the invention.
The invention also provides a method for preparing the liquid strain of the Phlebopus portentosus, which comprises the following steps: and (3) culturing the Phlebopus portentosus in a liquid manner, when the Phlebopus portentosus is in dominant growth in the culture solution, adding the fungus powder of the Trichoderma fungus SF-2 or the immobilized fungus powder of the Trichoderma fungus SF-2 into the culture solution, and continuously culturing to obtain the Phlebopus portentosus liquid strain.
In the method, 0.25-2g of the fungus powder or immobilized fungus powder of the trichoderma reesei SF-2 can be added into 150mL of the culture solution of the Phlebopus portentosus.
In the method, the improved PDA liquid culture medium can be adopted to carry out liquid culture on the Phlebopus portentosus; the formula of the culture medium is as follows: weighing 200g of peeled potatoes, cutting into small pieces, adding 1000mL of water, heating to boiling, maintaining for 5-10min, filtering with gauze while the hot, discarding filter residues, adding 20g of glucose, 20g of corn meal, 2g of yeast powder, 2PO4 0.1-0.5g、MgSO4 g of KH and 0.01-0.05g of Vc into the filtrate when the filtrate is cooled to 50-60 ℃, adding ddH 2 O, heating and dissolving, and fixing the volume to 1000mL.
In the method, the Phlebopus portentosus can be subjected to liquid culture at 28 ℃ and 160 r/min.
In the method, after the fungus powder of the trichoderma fungus SF-2 or the immobilized fungus powder of the trichoderma fungus SF-2 is added into the culture solution of the Phlebopus portentosus, the culture can be continued for 5 to 7 days, and the Phlebopus portentosus liquid strain is obtained.
The invention also provides a method for preparing the solid strain of the Phlebopus portentosus, which comprises the following steps: the method is used for preparing the liquid strain of the Phlebopus portentosus, inoculating the liquid strain into a solid culture medium, and culturing to obtain the solid strain of the Phlebopus portentosus.
The formula of the solid culture medium can be as follows: the fertilizer comprises, by mass, 10-15% of pinus massoniana wood dust, 5-10% of pinus massoniana pine needle powder, 10-20% of straw powder, 10-20% of cotton seed hulls, 10-15% of cow dung, 10-15% of corncob, 10-15% of perlite, 1-1.5% of glucose, 1-1.5% of gypsum, 0.01-0.05% of potassium dihydrogen phosphate, 0.001-0.005% of Vc, and adjusting the moisture content to 65-70% and the pH value to 7.2.
In the method, the liquid strain of the Phlebopus portentosus can be inoculated into a solid culture medium according to the inoculation amount of 1% (v/v), and the Phlebopus portentosus solid strain can be obtained by dark culture for 19-25 days at 24+/-1 ℃.
The invention utilizes the low-dose environment-friendly endophytic fungus trichoderma SF-2 with biocontrol function to co-culture with the ectomycorrhizal fungus Phlebsiella oxytoca to obtain excellent Phlebopus portentosus liquid strain and solid strain. Compared with the Phlebopus portentosus 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 Phlebopus portentosus which is in dominant growth, and the trichoderma SF-2 can effectively promote the growth of the Phlebopus portentosus and reduce pollution. If the liquid strain or the solid strain is inoculated to pine forest (such as natural pinus massoniana), the synergistic effect can be achieved, and trichoderma SF-2 can promote hypha growth and fruiting body development of the Phlebopus portentosus in the pine forest, so that the production period of the Phlebopus portentosus fruiting body is shortened, and the yield of the Phlebopus portentosus is improved. This would be an efficient semi-artificial cultivation method for Phlebopus portentosus.
The invention has the following beneficial effects:
(1) The trichoderma SF-2 provided by the invention can obviously improve the hypha growth speed and extracellular enzyme activity of the Phlebopus portentosus.
Experiments prove that after the sodium alginate sponge immobilized trichoderma powder is added into the culture solution of the boletus reticulatus according to the proportion of adding 0.5g of trichoderma SF-2 powder into each 150mL of culture solution, the mycelium dry weight of the boletus reticulatus can reach 15.05mg/mL, the laccase activity can reach 23.61U/mL, the polyphenol oxidase activity can reach 38.67U/mL, the neutral protease activity can reach 3.519U/mL, the amylase activity can reach 0.596U/mL, and the cellulase activity can reach 0.566U/mL. The trichoderma SF-2 is adopted to induce and prepare the liquid strain of the Phlebopus portentosus, and the obtained liquid strain of the Phlebopus portentosus has good growth vigor, fast spawn running, high hypha biomass and 6-7 days of culture and inoculation. After the obtained liquid strain of the Phlebopus portentosus is inoculated into a solid culture medium, hyphae grow well and are uniformly distributed in the medium, and the solid culture medium can be fully grown in 19-25 days.
