CN115074268B - Application of biological agent for promoting synthesis of truffle mycorrhiza - Google Patents

Application of biological agent for promoting synthesis of truffle mycorrhiza Download PDF

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CN115074268B
CN115074268B CN202210527804.1A CN202210527804A CN115074268B CN 115074268 B CN115074268 B CN 115074268B CN 202210527804 A CN202210527804 A CN 202210527804A CN 115074268 B CN115074268 B CN 115074268B
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mycorrhiza
truffle
synthesis
promoting
seedlings
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CN115074268A (en
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万山平
于富强
刘美菊
赵吉霞
王冉
黄兰兰
刘培贵
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Yunnan Agricultural University
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Yunnan Agricultural University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G18/00—Cultivation of mushrooms
    • A01G18/10—Mycorrhiza; Mycorrhizal associations
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G7/00—Botany in general
    • A01G7/06—Treatment of growing trees or plants, e.g. for preventing decay of wood, for tingeing flowers or wood, for prolonging the life of plants
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
    • A01P21/00—Plant growth regulators
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture

Abstract

The invention discloses an application of a biological agent for promoting the synthesis of truffle mycorrhiza, which comprises the following steps: selecting Burkholderia strain (Burkholderia sediminicola) as growth promoting bacteria, placing in TSB liquid culture medium, and performing propagation culture at 28deg.C for 3 days to obtain a concentration of 2.4X10 9 The cfu/mL bacterial inoculum is added as an exogenous microbial agent during the synthesis of the truffle mycorrhiza, and co-culture is carried out. The invention improves the growth of mycorrhizal seedlings, including plant height, ground diameter and root system development. The method has the advantages that the mycorrhiza infection rate is greatly increased, the mycorrhiza synthesis cost of the truffle is greatly reduced, the mycorrhiza synthesis time period is shortened, and the quality of the truffle mycorrhiza seedlings is improved.

