Psychrobacter zopfii zy-1-21 and application thereof

Abstract

The present application relates to cold bacillus ZY-1-21 and its application, belong to the field of microbial technology, the taxonomic name of the cold bacillus ZY-1-21 of the present application is Frigoribacterium sp., the preservation number is CGMCC No.35078, the preservation date is July 2, 2025, and the preservation unit is China General Microbiological Culture Collection Center.The cold bacillus ZY-1-21 of the present application is configured as a bacterial agent and irrigated in the rhizosphere of orchid, which can significantly improve the total phosphorus, soluble sugar, soluble protein, CAT, SOD and POD content of the plant and reduce the MDA content, effectively promote the growth of orchid;Through experimental verification, the strain can still play a growth-promoting effect under low-temperature stress conditions, promote the root system of dendrobium propagation seedling to be more developed, and improve the leaf crown and plant height, which can reduce the growth inhibition caused by low-temperature adversity and ensure the normal growth of orchid under low-temperature conditions.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to pyrrolizum bacillus ZY-1-21 and its applications. Background Technology

[0002] Orchids are widely used in the traditional Chinese medicine industry and ornamental horticulture. Most orchids thrive in warm environments and are sensitive to low temperatures. When subjected to prolonged low-temperature stress, seedlings accumulate large amounts of reactive oxygen species, leading to increased membrane lipid peroxidation and hindering the synthesis of osmotic regulators. This significantly inhibits normal growth, limiting large-scale understory cultivation and overwintering propagation of orchids. Current production practices primarily address low-temperature damage to orchids by using greenhouse insulation and spraying plant growth regulators. However, greenhouse cultivation is energy-intensive, and long-term application of chemical agents can damage the rhizosphere microbial community structure.

[0003] Rhizosphere functional bacteria can establish a symbiotic relationship with plant roots, alleviating abiotic stresses by regulating host osmotic regulators and antioxidant enzyme systems, representing an environmentally friendly approach. Currently, microbial agents used for orchids are mainly Bacillus and Pseudomonas, but these strains are mostly general growth-promoting bacteria and not the dominant rhizosphere or endophytic flora of orchids. Yunnan is an important suitable production area for orchids, where cultivation is mostly carried out in protected facilities or understory semi-wild models. During autumn and winter, this production area often experiences sudden, short-term low temperatures. Low-temperature stress inhibits the above-ground growth of orchid seedlings, causing wilting, inducing oxidative damage, and disrupting the metabolism of stress-related substances within the plant. Seedling growth is suppressed or even death occurs, posing a significant risk to the artificial cultivation of orchids. Therefore, starting from the rhizosphere microecology of healthy plants in the original production areas of orchids, exploring novel microbial resources that combine growth-promoting capabilities with low-temperature adaptability has significant application value for stress-resistant cultivation of orchids.

[0004] Frigoribacterium sp. is a group of Gram-negative bacteria mainly distributed in polar, deep-sea and high-altitude low-temperature environments. Its research mainly focuses on the mechanisms of adaptation to extreme environments, the development of novel enzyme systems and the discovery of bioactive substances. However, there is a relative scarcity of specific review materials on species of this genus in existing public literature databases.

[0005] To date, there are no reports, either domestically or internationally, on the isolation, colonization, or application as biofertilizer of *Microbacterium cyrtifolium* strains in orchids. The growth-promoting effects of this genus on orchids and its application prospects are entirely a technological gap. Summary of the Invention

[0006] To address the problems mentioned in the background art, this invention provides a strain of *Frigoribacterium* with significant growth-promoting effects obtained from orchid tissue. The taxonomic name of this *Frigoribacterium* ZY-1-21 is *Frigoribacterium* sp., with accession number CGMCC No. 35078, accession date July 2, 2025, and deposited at the China General Microbiological Culture Collection Center. This *Frigoribacterium* effectively increases the total phosphorus content, soluble sugar content, soluble protein content, SOD activity, CAT activity, and POD activity in orchids, or reduces MDA content, thus promoting the growth of orchids.

[0007] To achieve the above objectives, this invention provides a frostobacterium ZY-1-21 isolated from healthy Dendrobium nobile plants, taxonomically named Frigoribacterium sp., with accession number CGMCC No.35078, accession date July 2, 2025, and deposited at the China General Microbiological Culture Collection Center.

[0008] The present invention also provides a bacterial agent containing Bacillus pyriformis ZY-1-21.

[0009] The present invention also provides the application of pyrrolizobacterium ZY-1-21 or bacterial agents containing pyrrolizobacterium ZY-1-21 in the preparation of orchid growth-promoting products.

[0010] Furthermore, the orchids mentioned are Cymbidium goeringii or Dendrobium nobile.

[0011] The present invention also provides the application of pyrrolizibacillus ZY-1-21 or a bacterial agent containing pyrrolizibacillus ZY-1-21 in promoting the growth of orchids, wherein the orchids are Cymbidium or Dendrobium.

[0012] The present invention also provides a method for promoting the growth of orchids, wherein a fungal agent containing Bacillus pyriformis ZY-1-21 is applied to the rhizosphere of the orchid; wherein the orchid is Cymbidium goeringii or Dendrobium nobile.

[0013] This invention also provides the application of pyrrolizobacterium ZY-1-21 in the preparation of products that improve the cold resistance of Dendrobium.

