Application and method of galactomyces geotrichum NBG001 in improving alkaloid accumulation in lycoris radiata

Abstract

The application provides an application and method of Galactomyces geotrichum NBG001 in improving accumulation of medicinal alkaloids in Lycoris radiata, and belongs to the technical field of agricultural production. The Galactomyces geotrichum NBG001 is preserved in China Center for Type Culture Collection on July 7, 2025, and the preservation number is CCTCC NO: M 20251547. Through induction of expression of signal molecules and key genes in Lycoris radiata, simultaneous accumulation of galanthamine, lycoramine and narcissus cyclins in Lycoris radiata is significantly improved.

Description

Technical Field

[0001] This invention provides an application and method for using Galactobacillus niger NBG001 to increase the accumulation of alkaloids in Lycoris radiata, belonging to the field of agricultural production technology. Background Technology

[0002] Amaryllidaceae alkaloids are a class of unique secondary metabolites found only in plants of the Amaryllidaceae family, possessing a variety of medicinal activities. Galantamine, as a representative component, has been widely used in the treatment of Alzheimer's disease in its hydrobromide form; lycorine exhibits significant antitumor, antiviral, antibacterial, and anti-inflammatory activities; and narcissin demonstrates potent antitumor activity. In Asia, Amaryllidaceae alkaloids are mainly derived from Lycoris radiata (…). Lycoris radiata Alkaloids can be extracted from Lycoris radiata, but their content within Lycoris radiata is extremely low. Only about 60 grams of total alkaloids can be extracted from 15 kilograms of dried Lycoris radiata bulbs, making extraction difficult, requiring large amounts of organic solvents, and generating highly corrosive acidic and alkaline waste liquids. Therefore, how to efficiently promote the accumulation of medicinal alkaloids in Lycoris radiata is key to reducing its production costs and further promoting its development and utilization. However, currently, there is a lack of practical and efficient induction strategies.

[0003] Recent studies have shown that plant endophytic fungi play a crucial role in promoting the synthesis of secondary metabolites in host plants. They may promote the accumulation of specific bioactive components by secreting inducing factors, inducing plant defense responses, and activating key synthetic genes. This holds promise for overcoming the current bottlenecks of low alkaloid accumulation efficiency and high extraction costs. Summary of the Invention

[0004] In order to overcome the above-mentioned defects and deficiencies in the prior art, the present invention provides an application and method for using Galactomyces cerevisiae NBG001 to increase the accumulation of alkaloids in Lycoris radiata, which can significantly increase the accumulation of various alkaloids in Lycoris radiata.

[0005] To solve the above-mentioned technical problems, the present invention provides an application of Galactomyces cerevisiae NBG001 in increasing the accumulation of alkaloids in Lycoris radiata. Galactomyces cerevisiae NBG001 was deposited at the China Center for Type Culture Collection on July 7, 2025, with accession number CCTCC NO: M 20251547.

[0006] Furthermore, the alkaloids include galantamine, lycorine, and narcissine.

[0007] Furthermore, *Geotrichum galactosomalidase* NBG001 enhances alkaloid accumulation by inducing Lycoris radiata to produce signaling molecules and activating key genes for synthesis.

[0008] Furthermore, the signaling molecules include NO and H2O2.

[0009] Furthermore, the key genes include genes encoding phenylalanine ammonia-lyase. PAL Gene encoding tyrosine decarboxylase TYDC Gene encoding oxygen methyltransferase OMT .

[0010] In addition, the present invention also provides a method for increasing the accumulation of alkaloids in Lycoris radiata, wherein sterile Lycoris radiata bulbs are induced and cultured until they grow into complete plants, and a piece of Galactobacillus galactosidase NBG001 is taken and inoculated onto Lycoris radiata seedlings; the Galactobacillus galactosidase NBG001 was deposited at the China Center for Type Culture Collection on July 7, 2025, with accession number CCTCC NO: M20251547.

[0011] The specific process includes the following steps:

[0012] Sterilized Lycoris bulbs were longitudinally cross-cut into four equal pieces. Each piece was vertically inserted into MS medium containing 6 mg / L 6-BA, 0.5 mg / L NAA, 30 g / L sucrose, and 10 g / L agar to induce bulb formation. The resulting bulbs were then transplanted into MS medium containing 0.5 mg / L NAA, 30 g / L sucrose, and 10 g / L agar and cultured in a light incubator to induce bulblet formation. After multiple bulblets had formed, each bulblet was separated and transplanted into MS medium containing 0.5 mg / L NAA, 30 g / L glucose, and 10 g / L agar to induce the formation of complete plants. Experiments were conducted 8 weeks after transplanting. The light incubator was maintained at a 12-hour photoperiod, a light intensity of 3400 Lx, and a temperature cycle of 25 / 18°C.