Description

Application of biological agent for promoting synthesis of truffle mycorrhiza
Technical Field
The invention relates to the technical field of microorganisms, in particular to application of a biological agent for promoting synthesis of truffle mycorrhiza.
Background
Mycorrhizal formation is a prerequisite for artificial cultivation of truffles. The quantity and the yield of the mycorrhizal fungi are obviously positively correlated, and the growth of root systems, plant heights and ground diameters of the Huashan pine seedlings can be obviously promoted by the treatment of exogenously added mycorrhizal fungi growth promoting bacteria; most importantly, the treatment of adding mycorrhizal growth-promoting bacteria obviously promotes the formation of mycorrhizal and increases the mycorrhizal formation rate of the white truffle infected Chinese mountain pine seedlings. The addition of the microbial growth promoting microbial agent is proved by taking the truffle (Tuber huidongense) with high threat degree and economic value as a column, so that the growth of mycorrhizal seedlings, including plant height, ground diameter and root system development, can be greatly improved; greatly increases the mycorrhizal infection rate, thereby improving the quality of mycorrhizal seedlings and laying an important technology for realizing high yield by artificial planting.
Disclosure of Invention
Aiming at the problems, the invention provides application of a biological agent for promoting the synthesis of the truffle mycorrhiza, which improves the growth of mycorrhiza seedlings, including plant height, ground diameter and root system development. The method has the advantages that the mycorrhiza infection rate is greatly increased, the mycorrhiza synthesis cost of the truffle is greatly reduced, the mycorrhiza synthesis time period is shortened, and the quality of the truffle mycorrhiza seedlings is improved.
According to an object of the present invention, there is provided a method for preparing a biological agent for promoting synthesis of truffle mycorrhiza, comprising the steps of:
selecting Burkholderia strain as growth promoting bacteria, placing in TSB liquid culture medium, and performing propagation culture at 28deg.C for 3 days to obtain a concentrated solution with concentration of 2.4X10 9 cfu/mL of bacterial inoculum.
Further, the preparation method of the TSB liquid culture medium comprises the following steps:
tryptone 17.0g, soytone 3.0g, glucose 2.5g, naCl 5.0g and K 2 HPO 4 2.5g, distilled water to 1000mL, pH=7.0-7.4, and sterilizing at 121deg.C for 20min.
According to another object of the present invention, there is provided a biological agent for promoting the synthesis of truffle mycorrhiza, which is prepared by the above preparation method.
According to a third object of the present invention there is provided the use of a biological agent for promoting the synthesis of truffle mycorrhiza.
The beneficial effects of the invention are as follows:
the invention improves the growth of mycorrhizal seedlings, including plant height, ground diameter and root system development. The method has the advantages that the mycorrhiza infection rate is greatly increased, the mycorrhiza synthesis cost of the truffle is greatly reduced, the mycorrhiza synthesis time period is shortened, and the quality of the truffle mycorrhiza seedlings is improved.
Drawings
FIG. 1 is a macroscopic morphology of the ectomycorrhizal formed by Butyrosporum comosum and Pinus armandii in example 4 of the present invention;
FIG. 2 is a diagram showing the surface structure of mycorrhizal fungi formed by Butyrosporum tobirum and Pinus armandii in example 4 of the present invention;
FIG. 3 is a cross-sectional view of mycorrhizal fungi formed by Butyrosporum comosum and Pinus armandii in example 4 of the present invention;
FIG. 4 is a graph showing the comparison of the promotion of mycorrhiza formation by Burkholderia in example 4 of the present invention;
FIG. 5 is a graph showing the effect of Burkholderia on promoting the plant height of mycorrhizal seedlings in example 4 of the present invention;
FIG. 6 is a graph showing the effect of B.berkovicii on promoting root diameter of mycorrhizal seedlings in example 4 of the present invention.
Detailed Description
The technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
A preparation method of a biological agent for promoting the synthesis of truffle mycorrhiza comprises the following steps:
selecting Burkholderia strain as growth promoting bacteria, placing in TSB liquid culture medium, and performing propagation culture at 28deg.C for 3 days to obtain a concentrated solution with concentration of 2.4X10 9 cfu/mL of bacterial inoculum.
The preparation method of the TSB liquid culture medium comprises the following steps:
tryptone 17.0g, soytone 3.0g, glucose 2.5g, naCl 5.0g and K 2 HPO 4 2.5g, distilled water to 1000mL, pH=7.0-7.4, and sterilizing at 121deg.C for 20min.
Example 2
A biological agent for promoting the synthesis of truffle mycorrhiza is prepared by the preparation method of the embodiment 1.
Example 3
The application of the biological agent for promoting the synthesis of the truffle mycorrhiza is used for promoting the synthesis of the truffle mycorrhiza.
The specific operation steps are as follows
1. Screening and seedling raising of Chinese pine seeds
(1) Seed selection and disinfection
Taking healthy and full Huashan pine seeds within 1 year, sterilizing and accelerating germination, wherein 50% carbendazim 800 times of liquid is used for seed sterilization; soaking seeds in warm water at 50 ℃, naturally cooling, soaking for 24 hours, taking out and airing for standby.
(2) Seedling substrate
Mixing unsieved vermiculite and perlite at volume ratio of 1/1, adding water to water content of 30%, and sterilizing with high pressure steam at 121-126 deg.C for 1 hr.
(3) Sowing and seedling raising
And (3) sowing the seeds prepared in the step (1) into the matrix in the direction with the opening end facing downwards, wherein the sowing depth is 1.5-2cm. After sowing, the seedlings are covered with plastic films for moisture preservation and heat preservation so as to ensure that the seedlings are tidy. And selecting strong pine seedlings for standby after the pine seedlings grow to about 10cm in height.
2. Preparation of growth-promoting bacterial agent