[0014] The beneficial effects of this invention are:

[0015] The *Cryptobacterium pyridae* ZY-1-21 of this invention, when formulated as a fungal agent and applied to the rhizomes, rooted seedlings, or propagated seedlings of 'Chang Sheng Hong' and 'Zhu Sha Lan', significantly increases the content of total phosphorus, soluble sugar, soluble protein, CAT, SOD, and POD in the rhizomes, rooted seedlings, and propagated seedlings, while decreasing the content of MDA. Results from culturing rhizomes and rooted seedlings for 60 days and propagated seedlings for 90 days demonstrate that the promoting effect of *Cryptobacterium pyridae* ZY-1-21 on the rhizomes, rooted seedlings, and propagated seedlings of 'Chang Sheng Hong' and 'Zhu Sha Lan' is stable, proving that *Cryptobacterium pyridae* ZY-1-21 can significantly enhance the colonization ability of 'Chang Sheng Hong' and 'Zhu Sha Lan' and can be used as a rhizosphere growth-promoting bacterium for orchid plants.

[0016] The *Cryptobacterium pyridae* ZY-1-21 of this invention was formulated as an inoculum and applied to the rhizosphere of *Dendrobium nobile* and *Dendrobium officinale* seedlings. Growth was observed after 15, 25, and 35 days of cultivation under low-temperature stress. The results demonstrated that *Cryptobacterium pyridae* ZY-1-21 significantly promoted and protected the growth of *Dendrobium nobile* and *Dendrobium officinale* seedlings under low-temperature conditions. This promotional effect was mainly manifested in increasing plant height and crown width, increasing the content of soluble sugars and proteins in the plant, increasing the activity of CAT antioxidant enzymes, reducing MDA content, and alleviating cell membrane oxidative damage caused by low-temperature stress. It can serve as a functional microbial resource for cold-resistant and growth-promoting microbial inoculum for orchids. Attached Figure Description

[0017] Figure 1 This is a macroscopic morphological diagram of pyrrolizum bacillus ZY-1-21;

[0018] Figure 2 This is a microscopic morphological diagram of pyrrolizum bacillus ZY-1-21;

[0019] Figure 3 This is a phylogenetic tree of 16S rDNA sequences of pyrrolizobacter ZY-1-21 and related species;

[0020] Figure 4 The effect of pyrrolizum bacillus ZY-1-21 inoculum on the total phosphorus content of 'City Red' and 'Vermilion Orchid';

[0021] Figure 5 The effect of pyrrolizum bacillus ZY-1-21 inoculum on the soluble sugar content of 'Changchanghong' and 'Zhushalan' varieties;

[0022] Figure 6 The effect of pyrrolizum bacillus ZY-1-21 inoculum on the soluble protein content of 'Changchanghong' and 'Zhushalan' varieties;

[0023] Figure 7 The effect of pyrrolizum bacillus ZY-1-21 inoculum on the catalase (CAT) content of 'Changchanghong' and 'Zhushalan' varieties;

[0024] Figure 8 The effect of pyrrolizum bacillus ZY-1-21 inoculum on the superoxide dismutase (SOD) content of 'Changchanghong' and 'Zhushalan' varieties;

[0025] Figure 9 The effect of pyrrolizum bacillus ZY-1-21 inoculum on the peroxidase (POD) content of 'Changchanghong' and 'Zhushalan' varieties;

[0026] Figure 10 The effect of pyrrolizum bacillus ZY-1-21 inoculum on the malondialdehyde (MDA) content of 'Changchanghong' and 'Zhushalan' varieties;

[0027] Figure 11 This is a comparison of the plant conditions of the rhizomes in the experimental and control groups of 'Mayor Red' and 'Vermilion Orchid'.

[0028] Figure 12 This is a comparison of the plant conditions of the rooted seedlings in the experimental and control groups of 'Mayor Red' and 'Vermilion Orchid'.

[0029] Figure 13 This is a comparison of the plant conditions of the experimental and control groups of 'Mayor Red' and 'Vermilion Orchid' seedlings.

[0030] Figure 14 Effects of pyrrolizobacterium tumefaciens ZY-1-21 inoculum on plant height of Dendrobium nobile and Dendrobium officinale;

[0031] Figure 15 Effects of pyrrolizum bacillus ZY-1-21 inoculum on crown width of Dendrobium nobile and Dendrobium officinale;

[0032] Figure 16 Effects of Cynomorium frutescens ZY-1-21 inoculum on root length of Dendrobium nobile and Dendrobium officinale;

[0033] Figure 17 Effects of Cryptomylobacterium ZY-1-21 inoculum on soluble sugar content in Dendrobium nobile and Dendrobium officinale;

[0034] Figure 18 Effects of Cynomorium frutescens ZY-1-21 inoculum on soluble proteins in Dendrobium nobile and Dendrobium officinale;

[0035] Figure 19 Effects of Cynomorium cyrtogenes ZY-1-21 inoculum on catalase (CAT) in Dendrobium nobile and Dendrobium officinale;

[0036] Figure 20 Effects of Cynomorium frutescens ZY-1-21 inoculum on malondialdehyde (MDA) in Dendrobium nobile and Dendrobium officinale;

[0037] Figure 21 Comparison of growth status between the blank group and the experimental group of Dendrobium nobile experimental seedlings;

[0038] Figure 22 Comparison of growth status between the control group and the experimental group of Dendrobium officinale experimental seedlings;

[0039] Figures 4 to 10 In the image, A is 'Mayor's Red' and B is 'Vermilion Orchid'; Figures 11 to 13 In the diagram, A represents 'Vermilion Orchid', B represents 'City Red', a represents the control group, and e represents the frostbite group; Figures 14 to 20 In the image, A represents Dendrobium nobile and B represents Dendrobium officinale. Figure 21 and Figure 22 In the study, CK was the control group, HL was the cold bacillus ZY-1-21 group, and the numbers represented the number of days of stress. Detailed Implementation

[0040] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the technical solutions of this invention will be described in detail below. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are all within the scope of protection of this invention.

