Sporeless mutant of mycena basidiacola and application thereof

Abstract

This invention discloses a spore-deficient strain of *Pleurotus ostreatus* (also known as *Pleurotus ostreatus*). Agrocybe salicacicola T32, with accession number GDMCC NO:63850 at the Guangdong Provincial Center for Microbial Culture Collection; this fungus is a delicious edible fungus, obtained through basidiospore self-pollination, and has the advantage of not producing spores; using this strain for protoplast monokaryosis, the resulting regenerated monokaryotic strain can create a series of spore-deficient strains; this invention provides breeding parent material and breeding method for the creation of spore-free strains of *Pleurotus ostreatus*, and is applicable to the breeding of spore-free strains of *Pleurotus ostreatus* and the development and research of basidiospore development.

Description

Technical Field

[0001] This invention relates to a spore-deficient strain of *Pleurotus ostreatus* (also known as *Pleurotus ostreatus*). Agrocybe salicacicola The T32 strain and its applications belong to the field of microbial technology. Background Technology

[0002] Yangliu Tiantou Mushroom ( Agrocybe salicacicola *Agrocybe aegerita* is a delicious edible fungus species distributed in northwestern Yunnan Province. It is a similar species to *Agrocybe aegerita* and has been successfully cultivated through artificial domestication. During the cultivation of edible fungi, the fruiting bodies produce a large number of basidiospores. The large amount of spores released into the air causes respiratory diseases such as "mushroom lung" in mushroom harvesters. Furthermore, escaped spores reduce the diversity of similar fungi in the surrounding environment, attract spore-feeding pests, and degrade the quality of fruiting bodies. Therefore, cultivating non-basidiospore-producing varieties has become an important direction in edible fungus breeding. Summary of the Invention

[0003] This invention provides a spore-deficient strain of *Pleurotus ostreatus* (also known as *Pleurotus ostreatus*). Agrocybe salicacicola T32 was deposited on September 28, 2023, at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC NO:63850. The deposit address is: Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0004] Another objective of this invention is to apply the above-mentioned spore-deficient *Pleurotus ostreatus* T32 to the breeding of spore-free *Pleurotus ostreatus*, thereby obtaining *Pleurotus ostreatus* with basidiospore-deficient spores and solving the problem caused by spore dispersion.

[0005] The present invention achieves the above objectives using the following technical solution: 1. Collect the fruiting bodies of *Pleurotus ostreatus* from the decaying branches of willow trees in Dali City, Yunnan Province. Using the tissue isolation method, break the fruiting bodies longitudinally, pick out the tissue block at the junction of the stipe and cap, inoculate it onto YPD medium, and incubate it at 25℃. After white mycelium grows, further transfer and purification culture is carried out to obtain purified mycelium. 2. When the purified mycelium has grown to the edge of the YPD medium, pick out a mycelium block and inoculate it into a spawn bag containing culture medium for mycelial culture and fruiting. 3. Take mature fruiting bodies and collect basidiospores from the fruiting bodies to make a spore suspension. Spread the spore suspension onto YPD medium for culture. Pick single colonies and transfer them to YPD medium for culture. Pick mycelia and observe them under a microscope. Collect monokaryotic strains without clamping structures. 4. The monokaryotic strains were subjected to confrontation culture. The mature fruiting bodies produced by the monokaryotic strain T32 after hybridization with two complementary monokaryotic strains from the same batch had white gills. Microscopic observation showed that the hymenium did not produce basidiospores.

[0006] The monokaryotic strain T32 has white mycelium, neat colony edges, well-developed aerial hyphae with numerous branches, and no clamping structures. Based on the morphological characteristics and molecular analysis of the fruiting bodies obtained by hybridization with a mating-type complementary monokaryotic strain, this strain was ultimately identified as *Pleurotus ostreatus*. Agrocybe salicacicola ).

[0007] Another objective of this invention is to apply the aforementioned monokaryotic strain T32 to the breeding of basidiospore-deficient *Pleurotus ostreatus*. After hybridization of monokaryotic strain T32 with wild-type monokaryotic strains, approximately 50% of the progeny monospore-producing strains are non-basidiospore-producing defective strains. By using this monokaryotic strain to hybridize with other wild-type monokaryotic strains, isolate spores, and perform hybrid (backcross) hybridization, a large number of basidiospore-deficient strains can be created.

