Methods for achieving stable yield and improved quality in morel cultivation using violacein or violacein-producing microorganisms

Abstract

This invention relates to the field of biological seed production and breeding technology, specifically a method for achieving stable yield and improved quality in morel cultivation using violacein or violacein-producing microorganisms. The application involves first mixing violacein-producing microorganisms with morel mother cultures, primary cultures, or cultivars for large-scale cultivation, followed by artificial cultivation. Vibriolacein overcomes the problems of strong soil microbial activity and morel strain aging associated with sowing morels at higher temperatures, raising the sowing temperature from below 20°C in the current model to below 30°C. In the Central Plains region, sowing is moved forward from the current late October period to late August to early September, thus avoiding the impact of abnormal weather during spring fruiting in the current model. This achieves stable production of yellow morels through artificial cultivation and black morels, improving the antioxidant, antitumor, and antibacterial activities of morel products, and contributing to the sustainable and stable development of the morel industry.

Description

Technical Field

[0001] This invention belongs to the field of biological seed production and breeding technology, and specifically relates to a method for achieving stable yield and improved quality of morel cultivation using violacein or violacein-producing microorganisms. Background Technology

[0002] Currently, the outdoor "soil cultivation model" for morel mushrooms widely promoted and applied in my country mainly uses domesticated strains of the black morel lineage, including *Morchella importuna*, *Morchella sextileta*, and *Morchella eximia*. Patent 202111206603.3 discloses a method for the propagation and stable-yield cultivation of black-veined morel spawn. Compared with traditional morel tissue isolation breeding, simple spore breeding, and existing cultivation management techniques, this technology can increase production efficiency by 3-5 times, solving a series of problems in morel spawn production, such as unstable fruiting, low yield, and high costs, which are dependent on weather and seasonal cultivation. Morel cultivation is generally carried out during the low-temperature season when the maximum temperature is below 20℃ (late October to late November in the Central Plains region). After sowing, after nearly a winter of mycelial growth management, the fruiting management stage begins when the minimum temperature stabilizes above 4℃ from late February to early March of the following year. Due to the frequent occurrence of abnormal weather in spring in recent years (such as late spring cold snaps, hot and dry winds, temperature fluctuations, and early arrival of high temperatures), the production of morel mushrooms using traditional cultivation methods has become unstable. Every year, over 70% of production struggles to generate stable profits, resulting in significant economic losses for mushroom farmers. However, if sowing can be done about two months earlier, for example, during the season when the highest temperature is below 30℃ (late August to early September in the Central Plains region), production can be completed before the onset of winter, thus avoiding the adverse effects of abnormal weather during the spring fruiting season and improving the stability of morel mushroom cultivation. The main reasons why morel mushrooms cannot be sown earlier in the current production model are twofold: firstly, at high temperatures, the activity of other microorganisms (miscellaneous bacteria) in the soil is strong, antagonizing the growth of morel mycelium and reducing the mycelial biomass in the soil; secondly, at high temperatures, morel strains are prone to aging, manifested as weakened mycelial vitality, fewer sclerotia produced, less nutrient storage in the soil mycelial network, fewer mushrooms, and poorer mushroom quality. Patent 202310509446.6 discloses a microbial agent for correcting the continuous cropping obstacles in morel mushroom cultivation. It can improve the soil nutrient supply, enhance soil water retention, aeration, and mineral element content, thereby improving soil structure, further alleviating the continuous cropping obstacles in morel mushroom cultivation, promoting the dissolution and release of insoluble nutrients in the soil, effectively breaking up soil compaction, promoting the formation of aggregate structure, and converting ineffective fertilizers fixed in the soil into effective fertilizers. It improves the nutrient supply, aeration, and looseness of the soil, which can promote the growth of morel mushrooms, reduce the incidence of pests and diseases, enhance the stress resistance of morel mushrooms, and improve their quality. Summary of the Invention

[0003] This invention proposes a method for achieving stable yield and improved quality of morel cultivation using violacein or violacein-producing microorganisms. It solves the problems of unstable production and difficulty in profit for mushroom farmers in the existing "winter sowing and spring fruiting" artificial cultivation model of morel, and provides a brand-new scheme for morel spawn production and strain selection.

[0004] The technical solution of this invention is implemented as follows:

[0005] On the one hand, this invention proposes the application of violacein or violacein-producing microorganisms in morel cultivation, including any one of the following applications:

[0006] (1) Increase the cultivation temperature to 30℃;

[0007] (2) Achieve stable production of yellow morel mushrooms and black morel mushrooms;

[0008] (3) Enhance the antioxidant, antitumor and antibacterial activities of morel mushroom products.

[0009] Preferably, the above application is achieved by mixing violacein or violacein-producing microorganisms with morel mother cultures, primary cultures, or cultivated cultures.

[0010] On the other hand, the present invention proposes a method for achieving stable yield and improved quality of morel cultivation using violacein or violacein-producing microorganisms. First, violacein or violacein-producing microorganisms are mixed with morel mother culture, original culture or cultivation culture and then cultured on a large scale, and then artificially cultivated.

[0011] Preferably, the microorganisms that produce violacein are violacein-producing bacteria or engineered bacteria.

[0012] Preferably, the microorganism producing violetin is a deep blue-violet bacillus with the preservation number ATCC 12473.

[0013] Preferably, the ratio of the above-mentioned violacein-producing microorganisms to the morel mother culture, primary culture, or cultivated culture is 1 × 10⁶ violacein-producing bacteria per 500g of wet morel culture. 6 -1×10 12 indivual.