[0041] Example 1

[0042] The taxonomic name of the frostbite ZY-1-21 of this invention is Frigoribacterium sp., with accession number CGMCC No.35078, accession date July 2, 2025, and depositary institution is China General Microbiological Culture Collection Center.

[0043] Obtaining and identifying pyrrolizumab ZY-1-21:

[0044] Healthy *Dendrobium dentata* plants epiphytic on trees, grown in *Dendrobium* cultivation bases in Yunnan Province and free from artificial fertilization and pesticides for 5-7 years, were selected as isolation samples. Disease-free, non-browning parts were cut from the samples, rinsed with tap water for 20-30 minutes to remove surface debris, and then disinfected on a clean bench with 75% ethanol for 20-60 seconds, followed by disinfection with 0.1% mercuric chloride solution (or 3% NaClO) for 8-15 minutes. The samples were then rinsed 6-10 times with sterile water, and excess moisture was absorbed with sterile paper. The cleaned samples were then cut into 0.2-2 cm pieces using a sterile scalpel. 3 The cut samples were transferred to SDA medium and cultured at 25-38℃ for 6 days. After three repeated purification cultures, a pure culture was obtained and named strain ZY-1-21.

[0045] The strain ZY-1-21 was sent to the China Industrial Microbial Culture Collection Center of China National Research Institute of Food Fermentation Industries for identification. It was identified as *Frigoribacterium* sp. using the method FMIC-QO01-001-2015 Microbiological Detection Method for Multiphase Identification of Bacteria.

[0046] Strain ZY-1-21 was cultured in TSA medium at 25°C for 72 h. The macroscopic morphology of the strain is as follows: Figure 1 As shown: Colonies are yellow, round, moist, opaque, and have neat edges.

[0047] Gram staining results: Gram-positive, bacteria are rod-shaped, 0.5-0.7 μm × 1.1-2.0 μm, arranged singly or in pairs, microscopic morphology as shown in the attached figure. Figure 2 As shown.

[0048] The 16S rDNA of *Cryptospira* ZY-1-21 was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. A phylogenetic tree was constructed using MEGA software and the adjacent-position ligation method. After 1000 similarity replicates, the phylogenetic tree of ZY-1-21 and related species' 16S rDNA sequences was obtained as follows: Figure 3 As shown in the figure, the phylogenetic tree nodes only display Bootstrap values ​​greater than 50%, and the superscript "T" indicates the type strain. The results indicate that, based on the 16S rDNA sequence phylogenetic tree, *Cryptobacterium* ZY-1-21 and *Frigoribacterium faeni* NBRC 103066... T (BJUY01000064) clustered on the same branch. Based on the morphological and molecular biological identification results, strain ZY-1-21 was identified as belonging to the genus Frigoribacterium sp.

[0049] Example 2

[0050] The growth-promoting effect of Cymbidium falciparum ZY-1-21 on Cymbidium goeringii

[0051] Preparation of bacterial suspension: (1) Inoculate glycerol-preserved *Microbacterium cyrtifolium* ZY-1-21 onto YEA medium and culture at 25-30℃ for 2-3 days; (2) Pick a single colony from the well-grown *Microbacterium cyrtifolium* ZY-1-21 on YEA medium and inoculate it into YEB liquid medium, culture at 25-30℃ and 250 rpm for 2-3 days; (3) Take an appropriate amount of bacterial suspension and adjust the content of *Microbacterium cyrtifolium* ZY-1-21 to 1×10⁻⁶ with sterile water. 5 cfu / mL ~3×10 5 The desired bacterial culture was obtained by dispensing cfu / mL.

[0052] Experiment on the growth-promoting effect of Cymbidium goeringii:

[0053] Experimental group: Two Cymbidium varieties, 'Changcheng Hong' and 'Zhusha Lan', were selected. 15 mL of the above-mentioned bacterial solution was applied to the rhizomes, rooted seedlings (bottle seedlings), and propagated seedlings (potted seedlings) of both varieties, once every 7 days, for a total of 3 applications. Control group (CK): An equal volume of YEB liquid culture medium diluted with sterile water was applied to the rhizomes, rooted seedlings (bottle seedlings), and propagated seedlings (potted seedlings) of both varieties, once every 7 days, for a total of 3 applications. Rhizomes and rooted seedlings were cultured for 60 days, and propagated seedlings for 90 days before growth was measured.