[0013] Pick up a 5 mm diameter culture of *Gastromyxonomycetes NBG001* using an inoculation loop and streak it on PDA medium until a single colony forms. Use a sterile punch to take a 5 mm diameter culture disc and inoculate it onto a fresh PDA medium. Five days later, use a sterile punch to take a 5 mm diameter culture disc from the edge of the colony, with the mycelial side facing down and close to the *Lycoris radiata* plant, and inoculate it onto MS medium.

[0014] The beneficial technical effects achieved by the present invention are as follows: The present invention provides an application and method for improving the accumulation of alkaloids in Lycoris radiata by using Galactylum bisporus NBG001. Galactylum bisporus NBG001 can significantly promote the simultaneous accumulation of galantamine, lycorine, and narcissin in Lycoris radiata. Attached Figure Description

[0015] Figure 1 The nitric oxide content in Lycoris plants after inoculation with Galactobacillus oryzae for 3, 7, and 14 days in this embodiment of the invention;

[0016] Figure 2The hydrogen peroxide content in Lycoris plants after inoculation with Galactobacillus oryzae for 3, 7, and 14 days in this embodiment of the invention;

[0017] Figure 3 The relative expression levels of key genes for alkaloid synthesis in Lycoris plants 7 days after inoculation with Galactobacillus oryzae in this embodiment of the invention;

[0018] Figure 4 The present invention describes the content of galantamine, lycorine, and narcissin in Lycoris leaves, bulbs, and roots inoculated with Galactobacillus galactosidase in this embodiment of the invention.

[0019] The preservation information of the *Gastrodinium galactosomalidase* of this invention is as follows:

[0020] Galactobacillus cereus, classified and named Galactomyces geotrichum NBG001 is deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M 20251547, on July 7, 2025. Detailed Implementation

[0021] The present invention will be further described below with reference to specific embodiments. These embodiments are only used to more clearly illustrate the technical solutions of the present invention and should not be construed as limiting the scope of protection of the present invention.

[0022] The present invention will be further described below with reference to the accompanying drawings and embodiments. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all obtainable through conventional commercial channels. The experimental materials involved in the following embodiments are as follows:

[0023] Galactosomalidomyces cerevisiae ( Galactomycesgeotrichum The NBG001 strain was derived from a previously published strain in our laboratory (publication patent application number: 2025111688719). This strain was deposited at the China Center for Type Culture Collection on July 7, 2025, with accession number: CCTCC NO: M 20251547. The glycerol tube was stored at -80℃.

[0024] Example 1: Method for increasing alkaloids in Lycoris radiata using the Galactystoma NBG001 strain

[0025] Sterilized Lycoris bulbs were longitudinally cross-cut into four equal pieces. Each piece was vertically inserted into MS medium containing 6 mg / L 6-BA, 0.5 mg / L NAA, 30 g / L sucrose, and 10 g / L agar to induce bulblet formation. After multiple bulblets had formed, the individual bulblets were separated and transplanted into MS medium containing 0.5 mg / L NAA, 30 g / L glucose, and 10 g / L agar to induce them into complete plants. Experiments were conducted 8 weeks after transplanting. The photoperiod was 12 h, the light intensity was 3400 Lx, and the temperature cycle was 25 / 18°C (day / night).

[0026] Galactobacillus gnatus NBG001 was suspended in 20% glycerol and stored at -80°C for an extended period. During recovery culture, an inoculation loop was used to pick up NBG001 culture and streaked it on PDA medium at 28°C until single colonies formed. Using a sterile punch, 5 mm diameter mycelial discs were inoculated onto fresh PDA medium. Five days later, using a sterile punch, 5 mm diameter mycelial discs were taken from the edge of the colonies, hyphae side down, and inoculated onto MS medium, adhering closely to the Lycoris radiata plant. Lycoris radiata plants inoculated with 5 mm diameter PDA discs served as the control group.

[0027] Example 2: Effect of *Gastromyxonomycete* NBG001 strain on the content of signaling molecules in Lycoris radiata.

[0028] Following the method described in Example 1, Lycoris radiata was inoculated. Plants were harvested 3, 7, and 14 days after inoculation, flash-frozen in liquid nitrogen, and stored at -80°C. 0.1 g of plant tissue was accurately weighed and added to 500 μL of phosphate buffer (0.1 M, pH = 7.0-7.4). The tissue homogenate was obtained by grinding at 0°C and 70 Hz for 30 s in a cryogenic homogenizer. The homogenate was then centrifuged at 3500 rpm for 10 min to obtain the supernatant. The NO and H₂O₂ contents in the supernatant were determined. For NO determination, the supernatant was mixed with a nitrate colorimetric reagent to generate a light red azo compound. The absorbance was measured at 550 nm. The NO content in each sample was calculated based on a standard curve constructed using the sodium nitrite standard concentration and absorbance. The method for detecting H2O2 involves mixing the supernatant with ammonium molybdate solution to form a pale yellow complex. The absorbance is measured at 405 nm. The H2O2 content in each sample is determined by calculating a standard curve based on the concentration and absorbance of the H2O2 standard. The detection results are as follows: Figure 1 and Figure 2 As shown.

