An Arctic-derived strain of Vishnus victoriae and its volatile substances and applications

Abstract

The present invention discloses a strain of Victorian Vishniacozyma yeast of Arctic origin and its volatile substances and applications, belonging to the field of biotechnology. The strain of the present invention is Victorian Vishniacozyma yeast (Vishniacozyma victoriae) D41 with a deposit number of CCTCC NO: M 20221038, and the components of the volatile substances produced by it include ethanol, 2,5-dimethylfuran, isopentanol, n-valeric acid, isovaleric acid, 2-methylbutyric acid, valerolactone, 3-(2-furyl)-2-methyl-2-propenal, 2,5-dimethylbenzaldehyde, α-cedrene, cedrene, cedrol, and 2,5-di-tert-butylphenol. Victorian Vishniacozyma yeast D41 can promote the growth and flowering of Arabidopsis thaliana at both 20°C and 10°C. The present invention has a good application prospect in the promotion and cultivation of low-temperature plants.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to an Arctic-origin Vichysaccharomyces victoriae yeast, a volatile substance thereof and an application thereof. Background Art

[0002] Microbial volatile organic compounds (VOCs) are volatile organic byproducts produced by microorganisms (fungi and bacteria) during primary metabolism (synthesis of DNA, amino acids and fatty acids, etc.) and secondary metabolism. They are usually characterized by low molecular weight (<300Da), low boiling point and high vapor pressure. More than 2,000 microbial VOCs have been identified, including substances from chemical categories such as alkanes, alkenes, aldehydes, ketones, alcohols, terpenes, sulfur-containing compounds and nitrogen-containing compounds. Studies have found that volatile organic compounds synthesized and released by microorganisms in some flowers can serve as signal factors for plant growth, playing a role in nutrient acquisition, photosynthesis, plant hormone metabolism and hormone function, thereby promoting and improving plant growth and development processes, such as seed germination, growth, reproduction, etc.

[0003] Cryptococcus victoriae was first isolated from Victoria Land in Antarctica in 1999 and renamed Vishniacozyma victoriae in 2015. It has since been found in glaciers, mountains, the Arctic, caves, and even in urban air. Research on Vishniacozyma victoriae has primarily focused on its cold tolerance, with some strains found to produce cold-active enzymes, antifreeze proteins, unsaturated fatty acids, and specialized polysaccharides. These strains possess antimicrobial properties, can mitigate fungal diseases and rot in wheat, pears, and apples, and are even antagonists of the purinergic receptor P2X7, which can cause inflammation, and can degrade polyphenols and dyes.

[0004] The present invention is the first to study the volatile substances of the yeast Vishnikov's victoria and its promotion of low-temperature growth and flowering of plants. Summary of the Invention

[0005] The present invention provides an Arctic-derived strain of V. victoriae, its volatile compounds, and their applications. The V. victoriae strain is isolated from the flowers of Saxifraga cespitosa, and its volatile compounds can promote plant growth and flowering. This invention explores new ideas and research areas for promoting plant growth and flowering at low temperatures.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

[0007] In a first aspect, a strain of Vishniacozyma victoriae isolated from the flowers of Saxifraga cespitosa is provided, with a deposit number of CCTCC NO: M 20221038. After purification and identification, the strain had the highest similarity with the ITS, 26S rRNA gene, and 18S rRNA gene sequences of Vishniacozyma victoriae, at 99.04%, 99.52%, and 99.76%, respectively. The strain was named Vishniacozyma victoriae D41.

[0008] The bacterial size of Vishniacozyma victoriae D41 strain is 2.8-4.0×4.4-5.1 μm, and the colonies formed on YM plates are red and shiny.

[0009] In a second aspect, volatile substances produced by the Vishniacozyma victoriae yeast, deposited with CCTCC NO: M 20221038, are provided. Furthermore, a method for preparing the volatile substances comprises the following steps: inoculating the Vishniacozyma victoriae yeast strain D41 into a liquid culture medium, culturing the culture medium, and collecting the volatile substances from the culture medium. The method for collecting the volatile substances comprises solid-phase adsorption.

[0010] The components of the volatile substance include: ethanol, 2,5-dimethylfuran, isopentanol, n-valeric acid, isovaleric acid, 2-methylbutyric acid, valerolactone, 3-(2-furyl)-2-methyl-2-propenal, 2,5-dimethylbenzaldehyde, α-cedrene, cedrene, cedrol, and 2,5-di-tert-butylphenol.

[0011] In a third aspect, the invention provides a use of the volatile substances produced by the Vishnikov yeast strain in promoting plant growth and flowering.

[0012] The promotion of plant growth and flowering includes an increase in chlorophyll content, plant weight, root weight and / or number of flowers.