(2) The trichoderma SF-2 provided by the invention has good biocontrol effect and can reduce pollution.
Experiments prove that after the liquid strain of the Phlebopus portentosus is inoculated to a solid culture medium, under the condition that no chemical agent is used, when hyphae grow on the solid culture medium, 1-2 bags of 10 bags of solid culture have pollution on average.
The patent preservation information of the trichoderma SF-2 provided by the invention is as follows:
preservation name: trichoderma sp.SF-2
Preservation date: 2022, 06, 22 days
Preservation number: CCTCC NO: M2022944
Preservation mechanism: china center for type culture Collection
Address: chinese university of Wuhan and Wuhan.
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 the culture of Trichoderma SF-2 against a plate of Boletus reticulatus. The left side is a Phlebopus portentosus colony, and the right side is a Trichoderma SF-2 colony.
FIG. 4 shows Trichoderma SF-2 powder.
FIG. 5 shows the immobilized Trichoderma SF-2 powder of sodium alginate microspheres.
FIG. 6 shows the sodium alginate sponge immobilized Trichoderma SF-2 powder.
FIG. 7 shows liquid strains of Phlebopus portentosus induced by Trichoderma powder; CK is culture solution of Phlebopus portentosus of control group without adding trichoderma powder; the group A is a culture solution of Phlebopus portentosus added with trichoderma SF-2 bacteria powder; the group B is a Phlebopus portentosus culture solution added with sodium alginate microsphere immobilized trichoderma SF-2 bacteria powder; group C is Phlebopus portentosus culture solution added with sodium alginate sponge immobilized trichoderma SF-2 bacteria powder.
FIG. 8 shows solid strains of Phlebopus portentosus induced by Trichoderma powder; the CK group is a solid strain obtained by inoculating a solid matrix with a liquid strain of Phlebopus portentosus of a control group without adding trichoderma powder and culturing for 15 days; the A treatment group is a solid strain obtained by inoculating a solid matrix with a liquid strain of Phlebopus portentosus added with free trichoderma SF-2 bacteria powder and culturing for 15 days.
Detailed Description
The technical scheme of the present invention is described in detail below with reference to specific embodiments and drawings.
The Phlebopus portentosus used in the examples below was the strain isolated and stored in the laboratory. The public can obtain the strain from the college of biological science and technology of national university in Hubei, and the strain is only used for repeated experiments related to the invention and can not be used for other purposes. The implementation of the present invention is independent of the strain.
The media used in the following examples:
Improved PDA liquid medium: weighing peeled potato 200g, cutting into small pieces, placing into a pot, adding 1000mL of water, heating to boil, maintaining for 5-10min, filtering with 2 layers of gauze while hot, and discarding residues. Cooling the filtrate to 50-60deg.C, adding glucose 20g, corn flour 20g, yeast powder 2g, KH 2PO4 0.1-0.5g、MgSO4 g, vc 0.01-0.05g, ddH 2 O, heating to dissolve, fixing volume to 1000mL, packaging in 250mL glass triangular bottles, sterilizing at 150mL per bottle at 121deg.C for 20min, and keeping.
Improved PDA solid medium: 20g of agar powder is added on the basis of the modified PDA liquid medium.
The assay methods used in the following examples:
the method for measuring the hypha growth of the Phlebopus portentosus comprises the following steps: putting the sample into a 10mL centrifuge tube, centrifuging at 4 ℃ and 4000r/min for 10min, obtaining a precipitate after centrifugation, namely hypha, 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 Phlebopus portentosus by the dry weight of the hypha.
Method for measuring extracellular enzyme activity of boletus reticulatus: putting the sample into a 10mL centrifuge tube, centrifuging for 10min at 4 ℃ and 9000r/min, obtaining a supernatant as a crude enzyme solution, and measuring the extracellular enzyme activity in the crude enzyme solution.