[0054] Growth status measurement results:

[0055] (1) Determination of total phosphorus content

[0056] A tissue total phosphorus content detection kit was used. The kit was purchased from Beijing Box Biotechnology Co., Ltd., catalog number: AKIC003C, specification: 60T / 50S; specific procedures were followed according to the kit instructions. After treatment with *Microbacterium cyrtifolium* ZY-1-21, the total phosphorus content in the rhizomes, rooted seedlings, and propagated seedlings of 'Changcheng Hong' and 'Zhusha Lan' was significantly increased in all three growth stages. The results are as follows: Figure 4 (In the figure, A represents Mayor Red, and B represents Cinnabar Orchid.) Among them, Mayor Red: the total phosphorus of the rhizome increased by 116.6% (calculation method: (0.01297-0.0061) / 0.0061*100%), the total phosphorus of the rooted seedlings increased by 93.2%, and the total phosphorus of the propagated seedlings increased by 109.3%; Cinnabar Orchid: the total phosphorus of the rhizome increased by 135.1%, the total phosphorus of the rooted seedlings increased by 74.1%, and the total phosphorus of the propagated seedlings increased by 99.2%.

[0057] (2) Determination of soluble sugar content

[0058] A plant soluble sugar content detection kit was used. The kit was purchased from Beijing Box Biotechnology Co., Ltd., catalog number: AKPL008C, specification: 70T / 50S; specific procedures were followed according to the kit instructions. The results of soluble sugar content determination for different treatments are shown below. Figure 5 (As shown in the figure, A represents 'City Red' and B represents 'Cinnabar Orchid'.) Treatment with *Bacillus thuringiensis* ZY-1-21 significantly increased the soluble sugar content of rhizomes, rooted seedlings, and propagated seedlings of both 'City Red' and 'Cinnabar Orchid'. Specifically, for 'City Red', the soluble sugar content increased by 55.3% in rhizomes, 36.0% in rooted seedlings, and 60.7% in propagated seedlings; for 'Cinnabar Orchid', the soluble sugar content increased by 47.1% in rhizomes, 20.9% in rooted seedlings, and 48.7% in propagated seedlings.

[0059] (3) Determination of soluble protein content

[0060] The Bradford method protein content assay kit was used. The kit was purchased from Beijing Box Biotechnology Co., Ltd., catalog number: AKPR015, specification: 500T (2500T Micropore). Detailed procedures were followed according to the kit instructions. Results of soluble protein content determination under different treatments are shown below. Figure 6 (As shown in the figure, A represents 'City Red' and B represents 'Cinnabar Orchid'.) Treatment with *Microbacterium frutescens* ZY-1-21 significantly increased the soluble protein content of rhizomes, rooted seedlings, and propagated seedlings of both 'City Red' and 'Cinnabar Orchid'. Specifically, for 'City Red': the soluble protein content of rhizomes increased by 46.0%, that of rooted seedlings increased by 25.1%, and that of propagated seedlings increased by 82.4%; for 'Cinnabar Orchid': the soluble protein content of rhizomes increased by 39.8%, that of rooted seedlings increased by 15.6%, and that of propagated seedlings increased by 78.7%.

[0061] (4) Catalase (CAT) assay

[0062] The catalase (CAT) activity assay kit (ammonium molybdate colorimetric method) was used. The kit was purchased from Beijing Box Biotechnology Co., Ltd., catalog number: AKAO003-2C, specification: 120T / 50S; specific procedures were followed according to the kit instructions. CAT assay results for different treatments are shown below. Figure 7 (As shown in the figure, A represents *City Red* and B represents *Cinnamomum camphora*). Treatment with *Microbacterium tumefaciens* ZY-1-21 significantly increased the CAT content in the rhizomes, rooted seedlings, and propagated seedlings of both *City Red* and *Cinnamomum camphora*. Specifically, for *City Red*: CAT increased by 38.9% in rhizomes, 45.3% in rooted seedlings, and 40.3% in propagated seedlings; for *Cinnamomum camphora*: CAT increased by 34.5% in rhizomes, 54.4% in rooted seedlings, and 58.9% in propagated seedlings.

[0063] (5) Superoxide dismutase (SOD) assay

[0064] Superoxide dismutase (SOD) activity assay kits were used. The kits were purchased from Beijing Box Biotechnology Co., Ltd., catalog number: AKAO001C-50S, specification: 110T / 50S; specific procedures were followed according to the kit instructions. SOD measurement results for different treatments are shown below. Figure 8(As shown in the figure, A represents *City Red* and B represents *Cinnamomum camphora*). Treatment with *Microbacterium tumefaciens* ZY-1-21 significantly increased the SOD content in the rhizomes, rooted seedlings, and propagated seedlings of both *City Red* and *Cinnamomum camphora*. Specifically, for *City Red*: SOD increased by 48.4% in rhizomes, 55.1% in rooted seedlings, and 70.9% in propagated seedlings; for *Cinnamomum camphora*: SOD increased by 38.3% in rhizomes, 47.5% in rooted seedlings, and 63.1% in propagated seedlings.

[0065] (6) Peroxidase (POD) assay

[0066] The peroxidase (POD) activity assay kit (catalog number: AKAO005C, specification: 60T / 50S) was used; detailed procedures were followed according to the kit instructions. POD assay results for different treatments are shown below. Figure 9 (As shown in the figure, A represents *City Red* and B represents *Cinnamomum camphora*). Treatment with *Microbacterium tumefaciens* ZY-1-21 significantly increased the POD content in the rhizomes, rooted seedlings, and propagated seedlings of both *City Red* and *Cinnamomum camphora*. Specifically, for *City Red*: POD increased by 60.3% in rhizomes, 72.8% in rooted seedlings, and 77.7% in propagated seedlings; for *Cinnamomum camphora*: POD increased by 87.0% in rhizomes, 80.0% in rooted seedlings, and 54.3% in propagated seedlings.

