Application of mycorrhizal fungi in regulating carbon allocation in Dendrobium officinale plants

Abstract

This invention provides the application of mycorrhizal fungi in regulating carbon allocation in Dendrobium officinale plants, belonging to the field of orchid mycorrhizal symbiosis technology. The invention includes the following steps: preparing a mycorrhizal fungal inoculant, wherein the mycorrhizal fungi are one or both of *Cladosporium halophilum* or *Dendrobium nobile*; soaking the roots of Dendrobium officinale tissue culture seedlings in the mycorrhizal fungal inoculant, and then transplanting them into a culture medium for cultivation. This invention, by inoculating the roots of Dendrobium officinale with mycorrhizal fungi, promotes mycorrhizal symbiosis and growth of Dendrobium officinale, while also inducing the expression of certain specific genes in Dendrobium officinale, activating a series of secondary metabolic pathways, thereby causing changes in the carbon allocation of the aboveground parts and roots of Dendrobium officinale seedlings, further influencing and regulating the biosynthesis of Dendrobium officinale polysaccharides, which is beneficial for further elucidating the molecular regulatory mechanism of mycorrhizal symbiosis on sugar metabolism in Dendrobium officinale.

Description

Technical Field

[0001] This invention belongs to the field of orchid mycorrhizal symbiosis technology, and particularly relates to the application of a mycorrhizal fungus in regulating carbon allocation in Dendrobium officinale plants. Background Technology

[0002] Dendrobium officinale (Kimura et Migo), also known as "Black Joint Grass," is a perennial herbaceous plant belonging to the Orchidaceae family. Its medicinal use was first recorded in the pharmacopoeia *Shennong Bencao Jing*. Currently, over 1500 species of Dendrobium have been discovered worldwide, making it the second largest genus in the Orchidaceae family. my country has 74 species of Dendrobium (including 2 varieties).

[0003] Dendrobium officinale is an epiphytic orchid, a semi-parasitic plant that grows nakedly on rocks and tree trunks. Because it lacks endosperm and has difficulty germinating under normal conditions, it needs to combine with symbiotic fungi to form a mycorrhizal structure under suitable conditions. Studies have shown that mycorrhizal symbiosis has a strong promoting effect on the growth and development of Dendrobium officinale, its adaptation to the environment, and the accumulation of active metabolites.

[0004] In the study of Dendrobium mycorrhizae, subtractive cDNA libraries of Dendrobium officinale mycorrhizae induced by *Mycena sp.* and seed inhibition and dissipation libraries of Dendrobium officinale induced by *Sebacinasp.* have been established. Simultaneously, mycorrhizal symbiosis also participates in regulating the biosynthesis of bioactive substances such as polysaccharides, dendrobines, and terpenoids. However, how mycorrhizal fungi participate in the regulation of sugar metabolism in Dendrobium officinale, and how they improve the polysaccharide content and quality of Dendrobium officinale, remains unreported. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide an application of mycorrhizal fungi in regulating carbon allocation in Dendrobium officinale plants, thereby altering the carbon allocation between the aboveground and underground parts of Dendrobium officinale and regulating the biosynthesis of Dendrobium officinale polysaccharides.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] The application of mycorrhizal fungi in regulating carbon allocation in Dendrobium officinale plants, wherein the mycorrhizal fungi are one or both of Cladosporium halotolerans or Mycena dendrobii.

[0008] This invention also provides a method for regulating carbon allocation in Dendrobium officinale plants using mycorrhizal fungi, comprising the following steps:

[0009] Prepare mycorrhizal fungal inoculum, wherein the mycorrhizal fungus is one or both of salt-tolerant Cladosporium or Dendrobium nobile; soak the roots of Dendrobium officinale tissue culture seedlings in the mycorrhizal fungal inoculum, and then transplant them into a culture medium for cultivation.

[0010] Preferably, the preparation of the mycorrhizal fungal inoculant includes: inoculating the mycorrhizal fungi into PDA solid medium and activating them for 2-3 days, and then inoculating them into PDB liquid medium for fermentation culture for 7-10 days.

[0011] Preferably, the Dendrobium officinale tissue culture seedlings are hardened off for 3-7 days before being soaked in mycorrhizal fungal inoculant, and then transplanted into the culture medium for pre-culture for 5-10 days.

[0012] More preferably, the disinfection before transplanting includes: cleaning the substrate around the roots of the tissue culture seedlings, disinfecting with 0.1% carbendazim for 8-12 minutes, and then rinsing with sterile water 4-6 times.

[0013] Preferably, the roots are soaked in mycorrhizal fungal inoculant for 20-40 seconds.

[0014] Preferably, after transplanting, the remaining mycorrhizal fungal agent from the root soaking is poured into the culture medium.

