A fungus that promotes the growth of Saussurea involucrata seedlings

Abstract

A fungus that promotes the growth of Saussurea involucrata seedlings is Juxtiphoma sp. with a preservation number of CGMCC No.40985. The fungus CGMCC No.40985 can grow at 4-25°C and can colonize in the roots of Saussurea involucrata. When sowing Saussurea involucrata seeds or transplanting Saussurea involucrata seedlings at 10-20°C, adding the fungus CGMCC No.40985 to the substrate and culturing for 40-90 days can significantly increase the leaf dry weight and root dry weight of the seedlings, increase the root length, and promote the growth of the seedlings. The fungus CGMCC No.40985 has good application prospects for artificial cultivation of Saussurea involucrata.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering, and relates to a fungus capable of promoting the growth of Tianshan Saussurea involucrata seedlings. Background Art

[0002] Saussurea involucrata Kar.Et Kir., also known as snow lotus and Xinjiang snow lotus, is a medicinal plant of the genus Saussurea of ​​the Asteraceae family. The dried aerial parts of Saussurea involucrata can be used as medicine, namely the Uyghur medicinal material Saussurea involucrata. Uyghur medicine believes that Saussurea involucrata has the functions of nourishing the kidney and promoting blood circulation, strengthening tendons and bones, nourishing nerves, and regulating abnormal body fluids. It is used to treat rheumatoid arthritis, joint pain, lung cold cough, kidney and lower abdomen cold pain, excessive leucorrhea, etc. Traditional Chinese medicine believes that Saussurea involucrata has the functions of warming the kidney and assisting yang, dispelling wind and dampness, and promoting blood circulation. It is used to treat wind-cold dampness arthritis, rheumatoid arthritis, lower abdomen cold pain, and irregular menstruation. Saussurea involucrata has multiple pharmacological effects such as anti-rheumatic analgesic, antioxidant, anti-radiation, and anti-tumor, and has good application prospects in the research and development of medicines, health products, and cosmetics.

[0003] The Tianshan Snow Lotus is distributed in the Tianshan Mountains, Altai Mountains and Kunlun Mountains in the Xinjiang Uygur Autonomous Region of my country. It grows in weathering zones such as high-altitude glacial stone slopes, valleys, crevices, watersides, and meadows at an altitude of 2400-4100m. The average temperature of the hottest month in the growing environment is around 4°C. The Tianshan Snow Lotus is a perennial that blooms and bears fruit once, and its life cycle in the wild is generally 5 to 6 years. At present, the Tianshan Snow Lotus cannot be cultivated artificially on a large scale, and medicinal materials are completely dependent on wild resources. Due to the large-scale indiscriminate mining of the Tianshan Snow Lotus for a long time, the plant resources have been on the verge of exhaustion. In 2021, the Tianshan Snow Lotus was listed as a national second-level protected wild plant.

[0004] Endophytic fungi refer to a type of fungi that live in plant cells or in plant tissues during a certain period of their life history and do not cause obvious diseases in plant tissues. Dark septate endophytic fungi are a special type of endophytic fungi that colonize in plant root tissues. Their main morphological characteristics are: dark mycelium color, thick cell walls, and obvious septa. According to existing reports, dark septate endophytic fungi are common in plant root tissues in stress habitats such as low temperature, moisture, and salt. Their colonization is beneficial to the survival of host plants under adverse conditions. Studies have shown that there are a large number of endophytic fungi in the root tissues of Saussurea tianshanensis, among which dark septate endophytic fungi are the main group of endophytic fungi in the root tissues of Saussurea tianshanensis. The discovery of fungi that are beneficial to the survival and growth of Saussurea tianshanensis seedlings from the endophytic fungi in the roots of Saussurea tianshanensis and their application in the cultivation of Saussurea tianshanensis seedlings has a credible theoretical basis and good application prospects. Summary of the invention

[0005] The purpose of the invention is to provide a fungus which is beneficial to the survival of snow lotus seedlings and can promote the growth of snow lotus seedlings.

[0006] The fungus described in the present invention is identified as Juxtiphoma sp., with a deposit number of CGMCC No. 40985, and has been deposited in the China General Microbiological Culture Collection Center on November 17, 2023. The depository address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The deposit and survival certificate of the above strains is attached.

[0007] The culture characteristics of the fungus CGMCC No.40985 are as follows: the colony diameter is 7-7.5 cm after culturing on PDA medium at 15°C for 10 days, and the colony diameter is 6.4-7 cm after culturing at 20°C for 10 days; the colony is round with a flat edge, light grayish white at the beginning, and the color of the center of the colony becomes darker as the culture time increases, and finally becomes dark green, with a lighter color at the edge, grayish green to dark green, and the aerial hyphae are not well developed and are villi-like; the hyphae are dark and septate, and the middle of the hyphae swells to form thick-walled spores, or forms thick-walled spore chains.

[0008] The ribosome internal transcribed spacer (ITS) sequence, the second RNA polymerase II (RPB2) gene sequence and the β-tubulin (β-tublin) gene sequence of the fungus CGMCC No. 40985 of the present invention are shown in the sequence table.