① Laccase activity assay: laccase activity was determined by o-tolidine method, see literature (Chen Guimei. Intermate with exogenous mycorrhizal fungi [ D ]. University of North-west agriculture and forestry science and technology, 2009). ② Polyphenol oxidase activity assay: the polyphenol oxidase activity was determined by catechol method, see literature (Wang Weike, liu Na, zhou Zufa, song Jiling, yuan Weidong, a surname) static.8 extracellular enzymes activity changes at different growth stages of Lentinus edodes [ J ]. Zhejiang agricultural science, 2014 (4): 498-501). ③ Neutral protease activity assay: protease activity was determined using Fu Lin Fenfa, see (Bai Yan, wang Weixin. Methods for determining intestinal protease, amylase, lipase and cellulase activities of Stichopus japonicus [ J ]. Feed industry, 2012,33 (20): 28-32). ④ Measurement of amylase activity: amylase activity was determined by DNS colorimetry, see DNS colorimetry in literature (Zhang Hua, main code, biochemical experimental guidance (2 nd edition) [ M ]. Chinese university of agriculture press 2020). ⑤ Cellulase activity assay: the cellulase activity was determined by DNS colorimetric method, see DNS colorimetric method in literature (Huang Chunlin. Ba Keshen black umbrella 8 studies on the law of change of extracellular enzyme activity [ D ]. Xinjiang university of agriculture, 2018).
The data processing and analysis methods used in the following examples: test data were analyzed by one-way variance using SPSS STATISTICS software and expressed as mean ± standard deviation.
Unless otherwise indicated, all reagents used in the examples below are conventional in the art and are commercially available or formulated according to conventional methods in the art and are of laboratory scale. Unless otherwise indicated, the experimental procedures and conditions used in the following examples are those conventional in the art and may be referred to the 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 inventors of the present invention have unexpectedly obtained an endophytic fungus on a modified PDA solid medium during isolation of wild Phlebopus portentosus. The strain is cultured at 28 ℃ to obtain mycelium. The total DNA of mycelia was extracted using a fungus genomic DNA extraction kit (OMEGA, cat# D3471-00) according to the kit instructions. rDNA-ITS sequence PCR amplification is carried out by using a forward primer ITS1F (5'-CTTGGTCATTTTAGAGGAAGTAA-3') and a reverse primer ITS4B (5'-CAGGAGACTTGTACACGGTCCAG-3'), and the PCR purified product is subjected to bidirectional sequencing, so that the ITS sequence (shown as SEQ ID NO: 1) of the strain is obtained, and the similarity of the ITS sequence with the ITS sequence (GenBank accession number: MN 809171.1) of the registered Trichoderma Korean novel strain Trichoderma songyi sp.nov. In GenBank is 98.09%, so that the strain is identified as Trichoderma sp, and is named as Trichoderma SF-2.
The ITS sequence of Trichoderma SF-2 is as follows:
CCTTTTCTAGGGGAACTTCACGAGGGATCATTACCGAGTCCAGCAACTCCCAAACCCAATGTGAACCATACCAAACTGTTGCCTCGGCGAGGGTCACGCCCCGGGTGCGTCGCAGCCCCGGAACCAGGCGCCCGCCGGAGGGACCAACCAAACTCTTTACTGTAGTCCCCTCGCGGACGTTATTTCTTACAGCTCTGAGCAAAAATTCAAATGAATCAAAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCGCCAGTATTCTGGCGGGCATGCCTGTCCGAGCGTCATTTCAACCCTCGAACCCCTCCGGGGGGTCGGCGTTGGGGATCGGGAACCCCTCAGACGGGATCCCGGCCCCGAAATACAGTGGCGGTCTCGCCGCAGCCTCTCCTGCGCAGTAGTTTGCACAACTCGCACCGGGAGCGCGGCGCGTCCACGTCCGTAAAACACCCAACTTCTGAAATGTTGACCTAGGAACAGGTAGAAAAACCCGCTAAACTTACCCATATCAGTAACCGAAGAGAGAATC(SEQ ID NO:1)
Trichoderma SF-2 was cultured on a modified PDA solid medium, the mycelia of the strain were initially white, dense, felt-like (FIG. 1), yellow after the mycelia were aged, and the amount of produced spores was small, similar to that of Korean novel species Trichoderma songyi sp.nov. The phylogenetic tree of Trichoderma SF-2 constructed based on 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 counter culture of Phlebopus portentosus with Trichoderma SF-2
1 Piece of bacterial cake is picked up at the edge of the activated bacterial colony of the Phlebopus portentosus by a sterile puncher with the diameter of 6mm, inoculated in an improved PDA solid culture medium with the diameter of 90mm, placed in a 28 ℃ incubator for dark culture, when the bacterial colony of the Phlebopus portentosus is cultured to 1.5-2cm (after about 8-10d of culture), 1 piece of trichoderma SF-2 bacterial cake with the diameter of 6mm is inoculated at a position 30mm away from the bacterial colony of the Phlebopus portentosus, placed in the 28 ℃ incubator for dark culture continuously, and whether the Phlebopus portentosus and trichoderma fungi are opposite to grow or not is observed.
