A system for producing fruiting bodies of Blackwellomyces pseudomilitaris

Abstract

A system for the production of fruiting bodies of Blackwellomyces pseudomilitaris, comprising: an isolation unit configured to isolate pure fungal cultures from collected samples, the isolation unit comprising sterilization equipment for treating samples with 0.1% HgCl 2 and a device for washing with sterile water; a solid media cultivation unit configured to cultivate mycelial tissue on semi-solid media selected from the group consisting of PDA, SDAY, PDA with chicken egg yolk, and PDA with MgSO4, the solid media cultivation unit comprising incubation chambers in which a temperature of 19-25°C and a humidity of 80% are maintained; a liquid media cultivation unit configured to cultivate mycelium in liquid media selected from the group consisting of PD broth, SDY broth, and broth-I, the liquid media cultivation unit comprising a rotary shaker operating at 145 rpm; a lighting system configured to provide white light with an intensity of 300 lux for 8-9 hours; and a molecular identification unit configured to perform ITS primer-based identification and phylogenetic analysis using a computing device.

Description

FIELD OF THE INVENTION

[0001] The present disclosure relates to a system for producing fruiting bodies of Blackwellomyces pseudomilitaris. More specifically, the present invention relates to a system that facilitates the cultivation of mycelial tissue of B. pseudomilitaris in solid and liquid media and its growth into fruiting bodies. BACKGROUND OF THE INVENTION

[0002] Cordyceps militaris, a medicinal mushroom with antioxidant, anti-inflammatory, and anti-cancer properties, dominates the commercial market for entomopathogenic fungi. However, the Cordyceps industry relies primarily on two species, although the genus includes over 500 known species. Blackwellomyces pseudomilitaris, which is morphologically similar to Cordyceps militaris and contains similar bioactive compounds, including cordycepin, represents a promising alternative species with significant medicinal potential.

[0003] Existing cultivation systems face critical limitations: Entomopathogenic fungi are rare and difficult to obtain from natural habitats, species identification is problematic, and current isolation protocols are not universally applicable to Cordyceps species. While previous work has successfully isolated various Cordyceps species to the mycelial stage, few systems allow cultivation to fruiting bodies, which is essential for commercial viability. Available cultivation protocols are lengthy and complex, limiting industrial scalability.

[0004] B. pseudomilitaris, first discovered in Thailand and recently detected in India, produces characteristic red pigments in culture media and shares important morphological characteristics with the commercially valuable C. militaris. However, there is no integrated system for the efficient isolation, cultivation, and production of B. pseudomilitaris fruiting bodies using standardized media and controlled conditions.

[0005] There is a need for a comprehensive system that enables reliable isolation and cultivation of B. pseudomilitaris from wild specimens through fruiting body production, provides a cost-effective alternative to existing Cordyceps cultivation systems, and expands commercial opportunities in the medicinal mushroom market. Summary of the invention

[0006] The present disclosure relates to a system for producing fruiting bodies of Blackwellomyces pseudomilitaris. The present invention relates to a comprehensive system for producing fruiting bodies of B. pseudomilitaris, an entomopathogenic fungus with medicinal properties similar to those of Cordyceps militaris. The system integrates units for isolation, cultivation, identification, and analysis to enable complete production from wild collection to fruiting body development. Both solid and liquid media cultivation methods are used under controlled environmental conditions.

[0007] The present disclosure aims to provide a system for producing fruiting bodies of Blackwellomyces pseudomilitaris. The system comprises: an isolation unit (102) for isolating pure fungal cultures from collected samples, wherein the isolation unit (102) comprises sterilization equipment for treating the samples with 0.1% HgCl2 and a device for washing with sterile water; a solid media cultivation unit (104) for culturing mycelial tissue on semi-solid media selected from the group consisting of PDA, SDAY, PDA with chicken egg yolk, and PDA with MgSO4, wherein the solid media cultivation unit (104) comprises incubation chambers with a temperature of 19-25°C and a humidity of 80%;a liquid media cultivation unit (106) for cultivating mycelium in liquid media selected from the group consisting of PD broth, SDY broth, and broth-I, wherein the liquid media cultivation unit (106) comprises a rotary shaker operating at 145 rpm; an illumination system (108) configured to provide white light with an intensity of 300 lux for 8-9 hours; and a molecular identification unit (110) configured to perform ITS primer-based identification and phylogenetic analysis using a computer device.