[0029] from Figure 1The results show that on day 3 after inoculation, there was no significant difference in NO content between the MOCK control group and the NBG001 inoculated group of Lycoris radiata. However, on days 7 and 14 after inoculation, the NO content in the NBG001 inoculated group of Lycoris radiata was significantly higher than that in the control group. Therefore, inoculation with *Gastrodia galactosum* NBG001 significantly induced the release of NO in Lycoris radiata. NO is one of the markers of activated plant defense responses, which can activate downstream signals and promote the synthesis of alkaloids in the Amaryllidaceae family.

[0030] from Figure 2 The results show that on days 3, 7, and 14 after inoculation, the H2O2 content in the Lycoris radiata plants treated with NBG001 was significantly higher than that in the control group inoculated with PDA discs. This indicates that inoculation with Galactobacillus NBG001 significantly induced the release of H2O2 in Lycoris radiata. H2O2 is also one of the markers of plant defense response activation. It can synergistically work with NO to activate downstream signals and promote the synthesis of alkaloids in the Amaryllidaceae family.

[0031] Example 3: Effect of *Gastromyxobolus galactosomalidone* strain NBG001 on the expression levels of key genes involved in alkaloid synthesis in *Lycoris radiata*.

[0032] Following the method described in Example 1, inoculated Lycoris radiata were treated. Seven days after inoculation, NBG001 inoculated plants and control group plants were harvested and separated into leaves, bulbs, and roots using a sterile knife. After flash freezing in liquid nitrogen, the separated parts were stored at -80°C. Total RNA was extracted from each tissue of Lycoris radiata using the RNAprep Pure plant total RNA extraction kit and then analyzed by NanoDrop. TM RNA was quantified using a 1000 micro spectrophotometer. 1 μg of total RNA was treated with DNase and reverse transcribed into cDNA. The cDNA was then analyzed using SYBR Green. ® Premix Ex Taq TM Perform qRT-PCR reaction, to TIP41 As an internal reference gene, use 2 –ΔΔCT The relative expression levels of key genes in alkaloid synthesis were calculated; three key genes in the Amaryllidaceae alkaloid synthesis pathway were given special attention: PAL (Phenylalanine ammonia-lyase) TYDC (Tyrosine decarboxylase) OMT (O-methyltransferase), specific primer information is shown in Table 1, and the detection results are as follows: Figure 3 As shown.

[0033] Table 1 Primers used in qRT-PCR

[0034] Primer name Primer sequence Serial Number 5'-GTGATGTTGTTGTTGAGGAGGCGA-3' SEQ ID NO.1 5'-TAGGAGGACCAAGCAAGGAGGTGC-3' SEQ ID NO.2 5'-ACCATCGTCTCTGTTTCCATCTGTC-3' SEQ ID NO.3 5'-TTGCTTTCTCTCATTCTCATCCGTA-3' SEQ ID NO.4 5'-GGGAGGTGACTGAGAAACATGAAT-3' SEQ ID NO.5 5'-CCAAAGCGGTTGTGAGCAGAGAAT-3' SEQ ID NO.6 5'-AATGTGCAAGCAGGGCTAGTAA-3' SEQ ID NO.7 5'-GCAACCATCCAAAGTTTAACTGCT-3' SEQ ID NO.8

[0035] from Figure 3 It can be seen that: compared with the control group plants inoculated with PDA discs, the bulbs and roots of the Lycoris radiata in the NBG001 treatment group showed higher concentrations of the drug. PAL Gene expression levels were significantly increased, reaching 4.96 and 3.61 times that of the control, respectively; in the NBG001 treatment group, Lycoris radiata roots showed... TYDC Gene expression was significantly increased, reaching 2.45 times that of the control group; in the NBG001 treatment group, the expression levels of Lycoris bulbs and roots were significantly increased. OMT Gene expression levels were also significantly increased, reaching 1.66 and 1.46 times that of the control group, respectively. This demonstrates that *Galactomyces NBG001* can effectively increase the expression levels of key genes in *Lycoris radiata*.

[0036] Example 4: Effect of *Gastromyxonomycetes NBG001* strain on alkaloid accumulation in Lycoris radiata.