[0067] (7) Determination of malondialdehyde (MDA)

[0068] Malondialdehyde (MDA) content detection kit was used. The kit was purchased from Beijing Box Biotechnology Co., Ltd., catalog number: AKFA013C, specification: 60T / 50S; specific procedures were followed according to the kit instructions. MDA determination results for different treatments are shown below. Figure 10 (As shown in the figure, A represents 'City Red' and B represents 'Cinnabar Orchid'.) After treatment with *Microbacterium frutescens* ZY-1-21, the MDA content of rhizomes, rooted seedlings, and propagated seedlings of both 'City Red' and 'Cinnabar Orchid' was significantly reduced. Specifically, for 'City Red': MDA content in rhizomes decreased by 19.7%, in rooted seedlings by 32.0%, and in propagated seedlings by 24.1%; for 'Cinnabar Orchid': MDA content in rhizomes decreased by 26.1%, in rooted seedlings by 26.8%, and in propagated seedlings by 31.7%.

[0069] (8) Comparison of growth status of rhizomes, rooted seedlings (bottle seedlings) and propagated seedlings (pot seedlings)

[0070] The plant condition with rhizomes is shown in the attached figure. Figure 11 (A in the picture is Cinnamomum camphora, B in the picture is Cinnamomum cassia) as shown; the plant condition of the rooted seedlings is as attached. Figure 12 (A in the picture is Cinnamomum camphora, B in the picture is Cinnamomum cassia) as shown; the plant condition of the propagated seedlings is as attached. Figure 13 (As shown in the picture, A is Cinnabar Orchid and B is Mayor's Red). Figures 11 to 13 In the figure, a represents the control group and e represents the cytokine bacillus group.

[0071] pass Figures 11 to 13 It is evident that the bacterial solution containing Bacillus pyriformis ZY-1-21 has a significant growth-promoting effect on the rhizomes, rooted seedlings (bottle seedlings), and propagated seedlings (pot seedlings) of Cinnamomum camphora and Cinnamomum cassia.

[0072] Example 3

[0073] Effects of pyrrolizobacterium pyrrolizum (ZY-1-21) on Dendrobium growth under low temperature conditions

[0074] Experimental group: Dendrobium species were selected as Dendrobium nobile and Dendrobium officinale. 15 mL of the bacterial solution prepared in Example 2 was poured into the rhizosphere of the propagated seedlings of both species. After 10 days of incubation at room temperature, whole-plant samples were collected to determine their growth status. The propagated seedlings of the experimental group were then transferred to a low-temperature (10℃) environment and incubated for 15, 25, and 35 days, after which whole-plant samples were collected to determine their growth status.

[0075] Control group (CK): Equal volumes of YEB liquid culture medium diluted with sterile water were applied to the rhizosphere of the propagated seedlings of both types of Dendrobium. After 10 days of incubation at room temperature, whole-plant samples were collected to determine their growth status. The propagated seedlings of the control group were transferred to a low-temperature (10℃) environment and incubated for 15, 25, and 35 days, after which whole-plant samples were collected to determine their growth status.

[0076] (1) Plant height

[0077] Plant height results for different treatments, as follows Figure 14(Figure A represents *Dendrobium nobile*, and Figure B represents *Dendrobium officinale*). Under low-temperature stress, the cold-resistant bacterium ZY-1-21 significantly promoted the growth of *Dendrobium nobile* and *Dendrobium officinale* seedlings. In the initial sampling (0 day) after 10 days of pre-culture at room temperature, the seedlings of both *Dendrobium* species inoculated with ZY-1-21 were significantly taller than the control group. Specifically, the height of *Dendrobium nobile* seedlings increased by 182.6%, and that of *Dendrobium officinale* seedlings increased by 99.2%. The strain promoted longitudinal elongation of *Dendrobium* even at room temperature. After being transferred to a 10℃ low-temperature environment, with increasing stress time, the plant height of the control group was continuously inhibited by low temperature, while the plant height of the ZY-1-21 treatment group remained consistently higher than that of the control group. After 15 days of low-temperature treatment, the plant height of *Dendrobium nobile* increased by 136.4%, significantly higher than the control (CK), while the plant height of *Dendrobium officinale* increased by 17.2%, with no significant difference between the groups. After 25 days of low-temperature treatment, the plant height of the ZY-1-21 treatment groups reached their peak, with *Dendrobium nobile* increasing by 146.7% and *Dendrobium officinale* by 88.5%, both significantly higher than the control group. When the low-temperature treatment continued for 35 days, the plant height of *Dendrobium nobile* increased by 220.8% and *Dendrobium officinale* by 92.1%, and the plant height of both species remained significantly better than the blank control after inoculation with the strain. In summary, *Cryptobacter frutescens* ZY-1-21 can effectively alleviate the inhibition of low-temperature stress on the growth of *Dendrobium nobile* and *Dendrobium officinale* seedlings and promote longitudinal elongation of the plants.

[0078] Note: The low-temperature experiment of this invention is a destructive sampling method. The results of 0d, 15d, 25d, and 35d are the test results of different plants, and the same applies below.