[0008] An object of the present disclosure is to provide a system for producing fruiting bodies of B. pseudomilitaris.

[0009] A further objective of the present disclosure is to provide an integrated system that enables reliable isolation and cultivation of B. pseudomilitaris from wild specimens through the production of complete fruiting bodies, thus closing the current gap in cultivation systems for non-commercialized Cordyceps species.

[0010] Another object of the present disclosure is to cultivate mycelial tissue of Blackwellomyces pseudomilitaris in solid and liquid media and to allow it to grow to fruiting bodies.

[0011] Another object of the present disclosure is a system configured to produce highly efficient mycelium and fruiting bodies of B. pseudomilitaris using fewer resources.

[0012] To further clarify the advantages and features of the present disclosure, the invention will be explained in more detail with reference to specific embodiments illustrated in the accompanying drawings. These drawings show only typical embodiments of the invention and are therefore not to be construed as limiting its scope. The invention will be described and explained in more detail with reference to the accompanying drawings. SHORT DESCRIPTION OF THE FIGURE

[0013] These and other features, aspects, and advantages of the present disclosure will be better understood when the following detailed description is read with reference to the accompanying drawings, in which like characters represent like parts throughout. Fig. 1 shows a block diagram of a system for producing fruiting bodies of B. pseudomilitaris according to an embodiment of the present disclosure.

[0014] Those skilled in the art will also appreciate that the elements in the drawings are shown for convenience and are not necessarily to scale. For example, the flowcharts illustrate the method by key steps to enhance understanding of aspects of the present disclosure. Moreover, with respect to device construction, one or more components of the device may be represented in the drawings by conventional symbols, and the drawing may show only the specific details relevant to understanding embodiments of the present disclosure in order not to clutter the drawings with details that would be readily apparent to those skilled in the art after reading the present description. DETAILED DESCRIPTION:

[0015] For a better understanding of the principles of the invention, reference is made below to the embodiment illustrated in the drawings and described in specific language. However, the scope of the invention is not limited thereby. Changes and further modifications to the illustrated system, as well as further applications of the principles of the invention, are possible, as would normally occur to one skilled in the art to which the invention pertains.

[0016] It will be understood by those skilled in the art that the foregoing general description and the following detailed description are exemplary and explanatory of the invention and are not intended to be limiting thereof.

[0017] References in this specification to "one aspect," "another aspect," or similar expressions mean that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present disclosure. Therefore, the occurrences of the terms "in one embodiment," "in another embodiment," and similar expressions throughout this specification may or may not all refer to the same embodiment.

[0018] The terms "comprises," "having," or other variations thereof are intended to cover non-exclusive inclusion, such that a process or method comprising a list of steps not only includes those steps, but may also include other steps not expressly listed or inherent in such process or method. Likewise, the statement "comprises" with respect to one or more devices, subsystems, elements, structures, or components does not exclude, without further limitation, the existence of other devices, other subsystems, elements, structures, or components, or additional devices, additional subsystems, additional elements, additional structures, or additional components.

[0019] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. The system, methods, and examples provided herein are for illustrative purposes only and should not be considered limiting.

[0020] Embodiments of the present disclosure will be described in detail below with reference to the accompanying drawings.

[0021] Fig. 1 shows a block diagram of a system for producing fruiting bodies of Blackwellomyces pseudomilitaris according to an embodiment of the present disclosure.

[0022] Referring to Fig.1, the system (100) comprises: a) an isolation unit (102) configured to isolate pure fungal cultures from collected samples, wherein the isolation unit (102) comprises sterilization equipment for treating the samples with 0.1% HgCl2 and a device for washing with sterile water; b) a solid media cultivation unit (104) configured to cultivate mycelial tissue on semi-solid media selected from the group consisting of PDA, SDAY, PDA with chicken egg yolk, and PDA with MgSO4, wherein the solid media cultivation unit (104) comprises incubation chambers in which a temperature of 19-25°C and a humidity of 80% are maintained;c) a liquid media cultivation unit (106) configured to cultivate mycelium in liquid media selected from the group consisting of PD broth, SDY broth, and broth-I, the liquid media cultivation unit (106) comprising a rotary shaker operating at 145 rpm; d) an illumination system (108) configured to provide white light with an intensity of 300 lux for 8-9 hours; and e) a molecular identification unit (110) configured to perform ITS primer-based identification and phylogenetic analysis using a computer device.

