A strain of chalcogenide and its application

Abstract

This invention provides a *Ceratophyllum demersum* strain and its applications. The *Ceratophyllum demersum* strain has the accession number CCTCC NO: M20252398 at the China Center for Type Culture Collection, with a deposit date of October 30, 2025. The *Ceratophyllum demersum* strain of this invention can synergistically promote the multi-stage development of *Cymbidium* without requiring the replacement of different fungi, simplifying the propagation process; it reduces competition between fungal species and symbiotic instability caused by environmental factors, improving the consistency of germination rate and seedling survival rate. It eliminates the need for multi-strain staged inoculation and culture management, reducing operational steps and costs, making it suitable for greenhouse seedling cultivation and large-scale propagation.

Description

Technical Field

[0001] This invention relates to the field of plant microbial technology, and in particular to a chalcogenide strain and its application. Background Technology

[0002] Orchid seeds lack endosperm and the nutrients necessary for germination, making them unable to germinate independently under natural conditions. Therefore, during germination, they rely on specific symbiotic fungi to provide carbon, nitrogen, and phosphorus sources to activate protocorm formation and subsequent development. Beyond the germination stage, the entire growth and development of orchids is profoundly influenced by fungal symbiosis, requiring continuous nutrient support from fungi from seedling to mature plant.

[0003] However, existing research indicates that the symbiotic relationship between orchids and mycorrhizal fungi is not only highly species-specific but also exhibits significant developmental stage specificity. While some fungi can induce seed germination, they cannot maintain seedling growth or support the morphogenesis of mature plants, leading to a clear functional differentiation between germination-promoting and growth-promoting fungi. This characteristic means that orchids often rely on the dynamic replacement of symbiotic fungi at different stages throughout their natural life cycle. Current artificial propagation strategies mainly attempt to achieve a "multi-species relay" symbiosis by replacing different fungi at different stages, with each fungus undertaking germination-promoting and growth-promoting functions respectively. However, this approach has significant limitations: on the one hand, it requires phased management of the isolation, domestication, cultivation, and inoculation of different microorganisms, making the operation process complex and technically demanding, and difficult to implement stably under large-scale production conditions; on the other hand, the coexistence of multiple fungal groups is prone to competitive exclusion effects or unstable interactions, resulting in poor consistency of the symbiotic system, significant fluctuations in germination and seedling rates, and difficulty in establishing a long-term reliable propagation system. The aforementioned shortcomings have, to some extent, limited the promotion and application of artificial propagation techniques for orchids, and also highlighted the necessity of developing single-strain, multi-stage compatible, or highly efficient growth-promoting strains.

[0004] As an endemic species of the East Asian-South Asian biota, *Cymbidium lancifolium* is commonly found in urban green spaces and natural habitats. However, due to habitat fragmentation caused by urbanization and excessive harvesting, the wild population of this species continues to decline. At the same time, its high dependence on specific symbiotic fungi makes natural regeneration difficult, and a mature artificial breeding system has not yet been established, making conservation and restoration efforts significantly challenging.

[0005] Therefore, screening for symbiotic fungal resources that can promote the germination and growth of Cymbidium goeringii and constructing an artificial culture system suitable for its life cycle is of great significance for improving the efficiency of artificial breeding and enhancing the population recovery potential. Summary of the Invention

[0006] In view of the deficiencies in the prior art, the present invention proposes a hornbacterium strain and its application.

[0007] This invention provides a strain of *Castrolabebrion*, which has the accession number CCTCC NO: M20252398 at the China Center for Type Culture Collection and the accession date is October 30, 2025.

[0008] The application used Ceratobasidium strain ZSP3-1 was deposited on October 30, 2025, at the China Center for Type Culture Collection (CCTCC, address: Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, 430072, China). The strain name is... Ceratobasidium sp. ZSP3-1, classified as Ceratobasidium sp.

[0009] The present invention also provides the application of the aforementioned hornbacterium strain in promoting the growth and development of Cymbidium goeringii.

[0010] In some embodiments, the growth and development includes: seed germination, protocorm formation, seedling growth, root development, aboveground growth, and flower bud differentiation and flowering.