[0013] The plants include Arabidopsis thaliana.

[0014] Compared with the prior art, the present invention has the following advantages: at 20°C, the chlorophyll content of Arabidopsis thaliana can be increased by 112%, the plant weight by 90%, the root weight by 337%, and the flowering rate by 286%; at 10°C, the chlorophyll content of Arabidopsis thaliana can be increased by 60%, the plant weight by 39%, the plant height by 130%, and the flowering rate by 238%. The volatile substances produced by the yeast Vishnikov's victoria of the present invention have the effect of promoting the growth and flowering of Arabidopsis thaliana at low temperatures. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is the phylogenetic tree diagram of the ITS sequence of Vishniacozyma victoriae D41: the Neighbor-Joining phylogenetic tree of strain D41 constructed based on the ITS sequence alignment results with Sterigmatomyces halophilus (NR073302.1) as the outer branch.

[0016] Figure 2 This is the phylogenetic tree of the 26S rRNA gene sequence of Vishniacozyma victoriae D41: Neighbor-Joining phylogenetic tree of strain D41 constructed based on the 26S rRNA gene sequence alignment results with Rhodotorula glutinis (DQ377681.1) as the outer branch.

[0017] Figure 3 This is the phylogenetic tree of the 18S rRNA gene sequence of Vishniacozyma victoriae D41: Neighbor-Joining phylogenetic tree of strain D41 constructed based on the 18S rRNA gene sequence alignment results with Rhodotorula_glutinis (NG_062726.1) as the outer branch.

[0018] Figure 4 This figure compares the effects of Vishniacozyma victoriae D41 volatile compounds on the growth of Arabidopsis thaliana at 20°C. CK: control group, uninoculated with yeast; D41: inoculated with Vishniacozyma victoriae D41.

[0019] Figure 5This figure compares the effects of Vishniacozyma victoriae D41 volatile compounds on the growth of Arabidopsis thaliana at 10°C. CK: control group, uninoculated with yeast; D41: inoculated with Vishniacozyma victoriae D41.

[0020] Strain preservation information: The strain preservation date is July 5, 2022, the preservation number is CCTCC NO: M20221038, the classification name is Vishniacozyma victoriae D41, the preservation unit name is China Center for Type Culture Collection (CCTCC), and the address is Wuhan University, Wuhan, China. DETAILED DESCRIPTION

[0021] The features and advantages of the present invention can be further understood by referring to the following examples and accompanying drawings. The examples provided are merely illustrative of the methods of the present invention and are not intended to limit the scope of the present invention in any way. Specific experimental conditions and methods not specified in the following examples are generally in accordance with conventional methods, such as those described in J. Sambrook et al., eds., Molecular Cloning: A Laboratory Manual (3rd edition), Science Press, 1992; D.L. Spector et al.

[0022] The strain used was Vishniacozyma victoriae isolated from the flowers of Saxifraga cespitosa. The ITS, 26S rRNA gene, and 18S rRNA gene sequences of the strain are shown in SEQ ID NOs. 1-3, respectively. They have the highest similarity with Vishniacozyma victoriae, which are 99.04%, 99.52%, and 99.76%, respectively. Figure 1-3 ), which was named V. victoriae D41. This strain was deposited on July 5, 2022, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with the deposit number CCTCC NO: M 20221038.

[0023] Transmission electron microscopy revealed that strain D41 measured 2.8-4.0 × 4.4-5.1 μm in size. Its colonies on YM plates were red and glossy. It grew in a temperature range of 4°C-28°C, at a pH of 4.0-9.0, and tolerated 0%-5% NaCl, 0.1%-50% glucose, 0.1%-20% fructose, and 0.1%-60% sucrose.

[0024] Example 1 Extraction and Detection of Volatile Substances from Vichysoma victoriae

[0025] 1. Extraction of Volatile Compounds from Vichysoma victoriae D41

[0026] 1) The vichysoma vichysoma strain D41 was inoculated into a test tube containing 5 mL of artificial nectar medium (120 g of glucose, 120 g of fructose, 60 g of sucrose, 3 g of yeast extract, 3 g of malt extract, 5 g of peptone, 1 L of deionized water, natural pH) and cultured at 10°C and 20°C, respectively, to the late logarithmic phase.

[0027] 2) Centrifuge the bacterial suspension at 12,000 rpm for 5 minutes, transfer the supernatant to a 20 mL light-proof headspace adsorption bottle, and then add 200 ppm of internal standard (N,N-dimethylformamide) to each sample.

[0028] 3) Insert a 75 μm CAR / PDMS (carboxen / polydimethylsiloxane) solid-phase microextraction column vertically into the adsorption flask for adsorption. Incubate in a 60°C water bath for 6 hours.

