Simple Stropharia rugoso-annulata spore collection method

Abstract

The invention discloses a simple method for collecting spores of Stropharia rugosoannulata, and belongs to the technical field of edible mushroom breeding and microbial separation. According to the method, plastic-coated white printing paper is cut into a 10 cm * 10 cm plastic sheet as a spore bearing carrier (denoted as A), and a kraft paper box and a plastic box (denoted as B) which are disinfected by 75% alcohol are matched to form a closed collecting device; sterilizing the surface of the cap of the stropharia rugoso-annulata after opening the umbrella, then inversely covering the cap A with the cap B, and standing for more than 8 hours to form a clear spore mark; then the spores are scraped aseptically in an ultra-clean workbench, the spores are inoculated to a PDA culture medium plate through a three-area line drawing method, and the germination condition is observed through constant-temperature culture at the temperature of 25 DEG C; the method is simple and convenient to operate, low in cost, low in pollution rate, capable of efficiently obtaining single spores and remarkably improving the separation success rate of the single spores, and suitable for scientific research and edible mushroom breeding practice.

Description

1. Technical Field

[0001] This invention relates to the field of edible fungi breeding and microbial isolation technology, specifically to a method for *Agaricus bisporus* (Stropharia masticata). Stropharia rugosoannulata A method for efficient and simple collection and separation of single spores. 2. Background Technology

[0002] *Agaricus bisporus* is an important edible and medicinal fungus with high economic and ecological value. In breeding work, obtaining homozygous single spores is a crucial prerequisite for genetic analysis, hybridization, and strain improvement. Traditional spore collection methods often use ordinary filter paper, glass slides, or aluminum foil as receiving materials, which suffer from problems such as poor spore adhesion, susceptibility to moisture, difficulty in aseptic scraping, and high contamination rates. Furthermore, existing methods often require complex equipment or lengthy cultivation periods, are cumbersome, and have low single-spore isolation efficiency.

[0003] While some improved methods have attempted to collect spores directly in sterile petri dishes, these methods are costly and result in uneven spore distribution, which is detrimental to subsequent streak separation. Therefore, there is an urgent need for a simple, low-cost method that minimizes the risk of contamination and efficiently collects individual germinating spores. 3. Summary of the Invention

[0004] The purpose of this invention is to provide a simple, efficient, and low-cost method for collecting spores of Pleurotus ostreatus, solving the problems of high spore collection contamination rate, poor adhesion, and difficulty in single spore isolation in the prior art, thereby improving the single spore germination rate and the success rate of pure culture.

[0005] This invention is achieved through the following technical solution: Use laminated white printing paper cut into 10 cm × 10 cm plastic sheets (A). The surface is smooth, waterproof, sterile and easy to handle, which is conducive to the uniform settling of spores and subsequent scraping. Using 75% alcohol to disinfect the caps and all contact materials effectively reduces external microbial contamination. Using a combination of kraft paper boxes and plastic boxes (B) as a covering container ensures a certain degree of air permeability while preventing external pollution; Let it stand for at least 8 hours to ensure full release of spores and formation of clear imprints; Spores were aseptically scraped in a clean bench and inoculated onto PDA plates using the three-zone streak method to effectively dilute the spore density and increase the probability of single colony isolation. Inverted culture at a constant temperature of 25℃ optimizes germination conditions and facilitates observation of single-spore germination.

[0006] Compared with the prior art, the present invention has the following advantages: Easy to operate: No special equipment required, materials are readily available, and the steps are clear; Low cost: Uses common office supplies (plasticized paper, cardboard boxes) instead of expensive sterile supplies; Low contamination rate: The entire process is disinfected with alcohol and operated in a clean bench, effectively controlling bacteria. Spores adhere firmly and are evenly distributed: the coated surface facilitates spore sedimentation and prevents them from falling off easily; High success rate of single-spore isolation: Combining the three-zone streak method significantly improves the efficiency of obtaining single colonies; Suitable for scientific research and production: It can be widely used in edible fungi breeding, preservation and genetic research. 4. Detailed Implementation

[0007] The present invention will be further described below with reference to the embodiments, but the present invention is not limited to the following embodiments. Example

[0008] Take ordinary A4 white printing paper, send it to a printing shop for single-sided lamination, and after cooling, cut it into 10 cm × 10 cm square pieces, labeled as carrier A; Prepare one square kraft paper box and one transparent plastic box with an inner diameter slightly larger than 10 cm. When combined, they can completely cover the carrier A. This box is called the covering container B. Collect naturally opened large cap mushroom fruiting bodies from the wild, selecting those with intact caps and no insect damage. Wipe the surface of the cap with a cotton ball soaked in 75% alcohol for 30 seconds and let it air dry. On a clean laboratory bench, wipe the front and back of carrier A and the inner wall of the covered container B with 75% alcohol, and let it air dry. Place the sterilized cap with gills facing down in the center of carrier A, quickly cover it with container B, and let it stand in a dark, windless place for 12 hours. Dark brown circular spore prints were visible on carrier A after 12 hours; Transfer carrier A, cap and B together to a clean bench, sterilize with ultraviolet light for 30 minutes, and then light an alcohol lamp. Using a flame-sterilized and cooled inoculation loop, gently scrape the edge area of ​​the spore print (the area with lower spore density) and inoculate it onto a PDA plate using the three-zone streak method; Seal the plates with sealing film and invert them in a 25°C constant temperature incubator; After 48 hours, a single spore can be seen germinating to form a tiny colony, and a pure single-spore strain can be obtained after 7 days.

[0009] After repeated experiments, the success rate of single-spore isolation was >85%, and the contamination rate was <5%.

Claims

1. A simple method for collecting Stropharia rugosoannulata spores, characterized by, The method comprises the following steps: (1) The white printing paper is subjected to plastic treatment and cut into a plastic sheet with a size of 10 cm x 10 cm, serving as a spore receiving carrier A; a size-matched kraft paper box and a plastic box are prepared and combined to form a covering container B; (2) A large fruiting body of the mushroom is collected, and the cap is removed and disinfected by wiping the surface of the cap with 75% alcohol; (3) The disinfected cap is placed upside down in the center of the carrier A disinfected by 75% alcohol, and the covering container B disinfected by 75% alcohol is placed on the cap to form a closed space; (4) The spores are allowed to naturally settle on the surface of the carrier A for more than 8 hours to form a spore print; (5) The cap and the covering container B are removed, and sterile operation is performed in a clean bench, and a sterilized inoculation loop is used to scrape the spores on the spore print; (6) The scraped spores are inoculated on a PDA solid culture medium plate by a three-zone division line method; (7) The inoculated PDA plate is sealed and inverted and placed in a 25℃ constant temperature incubator for culture, and the spore germination and single colony formation are observed.

2. The method of claim 1, wherein: The carrier A is a plasticized white printing paper, which is smooth, waterproof, easy for spore adhesion and convenient for subsequent sterile scraping.

3. The method of claim 1, wherein: The covering container B is composed of a kraft paper box and a plastic box, which has both air permeability and anti-pollution functions and is surface-disinfected by 75% alcohol.

4. The method of claim 1, wherein: The spore collection time is not less than 8 hours to ensure the formation of a clear and dense spore print.

5. The method of claim 1, wherein: The whole process of spore scraping and streaking is completed in a clean bench, and strict sterile operation is performed to avoid bacterial contamination.