CN113265367B

CN113265367B - Culture medium for rapidly obtaining Morchella conidia and preparation method thereof

Info

Publication number: CN113265367B
Application number: CN202110536915.4A
Authority: CN
Inventors: 赵琪; 吕梦岚; 杨祝良; 木立恒; 巴鸿; 黄伟
Original assignee: Kunming Institute of Botany of CAS
Current assignee: Kunming Institute of Botany of CAS
Priority date: 2021-05-17
Filing date: 2021-05-17
Publication date: 2023-05-26
Anticipated expiration: 2041-05-17
Also published as: CN113265367A

Abstract

The invention provides a culture medium for rapidly obtaining Morchella conidium and a preparation method thereof. Taking Morchella (Morchella spp.) mother strain obtained by single spore, multiple spores or tissue separation, activating on a common PDA or CYM culture medium to obtain mother strain, and inoculating Morchella into the following formula by a conventional aseptic operation method: culturing in a medium comprising 0-20% of carbon source, 0-20% of nitrogen source, 0.1-2% of trace elements, 12-25% of agar and 5-8% of ph at 5-25deg.C for 14-30 days in dark place, and preserving at 2-8deg.C for use after macroscopic detection or microscopic detection of the culture result.

Description

Culture medium for rapidly obtaining Morchella conidia and preparation method thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a method for obtaining asexual spores by asepsis culture of edible fungi, which comprises the steps of medium proportion and culture conditions. More particularly, relates to a preparation method for rapidly obtaining Morchella conidia.

Background

Morchella spp is a world famous edible and medicinal fungus with the effects of resisting tumor, resisting oxidization, resisting bacteria, reducing blood fat, improving immunity and the like. In recent years, with the pursuit of green health by people, morchella is promoted by more people, the demand is increasingly increased, the number of wild morchella is drastically reduced, and artificial cultivation of morchella is increasingly becoming a research focus. In 2010, the popularization and spread of independent nutrition adding technology has led to the development of Morchella cultivation technology. However, nearly 70% of cultivars are still unable to stably gain annually. The main reasons are lack of basic biological researches such as morchella species sex, life history, nutrition metabolism, physiology and the like.

Conidia (conidia) is the most common type of asexual spores in hyphae-bearing fungi, the asexual propagation of most and all fungi of the phylum ascomycotina, mainly by wind, water and animal transmission. The characteristics of spore morphology, structure, size, color, arrangement and the like are different from species to species, and are important basis for strain identification. Part of the conidium can be used for breeding, preservation, inoculation expansion and the like.

The earliest report of Morchella conidia was in artificial cultivation experiments tried in Morlliard 1904, which found that a large amount of white mold could be formed on the surface of the inoculated soil layer. In 1982, a large number of conidia were also reported in the artificial morchella cultivation experiments of the lower, and the germination rate of the conidia on potato culture medium was indicated to be less than 1%. In 2005, masaphy described for the first time the asexual spore morphology during cultivation of morchella. Commercial cultivation of Morchella shows that: the production of conidia (commonly referred to as "fungus cream") has an important indicative effect on fruiting. Conidia are very easy to generate in the field cultivation process of Morchella esculenta (figure 4), but no report that Morchella esculenta mother strain stably and rapidly obtains conidia under other sterile conditions except Qi Peng and the like (2021) which adopt sterilized humus soil culture medium to obtain a small amount of conidia is available at present.

Disclosure of Invention

The invention aims to provide a pure culture method of Morchella conidia, aiming at the defects in the prior art. The method can obtain the conidium of the morchella under the aseptic condition, solves the problem that the conidium is difficult to obtain by pure culture of the morchella, and provides a standard for fine variety breeding of the morchella. Meanwhile, the culture medium for rapidly obtaining the Morchella conidium is provided, the limitations of difficult and easy pollution of the existing pure culture of Morchella are broken through, and a scientific method is provided for breeding improved Morchella varieties.

The above object of the present invention is achieved by the following technical solutions:

the preparation method comprises the steps of preparing a special culture medium, inoculating, culturing, detecting and the like, wherein Morchella mother seeds obtained by single-spore, multi-spore or tissue separation are activated on a common PDA or CYM culture medium to be mother seeds, and then the mother seeds are inoculated to a formula by conventional aseptic operation: culturing in a medium comprising 0-20% of carbon source, 0-20% of nitrogen source, 0.1-2% of trace elements, 12-25% of agar and 5-8% of ph at 5-25deg.C for 14-30 days in dark place, and preserving at 2-8deg.C for use after macroscopic detection or microscopic detection of the culture result.

According to the preparation method for rapidly obtaining Morchella conidium, the preparation method of the special culture medium comprises the following steps: 0-20% of carbon source, 0-10% of nitrogen source, 0.5-2% of trace elements, 12-25% of agar, 1000ml of water, pH of 5-8 and 121-126 ℃ for 15-90min, and preparing the culture medium by the conventional mother culture preparation method.

According to the preparation method for rapidly obtaining Morchella conid, the culture step is to culture for 14-30 days at 5-25 ℃ after inoculation until mycelia completely grow on a culture dish or a test tube.

According to the preparation method for rapidly obtaining Morchella conidium, the carbon source is one or a mixture of at least two of wheat, wood dust, cotton seed hulls, glucose, sucrose and fructose.

According to the preparation method for rapidly obtaining Morchella conid, the nitrogen source is one or a mixture of at least two of yeast powder, peptone, beef extract and soybean powder.

According to the preparation method for rapidly obtaining Morchella conid, the trace elements are one or a mixture of at least two of magnesium sulfate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate and potassium carbonate.