[0014] Furthermore, the preparation method of the above-mentioned engineered bacteria producing violacein is as follows:

[0015] (1) Construct a recombinant vector for the purple bacitracin synthesis gene cluster;

[0016] (2) The recombinant vector from step (1) was transferred into morel mycelial cells, and after verification, an engineered bacterium producing violacein was obtained.

[0017] Preferably, in step (1) above, the violacein synthesis gene cluster is vioABCDE, which is composed of five genes: vioA, vioB, vioC, vioD and vioE.

[0018] Preferably, the recombinant vector in step (1) above is a eukaryotic expression vector, such as the pCAMBIA series plasmids, such as pCAMBIA1301, pCAMBIA1302, pCAMBIA1303 and pCAMBIA1304.

[0019] Preferably, in step (2) above, the recombinant vector is transferred into morel mycelial cells using any one of the following methods: polyethylene glycol-mediated protoplast transformation, electroporation transformation, transposon method, gene gun method, restriction enzyme-mediated method, and Agrobacterium-mediated method.

[0020] The present invention has the following beneficial effects:

[0021] 1. The method of achieving stable yield and improved quality of morel cultivation using violacein or violacein-producing microorganisms is a conventional technique in the field of biology, which is easy to operate and implement. By applying it to sowing at higher temperatures, the artificial cultivation of yellow morel mushrooms and the stable production of black morel mushrooms have been successfully achieved, resulting in morel products with stronger health benefits. Ultimately, the dual goals of stable production and improved quality of artificially cultivated morel mushrooms are achieved, and the implementation effect is significant.

[0022] 2. This invention utilizes violacein or violacein-producing microorganisms to achieve the artificial cultivation of *Morchella tessella*, offering the following advantages: *Morchella tessella* mixed with violacein achieves winter fruiting, yielding approximately 1.0 kg of fresh mushrooms per square meter of planting area; the methanol extract of *Morchella tessella* mixed with violacein exhibits a half-maximal inhibitory concentration (MIC) of 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) radical scavenging at 146.50 μg / mL, 3.85 times that of the control morel; the minimum inhibitory concentrations (MICs) against *Pseudomonas aeruginosa* and *Staphylococcus aureus* are 1200 and 400 μg / mL, respectively; and the half-maximal inhibitory concentration (EC50) against the mold *Alternaria brassicae* is... 50 The concentration was 200 μg / ml; the inhibition rates against the growth of cervical cancer cells (HeLa), lung cancer cells (A549), breast cancer cells (MCF-7), and liver cancer cells (HepG2) were 61.6%, 64.5%, 35.43%, and 58.64%, respectively.

[0023] 3. This invention utilizes violacein or violacein-producing microorganisms to achieve the artificial cultivation of *Morchella esculenta*, offering the following advantages: *Morchella esculenta* mixed with violacein achieves winter fruiting, with approximately 0.15 kg of fresh mushrooms harvested per square meter of planting area; the methanol extract of *Morchella esculenta* mixed with violacein exhibits a half-maximal DPPH radical scavenging concentration of 86.47 μg / mL, which is 5.29 times that of the control morel; the MICs against *Pseudomonas aeruginosa* and *Staphylococcus aureus* are 800 and 300 μg / mL, respectively; and the EC50 against the mold *Alternaria brassicae*... 50 The concentration was 200 μg / ml; the inhibition rates against the growth of cervical cancer cells (HeLa), lung cancer cells (A549), breast cancer cells (MCF-7), and liver cancer cells (HepG2) were 69.81%, 68.62%, 43.78%, and 66.72%, respectively.

[0024] 4. This invention utilizes an engineered morel strain producing violacein to achieve the artificial cultivation of *Morchella esculenta*, which has the following advantages: the engineered morel strain enables winter fruiting, with approximately 0.2 kg of fresh mushrooms harvested per square meter of planting bed; the methanol extract of the engineered morel strain has a half-maximal DPPH radical scavenging concentration of 489.70 μg / mL; the MICs against *Pseudomonas aeruginosa* and *Staphylococcus aureus* are 1500 and 700 μg / mL, respectively; and the EC50 against the mold *Alternaria brassicae* has a high DPPH radical scavenging concentration. 50 The concentration was 500 μg / ml; the inhibition rates against the growth of cervical cancer cells (HeLa), lung cancer cells (A549), breast cancer cells (MCF-7), and liver cancer cells (HepG2) were 28.71%, 29.54%, 17.47%, and 18.50%, respectively. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 The images show the growth of *Bacillus violaceus* on CYM plates at 25°C for 5 days; the left image shows colony characteristics, and the right image shows cell morphology.

[0027] Figure 2 These are photos of the fruiting process of black morel cultivars; the left photo shows the fruiting process of black morel cultivars mixed with purple bacillus, and the right photo shows the fruiting process of the control black morel cultivars.

[0028] Figure 3These are photos of the fruiting process of the mother culture of *Morchella esculenta*; the left photo shows the fruiting process of *Morchella esculenta* mixed with *Porphyromonas purpureus*, and the right photo shows the fruiting process of the control *Morchella esculenta*.

[0029] Figure 4 The images show the fruiting process of the genetically engineered morel strain that produces violacein; the left image shows the fruiting process of the genetically engineered morel strain that produces violacein, and the right image shows the fruiting process of the control morel strain. Detailed Implementation

[0030] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0031] Unless otherwise specified, the experimental methods used in the following experimental examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0032] Violacein is a non-water-soluble secondary metabolite obtained by microorganisms through the oxidative condensation of tryptophan molecules as a precursor. Its chemical name is 3-[2-hydroxy-5-(5-hydroxy-1H-indole-3-yl)-1Hpyrrole]. Violacein-producing bacteria are mainly Gram-negative bacteria, including but not limited to *Chromobacterium* spp., *Janthinobacterium* spp., *Pseudoalteromonas* spp., and *Iodobacter* spp., which can be screened from environmental samples such as marine, glacier, river, and soil samples. The gene cluster (vioABCDE) controlling violacein synthesis consists of five genes: vioA, vioB, vioC, vioD, and vioE. Engineered strains expressing violacein can be constructed using genetic engineering and other techniques.