[0008] The beneficial effects of this invention are as follows: This invention, *Phyllostachys edulis* (also known as *Phyllostachys edulis*), is a type of mushroom grown in the field. Agrocybe salicacicola The T32 strain was obtained from wild *Pleurotus ostreatus* strains through basidiospore isolation and culture. Experiments have shown that after hybridization with wild-type monokaryotic strains, more than 50% of the progeny strains (monokinetic strains) obtained from this strain are spore-deficient strains. Among these strains, the fruiting bodies produced by the complementary mating strains do not produce basidiospores. Using this strain, spore-deficient strain lines were created, effectively increasing the breeding materials of *Pleurotus ostreatus*. This lays the foundation for the breeding of spore-free strains of *Pleurotus ostreatus* and the utilization and development of genes related to spore development. Attached Figure Description

[0009] Figure 1 This is a colony morphology diagram of the T32 strain of the present invention on YPD medium; Figure 2 Hyphae morphology of wild-type heterokine strain (Figure A) and T32 strain (Figure B); Figure 3 The images show agarose gel electrophoresis results of DNA extraction from strain T32 (Figure A) and amplified PCR products (Figure B) of this invention. Figure 4 Phylogenetic tree of strain T32 of this invention; Figure 5 Morphological diagram of fruiting bodies produced by backcrossing of strain T32 of this invention; Figure 6 Microscopic morphological diagram of the fruiting body produced by crossbreeding the wild-type strain and the spore-deleting strain of this invention. Detailed Implementation

[0010] The present invention will be further described in detail below with reference to embodiments and accompanying drawings. However, the scope of protection of the present invention is not limited to the contents described. Unless otherwise specified, the methods in the embodiments shall be carried out in accordance with conventional operations. Unless otherwise specified, the reagents used shall be conventionally purchased reagents or reagents prepared in accordance with conventional methods.

[0011] The reagents required for the experiment are: YPD medium (containing 0.2% yeast extract, 0.2% peptone, 2% glucose, and 2% agar), DNA extraction kit (purchased from Biomiga), and PCR reagents (purchased from Nanjing Novizan). Fruiting body culture medium: 98% cottonseed hulls, 1% gypsum, 1% quicklime, moisture content 60%; Example 1: Isolation and Identification of Strain T32 1. Collection and isolation of wild *Pleurotus ostreatus* dikaryotic strains The wild strain was collected from Dali City, Yunnan Province. The fruiting bodies grew on the decaying branches of willow trees. After the fruiting bodies were removed, the tissue isolation method was used to break the fruiting bodies longitudinally and pick out the tissue block at the junction of the stipe and the cap. The tissue block was inoculated onto YPD medium and cultured at 25°C. After white mycelium grew, it was further purified by transfer using YPD medium to obtain purified mycelium. 2. Fruiting body culture When the purified mycelium reaches the edge of the YPD medium, pick out a 1cm diameter mycelium block and inoculate it onto the fruiting medium. Inoculate 3 blocks per bag and culture at 25℃ in the dark until the mycelium fills the bag and yellow water appears. Then open the bag and transfer it to a greenhouse with 90% humidity and 22℃ for fruiting culture under 12 hours of light / 12 hours of darkness.

[0012] 3. Isolation and Identification of Single-Spore Strains After 45 days of cultivation, mature fruiting bodies were harvested, the stipes were removed, and the caps were placed on sterile tracing paper to collect basidiospores. The spores on the tracing paper were scraped onto sterile water using a sterile inoculation loop, mixed well, and then the spore solution was spread onto YPD medium. After culturing in an incubator at 25°C for 5 days, single colonies were picked with an inoculation needle and transferred to YPD medium for further cultivation. The mycelium was picked and observed under a microscope. Strains with clamp connections were heterokine strains, while those without were monokine strains. 53 monokine strains without clamp connections were collected. 4. Mating and fruiting body characteristics of single-spore strain T32 Monokaryotic strains were subjected to confrontation culture. Two 1cm diameter mycelial blocks from different monokaryotic strains were selected and transferred to YPD medium, spaced 4cm apart, and cultured at 25℃. When the mycelia of both strains grew together, mycelia were examined under a microscope at the junction. The presence of clamp structures indicated mating. The hybrid mycelia were used for fruiting culture. The mature fruiting bodies produced by the monokaryotic strain T32, after hybridization with two complementary monokaryotic strains from the same batch, exhibited white gills, and microscopic observation showed no basidiospores produced in the hymenium. Therefore, strain T32 was selected as the starting strain for subsequent hybridization verification.

[0013] 5. Mycelial morphology characteristics of strain T32 The T32 strain was inoculated onto YPD medium and cultured at 25°C for one week. Its mycelial characteristics were observed; the mycelium was white, the colony edges were neat, and aerial hyphae were well-developed. Figure 1 ), with many branches and no locking mechanism ( Figure 2 B) Figure 2 A represents the control wild-type heterokine strain, which exhibits clamp-like binding structures.