[0015] Preferably, the culture medium is prepared by volume proportions of 5%-15% large pine bark, 15%-25% small pine bark, 25%-35% pumice, 25%-35% celestial soil, and 5%-15% planting stone.

[0016] Preferably, during the culture period, the temperature is 27±1℃, the culture medium is kept moist, and the shade level is 70%-80%.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] This invention provides the application of mycorrhizal fungi in regulating carbon allocation in Dendrobium officinale plants and a method for regulating carbon allocation in Dendrobium officinale plants using mycorrhizal fungi. When different mycorrhizal fungi are inoculated into the roots of Dendrobium officinale, distinct mycorrhizal structures form on the roots. Cladosporium mycorrhizae exhibit beaded hyphal knots, while Dendrobium nobile mycorrhizae exhibit spiral hyphal knots.

[0019] This invention, through the determination of glucose, fructose, and sucrose concentrations in the aboveground and root parts of Dendrobium after inoculation with mycorrhizal fungi, demonstrates that different inoculation treatments affect sugar metabolism and carbon allocation in Dendrobium.

[0020] After a mycorrhizal symbiotic system was formed in the roots of Dendrobium officinale, the transcriptome sequencing results showed that the KEGG metabolic pathway was significantly enriched in the mycorrhizal roots. This enrichment included sugar metabolism pathways such as photosynthesis, photosynthetic carbon fixation, fructose and mannose metabolism, pentose and glucose interconversion, degradation of other polysaccharides, and polysaccharide synthesis and metabolism. This indicates that mycorrhizal symbiosis may affect the polysaccharide content by regulating the host's biosynthetic and consumption pathways, and the combined effect of these two pathways determines the accumulation of polysaccharides. Attached Figure Description

[0021] Figure 1 The image shows the microstructure of mycorrhizal fungi infecting the roots of Dendrobium officinale; in the image, A and D are blank controls; B and E are Dendrobium nobile infection and colonization on the roots of Dendrobium officinale; C and F are halophilic spores infection and colonization on the roots of Dendrobium officinale.

[0022] Figure 2 The figures show the detection results of different sugar contents in the aboveground parts and roots of Dendrobium officinale after inoculation with mycorrhizal fungi in Examples 1 and 2; in the figure, A is glucose in the aboveground parts; B is fructose in the aboveground parts; C is sucrose in the aboveground parts; D is glucose in the roots; E is fructose in the roots; and F is sucrose in the roots. Detailed Implementation

[0023] This invention provides the application of mycorrhizal fungi in regulating carbon allocation in Dendrobium officinale plants, wherein the mycorrhizal fungi are one or both of Cladosporium halotolerans or Mycena dendrobii.

[0024] The present invention also provides a method for regulating carbon allocation in Dendrobium officinale plants using mycorrhizal fungi, comprising the following steps: preparing a mycorrhizal fungal inoculant, wherein the mycorrhizal fungi are one or both of salt-tolerant Cladosporium or Dendrobium nobile; soaking the roots of Dendrobium officinale tissue culture seedlings in the mycorrhizal fungal inoculant, and then transplanting them into a culture medium for cultivation.

[0025] The preferred method for preparing mycorrhizal fungal inoculants according to this invention includes: inoculating mycorrhizal fungi into PDA solid medium for activation for 2-3 days, and then inoculating them into PDB liquid medium for fermentation culture for 7-10 days. As one possible implementation method, the mycorrhizal fungi of this invention, namely halophilic Cladosporium halotolerans or Mycena dendrobii, are preserved in glycerol at -80°C.

[0026] In this invention, it is preferred that Dendrobium officinale tissue culture seedlings be hardened off for 3-7 days before being soaked in mycorrhizal fungal inoculant, allowing them to adapt to natural light and temperature to improve the survival rate of subsequent transplanting, and then transplanted into the culture medium for pre-culture for 5-10 days; it is further preferred that the hardening off be carried out in a cool and ventilated place in a greenhouse for 5 days, followed by 7 days of pre-culture.

[0027] The present invention preferably involves disinfecting the tissue culture seedlings before transplanting and pre-culturing, including: cleaning the substrate around the roots of the tissue culture seedlings, disinfecting with 0.1% carbendazim for 8-12 minutes to prevent root rot, more preferably disinfecting for 10 minutes, and then rinsing with sterile water 4-6 times, more preferably 5 times.

[0028] The present invention preferably involves soaking the roots in mycorrhizal fungal inoculant for 20-40 seconds, and more preferably for 30 seconds.