The invention also provides a special culture medium for rapidly obtaining Morchella conidium, which comprises the following components in percentage by weight: 0-20% of carbon source, 0-10% of nitrogen source, 0.5-2% of trace elements, 12-25% of agar and 1000ml of water, wherein the pH value is 5-8.

A special culture medium for rapidly obtaining Morchella conidia, wherein the special culture medium is prepared by the following formula and method: 0-20% of carbon source, 0-10% of nitrogen source, 0.5-2% of trace elements, 12-25% of agar, 1000ml of water, pH of 5-8 and 121-126 ℃ for 15-90min, and preparing the culture medium by the conventional mother culture preparation method.

The invention provides a pure culture method of Morchella conidia, which is quick, low in cost, simple in procedure, high in repeatability and easy to observe, and simultaneously provides a culture medium for quickly obtaining Morchella conidia. The method can obtain the conidium of the morchella under the aseptic condition, solves the technical problem that the conidium is difficult to obtain by pure culture of the morchella in the prior art, and provides a standard for breeding improved variety of the morchella. Breaks through the limitations of difficult pure culture and easy pollution of the existing Morchella, and provides a scientific and strong-operability method for breeding improved Morchella varieties.

Drawings

FIG. 1 is a conidium of Morchella esculenta strain 13;

FIG. 2 shows conidia of Morchella conica 301 strain;

FIG. 3 shows conidia of Morchella esculenta 202 strain;

FIG. 4 shows Morchella conidia (commonly referred to as "fungus cream").

Detailed Description

The present invention will be further described with reference to the accompanying drawings, which are given by way of examples only, and are not intended to limit the scope of the invention.

Example 1:

the invention discloses a culture medium for rapidly obtaining Morchella conidium and a preparation method thereof, comprising the following steps: taking common Morchella esculenta 13 strain in the market as a material in 6 months and 10 days in 2020, adopting a common PDA culture medium to activate and then taking the strain as a mother strain, transferring the strain on a culture medium containing 20% of glucose, 5% of yeast powder, 0.1% of potassium carbonate, 0.3% of magnesium sulfate, 20% of agar and pH7 in 16 days in 6 months, and culturing the strain in the dark at 20 ℃ for 13 days to obtain the conidium shown in figure 1.

Example 2:

the invention discloses a culture medium for rapidly obtaining Morchella conidium and a preparation method thereof, comprising the following steps: and (3) taking common Morchella conica 301 strain in the market as a material in the period of 10 months in 2020, adopting a common CYM culture medium to activate the Morchella conica 301 strain to serve as a mother strain, transferring the Morchella conica 301 strain into a culture medium containing 10% of sucrose, 10% of glucose, 2% of yeast powder, 3% of peptone, 0.2% of potassium carbonate, 0.3% of magnesium sulfate, 18% of agar and pH7 in the period of 15 days, and culturing the Morchella conica in a dark place at 15 ℃ for 17 days to obtain the conidium shown in figure 2.

Example 3:

the invention discloses a culture medium for rapidly obtaining Morchella conidium and a preparation method thereof, comprising the following steps: and (3) taking common Morchella esculenta 202 strain in the market as a material in 10 months and 9 days in 2020, adopting a common PDA (personal digital assistant) culture medium to activate and then taking the strain as a mother strain, transferring the strain on a culture medium containing 8% of wheat, 2% of yeast powder, 0.1% of monopotassium phosphate, 0.1% of magnesium sulfate, 18% of agar and pH7 in 14 days in 10 months, and culturing the strain in the dark at 25 ℃ to obtain the conidium shown in figure 3.

Claims

1. The preparation method comprises the steps of preparing a special culture medium, inoculating, culturing and detecting, wherein Morchella strains obtained by single spore, multiple spores or tissue separation are activated on a common PDA or CYM culture medium to form mother seeds, and then the mother seeds are inoculated to a formula by conventional aseptic operation: 20% glucose, 5% yeast powder, 0.1% potassium carbonate, 0.3% magnesium sulfate, 20% agar, 1000ml water and pH7, culturing in dark at 15-25deg.C for 14-30 days, and preserving at 2-8deg.C for use after macroscopic or microscopic detection of the culture result.

2. The preparation method comprises the steps of preparing a special culture medium, inoculating, culturing and detecting, wherein Morchella strains obtained by single spore, multiple spores or tissue separation are activated on a common PDA or CYM culture medium to form mother seeds, and then the mother seeds are inoculated to a formula by conventional aseptic operation: 10% of sucrose, 10% of glucose, 2% of yeast powder, 3% of peptone, 0.2% of potassium carbonate, 0.3% of magnesium sulfate, 18% of agar, 1000ml of water and pH7 are cultured for 14-30 days in a dark place at 15-25 ℃, and after visual inspection or microscopic inspection of the culture result, the culture is preserved at 2-8 ℃ for later use.

3. The preparation method comprises the steps of preparing a special culture medium, inoculating, culturing and detecting, wherein Morchella strains obtained by single spore, multiple spores or tissue separation are activated on a common PDA or CYM culture medium to form mother seeds, and then the mother seeds are inoculated to a formula by conventional aseptic operation: 8% of wheat, 2% of yeast powder, 0.1% of monopotassium phosphate, 0.1% of magnesium sulfate, 18% of agar, 1000ml of water and pH7 are cultured for 14-30 days at 15-25 ℃ in a dark place, and after the culture result is detected by naked eyes or microscopy, the culture is preserved at 2-8 ℃ for later use.