[0037] The inoculated Lycoris radiata was treated according to the method in Example 1. After 28 days of inoculation, the plants were harvested and separated into leaves, bulbs, and roots using a sterile knife. The roots were dried at 50°C to constant weight and then ground into powder. 1 g of plant powder was accurately weighed and 5 mL of methanol was added. The mixture was extracted at 4°C for 24 h, with three 30 min ultrasonic water baths at 70 kHz each. Afterward, the supernatant was obtained by centrifugation at 8000 rpm and 4°C for 10 min. The supernatant was filtered through a 0.22 μm filter membrane and the contents of galantamine, lycorine, and narcissin were determined by high performance liquid chromatography (HPLC). A C18 reverse-phase column (4.6 mm × 250 mm × 5 μm) was used. The mobile phase was (A) 0.05% triethylamine aqueous solution and (B) acetonitrile. The gradient elution system was 0–20 min, 10–30% B, 20–35 min, 30–40% B, with a flow rate of 1 mL / min, a column temperature of 35°C, and a sample loading volume of 20 μL. The detection wavelengths for galantamine, lycorine, and narcissin were 280, 290, and 300 nm, respectively. The results are as follows: Figure 4 As shown.

[0038] from Figure 4 The results show that 28 days after inoculation, inoculation with *Gastromycosis galactocele* NBG001 significantly promoted the accumulation of galantamine in the roots of *Lycoris radiata*, reaching 6.72 times that of the control group inoculated with PDA discs; significantly promoted the accumulation of lycorine in the bulbs of *Lycoris radiata*, reaching 2.20 times that of the control group; and significantly promoted the accumulation of narcissin in the bulbs and roots of *Lycoris radiata*, reaching 2.43 and 5.62 times that of the control group, respectively.

[0039] The present invention has been disclosed above with reference to preferred embodiments, but it is not intended to limit the present invention. All technical solutions obtained by adopting equivalent substitutions or equivalent transformations fall within the protection scope of the present invention.

Claims

1. The application of *Gastrodia galactosomalidone* NBG001 in enhancing the accumulation of alkaloids in *Lycoris radiata*, characterized by: The *Gastromyces galactosomalidomia* NBG001 was deposited at the China Center for Type Culture Collection on July 7, 2025, with accession number CCTCC NO: M20251547. *Gastromyces galactosomalidomia* NBG001 increases the accumulation of alkaloids by inducing the synthesis of signaling molecules of Lycoris radiata and activating key genes for synthesis. The alkaloids are galantamine, lycorine, or narcissin.

2. The application according to claim 1, characterized in that: The signaling molecules include nitric oxide (NO) and hydrogen peroxide (H2O2).

3. The application according to claim 1, characterized in that: The key genes include those encoding phenylalanine ammonia-lyase. PAL Gene encoding tyrosine decarboxylase TYDC Gene encoding oxygen methyltransferase OMT .

4. A method for increasing the accumulation of medicinal alkaloids in Lycoris radiata, characterized in that: Aseptic Lycoris bulbs were induced to grow into complete plants, and NBG001 galactosomal yeast slices were inoculated onto the Lycoris seedlings. The NBG001 galactosomal yeast was deposited at the China Center for Type Culture Collection on July 7, 2025, with accession number CCTCC NO: M 20251547. The alkaloids were galantamine, lycorine, or narcissin.

5. The method for increasing the accumulation of medicinal alkaloids in Lycoris radiata according to claim 4, characterized in that, The specific process includes the following steps: Sterilized Lycoris bulbs were longitudinally cross-cut into four equal pieces. Each piece was vertically inserted into MS medium containing 6 mg / L 6-benzyladenine, 0.5 mg / L naphthaleneacetic acid, 30 g / L sucrose, and 10 g / L agar to induce bulb formation. The generated bulbs were then transplanted into Murashige-Skoog medium containing 0.5 mg / L NAA, 30 g / L sucrose, and 10 g / L agar and cultured in a light incubator. After multiple small bulbs were formed, the individual small bulbs were separated and transplanted into MS medium containing 0.5 mg / L NAA, 30 g / L glucose, and 10 g / L agar to induce complete plants. The light incubator was maintained at a photoperiod of 12 h, a light intensity of 3400 Lx, and a temperature cycle of 25 / 18°C. Using an inoculation loop, pick up a culture of *Gastromyxonomycetes NBG001* and streak it on potato dextrose agar until a single colony forms. Using a sterile punch, take a 5 mm diameter mycelial disc and inoculate it onto fresh PDA medium. Five days later, using a sterile punch, take a 5 mm diameter mycelial disc from the edge of the colony, with the mycelial side facing down, close to the *Lycoris radiata* plant, and inoculate it onto MS medium.

Images