[0029] In this invention, after transplanting, the remaining mycorrhizal fungal agent from root soaking is poured into the culture medium. Preferably, the culture medium is prepared by volume percentages of 5%-15% large pine bark, 15%-25% small pine bark, 25%-35% pumice, 25%-35% neem soil, and 5%-15% pumice. More preferably, it is prepared by volume percentages of 10% large pine bark, 20% small pine bark, 30% pumice, 30% neem soil, and 10% pumice. Even more preferably, the culture medium is autoclaved at 121°C for 20 minutes.

[0030] During the preferred incubation period, the temperature is 27±1℃, the culture medium is kept moist, and the shade level is 70%-80%. More preferably, the shade level is 75%. Even more preferably, sterile water is applied every 2 days.

[0031] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0032] Example 1

[0033] A method for regulating carbon allocation in Dendrobium officinale plants using mycorrhizal fungi includes the following steps:

[0034] (1) Dendrobium nobile (Md) preserved in glycerol at -80℃ was inoculated into PDA solid medium for 3 days for activation, and mycelial cakes of the same size were taken with a punch and fermented in PDB medium for 10 days.

[0035] (2) Select one-year-old sterile Dendrobium officinale tissue culture seedlings of the same batch and with consistent growth as experimental seedlings. Place the seedlings in a cool and ventilated place in the greenhouse for 5 days to harden them off and allow them to adapt to natural light and temperature in order to improve the survival rate of subsequent transplanting.

[0036] (3) Wash the root substrate of the sterile tissue culture seedlings, disinfect them with 0.1% carbendazim for 10 minutes to prevent root rot, then rinse the roots with sterile water 5 times to remove any residual carbendazim, and transplant the tissue culture seedlings into a culture medium that has been autoclaved at 121℃ for 20 minutes for 7 days of pre-culture; the culture medium is prepared by 10% large pine bark, 20% small pine bark, 30% pumice, 30% fairy soil and 10% planting stone;

[0037] (4) Dispense the fermented bacterial solution into 50mL centrifuge tubes, 5mL per tube. Take out the pre-cultured Dendrobium seedlings, soak them in the bacterial solution for 30s, and then take them out. Pour the remaining bacterial solution evenly into the prepared culture medium and place them in a greenhouse for cultivation. The greenhouse temperature is 27±1℃. Keep the culture medium moist and use a double-layer shade net for shading. The shade level is 75%. Water with sterile water every 2 days.

[0038] Example 2

[0039] A method for regulating carbon allocation in Dendrobium officinale plants using mycorrhizal fungi includes the following steps:

[0040] Similar to Example 1, except that the mycorrhizal fungus in step (1) is a salt-tolerant mycorrhizal fungus (Ch).

[0041] Example 3

[0042] A method for regulating carbon allocation in Dendrobium officinale plants using mycorrhizal fungi includes the following steps:

[0043] Similar to Example 1, except that the fermentation culture liquid in step (4) is a mixture of Dendrobium nobile (Md) fermentation liquid and salt-tolerant Cladosporium (Ch) fermentation liquid.

[0044] Example 4

[0045] A method for regulating carbon allocation in Dendrobium officinale plants using mycorrhizal fungi includes the following steps:

[0046] (1) Dendrobium nobile (Md) preserved in glycerol at -80℃ was inoculated into PDA solid medium for 3 days for activation, and mycelial cakes of the same size were taken with a punch and fermented in PDB medium for 8 days;

[0047] (2) Select one-year-old sterile Dendrobium officinale tissue culture seedlings of the same batch and with consistent growth as experimental seedlings. Place the seedlings in a cool and ventilated place in the greenhouse for 4 days to harden them off and allow them to adapt to natural light and temperature in order to improve the survival rate of subsequent transplanting.

[0048] (3) Wash the root substrate of the sterile tissue culture seedlings, disinfect with 0.1% carbendazim for 8 minutes to prevent root rot, then rinse the roots with sterile water 4 times to remove any residual carbendazim, and transplant the tissue culture seedlings into a culture medium that has been autoclaved at 121℃ for 20 minutes for 5 days for pre-culture; the culture medium is prepared by 15% large pine bark, 25% small pine bark, 30% pumice, 25% fairy soil and 5% planting stone;

[0049] (4) Dispense the fermented bacterial solution into 50mL centrifuge tubes, 5mL per tube. Take out the pre-cultured Dendrobium seedlings, soak them in the bacterial solution for 20s, and then take them out. Pour the remaining bacterial solution evenly into the prepared culture medium and place them in a greenhouse for cultivation. The greenhouse temperature is 27±1℃. Keep the culture medium moist and use a double-layer shade net for shading. The shade level is 70%. Water with sterile water every 2 days.

[0050] Example 5

[0051] A method for regulating carbon allocation in Dendrobium officinale plants using mycorrhizal fungi includes the following steps:

[0052] (1) Salt-tolerant Cladosporium (Ch) stored in glycerol at -80℃ was inoculated into PDA solid medium for 2 days to activate it. Then, the same size bacterial cakes were taken with a punch and fermented in PDB medium for 7 days.