[0033] In this application, the bacteria that produce violacein refers to a solid or liquid culture of the target bacteria, or a mixture of culture and protectant. It can be obtained by directly harvesting the culture after solid culture, or by directly harvesting the culture after liquid fermentation culture, or by adding an adsorbent to the liquid fermentation culture, or by centrifugation, adding a freeze-drying protectant, freeze-drying, pulverizing and packaging after liquid fermentation culture, or by adding a protectant to the liquid fermentation culture and then spray-drying at low temperature.

[0034] Morel mushrooms in this application include black morel lineages such as *Morchella esculenta*, *Morchella septemlobus*, *Morchella septemlobus*, *Morchella septemlobus*, *Morchella septemlobus*, *Morchella septemlobus*, *Morchella septemlobus*, *Morchella septemlobus*, *Morchella septemlobus*, *Morchella septemlobus*, *Morchella septemlobus*, *Morchella septemlobus*, *Morchella septemlobus*, etc., as well as red morel lineages such as *Morchella septemlobus*. Morel mother culture refers to mycelial culture obtained through tissue isolation, multispore isolation, single-spore isolation, etc. Morel primary culture refers to bottled or bagged spawn obtained from the mother culture. Morel cultivar refers to bottled or bagged spawn obtained from the primary culture.

[0035] Example 1

[0036] The method for achieving stable yield and improved quality of black morel cultivation using bacteria that produce violacein in this application is as follows:

[0037] 1. Activation culture of morel mushrooms and violetin-producing bacteria: *Morchella tessmannii* (strain preservation number: CCTCC AF 2021045) was selected as the starting strain for morel mushrooms, and *Janthinobacterium lividum* (strain preservation number: ATCC 12473) was selected as the starting strain for violetin-producing bacteria. *Janthinobacterium lividum* produced violetin colonies after 5 days of culture on CYM plates at 25℃. Figure 1 Left) Bacterial morphological characteristics as follows Figure 1 The image on the right shows a rod-shaped culture. Both morel mushroom and bacterial activation cultures were performed on complete culture medium (CYM) plates. The CYM medium formula was: 2g peptone, 3g yeast extract, 0.5g magnesium sulfate, 0.45g potassium dihydrogen phosphate, 1g dipotassium hydrogen phosphate, 20g glucose, 20g agar, and 1000mL deionized water, sterilized at 121℃ for 25min. The starting morel mushroom strain was inoculated onto CYM plates using an inoculation hook, while the starting bacterial strain was streaked onto CYM plates using an inoculation loop. The plates were incubated upside down in the dark at 25℃ for 3-4 days. Morel mycelium filled the plates, while the bacteria showed significant mycelial growth.

[0038] 2. Morel spawn expansion culture: Morel spawn, spawn, and nutrient bag substrate formula (by weight percentage): wheat 60%, corn cob or broadleaf tree wood chips 39%, gypsum 1%, moisture content 60%. Morel mother spawn is inoculated with the original spawn (750mL original spawn bottle), and the original spawn is transferred to the spawn (15cm×30cm polypropylene plastic bag). Both the original spawn and the spawn are cultured at 25℃ in the dark until the mycelium fully occupies the culture medium.

[0039] 3. Fermentation culture of *Porphyromonas violaceus*: Bacterial expansion was carried out using CYM liquid medium (agar-free) in shake flasks. 200 mL of liquid medium was added to each 500 mL Erlenmeyer flask after sterilization, and three inoculation loops of bacterial growth were inoculated. The flasks were incubated at 25°C with shaking at 150 rpm for 4 days, until the culture became noticeably turbid. The bacterial count was determined using the pour plate method, and the bacterial culture was diluted with sterile physiological saline to a concentration of 1 × 10⁻⁶ bacteria. 6 per mL.

[0040] 4. Mixing bacteria with morel mushroom spawn: Crush the morel mushroom spawn into small pieces the size of peanuts, and mix 30 mL of bacterial culture (3 × 10⁻⁶) with every 500 g of wet morel mushroom spawn. 7 Mix the bacterial culture with the morel mushroom spawn at a ratio of 1:1 (each) and stir thoroughly.

[0041] 5. Morel spawn mixed with violacein is used for artificial cultivation:

[0042] (1) Site selection: Plan and select land 1-2 months in advance before sowing. Select arable land with a soil thickness of more than 20cm, sandy soil, and soil pH of around 7.0.

[0043] (2) Erecting a shed: Erect a shed with a top height of 2.5m and a shoulder height of 1.0m (build the frame first).

[0044] (3) Land preparation: Clear away debris, spread quicklime at a rate of 300 jin per mu (each jin equals 500 grams, the same below), rotary till to a depth of 20-30 cm, and water.

[0045] (4) Sowing: Shading nets (90% shading rate) are hung on the greenhouse frame in advance. Sowing takes place on September 2nd, when the average monthly temperature is 25℃, the highest temperature is below 30℃, and the soil temperature is around 25℃. The soil should be clump-like when squeezed in the hand but crumble easily when dropped, with a moisture content of approximately 26%. Sowing is done by broadcasting, using approximately 300 catties of seeds per mu (approximately 0.067 hectares). After sowing, cover with soil to ensure most of the inoculum is covered. Then, dig furrows to form raised beds, 1.2m wide, with furrows 30cm wide and 10cm deep. Finally, cover the ground with shading nets (90% shading rate).