Burkholderia (Burkholderia sediminicola) was selected as a growth-promoting bacterium. The culture was carried out in a TSB liquid medium (tryptone 17.0g, soytone 3.0g, glucose 2.5g,NaCl 5.0g,K2HPO42.5 g, distilled water to 1000mL, pH=7.0-7.4, sterilized at 121℃for 20 min) at 28℃for 3 days. Bacterial inoculum was prepared at a concentration of 2.4X109 cfu/mL for use.
3. Preparation of truffle microbial inoculum
Mature truffle produced in 12-2 months is selected 4 months each year, the species identity is determined through molecular identification and morphological observation, mature ascus fruit (the ratio of black brown spores to immature spores is 95:5, and is mature) is selected by using morphological data, the ascus fruit is washed 3 times by using sterile water, and the ascus fruit is frozen and preserved at the temperature of minus 24 ℃ for no more than 6 months for standby. Before the preparation of the microbial inoculum, frozen ascocarps are taken out, naturally thawed and cut into a plurality of pieces, the pieces are placed in a stirrer, crushed by adding sterile water, microscopic examination is carried out, 95% of ascocarps are determined to be free, 5% of spores are separated from the ascocarps, and the concentration of the microbial inoculum is 1.2X107 spores/strain inoculation concentration according to the method of the attached table 1.
4. Inoculation matrix preparation and pH adjustment
Humus (produced in needle-broad leaf miscibility forest, naturally decomposed outdoors for 2 years)/vermiculite/sand/perlite: 1/1/0.5/0.25, sterilizing humus for 3h, sterilizing sand for 1h, and sterilizing the rest components. Adding lime powder, mixing, and adjusting pH to 7.5.
5. Inoculation of
(1) According to step 3, 1.2X10 strains were inoculated 7 The amount of spores was calculated as the volume of each suspension of the truffle inoculum to be inoculated.
(2) According to step 4, a concentration of 2.4X10 is prepared 9 15mL of cfu/mL of growth-promoting bacterial agent.
(3) Adding the truffle bacterial agent and the growth promoting bacterial agent in the steps 5 (1) and 5 (2) into the substrate in the step 4, and fully and uniformly mixing.
(4) Taking out the seedlings in step 1 (3), removing root ends, and implanting 5 (3) into a matrix seedling bag mixed with the truffle and the growth promoting bacterial agent.
6. Cultivation and management of mycorrhizal seedlings
After the truffle mycorrhiza seedlings of the Chinese pine are cultivated for 5-6 months, the mycorrhiza seedlings are observed in a macroscopic and anatomical form, molecular biology is assisted for identification, and after the truffle mycorrhiza is confirmed to be the target truffle mycorrhiza, the mycorrhiza formation condition is calculated and counted, and the mycorrhiza formation rate reaches more than 90%, so that the transplanting can be realized.
Example 4
Mycorrhizal formation is a prerequisite for artificial cultivation of truffles. In general, mycorrhizal numbers have a clear positive correlation with yield. Compared with a control group inoculated with only truffle, the embodiment proves that the treatment of exogenously added mycorrhizal growth promoting bacteria can obviously promote the growth of root systems, plant heights and ground diameters of the Chinese pine seedlings; most importantly, the treatment of adding mycorrhizal growth-promoting bacteria obviously promotes the formation of mycorrhizal and increases the mycorrhizal formation rate of the white truffle infected Chinese mountain pine seedlings.
The addition of the microbial growth promoting agent is proved by taking the truffle (T.huidongense) as a column, and the growth of mycorrhizal seedlings, including plant height, ground diameter and root system development, can be greatly improved; greatly increases the mycorrhizal infection rate, thereby improving the quality of mycorrhizal seedlings and laying an important technology for realizing high yield by artificial planting.
Treatment group: the truffle inoculant is used for inoculating the pine seedlings of the mountain and simultaneously exogenously adding a growth promoting bacterium, namely, burkholderia (B.sediminicola).
CK group: the truffle inoculant is used for inoculating the Chinese pine seedlings, and no exogenous growth promoting bacteria are generated.
The results show that:
1. as shown in fig. 1-3, all the pinus huashanensis bacteria formed truffle roots by morphological identification.
2. Effect of added Burkholderia (B.sedimictola) on the formation of P.huashanensis seedlings and mycorrhiza
(1) As shown in FIG. 4, the formation of mycorrhizal of the Larix Gmelini-Lendong Tuber was significantly promoted
After adding burkholderia (B.sedimictola) as the growth promoting bacteria, the average number of mycorrhizas is 1205.4 per strain, and the number of mycorrhizas formed by the control CK is 719.2 per strain, so that compared with the CK, the addition of the microbial growth promoting bacteria obviously improves the mycorrhizal infection rate.
(2) As shown in FIG. 5, the plant height of the Huashansong is remarkably promoted
After adding burkholderia (B.sedimictola) as a growth promoting bacterium, the average value of the plant height of the Huashansong is 9.08 inches, the average value of the plant height of a control CK is 7.22 inches, and compared with the CK, the addition of the microbial growth promoting bacterium obviously improves the plant height of mycorrhizal seedlings.
(3) As shown in FIG. 6, the growth of the ground diameter of the Huashansong is remarkably promoted
After adding burkholderia (B.sedimictola) as a growth promoting bacterium, the average value of the ground diameter of the Huashansong is 3.97 mm, the average value of the ground diameter of a contrast CK is 2.56 mm, and compared with the CK, the addition of the microbial growth promoting bacterium obviously improves the growth of the ground diameter of the mycorrhizal seedlings.
The exogenous addition of the microbial agent greatly improves the growth of mycorrhizal seedlings, including plant height, ground diameter and root system development. The method has the advantages that the mycorrhiza infection rate is greatly increased, the mycorrhiza synthesis cost of the truffle is greatly reduced, the mycorrhiza synthesis time period is shortened, and the quality of the truffle mycorrhiza seedlings is improved. Optimizing the truffle mycorrhiza synthesis technology. The method is simple to operate, is convenient to popularize and apply, and provides a key technology with higher efficiency and economy for artificial cultivation of the truffle.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.