The results show that: the hyphae of the Phlebopus portentosus grow slowly on the modified PDA solid culture medium, and the hyphae are dense and strong. After inoculation of trichoderma SF-2, trichoderma wires grow faster, and hyphae are fine and loose. After 2d co-cultivation of Phlebopus portentosus with Trichoderma SF-2, phlebopus portentosus exhibited cross growth with Trichoderma SF-2, phlebopus portentosus growth was not affected by Trichoderma SF-2, and no obvious antagonistic line was formed at the cross growth (FIG. 3).
Example 3 preparation of liquid strain of Phlebopus portentosus
1. Liquid culture of bacterial species
(1) Liquid culture of Phlebopus portentosus
Selecting Phlebopus portentosus with good hyphae growth vigor from the improved PDA solid culture medium, taking bacterial cakes at the edge of bacterial colonies by using a sterile puncher with the diameter of 6mm, inoculating the bacterial cakes into 150mL of the improved PDA liquid culture medium (3 blocks/bottle and 6 mm/block in diameter), and culturing for 7-10d at the temperature of 28 ℃ under 160r/min in an oscillating way to obtain the liquid culture solution of Phlebopus portentosus.
(2) Liquid culture of trichoderma fungi
Selecting Trichoderma SF-2 with good hypha growth vigor from the improved PDA solid culture medium, taking bacterial cakes at the edge of bacterial colonies by using a sterile puncher with the diameter of 6mm, inoculating the bacterial cakes into 150mL of the improved PDA liquid culture medium (3 blocks/bottle, 6 mm/block), and carrying out shake culture for 3-5d at the temperature of 28 ℃ and the temperature of 160r/min to obtain the 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 fungus mycelia, washing the mycelia with sterile water for 2 times, oven drying at 60+ -1deg.C for inactivation (no growth activity by PDA plate culture detection), grinding the mycelia into powder, sieving with 60 mesh sieve to obtain Trichoderma SF-2 mycelia powder, and sealing for use.
3. Optimization of trichoderma powder addition amount
Treatment group: placing the inactivated Trichoderma SF-2 bacterial powder in an ultra-clean bench, sterilizing with ultraviolet rays for 30min, adding the inactivated Trichoderma SF-2 bacterial powder into a liquid culture solution of the cultured Phlebopus portentosus for 3-5d (at the moment, the Phlebopus portentosus is in dominant growth and appears as mycelium pellets with the size of rice grains in the culture solution) at different gradient concentrations (0 g/150mL, 0.25g/150mL, 0.5g/150mL, 1g/150mL, 1.5g/150mL and 2g/150mL are arranged), and then performing shaking culture at 28 ℃ and 160 r/min. After the trichoderma SF-2 bacteria powder is added, a sterile operation technology is adopted to absorb culture fluid samples each day, 10mL of culture fluid samples are sampled each time, each treatment is repeated for 3 times, continuous sampling is carried out for 7d, and the dry weight of the mycelium of the Phlebopus portentosus in the samples is measured.
Control group (CK group): no Trichoderma SF-2 bacteria powder is added, and the rest is the same as the treatment group.
The results are shown in Table 1, in which the dry weight of the mycelium of the treated group to which different amounts of Trichoderma powder were added was significantly increased compared to the CK group to which no Trichoderma powder was added, and the dry weight of the mycelium of the 6 d-th mycelium reached the maximum after the addition of Trichoderma powder, wherein the synergy was most significant with the dry weight of the mycelium of the treated group to which 0.5g/150mL of Trichoderma powder was added, and the maximum dry weight of the mycelium reached 12.89mg/mL.
TABLE 1 influence of different addition amounts of Trichoderma powder on dry weight of mycelium of Phlebopus portentosus
Each value in the table is expressed as the mean ± Standard Deviation (SD) of three tests, and the different letters in the same column represent significant differences (P < 0.05).
4. Optimizing adding mode of trichoderma powder
(1) Preparation of trichoderma powder
① Trichoderma powder: preparing trichoderma SF-2 bacterial powder by the method in the step 2.