[0014] 6. Molecular identification of strain T32 The T32 strain was inoculated onto PDA medium and cultured at 25°C for 7 days. A suitable amount of mycelium was picked, and DNA was extracted using a DNA extraction kit (purchased from Biomiga). 2 µL of the extracted DNA was electrophoresed on a 1% agarose gel to determine its quality and concentration. Figure 3 A).

[0015] Using DNA as a template, PCR amplification was performed using primers ITS1 (5-TCCGTAGGTGAACCTGCGC-3) and ITS4 (5-TCCTCCGCTTATTGATATGC-3). The PCR reaction system (25µL) consisted of: 12.5µL of 2×PCRmix (Nanjing Novizan Biotech), 1µL each of 10µmol / L ITS1 and ITS4 primers, 1µL of DNA template, and 9.5µL of ddH2O.

[0016] The PCR amplification program was as follows: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 54℃ annealing for 30 s, 72℃ extension for 60 s, 30 cycles; 72℃ extension for 10 min. The PCR products were sent to Yunnan Qingke Biotechnology Co., Ltd. for sequencing. Figure 3 B), perform BLAST in the NCBI database, download sequences with high sequence similarity, and construct a hierarchical cluster tree (B). Figure 4 Based on the morphological characteristics and molecular analysis of the fruiting bodies obtained by hybridization with mating-type complementary mononuclear bodies, T32 was identified as *Pleurotus ostreatus*. Agrocybe salicacicola ).

[0017] Example 2: Constructing a basidiospore-deleting heterokine strain using strain T32 1. Crossbreeding of T32 strain with wild-type strain 1 cm diameter mycelial blocks of strain T32 and wild-type mononuclear strain were selected and inoculated onto YPD medium for confrontation culture at 25°C. When the mycelia of the confrontation cultured strains grew together and produced confrontation binding lines, the mycelia at the binding lines were picked for microscopic observation. The presence of clamp-like binding structures indicated that the strains were mating complementary strains.

[0018] 2. Isolation and backcrossing of progeny strains Mycelia at the junction of the mating complementary strain were inoculated onto the fruiting body culture medium. After colonization, the mycelia were transferred to a 25°C, dark environment for cultivation. When the mycelia had fully colonized the bag and yellow water appeared on the surface of the mycelia, the bag was opened and the bag was transferred to a greenhouse at 22°C and 90% humidity for 12 hours of light / 12 hours of darkness for fruiting cultivation. Mature fruiting bodies were collected, basidiospores were collected, and a spore suspension was prepared and spread on YPD plates. Single colonies were picked and cultured, and the clamp connection structure was observed under a microscope. Monokaryotic strains without clamp connection structures were selected. The obtained monokaryotic strains were then confronted with the T32 strain to produce clamp-structured mycelia for fruiting analysis.

[0019] 3. Identification of fruiting populations of crossbred strains Seventy-nine strains exhibiting clamp structures during confrontation culture were transferred to fruiting medium and cultured. The resulting open-cap fruiting bodies were observed under a microscope. For microscopic observation, mature fruiting bodies were taken, and 5-6 gills were longitudinally cut along the gills with a clean blade. The gills were placed in freshly prepared Carnoy's fixative (anhydrous ethanol and glacial acetic acid mixed evenly in a volume ratio of 3:1) for 5 hours, then transferred to 35% ethanol for 15 minutes, and rinsed with deionized water for 15 minutes. The fruiting bodies were then hydrolyzed in 1 mol / L HCl solution for 6-8 minutes, rinsed with deionized water for 15 minutes, and then soaked in pH 7 phosphate buffer (0.68 g potassium dihydrogen phosphate added to 29.1 mL of 0.1 mol / L sodium hydroxide solution, diluted with water to 100 mL) for 10 minutes. The fruiting bodies were then stained with Gimza stain (1.5 g Gimza powder dissolved in 50 mL of glycerol, then mixed with 50 mL of neutral methanol) for 2 hours. The fruiting bodies were then covered with coverslips and observed under a microscope to observe the production of basidiospores. The results showed that 37 paired strains produced basidiospores ( Figure 5 A / B / C), the remaining 42 plants (53.17%) did not produce basidiospores ( Figure 5 D / E / F), microscopic observation showed that basidiospores were present in the hymenium of the wild-type strain ( Figure 6 A), non-basidiospore-producing strains lack basidiophores and spores in the hymenium ( Figure 6 B).

Claims

1. A spore-deficient strain of *Pleurotus ostreatus* ( Agrocybe salicacicola T32, whose accession number at the Guangdong Provincial Center for Microbial Culture Collection is GDMCC NO:63850.

2. The application of the spore-deleted *Pleurotus ostreatus* T32 as described in claim 1 in the breeding of sporeless *Pleurotus ostreatus*.

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