[0046] (5) Spreading management: After sowing, cover the greenhouse roof with shade netting to prevent temperatures above 30℃; prevent rain erosion, and prevent animals from trampling and grazing on the soil. If the seedbed is dry and lacks water, spray water as needed to keep the soil moist.

[0047] (6) Placement of external nutrient bags: Use 15cm×30cm polypropylene plastic bags to pack the nutrient bag materials, sterilize and let cool before use. Place the external nutrient bags 7 days after sowing, with a quantity of about 5000 catties per mu. Remove the shade net before placing. Nutrient bag opening method: Make a three-sided ring cut with a knife, and pull the uncut strips to one side by hand to fully expose the nutrient material. The cut should be 3-5cm wide and 10-15cm long. Quickly place the cut material surface on the bed surface and press it slightly by hand so that the mycelium in the soil can quickly contact the nutrient material. After setting up one shed, immediately cover the bed surface with the shade net again.

[0048] (7) Mushroom Induction and Management: After about 50 days of mycelial growth, induction of mushrooms is carried out in late October. At this time, the nutrient bags become noticeably lighter, the substrate inside the bags begins to turn bluish-brown, the mycelium on the bed surface is yellowish-brown, and morel primordia appear in the moist soil clods. The greenhouse roof is covered with a thin film, and then a shade net is placed on top. Before the induction operation, the greenhouse film is opened to allow the bed surface to be stimulated by low temperature for about 10 days. The day before watering for induction, the shade net covering the ground is removed from the greenhouse, and the ventilation openings on both sides of the greenhouse are opened for natural ventilation for 24 hours. Then, water is sprayed by mist spraying, watering thoroughly to a depth of 20cm in the soil surface. After 4 pm, the greenhouse door is closed, leaving a small number of ventilation openings (10-20cm wide). After induction, a small arched shed is built on the bed surface, covered with white plastic film, and perforated with a diameter of 4cm and a spacing of 30-40cm. After inducing mushroom growth, maintain a balance of temperature, light, air, and humidity within the greenhouse. Keep the temperature between 10-15℃, near-ground humidity between 80%-90%, and ensure even and bright diffused light and fresh air. In windy weather, close the side ventilation openings slightly; in rainy weather, open all side ventilation openings; in case of a significant drop in temperature, take measures to maintain warmth, such as closing the greenhouse doors, reducing ventilation, decreasing watering, and increasing covering; in hot weather, open the ventilation openings and spray water into the greenhouse for short periods, frequently but sparingly, or use roof spraying to cool the area. Do not spray water directly onto the primordia or small mushrooms; combine water spraying with ventilation.

[0049] (8) Harvesting and processing: Harvest morel mushrooms when the ascocarps stop growing, the cap ridges and pits are clearly defined, the ridges are smooth, and the flesh is thick and elastic. After harvesting, they can be sun-dried or oven-dried. Dried products should be sealed in plastic bags for storage, while fresh mushrooms should be stored at low temperatures.

[0050] 6. Results of artificial cultivation: The control group of *Morchella tessella*, which did not contain bacteria producing violacein, did not produce fruiting mushrooms. Figure 2 (Right), while the morel mushrooms mixed with violacein achieved winter fruiting ( Figure 2 (Left) Approximately 1.0 kg of fresh mushrooms are harvested per square meter of raised bed, and the morel mushrooms have thick ascocarps, rich aroma, and are of superior quality.

[0051] 7. Bioactivity of Morel Mushrooms: Using the same variety of morel mushrooms produced under conventional methods as a control, the antioxidant, antibacterial, antifungal, and antitumor activities of morel mushrooms mixed with violacein were determined. After drying, the morel mushroom samples were pulverized using a grinder and passed through an 80-mesh sieve. 5.0 g of the sample powder was weighed and placed in an Erlenmeyer flask. Methanol solution was added at a solid-liquid ratio of 1:5, and the mixture was ultrasonically extracted for 30 min. The extraction was then filtered, and the above steps were repeated twice. The filtrates from the three extractions were combined and vacuum dried to obtain the morel mushroom methanol extract. 0.1 g of the methanol extract was weighed and diluted to a 10 mL volumetric flask with methanol as a stock solution for later use.

[0052] The ability of the methanol extract to scavenge 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) free radicals was determined. It was found that the half-maximal scavenging concentration (IC50) of the mixed violacein morel was 146.50 μg / mL, while that of the control morel was 563.72 μg / mL. This means that the DPPH free radical scavenging ability of the morel produced by the mixed bacteria was 3.85 times that of the control morel.

[0053] The antibacterial activity of morel extracts was determined by microdilution. The results showed that the methanol extract of morel mixed with violacein had a minimum inhibitory concentration (MIC) of 1200 μg / ml against Pseudomonas aeruginosa and 400 μg / ml against Staphylococcus aureus, while the control morel extract showed no antibacterial activity.

[0054] The antifungal activity of morel extracts was determined using the mycelial growth rate method. The results showed that the methanol extract of morels mixed with violacein had a half-maximal effective concentration (EC50) against the mold Alternaria brassicae. 50 The concentration of the active ingredient was 200 μg / ml, while the methanol extract of the control morel mushroom showed no antifungal activity.

[0055] The antitumor activity of 200 μg / ml morel methanol extract was determined by the MTT assay. The results showed that the methanol extract of morel mixed with violacein inhibited the growth of cervical cancer cells (HeLa), lung cancer cells (A549), breast cancer cells (MCF-7), and liver cancer cells (HepG2) by 61.6%, 64.5%, 35.43%, and 58.64%, respectively, while the control morel methanol extract showed inhibition rates of 12.68%, 13.24%, 4.66%, and 9.87%, respectively. This indicates that the morel produced by this method has stronger antitumor activity than conventionally produced morel products.