[0011] In some embodiments, the application involves symbiotic culture of the aforementioned chalcogenide strain with Cymbidium goeringii seeds.

[0012] The present invention also provides a method for promoting the germination of orchid seeds, comprising the following steps:

[0013] (1) Preparation of culture medium;

[0014] (2) Inoculate the aforementioned chalcogenide strain into the culture medium;

[0015] (3) Perform surface disinfection treatment on the seeds of Cymbidium goeringii;

[0016] (4) Place the sterilized Cymbidium goeringii seeds on the culture medium containing the Cymbidium spp. for symbiotic culture.

[0017] In some embodiments, the culture medium is OMA medium, MS medium, or soil-based substrate.

[0018] In some embodiments, the substrate-containing soil substrate is a combination of coconut coir and sawdust.

[0019] The present invention also provides the application of the aforementioned chalcogenide strain in the preparation of Cymbidium faberi culture system.

[0020] In summary, compared with the prior art, the present invention achieves the following technical effects:

[0021] (1) The ZSP3-1 of the present invention can synergistically promote the multi-stage development of Cymbidium goeringii without the need to replace different fungi, simplifying the breeding process; it reduces the competition between fungal species and the symbiotic instability caused by environmental factors, and improves the consistency of germination rate and seedling rate. It eliminates the need for multi-species staged inoculation and culture management, reduces operation links and costs, and is suitable for greenhouse seedling cultivation and large-scale breeding.

[0022] (2) The ZSP3-1 of the present invention can achieve effective symbiosis in solid or soil substrates and has broad application prospects in the fields of resource protection, garden seedling cultivation and species reintroduction. Attached Figure Description

[0023] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 This is a colony diagram of ZSP3-1 of the present invention on a plate.

[0025] Figure 2 This is a mycelial diagram of ZSP3-1 of the present invention.

[0026] Figure 3 To verify the promoting effect of ZSP3-1 on Cymbidium goeringii using the plate culture method of this invention.

[0027] Figure 4 To verify the fungal infection of the base (60 days) and roots of seedlings (90 days) using tsanthemum blue staining, this invention was developed.

[0028] Figure 5 The soil-seeding method of this invention was used to cultivate protocorms (30 days) and seedlings (200 days) of Cymbidium goeringii.

[0029] Figure 6 This is to verify the specificity of the ZSP3-1 of the present invention for the orchid.

[0030] Figure 7 The biomass of *Cymbidium goeringii* after ZSP treatment for 90d, 180d, 270d, and 360d according to this invention. Detailed Implementation

[0031] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0032] 1. Obtaining Cymbidium goeringii protocorms through in-situ symbiotic germination

[0033] The seeds were placed in a lawn area with *Cymbidium goeringii* on the Natural Sciences campus of Sun Yat-sen University, Shenzhen. 4cm x 4cm seed packets were made of nylon fabric. The experiment was conducted during the rainy season. The day after the rain, approximately 200 *Cymbidium goeringii* seeds were placed in the seed packets and shallowly buried (approximately 1.5–2.5cm deep) in the topsoil of the lawn within a 30cm radius around the target *Cymbidium goeringii* plants. The soil was then backfilled with topsoil and turf. A marker was placed near the burial site for later retrieval. The burial period was set at three months for retrieval and verification. The recovered germinated individuals (protocorms or early seedlings) were immediately taken back to the laboratory for symbiotic fungal isolation, or stored at 4°C for later isolation.

[0034] 2. Isolation, pure culture, and identification of endophytic fungi

[0035] (1) Sample processing and separation:

[0036] Following the procedure described by Bayman et al. (2016), the collected Cymbidium protocorms were thoroughly washed under running tap water to remove any attached material such as mud and sand. Surface disinfection was then performed using a 1% NaClO solution for approximately 10 minutes. After disinfection, small tissue fragments containing fungal hyphae clusters in the cortex were grasped and separated using sterile forceps under a microscope.