Claims (1)

1. The application of a biological agent for promoting the synthesis of the truffle mycorrhiza of the huashanensis is characterized in that the biological agent is used for promoting the synthesis of the truffle mycorrhiza of the huashanensis, and a burkholderia strain Burkholderia sediminicola is selected as a growth promoting bacterium;
the preparation method of the biological agent comprises the following steps:
selecting Burkholderia strain as growth promoting bacteria, placing in TSB liquid culture medium, and performing propagation culture at 28deg.C for 3 days to obtain a concentrated solution with concentration of 2.4X10 9 cfu/mL of bacterial inoculum;
the preparation method of the TSB liquid culture medium comprises the following steps:
tryptone 17.0g, soytone 3.0g, glucose 2.5g, naCl 5.0g and K 2 HPO 4 2.5g, distilled water to 1000mL, pH=7.0-7.4, and sterilizing at 121deg.C for 20min.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101328464A (en) * 2008-05-27 2008-12-24 中国科学院昆明植物研究所 Synthetic method of truffle and bacteriorhiza
CN111849815A (en) * 2020-07-21 2020-10-30 广西民族大学 Plant growth promoting rhizosphere strain Gxun-20 and application thereof in plant growth promotion
WO2021030057A1 (en) * 2019-08-09 2021-02-18 Carolina Truffiéres Llc Methods and compositions for the production of ectomycorrhizal mycelia and methods of use thereof
CN112625970A (en) * 2020-12-31 2021-04-09 华南农业大学 Burkholderia cepacia JT79 and application thereof

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101328464A (en) * 2008-05-27 2008-12-24 中国科学院昆明植物研究所 Synthetic method of truffle and bacteriorhiza
WO2021030057A1 (en) * 2019-08-09 2021-02-18 Carolina Truffiéres Llc Methods and compositions for the production of ectomycorrhizal mycelia and methods of use thereof
CN111849815A (en) * 2020-07-21 2020-10-30 广西民族大学 Plant growth promoting rhizosphere strain Gxun-20 and application thereof in plant growth promotion
CN112625970A (en) * 2020-12-31 2021-04-09 华南农业大学 Burkholderia cepacia JT79 and application thereof

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