[0056] Example 2

[0057] The method for achieving stable yield and improved quality of yellow morel cultivation using bacteria that produce violacein in this application is as follows:

[0058] 1. Activation culture of morel mushrooms and violetin-producing bacteria: *Morchella esculenta* (strain preservation number: ATCC 10968) was selected as the starting strain for the morel mushrooms, and *Janthinobacterium lividum* (strain preservation number: ATCC 12473) was selected as the starting strain for the violetin-producing bacteria. The morel mushroom starting strain was inoculated onto CYM plates in a wavy pattern, and the bacterial starting strain was streaked onto the CYM plates. The plates were incubated at 25°C inverted in the dark until the morel mycelium completely covered the plate and obvious bacterial growth was observed.

[0059] 2. Morel mushroom and bacterial antagonistic culture: Inoculate a block of activated morel mushroom mycelium (4 mm in diameter, with a small amount of culture medium) on one side of a CYM plate. Then, streak purple bacteria 1 cm wide in an area 2 cm away from the outer edge of the morel mushroom mycelium block. After inoculation, incubate the plate upside down at 25°C for 1 week. Take morel mushroom cultures that have grown beyond the bacterial streak area and transfer them to a CYM slant. After the tube has filled, it becomes a mother culture of morel mushrooms mixed with violacein-producing bacteria.

[0060] 3. Morel spawn expansion culture: Morel spawn, spawn, and nutrient bag substrate formula (by weight percentage): wheat 60%, corn cob or broadleaf tree wood chips 39%, gypsum 1%, moisture content 60%. Morel mother spawn is inoculated with the original spawn (750mL original spawn bottle), and the original spawn is transferred to the spawn (15cm×30cm polypropylene plastic bag). Both the original spawn and the spawn are cultured at 25℃ in the dark until the mycelium fully occupies the culture medium.

[0061] 4. Morel mushrooms mixed with violacein are used for artificial cultivation:

[0062] (1) Site selection: Plan and select land 1-2 months in advance before sowing. Select arable land with a soil thickness of more than 20cm, sandy soil, and soil pH of around 7.0.

[0063] (2) Erecting a shed: Erect a shed with a top height of 2.5m and a shoulder height of 1.0m (build the frame first).

[0064] (3) Land preparation: Clear away debris, spread quicklime at a depth of 300 catties per mu, rotary till at a depth of 20-30 cm, and water.

[0065] (4) Sowing: Shading nets (90% shading rate) are hung on the greenhouse frame in advance. Sowing takes place on September 2nd, when the average monthly temperature is 25℃, the highest temperature is below 30℃, and the soil temperature is around 25℃. The soil should be clump-like when squeezed in the hand but crumble easily when dropped, with a moisture content of approximately 26%. Sowing is done by broadcasting, using approximately 300 catties of seeds per mu (approximately 0.067 hectares). After sowing, cover with soil to ensure most of the inoculum is covered. Then, dig furrows to form raised beds, 1.2m wide, with furrows 30cm wide and 10cm deep. Finally, cover the ground with shading nets (90% shading rate).

[0066] (5) Spreading management: After sowing, cover the greenhouse roof with shade netting to prevent temperatures above 30℃; prevent rain erosion, and prevent animals from trampling and grazing on the soil. If the seedbed is dry and lacks water, spray water as needed to keep the soil moist.

[0067] (6) Placement of external nutrient bags: Use 15cm×30cm polypropylene plastic bags to pack the nutrient bag materials, sterilize and let cool before use. Place the external nutrient bags 7 days after sowing, with a quantity of about 5000 catties per mu. Remove the shade net before placing. Nutrient bag opening method: Make a three-sided ring cut with a knife, and pull the uncut strips to one side by hand to fully expose the nutrient material. The cut should be 3-5cm wide and 10-15cm long. Quickly place the cut material surface on the bed surface and press it slightly by hand so that the mycelium in the soil can quickly contact the nutrient material. After setting up one shed, immediately cover the bed surface with the shade net again.

[0068] (7) Mushroom Induction and Management: After about 50 days of mycelial growth, induction of mushrooms is carried out in late October. At this time, the nutrient bags become noticeably lighter, the substrate inside the bags begins to turn bluish-brown, the mycelium on the bed surface is yellowish-brown, and morel primordia appear in the moist soil clods. The greenhouse roof is covered with a thin film, and then a shade net is placed on top. Before the induction operation, the greenhouse film is opened to allow the bed surface to be stimulated by low temperature for about 10 days. The day before watering for induction, the shade net covering the ground is removed from the greenhouse, and the ventilation openings on both sides of the greenhouse are opened for natural ventilation for 24 hours. Then, water is sprayed by mist spraying, watering thoroughly to a depth of 20cm in the soil surface. After 4 pm, the greenhouse door is closed, leaving a small number of ventilation openings (10-20cm wide). After induction, a small arched shed is built on the bed surface, covered with white plastic film, and perforated with a diameter of 4cm and a spacing of 30-40cm. After inducing mushroom growth, maintain a balance of temperature, light, air, and humidity within the greenhouse. Keep the temperature between 10-15℃, near-ground humidity between 80%-90%, and ensure even and bright diffused light and fresh air. In windy weather, close the side ventilation openings slightly; in rainy weather, open all side ventilation openings; in case of a significant drop in temperature, take measures to maintain warmth, such as closing the greenhouse doors, reducing ventilation, decreasing watering, and increasing covering; in hot weather, open the ventilation openings and spray water into the greenhouse for short periods, frequently but sparingly, or use roof spraying to cool the area. Do not spray water directly onto the primordia or small mushrooms; combine water spraying with ventilation.