[0023] In one embodiment, the isolation unit (102) further comprises flame-sterilized forceps configured to vertically transect the stroma and separate filamentous mycelial structures from within the stroma.

[0024] In one embodiment, the solid media cultivation unit (104) is configured to maintain natural pH conditions and includes an antibiotic delivery device for adding streptomycin to the media.

[0025] In one embodiment, the liquid media cultivation unit (106) is configured to enable mass production of mycelium secreting red pigment in the medium.

[0026] In one embodiment, the liquid media cultivation unit (106) is configured to operate the rotary shaker for 2-5 days.

[0027] In one embodiment, the system (100) further comprises a biochemical analysis unit (112) configured to analyze the red pigment secreted by the isolated cultures, wherein the biochemical analysis unit (112) comprises a filtering equipment with Whatman filter paper and a mixing device for combining the filtered pigment with 70% methanol in a 1:1 ratio.

[0028] In one embodiment, the biochemical analysis unit (112) further comprises ultrasonic equipment configured for 30 minutes of operation and centrifugation equipment configured for operation at 10,000 rpm for supernatant separation.

[0029] In one embodiment, the solid media cultivation unit (104) comprises autoclave equipment configured to sterilize media at a pressure of 15 pounds and media preparation equipment configured to prepare PDA at 0.3 g MgSO4 / L and SDAY with specific compositions of dextrose, peptone, and agar.

[0030] In one embodiment, the liquid media cultivation unit (106) comprises media preparation equipment configured to prepare liquid media with specific nutrient compositions, including potatoes, dextrose, peptone, yeast extract, MgSO4, VitB1, glucose, and KH2PO4.

[0031] The present invention provides a comprehensive system for the production of fruiting bodies of Blackwellomyces pseudomilitaris, a medicinal fungus of the genus Cordyceps with similar therapeutic properties to the commercially established Cordyceps militaris. The system addresses the urgent need for standardized cultivation protocols for non-commercialized entomopathogenic fungi that possess significant medicinal potential but lack practical production systems. The system comprises several integrated units, with the isolation unit employing sterile techniques such as sterilization with HgCl2 and washing with sterile water to obtain pure fungal cultures from field-collected samples. This unit contains specialized equipment such as flame-sterilized forceps for the precise separation of the mycelial structures from the stroma, ensuring contamination-free isolation of the viable fungal material.The solid medium cultivation unit allows cultivation in a controlled environment using various culture media, including PDA, SDAY and special formulations with egg yolk and MgSO . 4-Supplements. This unit ensures precise environmental conditions with temperature control from 19–25°C and 80% humidity, creating optimal conditions for mycelium growth and development. The system features antibiotic supplementation to prevent bacterial contamination during extended cultivation periods. The solid media system and liquid media cultivation unit enable mass production of mycelium using specialized broths, including PD broth, SDY broth, and custom nutrient formulations. The unit features a rotary shaker that runs at 145 rpm for 2–5 days, promoting optimal nutrient distribution and oxygenation for improved mycelium biomass production. The liquid cultivation system promotes the production of characteristic red pigments, which serve as quality indicators for successful B. pseudomilitaris cultivation.The lighting system enables critical photoperiod control with white light irradiation for 8-9 hours daily at 300 lux, thus mimicking natural light conditions essential for proper fungal development and fruiting body formation. This controlled lighting system is integrated into the cultivation units to ensure consistent environmental conditions throughout the entire production cycle. The molecular identification unit ensures species verification and quality control through ITS primer-based genetic analysis. This unit includes DNA extraction, PCR amplification, and phylogenetic analysis functions using Bioedit and Mega 11 software. This enables unambiguous species confirmation and prevents misidentification of cultured samples. The biochemical analysis unit enables comprehensive characterization of bioactive compounds, particularly the red pigments produced by B. pseudomilitaris.The unit includes filter systems, methanol extraction devices, ultrasonic devices, and 10,000 rpm centrifugation systems for effective isolation and purification of the compounds. The analytical functions support the quality assessment and standardization of the produced biomass for potential commercial applications.