[0079] (2) Crown width

[0080] The results are as follows Figure 15(Figure A represents *Dendrobium nobile*, and Figure B represents *Dendrobium officinale*). Under low-temperature stress, the cold-resistant bacterium ZY-1-21 significantly promoted the crown growth of *Dendrobium nobile* and *Dendrobium officinale* seedlings. In the initial sampling (0 day) after 10 days of pre-culture at room temperature, the crown width of both *Dendrobium* seedlings inoculated with ZY-1-21 was lower than that of the control group. It is speculated that the strain preferentially promoted the longitudinal height growth of the plant in the early stage of rhizosphere colonization, producing a short-term inhibitory effect on crown width. The longitudinal growth of the plant was dominant, thus reducing the crown width. After being transferred to a 10℃ low-temperature environment, with the increase of stress time, the crown width of the plants in the ZY-1-21 treatment group gradually exceeded that of the control group. After 15 days of low-temperature treatment, the crown width of *Dendrobium officinale* increased by 48.6%, significantly higher than the control (CK), while that of *Dendrobium nobile* increased by 32.6%, with no significant difference between groups. After 25 days of low-temperature treatment, the crown width of all groups treated with ZY-1-21 reached its peak, with *Dendrobium nobile* increasing by 107.3% and *Dendrobium officinale* by 87.0%, both significantly higher than the control group. After 35 days of continued low-temperature treatment, the growth-promoting effect slightly decreased, with *Dendrobium nobile* crown width increasing by 97.4% and *Dendrobium officinale* by 34.7%, but the crown width of both *Dendrobium* species inoculated with the strain was still significantly better than the blank control. In summary, *Cryptobacter frutescens* ZY-1-21 can effectively alleviate the inhibition of *Dendrobium nobile* and *Dendrobium officinale* seedling growth under 10℃ low-temperature stress, promoting crown expansion and verifying the growth-promoting potential of this strain on *Dendrobium* under low-temperature conditions.

[0081] (3) Root length

[0082] The results are as follows Figure 16(Figure A represents *Dendrobium nobile*, and Figure B represents *Dendrobium officinale*.) As shown, the cold-resistant bacterium ZY-1-21 significantly promoted root growth in *Dendrobium nobile* and *Dendrobium officinale* seedlings under low-temperature stress. In the initial sampling (0 day) after 10 days of pre-culture at room temperature, the root length of *Dendrobium nobile* seedlings inoculated with ZY-1-21 increased by 109.1%, a highly significant difference, while the root length of the *Dendrobium officinale* treatment group increased by 12.1%. After being transferred to a 10℃ low-temperature environment, the root length of the plants in the ZY-1-21 treatment group remained higher than that of the control group with increasing stress time. After 15 days of low-temperature treatment, the root length of *Dendrobium nobile* in the ZY-1-21 treatment group increased by 41.8%, and that of *Dendrobium officinale* increased by 93.2%. After 25 days of low-temperature treatment, the root length of *Dendrobium nobile* in the ZY-1-21 treatment group increased by 92.8%, and that of *Dendrobium officinale* increased by 74.3%, both significantly higher than the control group. After the low-temperature treatment continued for 35 days, the root length of *Dendrobium nobile* increased by 90.3%, significantly higher than the control group, and the root length of *Dendrobium officinale* increased by 72.6%, highly significantly higher than the control group. The results show that *Cryptospira* ZY-1-21 can effectively alleviate the inhibition of root growth in *Dendrobium nobile* and *Dendrobium officinale* seedlings under 10℃ low-temperature stress, and promote root elongation, verifying the growth-promoting potential of this strain on *Dendrobium* under low-temperature conditions. Moreover, the root-promoting effect of the strain can be observed in *Dendrobium nobile* at room temperature, while the significant root-promoting effect of *Dendrobium officinale* gradually appears with the duration of low-temperature treatment.

[0083] (4) Soluble sugar content

[0084] A plant soluble sugar content detection kit was used. The kit was purchased from Beijing Box Biotechnology Co., Ltd., product number: AKPL008C, specification: 70T / 50S; the specific procedure was followed according to the kit instructions. Results are as follows: Figure 17(Figure A represents *Dendrobium nobile*, and Figure B represents *Dendrobium officinale*.) As shown, the cold-resistant bacterium ZY-1-21 has a regulatory effect on the accumulation of soluble sugars in *Dendrobium nobile* and *Dendrobium officinale* seedlings under low-temperature stress. In the initial sampling (0 day) after 10 days of pre-culture at room temperature, the soluble sugar content of *Dendrobium nobile* seedlings inoculated with ZY-1-21 increased by 93.0%, which was significantly higher than that of the control group, while the soluble sugar content of the *Dendrobium officinale* treatment group increased by 12.7%. After being transferred to a low-temperature environment of 10℃, with the increase of stress time, the soluble sugar content of plants in the ZY-1-21 treatment group was generally higher than that of the control group. After 15 days of low-temperature treatment, the soluble sugar content of *Dendrobium nobile* in the ZY-1-21 treatment group increased by 75.8%, which was significantly higher than that of the control group, while the soluble sugar content of *Dendrobium officinale* increased by 3.6%. After 25 days of low-temperature treatment, the soluble sugar content of *Dendrobium nobile* in the ZY-1-21 treatment group increased by 169.3%, which was significantly higher than that of the control group, while the soluble sugar content of *Dendrobium officinale* increased by 55.9%. After the low-temperature treatment continued for 35 days, the soluble sugar content of *Dendrobium nobile* increased by 36.3%, which was significantly higher than that of the control group, while the levels of *Dendrobium officinale* in the treatment group were similar to those in the control group. In summary, *Cryptospira* ZY-1-21 can effectively induce the continuous accumulation of soluble sugars in *Dendrobium nobile* seedlings, enhance osmotic regulation, and reduce the damage caused by 10℃ low-temperature stress. *Dendrobium officinale* only showed a significant enhancement effect after 25 days of low-temperature treatment, verifying species-specific differences in the regulation of soluble sugar accumulation by this strain. The induction effect of the strain was evident in *Dendrobium nobile* at room temperature, while the significant enhancement effect of soluble sugars in *Dendrobium officinale* only appeared in the middle of the low-temperature treatment.