[0069] (8) Harvesting and processing: Harvest morel mushrooms when the ascocarps stop growing, the cap ridges and pits are clearly defined, the ridges are smooth, and the flesh is thick and elastic. After harvesting, they can be sun-dried or oven-dried. Dried products should be sealed in plastic bags for storage, while fresh mushrooms should be stored at low temperatures.

[0070] 5. Results of artificial cultivation: The control group of yellow morel mushrooms, which did not contain bacteria that produce violacein, did not produce fruiting mushrooms. Figure 3 (Right), while the yellow morel mushrooms mixed with purple bacillus achieved winter fruiting ( Figure 3 (Left) Approximately 0.15 kg of fresh mushrooms are harvested per square meter of raised bed, and the morel mushrooms have a rich aroma and are of excellent quality.

[0071] 6. Bioactivity of Morel Mushrooms: Using wild-caught yellow morel mushrooms as a control, the DPPH free radical scavenging ability of yellow morel mushrooms cultivated with mixed bacitracin was determined. The half-maximal concentration (IC50) of the mixed bacitracin-treated morel mushrooms was 86.47 μg / mL, while that of the control morel mushrooms was 457.63 μg / mL, meaning the DPPH free radical scavenging ability of the mixed bacitracin-treated morel mushrooms was 5.29 times that of the control morel mushrooms. The antibacterial activity of the methanol extract of morel mushrooms was determined using the microdilution method. The MICs of the methanol extract of yellow morel mushrooms cultivated with mixed bacitracin against *Pseudomonas aeruginosa* and *Staphylococcus aureus* were 800 and 300 μg / mL, respectively, while the control morel mushroom extract showed no antibacterial activity. The antifungal activity of the methanol extract of morel mushrooms was determined using the mycelial growth rate method. The EC50 of the methanol extract of yellow morel mushrooms cultivated with mixed bacitracin against the mold *Alternaria brassicae* was... 50 The concentration of 150 μg / mL was higher than that of the control morel extract, which showed no antifungal activity. The antitumor activity of the 200 μg / mL morel methanol extract was determined using the MTT assay. The results showed that the methanol extract of the mixed violacein-treated yellow morel inhibited the growth of cervical cancer cells (HeLa), lung cancer cells (A549), breast cancer cells (MCF-7), and liver cancer cells (HepG2) by 69.81%, 68.62%, 43.78%, and 66.72%, respectively, while the control morel methanol extract showed inhibition rates of 22.91%, 21.33%, 9.87%, and 16.62%, respectively. This indicates that the yellow morel produced with the mixed violacein has stronger antitumor activity than conventionally produced morel products.

[0072] Example 3

[0073] The method for achieving stable yield and improved quality of black morel cultivation using a genetically engineered strain of morel mushroom that produces violacein in this application is as follows:

[0074] 1. Culture of donor and recipient strains for genetic engineering: *Morchella tessmannii* (strain preservation number: CCTCCAF 2021045) was selected as the recipient strain for genetic engineering, and *Janthinobacterium lividum* (strain preservation number: ATCC 12473) was selected as the donor strain. A piece of the *Morchella tessmannii* starting strain was inoculated onto a CYM plate. The bacterial starting strain was then streaked onto the CYM plate in a wavy pattern and incubated at 25°C inverted in the dark until the *Morchella tessmannii* mycelium completely covered the plate and obvious bacterial growth was observed.

[0075] 2. Obtaining the Viocin Synthesis Gene Cluster: The viocin synthesis gene cluster was obtained from the genome of *Viocinobacter violaceus* using a specific primer amplification method. First, genomic DNA of *Viocinobacter violaceus* was extracted using a kit. Second, using the *Viocinobacter violaceus* genomic DNA as a template, the VioA, VioB, VioC, VioD, and VioE gene fragments were amplified using the primers shown in Table 1. The PCR amplification system was as follows: 5 μL of 10×Taq buffer (magnesium-free), 5 μL of 25 mM magnesium chloride, 2.5 μL of dimethyl sulfoxide, 4 μL of 2.5 mM dNTPs, 4 μL of forward primer, 4 μL of reverse primer, 1 μL of Taq DNA polymerase, 1 μL of template DNA, and sterile double dehydrator to a final volume of 50 μL. The PCR amplification reaction parameters were as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 64℃ annealing for 45 s, 72℃ extension for 1 min, 12 cycles, with a decrease of 0.5℃ per cycle; 94℃ pre-denaturation for 30 s; 61℃ annealing for 45 s, 72℃ extension for 1 min, 25 cycles; and a final extension at 72℃ for 30 min.

[0076] Table 1 Primers used to amplify the VioABCDE gene cluster sequence.

[0077]

[0078] 3. Construction of Recombinant Expression Vector: The expression vector was constructed based on the commercial vector pCAMBIA1302 plasmid. This expression vector contains the eukaryotic reporter gene hygromycin resistance (HygR) and the prokaryotic reporter gene kanamycin resistance, which can be used to screen for positive transformants in *Morchella esculenta* and *Agrobacterium tumefaciens*, respectively. The histone H2B expression sequence, lacking a stop codon, was inserted into the multiple cloning site of the vector. The five violacein synthesis gene fragments amplified in step 2 were sequentially ligated into the expression vector. The histone expression sequence was linked upstream of the *violacein* synthesis gene cluster sequence. The constructed recombinant fusion protein expression sequence has a nuclear localization signal and is used to express the recombinant fusion protein. The first and second promoters, the *Morchella esculenta* glyceraldehyde-3-phosphate dehydrogenase gene promoter (GPD) and the *Morchella esculenta* ubiquitin gene promoter (Ubiquitin-pro), respectively, replaced the original plasmid's CaMV35S promoter and were placed upstream of the expression gene; the *Aspergillus nidulans* tryptophan C terminator (TtrpC) was placed downstream of the expression gene.