[0032] The integrated system design enables scalable production suitable for both research applications and potential commercial cultivation. The modular architecture allows system expansion and modification according to production requirements while maintaining standardized protocols for consistent results. The system addresses current limitations in the cultivation of entomopathogenic fungi by providing comprehensive solutions from isolation to final product analysis, thus laying the foundation for the expanded use of B. pseudomilitaris as a viable alternative to existing Cordyceps species in medical applications.

[0033] In one embodiment, the material is collected in a separate plastic bag and container to prevent contamination, with the collected material being fed into the system.

[0034] In one implementation, the system's isolation unit performs the isolation of a pure fungal culture. The isolation unit is configured to perform sterilization with 0.1% HgCl2, followed by three washes in sterile distilled water. Stromal or stalk pieces are placed in a sterile Petri dish and cut vertically to separate small thread-like structures from within the stroma using flame-sterilized forceps. Inoculation then occurs on sterile medium, with various fungal structures, namely mycelial threads, being inoculated into the stroma. The system's solid cultivation unit is configured to culture the tissue on semi-solid medium. Two to three wild-collected samples are isolated on PDA (potato dextrose agar), SDAY (Sabouraud dextrose agar with yeast extract), PDA, and PDA + chicken egg yolk. Himedia granulated PDA with 0.3 g MgSO 4 / L, Himedia granulated PDA + 25 ml chicken egg yolk, SDAY (dextrose 20 g / L, peptone 5 g / L, type I agar 15 g / L + 0.3 g / L) are prepared and autoclaved at 15 lbs. The medium is poured with the antibiotic streptomycin, maintaining a natural pH. The medium is stored at a temperature of 19-25°C and 80% humidity. The egg yolk is shaken when the components are added, shaken again after autoclaving, and then poured. The lighting system allows the isolated culture plates, slats, and flasks to be stored at 19-25°C and 80% humidity with a regular 8-9 hour period of white light at 300 lux intensity.The liquid cultivation unit facilitates the cultivation of mycelium in liquid media, whereby the produced mycelium secretes red pigment into the medium, and the liquid media used include: PD broth (potato 20 g, dextrose and peptone 0.02 g, yeast extract 0.3 g, MgSO4 0.1 g, VitB1 50 mg, glucose 3 g, KH2PO4 0.1 g in 100 ml distilled water), SDY broth (dextrose 2 g, peptone 0.5 g, yeast extract 0.5 g, MgSO4 0.1 g in 100 ml DW), Broth-I Peptone 0.02 g, yeast extract 0.062 g, MgSO4 0.0125 g, VitB1 0.0025 g, glucose 1.25 g, sucrose 1.25 g, KH2PO4 0.0025 g) and placed on a rotary shaker at 145 rpm for 2-5 days.

[0035] In one embodiment, a system is configured to facilitate molecular identification and biochemical analysis. Fungal identification is performed using an ITS primer, the phylogenetic tree is constructed using a computer device, and biochemical analysis is performed. Isolated cultures secrete red pigment into the solid and liquid medium, which is filtered using Whatman filter paper 1 in a 1:1 ratio with 70% methanol. The mixture is shaken on a rotary shaker at 145 rpm for 2-3 days. After 2-3 days, no mycelial balls were visible in the mixture. It is then sonicated for 30 minutes and centrifuged at 10,000 rpm. The supernatant is used for biochemical analysis.

[0036] In one embodiment, Blackwellomyces pseudomilitaris (Hywel-Jones and Sivichai) Spatafora and Luangsa-ard, formerly known as Cordyceps pseudomilitaris, was collected on lepidopteran larvae from Maharashtra, India, and is characterized by a 25–50 mm long stalk and 12–25 mm long stromata. The perithecia are more or less superficial, submerged at the base with a prominent apex, oblong-ellipsoid or oblong-ovate, 289–574 µm long and 122–241 µm wide, with hyaline walls. The asci measure 210–395 × 5–6 µm, with an ascus cap, and ascospores do not fragment into partial spores.

[0037] In one embodiment, the isolated culture showed white mycelium, with colony growth beginning within 2 days on PDA, reaching 15–25 mm in diameter, circular with a knobby protrusion and ruffled edges, and slightly pink with red pigmentation in the medium. On SDAY, the colony diameter varied between 20 and 30 mm with similar characteristics. On PDA + egg medium, rapid growth was observed, reaching 35–40 mm within 2 days, and the colony structure remained consistent across all three media. Maximum pigment production occurred in egg medium. Pigmentation was observed not only on agar, but also in PD broth, SDY broth, and Broth I, with high mycelial growth in PD and SDY broth and less in Broth I. After an 8-day dark period, exposure to 8–9 hours of white light induced the formation of fruiting bodies identical to natural ones and comprising perithecia.pseudomilitaris produced red pigment in culture media.