② Preparation of sodium alginate microsphere immobilized trichoderma powder
Preparing 20mL of sodium alginate solution with the concentration of 2-3% (g/mL), adding 0.5g of trichoderma SF-2 bacteria powder, stirring uniformly, slowly dripping the mixed solution into 100mL of 2-4% (g/mL) CaCl 2 solution by using a 20-30mL sterile syringe, fixing for 3-5h, filtering by using sterile gauze, obtaining the sodium alginate microsphere immobilized trichoderma powder, washing 2 times by using sterile water, drying at 37+/-1 ℃, sealing, and preserving at 4 ℃ for later use.
③ Preparation of sodium alginate sponge immobilized trichoderma powder
Preparing 20mL of 1-2% (g/mL) sodium alginate solution by using commercially available defatted milk, adding 0.5g of trichoderma SF-2 bacteria powder, stirring and mixing, standing at room temperature for a period of time to discharge air in the mixed solution, standing at-20 ℃ for 3-5h, and transferring to-80 ℃ for vacuum freeze drying for 24-48h. And (3) placing the vacuum freeze-dried sample into 100mL of 1-3% (g/mL) CaCl 2 solution for crosslinking for 3-5 hours to obtain sodium alginate sponge immobilized trichoderma powder, washing the trichoderma powder with sterile water for 2 times, drying at 37+/-1 ℃ and sealing for later use.
④ Preparation of sodium alginate microspheres
Preparing 20mL of sodium alginate solution with the concentration of 2-3% (g/mL), adding no trichoderma powder, slowly dripping the sodium alginate solution into 100mL of 2-4% (g/mL) CaCl 2 solution by using a 20-30mL sterile syringe, fixing for 3-5h, filtering by using sterile gauze, obtaining sodium alginate microspheres, washing for 2 times by using sterile water, drying at 37+/-1 ℃, sealing, and preserving at 4 ℃ for later use.
⑤ Preparation of sodium alginate sponge
20ML of 1-2% (g/mL) sodium alginate solution is prepared from commercial skim milk, trichoderma powder is not added, the solution is placed at room temperature for a period of time to discharge air in the solution, the solution is frozen at the temperature of minus 20 ℃ for 3-5h, and then the solution is transferred to the temperature of minus 80 ℃ for vacuum freeze drying for 24-48h. And (3) placing the vacuum freeze-dried sample into 100mL of 1-3% (g/mL) CaCl 2 solution for crosslinking for 3-5 hours to obtain sodium alginate sponge, washing with sterile water for 2 times, drying at 37+/-1 ℃ and sealing for later use.
(2) Effect detection of different adding modes of trichoderma powder
The following experimental groups were set up:
Treatment group a: the Trichoderma SF-2 powder prepared in ① above was added to the liquid culture broth of Boletus reticulatus which had been cultured for 3-5d in a ratio of 0.5g of Trichoderma SF-2 powder per 150mL of the culture broth.
Treatment group B: the sodium alginate microsphere immobilized trichoderma powder prepared in ② is added into the liquid culture solution of the Phlebopus portentosus which is cultured for 3-5d according to the proportion of 0.5g of trichoderma SF-2 powder added into each 150mL of culture solution.
Treatment group C: the sodium alginate sponge immobilized trichoderma powder prepared in ③ above was added to the liquid culture broth of the Phlebopus portentosus which had been cultured for 3-5d in a proportion of 0.5g trichoderma SF-2 powder per 150mL of the culture broth.
B-ase:Sub>A control group: the sodium alginate microspheres prepared in ④ above, in an amount equivalent to that of the B-treated group, were added to the liquid culture broth of Boletus reticulatus which had been cultured for 3-5 d.
C-se:Sub>A control group: the sodium alginate sponge prepared in ⑤ above was added to the liquid culture broth of the Phlebopus portentosus which had been cultured for 3-5d in an amount equivalent to that of the C-treated group.
CK control group: the liquid culture solution of the Phlebopus portentosus which is not added with trichoderma reesei powder and sodium alginate and is cultured for 3-5 d.
The culture solution of Phlebopus portentosus of each experimental group was subjected to shaking culture at 28℃and 160 r/min. And (3) absorbing a culture solution sample of the Phlebopus portentosus by adopting a sterile operation technology every day, sampling 10mL each time, repeating 3 times of each experimental group, continuously sampling for 7 days, and measuring the hypha growth amount (measured by hypha dry weight) and extracellular enzyme activity of the Phlebopus portentosus in the culture solution, thereby selecting the optimal trichoderma powder adding mode for liquid strain culture of the Phlebopus portentosus.