[0079] 4. Introduction of recombinant vector into morel mycelial cells: An expression vector containing a violacein synthesis gene cluster was constructed and transformed into the recipient strain of *Morchella tessmannii* using an Agrobacterium-mediated transformation method. Positive transformants were initially screened for hygromycin resistance, and the transformation was further verified by PCR. The results showed that the transformation rate after the first screening was 7.5%. Ten positive transformants were randomly selected and cultured for five consecutive generations. Morel transformants that retained the hygromycin resistance trait were considered genetically stable transformants.

[0080] 5. Verification of violacein production by genetically engineered morel strains: Five genetically stable morel strains were randomly selected, and morel mycelia were obtained by cellophane covering plate method. After drying, the mycelia were pulverized through a 100-mesh sieve to obtain mycelial powder. Vibriolacein was extracted from the mycelia using methanol extraction method (solid-to-liquid ratio 1:5), and the content of violacein in the extract was determined by spectrophotometry (the extinction coefficient of violacein is 0.05601 mL / μg·cm). It was found that the violacein content of the morel strain mycelia was approximately 0.025% (w / w).

[0081] 6. Artificial Cultivation of Morel Mushroom Genetically Engineered Strains Producing Violacrimal: First, the strain is expanded. The substrate formula (by weight percentage) for the morel spawn, culture spawn, and nutrient bags is: wheat 60%, corn cob or broadleaf tree wood chips 39%, gypsum 1%, and moisture content 60%. The mother culture is inoculated with the mother culture (750mL spawn bottle), and the mother culture is then transferred to the culture spawn (15cm×30cm polypropylene plastic bag). Both the mother culture and the culture spawn are cultured at 25℃ in the dark until the mycelium fully colonizes the substrate. The artificial cultivation process of the morel mushroom genetically engineered strain producing violacrimal is as follows:

[0082] (1) Site selection: Plan and select land 1-2 months in advance before sowing. Select arable land with a soil thickness of more than 20cm, sandy soil, and soil pH of around 7.0.

[0083] (2) Erecting a shed: Erect a shed with a top height of 2.5m and a shoulder height of 1.0m (build the frame first).

[0084] (3) Land preparation: Clear away debris, spread quicklime at a depth of 300 catties per mu, rotary till at a depth of 20-30 cm, and water.

[0085] (4) Sowing: Shading nets (90% shading rate) are hung on the greenhouse frame in advance. Sowing takes place on September 2nd, when the average monthly temperature is 25℃, the highest temperature is below 30℃, and the soil temperature is around 25℃. The soil should be clump-like when squeezed in the hand but crumble easily when dropped, with a moisture content of approximately 26%. Sowing is done by broadcasting, using approximately 300 catties of seeds per mu (approximately 0.067 hectares). After sowing, cover with soil to ensure most of the inoculum is covered. Then, dig furrows to form raised beds, 1.2m wide, with furrows 30cm wide and 10cm deep. Finally, cover the ground with shading nets (90% shading rate).

[0086] (5) Spreading management: After sowing, cover the greenhouse roof with shade netting to prevent temperatures above 30℃; prevent rain erosion, and prevent animals from trampling and grazing on the soil. If the seedbed is dry and lacks water, spray water as needed to keep the soil moist.

[0087] (6) Placement of external nutrient bags: Use 15cm×30cm polypropylene plastic bags to pack the nutrient bag materials, sterilize and let cool before use. Place the external nutrient bags 7 days after sowing, with a quantity of about 5000 catties per mu. Remove the shade net before placing. Nutrient bag opening method: Make a three-sided ring cut with a knife, and pull the uncut strips to one side by hand to fully expose the nutrient material. The cut should be 3-5cm wide and 10-15cm long. Quickly place the cut material surface on the bed surface and press it slightly by hand so that the mycelium in the soil can quickly contact the nutrient material. After setting up one shed, immediately cover the bed surface with the shade net again.

[0088] (7) Mushroom Induction and Management: After about 50 days of mycelial growth, induction of mushrooms is carried out in late October. At this time, the nutrient bags become noticeably lighter, the substrate inside the bags begins to turn bluish-brown, the mycelium on the bed surface is yellowish-brown, and morel primordia appear in the moist soil clods. The greenhouse roof is covered with a thin film, and then a shade net is placed on top. Before the induction operation, the greenhouse film is opened to allow the bed surface to be stimulated by low temperature for about 10 days. The day before watering for induction, the shade net covering the ground is removed from the greenhouse, and the ventilation openings on both sides of the greenhouse are opened for natural ventilation for 24 hours. Then, water is sprayed by mist spraying, watering thoroughly to a depth of 20cm in the soil surface. After 4 pm, the greenhouse door is closed, leaving a small number of ventilation openings (10-20cm wide). After induction, a small arched shed is built on the bed surface, covered with white plastic film, and perforated with a diameter of 4cm and a spacing of 30-40cm. After inducing mushroom growth, maintain a balance of temperature, light, air, and humidity within the greenhouse. Keep the temperature between 10-15℃, near-ground humidity between 80%-90%, and ensure even and bright diffused light and fresh air. In windy weather, close the side ventilation openings slightly; in rainy weather, open all side ventilation openings; in case of a significant drop in temperature, take measures to maintain warmth, such as closing the greenhouse doors, reducing ventilation, decreasing watering, and increasing covering; in hot weather, open the ventilation openings and spray water into the greenhouse for short periods, frequently but sparingly, or use roof spraying to cool the area. Do not spray water directly onto the primordia or small mushrooms; combine water spraying with ventilation.