[0038] In one embodiment, DNA isolation resulted in a concentration of 1355.1–1360.3 µg / ml. A 564 bp ITS sequence was obtained and deposited in GenBank under accession number OR259389. It exhibits a similarity of 98.57% to B. pseudomilitaris (MT000700.1) and 98.55% to C. pseudomilitaris (JN943305.1). Phylogenetic analysis using MEGA11 software assigned all retrieved ITS sequences, and the resulting phylogenetic tree confirmed the identity as B. pseudomilitaris.

[0039] In one embodiment, FT-IR analysis of methanolic extracts from both natural fruiting bodies and pure cultured mycelium revealed similar peaks as in previous Cordyceps reports, such as CO stretching at 1020.89 cm -1 (Mannitol), CN stretching at 1116 cm -1 , C=C aromatics at 1469 cm -1 , C=O amide at 1643 cm -1, -CH-aldehyde stretching at 2862 cm -1 and -OH stretching at 3288 cm -1, which shows agreement with the peaks reported for C. militaris. GC-MS / MS analysis identified several compounds with significant biological activity. 1-Tridecene, which occurs at R. Time 7.404 and covers 1.66% of the area, is known for its antibacterial activity. 1-Hexadecanol (R. Time 8.802, 10.048, 10.195, 11.309) covers approximately 7.00% of the area and exhibits antimicrobial and anti-inflammatory activity. Cyclooctasiloxane, hexadecamethyl (R. Time 8.930, 11.445) covers 2.42% of the area and is antimicrobial. 1-Nonadecene (R. times 11.171, 12.193) covers 7.05% of the area with the highest peak and exhibits antituberculosis and anticancer, antioxidant, and antimicrobial properties. Cyclodecasiloxane eicosamethyl, detected at several R. times (10.694, 12.745, 13.387, 14.861, 15.781, 16.903, 18.296), covers approximately 12.07% of the area and exhibits antimicrobial properties.

[0040] In one embodiment, compounds with antibacterial activity include 1-dodecene, dodecane, cyclohexasiloxane dodecamethyl, 1-tridecene, 1-hexadecanol, hexadecane, heptadecane, dibutyl phthalate, nonacosane, 1-heptacosanol, and diisooctyl phthalate. Compounds with antimicrobial activity include 2,4-dichloro-5-fluoroacetophenone, tetradecane, 1-hexadecanol, and cyclooctasiloxane hexadecamethyl, dibutyl phthalate, nonacosane, and heptriacontane. Antioxidant activity is associated with n-hexadecanoic acid, 1-nonadecene, tetratetracontane, 4-methyldocosane, and octacosanol. Anti-cancer effects have been observed for dibutyl phthalate, 1-nonadecene, 2-methylhexacosane, 4-methyldocosane, and tetrapentacontane, while hexestrol is used in prostate cancer and octacosane is effective against slowly invasive melanomas. Anti-inflammatory properties have been observed for hentriacontane, 1-hexadecanol, 4-methyldocosane, and octacosanol.Antifungal compounds detected included dodecane, cyclononasiloxane octadecamethyl, heptacosane, and eicosane; azoxystrobin was also identified as a broad-spectrum fungicide.

[0041] In one embodiment, the pharmaceutical relevance includes the antidiabetic activity of 2-methylhexacosane and tetrapentacontane; octacosanol exhibited anti-fatigue, antihypoxia, antioxidant, and antitumor activities. Eicosane is used for throat disorders, and methanolic extracts exhibited potent antioxidant and antiproliferative activities. Fruiting bodies of B. pseudomilitaris were successfully cultured in vitro; FT-IR results show similarities in the functional groups of the cultured and natural fruiting bodies. B. pseudomilitaris is morphologically identical to Cordyceps militaris, a widely valued medicinal mushroom. GC-MS / MS data indicate that B. pseudomilitaris possesses similar biological potential, including antibacterial, antifungal, antioxidant, antimicrobial, antidiabetic, and antitumor activities, with some compounds already used in pharmaceutical formulations.These results indicate the biological potential of Blackwellomyces pseudomilitaris.