[0053] (2) Select one-year-old sterile Dendrobium officinale tissue culture seedlings of the same batch and with consistent growth as experimental seedlings. Place the seedlings in a cool and ventilated place in the greenhouse for 3 days to harden them off and allow them to adapt to natural light and temperature in order to improve the survival rate of subsequent transplanting.

[0054] (3) Wash the root substrate of the sterile tissue culture seedlings, disinfect them with 0.1% carbendazim for 12 minutes to prevent root rot, then rinse the roots with sterile water 6 times to remove any residual carbendazim, and transplant the tissue culture seedlings into a culture medium that has been autoclaved at 121℃ for 20 minutes for 10 days of pre-culture; the culture medium is prepared by 5% large pine bark, 15% small pine bark, 33% pumice, 32% fairy soil and 15% planting stone;

[0055] (4) Dispense the fermented bacterial solution into 50mL centrifuge tubes, 5mL per tube. Take out the pre-cultured Dendrobium seedlings, soak them in the bacterial solution for 40s, and then take them out. Pour the remaining bacterial solution evenly into the prepared culture medium and place them in a greenhouse for cultivation. The greenhouse temperature is 27±1℃. Keep the culture medium moist and use a double-layer shade net for shading. The shade level is 80%. Water with sterile water every 2 days.

[0056] Comparative Example 1

[0057] The fermentation culture was replaced with sterile water as a blank control.

[0058] In Examples 1 and 2, the infection rate of Dendrobium officinale roots was measured at 0, 3, 6, and 9 days after infection with Md and Ch, respectively. At the same time, the treatment group and the blank group on the 9th day were sent for transcriptomics analysis. The sugar content of different parts of Dendrobium officinale aboveground and underground parts was measured 40 days after infection.

[0059] Results Analysis: Figure 1As can be seen from B and E, *Dendrobium nobile* fungus Md has colonized the roots of *Dendrobium officinale* and formed spiral-shaped mycelial knots. Figure 1 C and F show that Cladosporium Ch has colonized the roots of Dendrobium officinale and formed beaded mycelial knots; and the mycorrhizal infection rate of both fungi reached 100% 9 days after inoculation.

[0060] Depend on Figure 2 It can be seen that after Md infection, the glucose and sucrose content in the aboveground parts of Dendrobium officinale decreased significantly (glucose), while the glucose and sucrose content in the roots increased significantly; after Md and Ch infection, the fructose content in both the aboveground parts and roots increased significantly.

[0061] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for regulating carbon allocation in Dendrobium officinale plants using mycorrhizal fungi, characterized in that, Includes the following steps: Prepare mycorrhizal fungal inoculant, wherein the mycorrhizal fungus is a salt-tolerant cladoceran; soak the roots of Dendrobium officinale tissue culture seedlings in the mycorrhizal fungal inoculant, and then transplant them into a culture medium for cultivation; The roots are soaked in mycorrhizal fungi inoculant for 20-40 seconds; after transplanting, the remaining mycorrhizal fungi inoculant is poured into the culture medium.

2. The method for regulating carbon allocation in *Dendrobium officinale* plants using mycorrhizal fungi according to claim 1, characterized in that, The preparation of the mycorrhizal fungal inoculant includes: inoculating the mycorrhizal fungi into PDA solid medium and activating them for 2-3 days, and then inoculating them into PDB liquid medium for fermentation culture for 7-10 days.

3. The method for regulating carbon allocation in *Dendrobium officinale* plants using mycorrhizal fungi according to claim 1, characterized in that, Before soaking the Dendrobium officinale tissue culture seedlings in mycorrhizal fungal inoculant, the seedlings were hardened off for 3-7 days and then transplanted into the culture medium for pre-culture for 5-10 days.

4. The method for regulating carbon allocation in *Dendrobium officinale* plants using mycorrhizal fungi according to claim 3, characterized in that, The disinfection process before transplanting includes: cleaning the substrate around the roots of the tissue culture seedlings, disinfecting with 0.1% carbendazim for 8-12 minutes, and then rinsing with sterile water 4-6 times.

5. The method for regulating carbon allocation in *Dendrobium officinale* plants using mycorrhizal fungi according to claim 1, characterized in that, The culture medium is prepared by volume percentage of 5%-15% large pine bark, 15%-25% small pine bark, 25%-35% pumice, 25%-35% fairy soil, and 5%-15% planting stone.

6. The method for regulating carbon allocation in *Dendrobium officinale* plants using mycorrhizal fungi according to claim 1, characterized in that, During the culture period, the temperature was 27±1℃, the culture medium was kept moist, and the shade level was 70%-80%.

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