[0029] 2. Detection of Volatile Compounds in Vichysoma victoriae D41

[0030] 1) Insert the solid phase microextraction adsorption column after adsorption into a gas chromatography-mass spectrometer (GCMS).

[0031] 2) The adsorbed substances were detected by gas chromatography-mass spectrometry (GCMS) under the following conditions: chromatographic interface temperature of 250°C; starting temperature set at 45°C, held at this temperature for 5 minutes; heating at a rate of 10°C / min to 180°C without holding; heating at a rate of 20°C / min to 230°C, held for 5 minutes; and finally heating at a rate of 20°C / min to 250°C, held for 5 minutes. The total heating time was 32 minutes.

[0032] 3) The test results are shown in Table 1. The volatile components of strain D41 include ethanol, 2,5-dimethylfuran, isopentanol, n-valeric acid, isovaleric acid, 2-methylbutyric acid, valerolactone, 3-(2-furyl)-2-methyl-2-propenal, 2,5-dimethylbenzaldehyde, α-cedrene, cedrene, cedrol, and 2,5-di-tert-butylphenol.

[0033] Table 1 Mass spectrometry peak contents of volatile components of V. victoriae D41

[0034]

[0035] The mass peak contents in this table are multiples of the mass peak of the internal standard substance (N,N-dimethylformamide) DMF at 200 ppm.

[0036] Example 2 Application of Victoria Vishniki yeast volatile substances

[0037] 1. Construction of the V. victoriae D41-plant co-culture system

[0038] 1) In a biosafety cabinet, pour approximately 10 mL of 1 / 2 MS plant culture medium (Coolaber, Catalog No. PM1060-50L) into a sterile, light-transmitting test tube (2 cm diameter, 18 cm height). After sufficient cooling, inoculate one Arabidopsis thaliana seed, disinfected with sodium hypochlorite, onto the surface of the medium.

[0039] 2) Hang an open EP tube inoculated with the yeast Vishnikov D41 on top of the test tube and close the cap (the opening of the EP tube should be approximately 1 cm from the cap). A control tube containing the same system but containing only culture medium without yeast D41 was used as a control. Set up 10 replicate tubes for each treatment.

[0040] 3) After sealing with a double-layer sealing film, place it in a constant temperature and light incubator, set the temperature (10°C and 20°C) and light period (light: dark = 15h:9h).

[0041] 2. Determination and calculation of plant growth parameters

[0042] After the plants have fully grown, they are gently removed and relevant growth parameters are measured, including plant fresh weight, fresh weight of the supraroot portion, fresh weight of the root system, height of the supraroot portion, number of leaves, number of open flowers, and chlorophyll content (total chlorophyll content, chlorophyll a content, and chlorophyll b content). Plant fresh weight, fresh weight of the supraroot portion, and fresh weight of the root system are measured using an analytical balance, while supraroot height is measured using a vernier caliper. The extraction, content determination and calculation methods of chlorophyll were referenced (1. Richard Hung, Samantha Lee W., Bennett Joan. 2013. Arabidopsisthaliana as a model system for testing the effect of Trichoderma volatileorganic compounds. Fungal Ecology[J], 6: 19-26. 2. Zhou Yong, Fan Xiaolei, LinYongjun, Chen Hao. 2018. Determination of cholophyll of rice. Bio-Protocol[J], 1010147.).

[0043] 3. Promoting Effects of the Victoria yeast D41 Strain and Its Volatile Substances on Arabidopsis

[0044] result( Figure 4 、 5 Table 2 shows that the V. victoriae D41 strain and its volatile compounds can increase chlorophyll content in Arabidopsis thaliana by 112%, plant weight by 90%, root weight by 337%, and flowering by 286% at 20°C. At 10°C, they can increase chlorophyll content in Arabidopsis thaliana by 60%, plant weight by 39%, plant height by 130%, and flowering by 238%. This indicates that the strain promotes growth and flowering of Arabidopsis thaliana at low temperatures.

[0045] Table 2 Effects of volatile substances from the victoria yeast strain D41 on the growth of Arabidopsis thaliana

[0046]

[0047] CK: control (no yeast D41 was inoculated in the co-culture system); D41: V. victoriae D41 strain.

Claims

1. A strain of Vishnus victoriae yeast, characterized in that It is the Victoria Vishniki yeast with a deposit number of CCTCC NO: M20221038 ( Vishniacozyma victoriae )D41.

2. The use of the Vishnikov yeast strain according to claim 1 in promoting plant growth and flowering, characterized in that: The plant is Arabidopsis thaliana.

3. The use according to claim 2, characterized in that The promoting of plant growth and flowering includes promoting the increase of chlorophyll content, plant weight, root weight and flowering number.

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