[0085] (5) Determination of soluble protein content

[0086] The Bradford method protein content assay kit was used. The kit was purchased from Beijing Box Biotechnology Co., Ltd., catalog number: AKPR015, specification: 500T (2500T Micropore). Detailed procedures were followed according to the kit instructions. Results of soluble protein content determination under different treatments are shown below. Figure 18(Figure A represents *Dendrobium nobile*, and Figure B represents *Dendrobium officinale*). Under low-temperature stress, the cold-resistant bacterium ZY-1-21 exhibits a differential regulatory effect on the accumulation of soluble proteins in *Dendrobium nobile* and *Dendrobium officinale* seedlings. In the initial sampling (0 day) after 10 days of pre-culture at room temperature, the soluble protein content of *Dendrobium nobile* seedlings inoculated with ZY-1-21 increased by 16.4%, significantly higher than the control group, while the soluble protein content of the *Dendrobium officinale* treatment groups showed no significant difference. After being transferred to a 10℃ low-temperature environment, with increasing stress time, the soluble protein content of the strain treatment groups showed different changes among species. After 15 days of low-temperature treatment, there was no significant difference in soluble protein between the Dendrobium nobile ZY-1-21 treatment group and the control group, while the soluble protein in the Dendrobium officinale treatment group increased by 18.3%, significantly higher than the control group. After 25 days of low-temperature treatment, the soluble protein in the Dendrobium nobile ZY-1-21 treatment group increased by 16.9%, extremely significantly higher than the control group, while the soluble protein in the Dendrobium officinale treatment group was lower than the control group. After the low-temperature treatment continued for 35 days, there was no significant difference between the Dendrobium nobile strain treatment group and the control group, while the soluble protein in the Dendrobium officinale treatment group increased by 6.9%. In summary, *Cryptospira* ZY-1-21 can regulate the metabolism of soluble proteins in both Dendrobium species to cope with low-temperature stress, but the response patterns show significant species differences. *Dendrobium nobile* showed a strain-induced effect at room temperature, with a significant increase only after 25 days of low temperature. *Dendrobium officinale* showed a significant upregulation of soluble proteins under long-term low-temperature stress (15 days and 35 days), with a transient decrease after 25 days of low temperature.

[0087] (6) Catalase (CAT) assay

[0088] The catalase (CAT) activity assay kit (ammonium molybdate colorimetric method) was used. The kit was purchased from Beijing Box Biotechnology Co., Ltd., catalog number: AKAO003-2C, specification: 120T / 50S; specific procedures were followed according to the kit instructions. Results are as follows: Figure 19(A in the figure represents *Dendrobium nobile*, and B represents *Dendrobium officinale*). Under low-temperature stress, the cold-resistant bacterium ZY-1-21 exhibited a differential regulatory effect on the CAT activity of *Dendrobium nobile* and *Dendrobium officinale* seedlings. In the initial sampling (0 day) after 10 days of pre-culture at room temperature, the CAT activity of *Dendrobium officinale* inoculated with ZY-1-21 increased by 18.7%, while the difference in CAT activity of *Dendrobium nobile* was not significant. After being transferred to a 10℃ low-temperature environment, the changes in CAT activity among the strain treatment groups showed species-specific differences with increasing stress time. After 15 days of low-temperature treatment, the CAT activity in the *Dendrobium nobile* ZY-1-21 treatment group decreased, while the CAT activity in the *Dendrobium officinale* treatment group increased by 25.2%. After 25 days of low-temperature treatment, the CAT activity in the *Dendrobium nobile* ZY-1-21 treatment group increased by 16.6%, and the CAT activity in the *Dendrobium officinale* treatment group increased by 9.6%. After the low-temperature treatment continued for 35 days, the CAT activities of both the *Dendrobium nobile* and *Dendrobium officinale* treatment groups were significantly higher than the blank control, with the CAT activity in the *Dendrobium nobile* ZY-1-21 treatment group increasing by 29.2% and the CAT activity in the *Dendrobium officinale* treatment group increasing by 28.8%. In summary, the results show that *Cryptospira* ZY-1-21 can regulate the CAT antioxidant system of two Dendrobium species in response to low-temperature oxidative stress, but the response patterns show significant species differences. Under long-term low-temperature stress (35 days), the strain can significantly enhance the CAT activity of both Dendrobium species, improve hydrogen peroxide scavenging ability, and reduce low-temperature-induced oxidative damage. In the early stage of low temperature (15 days), the CAT activity of *Dendrobium nobile* was temporarily inhibited, while the CAT activity of *Dendrobium officinale* was upregulated in the early stage of low temperature.