[0089] (8) Harvesting and processing: Harvest morel mushrooms when the ascocarps stop growing, the cap ridges and pits are clearly defined, the ridges are smooth, and the flesh is thick and elastic. After harvesting, they can be sun-dried or oven-dried. Dried products should be sealed in plastic bags for storage, while fresh mushrooms should be stored at low temperatures.

[0090] 7. Artificial cultivation effect: The non-genetically engineered control strain did not produce fruit ( Figure 4 (Right), while the morel mushroom genetically engineered strain successfully achieved winter fruiting ( Figure 4 (Left) Approximately 0.2 kg of fresh mushrooms are harvested per square meter of raised bed.

[0091] 8. Bioactivity of Morel Mushrooms: Using the same variety of morel mushrooms produced under conventional methods as a control, the antioxidant, antibacterial, antifungal, and antitumor activities of morel mushrooms artificially cultivated by genetically engineered strains were determined. The results showed that the half-maximal scavenging concentration (MCC) of morel mushrooms cultivated by the genetically engineered strain against DPPH free radicals was 489.70 μg / mL, while that of the control morel mushroom was 563.72 μg / mL, indicating that the DPPH free radical scavenging ability of the genetically engineered strain was stronger than that of the control morel mushroom. The methanol extract of morel mushrooms cultivated by the genetically engineered strain showed minimum inhibitory concentrations (MICs) of 1500 and 700 μg / mL against *Pseudomonas aeruginosa* and *Staphylococcus aureus*, respectively, indicating certain antibacterial activity, while the extract of the control morel mushroom showed no antibacterial activity. The half-maximal effective concentration (EC50) of the methanol extract of morel mushrooms cultivated by the genetically engineered strain against the fungus *Alternaria brassicae* was...50 The methanol extract of morel mushrooms produced by the genetically engineered strain at 500 μg / ml showed certain antifungal activity, while the methanol extract of the control morel mushrooms showed no antifungal activity. The antitumor activity of the methanol extract of morel mushrooms at 200 μg / ml was determined using the MTT assay. The results showed that the methanol extract of morel mushrooms produced by the genetically engineered strain inhibited the growth of cervical cancer cells (HeLa), lung cancer cells (A549), breast cancer cells (MCF-7), and liver cancer cells (HepG2) by 28.71%, 29.54%, 17.47%, and 18.50%, respectively, while the control morel mushroom methanol extract showed inhibition rates of 12.68%, 13.24%, 4.66%, and 9.87%, respectively. This indicates that morel mushrooms produced by the genetically engineered strain of *Morchella tessmannii* have stronger antitumor activity than conventionally produced morel mushrooms.

[0092] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Use of a microorganism producing a pyorubin for cultivation of a Morel, characterized in that, The application comprises any of the following applications: (1) increasing the cultivation temperature to 30℃; (2) realizing stable production of yellow and black Morel; (3) improving the antioxidant, antitumor and antibacterial activities of Morel products; The purple-pigmented bacillomycin-producing microorganism is Chromobacterium violaceum with the accession number of ATCC 12473.

2. A method for realizing stable yield and quality improvement of Morel cultivation by using a microorganism producing violacein, characterized in that: The purple-pigmented bacillomycin-producing microorganism is mixed with Morel cultivation species for expansion culture, and then for artificial cultivation. The purple-pigmented bacillomycin-producing microorganism is Chromobacterium violaceum with the accession number of ATCC 12473.

3. The method for realizing stable yield and quality improvement of Morel cultivation by using the microorganism producing violacein according to claim 2, characterized in that: The ratio of the purple nitrile producing microorganism mixed with the Morchella cultivation species is 1 x 10 6 -1x10 12 microorganisms per 500 g of wet Morchella cultivation species.

4. The method for realizing stable yield and quality improvement of Morel cultivation by using the microorganism producing violacein according to claim 3, characterized in that: The purple-pigmented bacillomycin-producing microorganism is a purple-pigmented bacillomycin-producing bacterium or an engineered bacterium.

5. The method for realizing stable yield and quality improvement of Morel cultivation by using the microorganism producing violacein according to claim 4, characterized in that, The preparation method of the engineered bacterium is: (1) constructing a recombinant vector of a purple-pigmented bacillomycin synthesis gene cluster; (2) transferring the recombinant vector of step (1) into Morel mycelium cells, and obtaining the engineered bacterium producing purple-pigmented bacillomycin after verification.

6. The method for realizing stable yield and quality improvement of Morel cultivation by using the microorganism producing violacein according to claim 5, characterized in that: The gene cluster for the synthesis of violacein in step (1) is vioABCDE consisting of vioA , vioB , vioC , vioD and vioE the five genes.

7. The method for realizing stable yield and quality improvement of Morel cultivation by using iocin-producing microorganisms according to claim 6, characterized in that: The recombinant vector in step (1) is a eukaryotic expression vector.

8. The method for realizing stable yield and quality improvement of Morel cultivation by using the microorganism producing violacein according to claim 7, characterized in that: In step (2), the recombinant vector is transferred into Morel mycelium cells by using any one of the following methods: polyethylene glycol-mediated protoplast transformation, electric transformation, transposon method, gene gun method, restriction enzyme-mediated method and Agrobacterium-mediated method.

Images