Experimental results:
mycelium growth amount of Phlebopus portentosus:
TABLE 2 influence of different addition modes of Trichoderma powder on the growth of mycelium of Phlebopus portentosus
Each value in the table is expressed as the mean ± Standard Deviation (SD) of three tests, and the different letters in the same column represent significant differences (P < 0.05).
As can be seen from table 2, the hyphase:Sub>A growth rate of the hepaticase:Sub>A was significantly improved in the ase:Sub>A-treated group, the B-treated group, and the C-treated group, to which the trichodermase:Sub>A powder was added, compared to the B-ase:Sub>A control group, the C-ase:Sub>A control group, and the CK control group to which the trichodermase:Sub>A powder was not added. After the trichoderma powder is added for induction culture for 2d, the growth vigor of the mycelium of the Phlebopus portentosus is obviously increased, the dry weight of the mycelium reaches the maximum value at the time of 6d, and then the mycelium starts to age and the dry weight of the mycelium slowly decreases along with the extension of the culture time. Among the treatment groups, the mycelium growth potential of Phlebopus portentosus in the treatment group C is best, and the mycelium dry weight is as high as 15.05mg/mL, which is significantly higher than that of other treatment groups.
Extracellular enzyme activity of boletus reticulatus:
The bolete can produce laccase, polyphenol oxidase, neutral protease, amylase, cellulase and other extracellular enzymes for decomposing complex nutrients. These enzymes play a very important role in the heavy parasitism, degradation of lignin and antagonism with surrounding microorganisms, and they can not only make the bolete mesenchyma absorb and utilize simple organic matters, but also can absorb and utilize complex organic matters such as plant or fungus residues.
TABLE 3 influence of different addition modes of Trichoderma powders on laccase Activity of Phlebopus portentosus culture solution
Each value in the table is expressed as the mean ± Standard Deviation (SD) of three tests, and the different letters in the same column represent significant differences (P < 0.05).
As can be seen from Table 3, the laccase activities in the culture solutions of Phlebsiellase:Sub>A rolls of the A treatment group, the B treatment group and the C treatment group were significantly higher than those of the B-A control group, the C-A control group and the CK control group. The laccase activities of the A treatment group, the B treatment group and the C treatment group have the same variation trend as the laccase activity of the CK control group, the maximum value is reached at the 6 th d of the induction culture of adding trichoderma powder, and the decrease starts at the 7 th d. Among the treatment groups, the treatment group C has the optimal effect, and the laccase activity of the Phlebopus portentosus reaches the maximum value of 23.61U/mL at the 6d of culture, which is obviously higher than that of other treatment groups.
TABLE 4 influence of different addition modes of Trichoderma powders on polyphenol oxidase activity of Boletus mexicanus culture solution
Each value in the table is expressed as the mean ± Standard Deviation (SD) of three tests, and the different letters in the same column represent significant differences (P < 0.05).
As can be seen from Table 4, the activity of polyphenol oxidase in the culture broth of Boletus meticulosus in the treated group was significantly higher than that in the B-A control group, the C-A control group and the CK control group. The change trend of the 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 decrease at the 7 th d, while the polyphenol oxidase activity of the C treatment group is in a strong rising trend at the 1 st to 7 th days, the 7 th day is as high as 38.67U/mL, and the C treatment group starts to decline at the 8 th day. The polyphenol oxidase activity of the C-treated group was significantly higher than that of the a-treated group and the B-treated group.
TABLE 5 influence of different addition modes of Trichoderma powders on neutral protease Activity of Phlebopus portentosus culture solution
Each value in the table is expressed as the mean ± Standard Deviation (SD) of three tests, and the different letters in the same column represent significant differences (P < 0.05).
As is clear from Table 5, the neutral protease activity of the culture broth of the Phlebopus portentosus was significantly changed after the induction treatment with the Trichoderma powder. After the induction treatment of trichoderma powder, the neutral protease activity of the culture solution is rapidly improved in the 1 st to 4 th days, the neutral protease activity is kept in a high and stable state in the 4 th to 6 th days, and the neutral protease activity starts to be reduced in the 7 th days. Compared with the trichoderma in different addition mode groups, the neutral protease activity of the culture solution in the C treatment group is obviously higher than that in the A treatment group and the B treatment group in the whole culture process, and the neutral protease activity is up to 3.519U/mL in the 6 d.