[0042] The present invention provides a system for culturing Blackellomyces pseudomilitaris, which uses fewer resources and yields highly efficient mycelium and fruiting bodies that, at first glance, look like Cordyceps militaris but are actually a species of Blackwellomyces. The system facilitates the isolation of the Cordyceps-related species, which is expensive and time-consuming, and the present system simplifies the process of culturing Blackwellomyces, revealing its primitive potential with GCMS / MS studies.

[0043] The drawings and the foregoing description illustrate examples of embodiments. Those skilled in the art will recognize that one or more of the described elements may well be combined to form a single functional element. Alternatively, certain elements may be separated into multiple functional elements. Elements of one embodiment may be added to another embodiment. For example, the order of the processes described herein may be changed and is not limited to the manner described herein. Furthermore, the actions of a flowchart need not be implemented in the order shown; nor do all actions need to be performed. Also, actions that are not dependent on other actions may be performed in parallel with the other actions. The scope of the embodiments is in no way limited by these specific examples.Numerous variations, whether explicitly stated in the specification or not, such as differences in structure, dimensions, and use of materials, are possible. The scope of the embodiments is at least as broad as indicated in the following claims.

[0044] Advantages, further benefits, and solutions to problems have been described above with reference to specific embodiments. However, the advantages, advantages, solutions to problems, and any components that may result in an advantage, advantage, or solution occurring or becoming more apparent are not to be construed as critical, required, or essential features or components of any or all of the claims. REFERENCES 100 The system comprises: A) An isolation unit. 102 An isolation unit 104 Solid Culture Media Cultivation Unit 106 Liquid Media Cultivation Unit 108 Lighting system 110 Molecular Identification Unit 112 Biochemical Analysis Unit

Claims

[1] A system for producing fruiting bodies of Blackwellomyces pseudomilitaris, comprising: an isolation unit configured to isolate pure fungal cultures from collected samples, the isolation unit comprising sterilization equipment for treating samples with 0.1% HgCl 2 and a device for washing with sterile water; a solid media cultivation unit configured to cultivate mycelial tissue on semi-solid media selected from the group consisting of PDA, SDAY, PDA with chicken egg yolk, and PDA with MgSO4, the solid media cultivation unit comprising incubation chambers in which a temperature of 19-25°C and a humidity of 80% are maintained; a liquid media cultivation unit configured to cultivate mycelium in liquid media selected from the group consisting of PD broth, SDY broth, and broth-I, the liquid media cultivation unit comprising a rotary shaker operating at 145 rpm; a lighting system configured to provide white light with an intensity of 300 lux for 8-9 hours; and a molecular identification unit configured to perform ITS primer-based identification and phylogenetic analysis using a computing device. [2] The system of claim 1, wherein the isolation unit further comprises flame-sterilized forceps configured to vertically transect the stroma and separate filamentous mycelial structures from within the stroma. [3] The system of claim 1, wherein the solid media cultivation unit is configured to maintain natural pH conditions and comprises an antibiotic delivery device for adding streptomycin to the media. [4] The system of claim 1, wherein the liquid media cultivation unit is configured to enable mass production of mycelium secreting red pigment in the medium. [5] The system of claim 1, wherein the liquid media cultivation unit is configured to operate the rotary shaker for a period of 2-5 days. [6] The system of claim 1, further comprising a biochemical analysis unit configured to analyze the red pigment secreted by the isolated cultures, the biochemical analysis unit comprising a filtering kit including Whatman filter paper and a mixing device for combining the filtered pigment with 70% methanol in a 1:1 ratio. [7] The system of claim 1, wherein the biochemical analysis unit further comprises ultrasonic equipment configured to operate for 30 minutes and centrifugation equipment configured to operate at 10,000 rpm for separating the supernatant. [8] The system of claim 1, wherein the solid media cultivation unit comprises autoclave equipment configured to sterilize media at a pressure of 15 pounds, and media preparation equipment configured to prepare PDA at 0.3 g MgSO 4 / L and SDAY with specific compositions of dextrose, peptone, and agar. [9] The system of claim 1, wherein the liquid media cultivation unit comprises media preparation equipment configured to prepare liquid media with specific nutrient compositions including potatoes, dextrose, peptone, yeast extract, MgSO4, VitB1, glucose, and KH2PO4.

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