[0009] The fungus described in the present invention can be added to the substrate when sowing Saussurea tianshanica seeds, or can be added to the substrate when transplanting or transplanting Saussurea tianshanica seedlings. The fungus described in the present invention is beneficial to the survival of seedlings and promotes the growth of seedlings under the above conditions. The beneficial effect of the fungus described in the present invention is that it is beneficial to the survival of Saussurea tianshanica seedlings at 10-20°C, increases the biomass of Saussurea tianshanica seedlings, thereby enhancing the adaptability of the seedlings to the environment, and has application value for the artificial cultivation of Saussurea tianshanica. DETAILED DESCRIPTION

[0010] Example 1. Low temperature adaptability of fungus CGMCC No.40985

[0011] The 1 cm diameter fungus CGMCC No.40985 PDA bacterial sheet was inoculated on the PDA plate. After 10 days of culture at 4℃, the colony diameter was (2.91±0.04) cm; after 17 days of culture, the colony diameter was (4.32±0.22) cm. After 10 days of culture at 10℃, the colony diameter was (5.18±0.27) cm. After 10 days of culture at 15℃, the colony diameter was (7.25±0.29) cm. After 10 days of culture at 20℃, the colony diameter was (6.68±0.32) cm. After 10 days of culture at 25℃, the colony diameter was (3.87±0.15) cm; after 17 days of culture, the colony diameter was (5.79±0.32) cm. It can be seen that the fungus CGMCC No.40985 has low temperature adaptability, grows faster at 10-20℃, and can still grow at 4℃.

[0012] Example 2. Preparation of solid fungus strain CGMCC No.40985

[0013] Sawdust and wheat bran are evenly mixed in a volume ratio of 3:1, and water is added to mix well. The water content of the matrix is ​​about 35%. The matrix is ​​divided into polypropylene seed bottles, and the matrix filling amount is about 75% of the volume of the seed bottle. The matrix is ​​steam sterilized at 121℃ for 2 hours, and after cooling, it is inoculated with the PDA bacterial sheet of fungus CGMCC No.40985, and placed in dark culture at (20±2)℃ for 25-35 days. It is used when the mycelium covers half to two-thirds of the volume of the matrix.

[0014] Example 3. Effect of fungus CGMCC No.40985 on the survival rate of Saussurea involucrata seedlings

[0015] Mix Pinnacle matrix and vermiculite in a volume ratio of 3:1, add water and mix well, sterilize at 121℃ for 3 hours, and use it as a matrix for transplanting and cultivating seedlings after cooling. Pack the matrix into 8×12 high-profile nutrient pots. The height of the matrix should be 1-2 cm lower than the edge of the nutrient pot.

[0016] Seedlings of Saussurea involucrata with 2-3 leaves and root length of 0.8-2 cm were transplanted onto the substrate, with 6 seedlings transplanted into each nutrient pot. The control group (CK) only transplanted seedlings, and the treatment group (T) was inoculated with solid fungus CGMCC No.40985 at a distance of 0.5-1.5 cm from the seedlings. The nutrient pots were cultured in an incubator at 20℃ for 12h of light / 10℃ for 12h of darkness, with a light intensity of 5000-6000lx. Water was applied regularly during the culture period. After 28 days of culture, the average number of seedlings in the CK group and the T group were (6.0±0.0) plants / pot and (5.8±0.4) plants / pot, respectively, with no significant difference between the two groups (P>0.05). After 63 days of cultivation, the average number of seedlings in the CK group and the T group were (3.7±1.0) plants / pot and (4.8±0.8) plants / pot, respectively. There were significant differences between the two groups (P<0.05). The number of seedlings in the T group was 1.3 times that of the CK group.

[0017] The seeds of Saussurea involucrata were shallowly sown on the substrate, and 8 seeds were sown in each nutrient pot. The control group (CK) was only sown, and the treatment group (T) was inoculated with the solid fungus CGMCC No.40985 at a distance of 0.5-1.5 cm from the seeds after sowing. The nutrient pots were placed in an incubator with a light intensity of 5000-6000 lx at 20℃ for 12h / darkness at 10℃. Water was applied regularly during the culture period. After 35 days of culture, the average number of seedlings in the CK group and the T group was (5.7±1.4) plants / pot and (5.7±0.8) plants / pot, respectively, and there was no significant difference between the two groups (P>0.05). After 90 days of culture, the average number of seedlings in the CK group and the T group was (2.0±1.5) plants / pot and (3.5±1.6) plants / pot, respectively, and there was a significant difference between the two groups (P<0.05). The number of seedlings in the T group was 1.75 times that of the CK group.

[0018] Example 4. Colonization of the fungus CGMCC No. 40985 in the roots of Saussurea involucrata seedlings when cultured at 15°C

[0019] Mix Pinnacle matrix, vermiculite and sawdust in a volume ratio of 3:1:1, add water and mix well, and pack them in PC plastic tissue culture bottles with a filling height of 3-3.5 cm. Sterilize the packed matrix with steam at 121℃ for 3 hours, and use it as the culture matrix after cooling.