TABLE 6 influence of different addition modes of Trichoderma powders on amylase activity of Phlebopus portentosus culture solution
Each value in the table is expressed as the mean ± Standard Deviation (SD) of three tests, and the different letters in the same column represent significant differences (P < 0.05).
As can be seen from Table 6, in the liquid culture of Boletus reticulata 1-4d, the amylase activities of both the Trichoderma powder induction treatment group and the CK control group were increased to different degrees, and the amylase activities of both the induction treatment group and the CK control group were higher. The amylase activity peaked on culture 4d, and then began to decline slowly. The amylase activity of the broth during the whole culture was significantly higher in the C-treated group than in the A-treated group and the B-treated group, up to 0.596U/mL at 4 d.
TABLE 7 influence of different addition modes of Trichoderma powders on cellulase Activity of Boletus reticulatus culture solution
Each value in the table is expressed as the mean ± Standard Deviation (SD) of three tests, and the different letters in the same column represent significant differences (P < 0.05).
As can be seen from table 7, the cellulase activity of the induction treated group and the CK control group showed the same trend in the whole culture. The cellulase activity of the culture solution at 1-3d increases rapidly, reaches a maximum at 3d, then starts to decline slowly, and slightly returns at 5d, but tends to decline as a whole. The cellulose activity of the culture broth during the whole culture process was significantly higher in the C-treated group than in the A-treated group and the B-treated group, up to 0.566U/mL in 3 d.
Example 4 preparation of solid strain of Phlebopus portentosus
The solid culture medium is prepared according to the conventional method, and the formula is as follows: the fertilizer comprises, by mass, 10-15% of pinus massoniana wood dust, 5-10% of pinus massoniana pine needle powder, 10-20% of straw powder, 10-20% of cotton seed hulls, 10-15% of cow dung, 10-15% of corncob, 10-15% of perlite, 1-1.5% of glucose, 1-1.5% of gypsum, 0.01-0.05% of potassium dihydrogen phosphate, 0.001-0.005% of Vc, and adjusting the moisture content to 65-70% and the pH value to 7.2.
The liquid strain of Boletus reticulatus obtained by culturing the A-treated group, the B-treated group, the C-treated group, the B-A control group, the C-A control group and the CK control group in section 4 of example 3 was inoculated into the prepared solid culture medium at an inoculum size of 1% (v/v) (i.e., the volume ratio of the culture solution of Boletus reticulatus to the solid culture medium was 1:100), and was dark-cultured in ase:Sub>A culture chamber at 24.+ -. 1 ℃ until myceliase:Sub>A were grown on the solid culture medium. Observing hypha growth vigor of the boletus of each group, inoculating 10 bags of solid culture medium into each group, repeating the experiment for 3 times, and selecting the optimal trichoderma powder adding mode for culturing the solid strain of the boletus of the group.
As shown in Table 8, the solid culture medium mainly comprising wood dust and needle powder of Pinus massoniana is used for preparing solid strain of Boletus reticulata, and Trichoderma SF-2 strain powder can remarkably promote the growth of Boletus reticulata in the solid culture medium, and mycelia are dense and strong and have less pollution. Wherein hyphae of the Phlebopus portentosus of the A treatment group grow stronger and grow fastest, about 19 days is full of solid culture medium, and about 1 bag is polluted in 10 bags on average; the hypha of the Phlebopus portentosus in the treatment group B grows stronger, the solid culture medium grows fully for about 25 days, and about 2 bags are polluted in 10 bags on average; the mycelium of Phlebopus portentosus in the group C had the strongest growth, and approximately 22 days had grown over the solid culture medium, with an average of approximately 2 bags out of 10 bags being contaminated. The hyphae of the B-A control group, the C-A control group and the CK control group without adding trichodermase:Sub>A SF-2 bacteriase:Sub>A powder grow poorly, the solid matrix can be grown up for about 36-37 days, and 3 bags are polluted on average in 10 bags.
Table 8 influence of different addition modes of trichoderma powders on the growth of solid strain of boletus reticulatus
Note that: "+++" "means the hypha grows well and has strong vigor," +++ "indicates hyphae the growth potential is relatively strong, and the growth potential is relatively high," + "indicates weak hyphae growth. Each value in the table is expressed as the mean ± Standard Deviation (SD) of three experimental tests, with different letters in the same column representing significant differences (P < 0.05).