[0089] (7) Determination of malondialdehyde (MDA)

[0090] MDA is the end product of lipid peroxidation and reflects the degree of cell membrane damage. The lower the MDA content, the better the membrane structure integrity. If the inoculated strain can reduce the MDA content, it proves that it has alleviated membrane damage caused by low temperature.

[0091] The malondialdehyde (MDA) content detection kit was used. The kit was purchased from Beijing Box Biotechnology Co., Ltd., catalog number: AKFA013C, specification: 60T / 50S; specific procedures were followed according to the kit instructions. Results are as follows: Figure 20(A in the figure represents *Dendrobium nobile*, and B represents *Dendrobium officinale*). Under low-temperature stress, the cold-resistant bacterium ZY-1-21 exhibited a differential regulatory effect on the MDA content of *Dendrobium nobile* and *Dendrobium officinale* seedlings. In the initial sampling (0 day) after 10 days of pre-culture at room temperature, the MDA content of both *Dendrobium nobile* and *Dendrobium officinale* seedlings inoculated with ZY-1-21 was higher than that of the control group. After being transferred to a 10℃ low-temperature environment, the response patterns of MDA in the two *Dendrobium* species to the strain treatment differed with increasing stress time. After 15 days of low-temperature treatment, the MDA content of the Dendrobium nobile ZY-1-21 treatment group decreased by 5.6%, and the MDA content of the Dendrobium officinale treatment group decreased by 53.6%, both significantly lower than the control group. After 25 days of low-temperature treatment, the MDA content of the Dendrobium nobile ZY-1-21 treatment group decreased by 41.9%, significantly lower than the control group, while the MDA content of the Dendrobium officinale treatment group was slightly higher than the control group, with no significant difference between the groups. After the low-temperature treatment continued for 35 days, the MDA levels of the Dendrobium nobile strain treatment group and the control group were similar, with no significant difference, while the MDA content of the Dendrobium officinale treatment group was significantly higher than the blank control. The results showed that *Cryptobacter cytogenes* ZY-1-21 significantly reduced the MDA content of both Dendrobium species during the early stage of low-temperature treatment (15 days), alleviating low-temperature-induced lipid peroxidation damage to the cell membrane. However, this protective effect was time-dependent and species-specific. The alleviating effect of *Dendrobium nobile* lasted until 25 days of low-temperature treatment, while *Dendrobium officinale* showed a reduction in membrane damage after 15 days of low-temperature treatment.

[0092] (8) Comparison of growth status of Dendrobium nobile and Dendrobium officinale seedlings in the control and experimental groups under low temperature stress

[0093] Figure 21 This study compares the growth status of Dendrobium nobile seedlings in the *Cryptobacter pyridostellaria heterophylla* ZY-1-21 group and the control group under a low-temperature condition of 10℃. In the figure, CK represents the control group, HL represents the *Cryptobacter pyridostellaria heterophylla* ZY-1-21 group, and the numbers represent the number of days of stress. The comparison shows that under low-temperature conditions, *Cryptobacter pyridostellaria heterophylla* ZY-1-21 can promote the development of Dendrobium nobile seedlings. Specifically, the inoculated plants have better root development, are more robust, and their leaves remain fully expanded. In contrast, the control group seedlings are prone to leaf wilting and root decay after low-temperature stress. This indicates that inoculation with ZY-1-21 can alleviate the damage of low temperature to Dendrobium nobile seedlings and maintain the normal development of the plants.

[0094] Figure 22This study compares the growth status of Dendrobium officinale seedlings in the *Cryptospira pyrenoidosa* ZY-1-21 group and the control group under a low temperature of 10℃. In the figure, CK represents the control group, HL represents the *Cryptospira pyrenoidosa* ZY-1-21 group, and the numbers represent the number of days of stress. The comparison shows that under low temperature conditions, *Cryptospira pyrenoidosa* ZY-1-21 can promote the development of Dendrobium officinale seedlings, specifically manifested in more developed root systems, better plant growth, and better leaf integrity than the control group.

[0095] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. *Cryptospira* ZY-1-21, characterized in that... The taxonomic name of the frostbite ZY-1-21 is Frigoribacterium sp., with accession number CGMCC No.35078, accession date July 2, 2025, and depositary institution is China General Microbiological Culture Collection Center.

2. A microbial agent, characterized in that, It contains the pyrrolizum bacillus ZY-1-21 as described in claim 1.

3. The application of the pyrrolizobacterium ZY-1-21 as described in claim 1 or the bacterial agent as described in claim 2 in the preparation of orchid growth-promoting products.

4. The application according to claim 3, characterized in that, The orchids mentioned are Cymbidium goeringii or Dendrobium nobile.

5. The application of the pyrrolizobacterium ZY-1-21 as described in claim 1 or the bacterial agent as described in claim 2 in promoting the growth of orchids, wherein the orchid is Cymbidium goeringii or Dendrobium nobile.

6. A method for promoting orchid growth, characterized in that, The fungal agent according to claim 2 is applied to the rhizosphere of orchids; the orchids are Cymbidium or Dendrobium.

7. The use of the pyrrolizobacterium ZY-1-21 as described in claim 1 in the preparation of products that improve the cold resistance of Dendrobium.

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