[0020] Shallowly sow the surface-sterilized sterile Saussurea involucrata seeds on the substrate, and then inoculate the PDA plate strain of the fungus CGMCC No.40985 at a distance of 0.5-1.5 cm from the seeds.

[0021] Sterile Tianshan Saussurea involucrata seedlings with 2 leaves and a root length of 1-1.5 cm were transplanted onto a sterile substrate, and then the PDA plate strain of the fungus CGMCC No.40985 was inoculated at a distance of 0.5-1.5 cm from the seedlings.

[0022] The tissue culture bottles were placed in a constant temperature of 15°C, with a light intensity of 3000-4000lx, a photoperiod of 12h / 12h, and cultured for 35 days. The seedlings were taken out, the roots were cut into small sections of 4-6mm, stained with acid fuchsin, and the fungal colonization in the root tissue was observed under an optical microscope to calculate the infection rate. The results showed that the fungal infection rate in the roots of the seedlings with sowing and bacteria was above 80%; the fungal infection rate in the roots of the seedlings with transplanting and bacteria was above 90%.

[0023] Example 5. Effect of fungus CGMCC No.40985 on the growth of Saussurea involucrata seedlings when cultured at 15°C

[0024] Mix Pinnacle matrix, vermiculite and sawdust in a volume ratio of 2:1:0.5, add water and mix well, and pack them in PC plastic tissue culture bottles with a filling height of 3-3.5 cm. Sterilize the packed matrix with steam at 121℃ for 3 hours, and use it as the culture matrix after cooling.

[0025] Aseptic seedlings of Saussurea involucrata with 2 leaves and a root length of 1-1.5 cm were transplanted onto a sterile substrate. Only aseptic seedlings were transplanted into the tissue culture bottles of the control group (CK). The PDA plate strain of the fungus CGMCC No. 40985 was inoculated in the treatment group (T) at a distance of 0.5-1.5 cm from the seedlings. The culture bottles were placed at a constant temperature of 15°C, with a light intensity of 2500-3000lx and a photoperiod of 12h / 12h. After 40 days of cultivation, the survival rate of the seedlings was calculated. The leaf fresh weight, root fresh weight and total root length of the surviving seedlings were measured; the leaves and roots were dried at 60°C, the dry weight was weighed, and the dry weight ratio and root-crown ratio were calculated. The results are shown in the table below.

[0026]

[0027] *: Significant difference compared with CK (P < 0.05); **: Very significant difference compared with CK (P < 0.01).

[0028] After 40 days of cultivation at 15℃, the survival rate of seedlings in the T group was 75%, which was twice that of the CK group. The leaf dry weight and root dry weight of the T group were 2.4 times and 4.2 times that of the CK group, respectively, and the root length was 2.3 times that of the CK group, and the results were significantly different (P < 0.05). In addition, compared with the CK group, the dry weight of leaves and roots in the T group was also significantly increased (P < 0.05).

[0029] Example 6. Effect of fungus CGMCC No.40985 on the growth of Saussurea involucrata seedlings when cultured at 20°C

[0030] Mix Pinnacle matrix, vermiculite and sawdust in a volume ratio of 3:1:1, add water and mix well, and pack them in PC plastic tissue culture bottles with a filling height of 3-3.5 cm. Sterilize the packed matrix with steam at 121℃ for 3 hours, and use it as the culture matrix after cooling.

[0031] Sow the seeds of Saussurea involucrata shallowly on the substrate, and sow 6 seeds in each tissue culture bottle. The control group (CK) was only sown, and the solid strain of fungus CGMCC No.40985 was inoculated 0.5-1.5cm away from the seeds after sowing in the treatment group (T). The tissue culture bottles were placed in a constant temperature culture at 20℃, with a light intensity of 4000-5000lx and a photoperiod of 12h / 12h. After 63 days of culture, the survival rate of the seedlings was calculated. The leaf fresh weight, root fresh weight and total root length of the surviving seedlings were measured; the leaves and roots were dried at 60℃, the dry weight was weighed, and the dry rate and root-crown ratio were calculated. The results are shown in the table below.

[0032]

[0033] *: Significant difference compared with CK (P < 0.05); **: Very significant difference compared with CK (P < 0.01).

[0034] After 63 days of cultivation at 20℃, the leaf dry weight and root dry weight of the T group were 1.7 and 2.8 times that of the CK group, respectively, and the root length was 1.8 times that of the CK group, with extremely significant differences (P<0.01). In addition, compared with the CK group, the root-to-crown ratio of the T group was also significantly increased (P<0.05).

Claims

1. A fungus for promoting the growth of Saussurea involucrata seedlings, characterized in that: The fungus has a preservation number of CGMCC No. 40985 and is classified as Juxtiphoma sp. The fungus can promote the growth of snow lotus seedlings under the condition of 10-20° C. and can be used for the artificial cultivation of snow lotus.

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