The above embodiments are only some, but not all, embodiments of the invention. The above embodiments are only for explaining and explaining the technical solution of the present invention, and are not for limiting the scope of the present invention. Any modification or variation of the above-described embodiments, which are within the scope of the present disclosure, are intended to be encompassed by the present invention.
Claims (10)
1. A Trichoderma atroviride endophytic fungus with a preservation number of CCTCC NO: M2022944.
2. A microbial agent comprising the trichoderma reesei fungus of the species boletus reticulatus of claim 1.
3. The microbial agent of claim 2, wherein the microbial agent comprises a microbial powder of the trichoderma fungus; the preparation method of the bacterial powder comprises the following steps: culturing the Trichoderma fungus in liquid for 3-5 days, aseptically filtering the obtained Trichoderma fungus culture solution to obtain Trichoderma fungus mycelia, washing with sterile water, oven drying at 60+ -1deg.C for inactivation, grinding the mycelia into powder, and sieving with 60 mesh sieve.
4. The microbial agent of claim 2, wherein the microbial agent comprises an immobilized microbial powder of the trichoderma fungus; the preparation method of the sodium alginate microsphere immobilized bacterial powder comprises the following steps: preparing 2-3% g/mL sodium alginate solution, adding 0.25-2 g of the bacterial powder in claim 3 into 20 mL, stirring uniformly, slowly dripping the mixed solution into 2-4% g/mL CaCl 2 solution, fixing 3-5 h, performing sterile filtration to obtain sodium alginate microsphere immobilized bacterial powder, washing with sterile water, and drying at 37+/-1 ℃.
5. The microbial agent of claim 2, wherein the microbial agent comprises an immobilized microbial powder of the trichoderma fungus; the immobilized bacteria powder is sodium alginate sponge immobilized bacteria powder; the preparation method of the sodium alginate sponge immobilized bacterial powder comprises the following steps: preparing 1-2% g/mL sodium alginate solution by using skimmed milk, adding 0.25-2 g of the bacterial powder of claim 3 into each 20mL, stirring, mixing, standing at room temperature for a period of time to discharge air in the mixed solution, freezing at-20deg.C for 3-5 h, and vacuum freeze-drying at-80deg.C for 24-48 h; and (3) putting the vacuum freeze-dried sample into 1-3% g/mL CaCl 2 solution to crosslink 3-5 h, obtaining sodium alginate sponge immobilized bacteria powder, washing with sterile water, and drying at 37+/-1 ℃.
6. Use of a trichoderma reesei fungus within boletus reticulatus according to claim 1 or a microbial inoculum according to any one of claims 2-5 for the preparation of a liquid strain of boletus reticulatus or a solid strain of boletus reticulatus.
7. A method for preparing a liquid strain of reticulum boletus, comprising the steps of: liquid culturing Phlebopus portentosus, when Phlebopus portentosus is in dominant growth in the culture solution, adding the bacterial powder described in claim 3 or the immobilized bacterial powder described in claim 4 or 5 into the culture solution, and continuing culturing to obtain Phlebopus portentosus liquid strain.
8. The method according to claim 7, wherein the bacterial powder according to claim 3 or the immobilized bacterial powder according to claim 4 or 5 is added per 150 mL of the culture broth in an amount of 0.25-2 g.
9. The method of claim 7, wherein the modified PDA liquid medium is used to liquid culture bolete with reticulata; the formula of the culture medium is as follows: weighing peeled potato 200 g, cutting into small pieces, adding water 1000 mL, heating to boil, maintaining 5-10 min, filtering with gauze while hot, discarding residue, cooling to 50-60deg.C, adding glucose 20 g, corn flour 20 g, yeast powder 2g, KH 2PO4 0.1-0.5 g、MgSO4 1 g, vc 0.01-0.05 g, adding ddH 2 O, heating to dissolve, and fixing volume to 1000 mL.
10. A method for preparing a solid strain of boletus reticulatus, comprising the steps of: the method of any one of claims 7-9 is used for preparing a liquid strain of portraitia reticulata, the liquid strain is inoculated into a solid culture medium, and the solid strain of portraitia reticulata is obtained through culture.
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| CN103430773A (en) * | 2013-08-28 | 2013-12-11 | 云南省热带作物科学研究所 | Method for preserving deep brown boletus reticulatus strains |
| CN110204627A (en) * | 2019-06-26 | 2019-09-06 | 西华师范大学 | A kind of U.S. Open Tennis handle speciosus polysaccharide and its preparation method and application |
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