[0037] (2) Purification, culture and preservation:

[0038] The isolated cortical tissue fragments were agar-agar (PDA) plates. The plates were incubated at 25°C in the dark. After 3–7 days, newly formed hyphae from the edges of single colonies on the plates were selected and transferred to new PDA plates for streak isolation and purification. The purification process was repeated at least three times to ensure the acquisition of a pure strain free from contamination. The purified strain was then transferred to PDA slant plates and stored at 4°C, named ZSP3-1. Figure 1 The image shows the colonies of ZSP3-1 on a plate, which are round, white to pale yellow colonies with neat edges and a fluffy texture.

[0039] (3) Morphological characteristics:

[0040] Morphological analysis was first performed on strain ZSP3-1. Detailed records of the ZSP3-1 colony phenotype (color, morphology, texture, and spread rate) were recorded on the PDA. Figure 1 ); Mycelial morphology was observed using slide culture technique (Prakash & Bhargava 2016).

[0041] The results showed that ZSP3-1 colonies in the culture medium were brownish-yellow with a small amount of aerial hyphae and grew rapidly (1 cm / d). Under a microscope, the ZSP3-1 hyphae were brownish-yellow, with a diameter of 2±0.6 μm, septate hyphae, and branching angles of 40-90°. Figure 2 It is sporeless, consistent with the characteristics of fungi in the genus *Caulis Basidiomycetes*.

[0042] (4) Molecular identification:

[0043] The activated and preserved strain was aseptically transferred, using an inoculation hook, to Erlenmeyer flasks containing sterile PD liquid medium. The flasks were then placed in a shaker at 25±1℃ (180 rpm) for incubation. The incubation time was adjusted according to the growth status of the strain, typically 4–7 days. The mycelium (approximately 100 mg wet weight) was collected by filtration. Genomic DNA was extracted from the fungus using the DNeasy Plant Mini Kit (Qiagen 69104), following the manufacturer's instructions.

[0044] The ITS region (containing ITS1, 5.8S, and ITS2) of fungal ribosomal DNA was amplified by PCR using the universal primer pair ITS1 (White et al. 1990), with an expected product size of approximately 550 bp. The amplified product was sequenced and then identified by sequence alignment.

[0045] 3. Validation of fungal growth-promoting function

[0046] Plate culture: The symbiotic germination experiment on artificial culture media followed the established protocol of Zhou & Gao (2016) in our laboratory. Three control culture media were set up: OMA medium without inoculation as an oligotrophic control, MS medium without inoculation (Murashige & Skoog, 1962) as a nutrient-rich control, and OMA + Tulasnella colaspora (a fungus that can promote the germination of various orchid plants) as a control.

[0047] Sterilized nylon mesh (4×4 cm) with a 45 μm aperture was laid on the surface of each culture medium. Mature, unopened capsules of *Cymbidium goeringii* were selected and disinfected by wiping with 75% ethanol. Under aseptic conditions, the capsules were opened, and the seeds were scattered into sterile bottles. The seeds were resuspended in 50 mL of sterile aqueous solution containing 0.1% agar to prepare a homogeneous suspension. 300 μL of the seed suspension (containing approximately 150 seeds) was evenly dropped onto the nylon mesh.

[0048] The tested strains ZSP3-1 and Tulasnella colaspora were activated on PDA plates (25±1℃, in the dark, for 8-10 days), and approximately 1 cm of the mycelial growth was cut from the edge using a sterile knife. 3 The mycelial blocks were inoculated into the center of nylon mesh. For the control treatment, blank PDA agar blocks of the same size were placed in the center of the mesh. Each treatment group had 15 independent replicates of culture dishes. The culture dishes were placed in a controlled environment incubator with the following conditions: temperature 25±1℃, relative humidity 75±5%, and a light / dark cycle of 12 hours / 12 hours. Germination was assessed after 60 days. Based on the standard germination stages (0-5 stages, see Table 1, modified from Arditti (1967)), the following were observed and recorded: seed germination (seed coat rupture), and the number and morphological changes of protocorms formed.

[0049] Table 1. Seed germination stages of orchid plants

[0050]

[0051] Figure 3 One-way ANOVA was performed on each stage. The results showed that: Stages 1-5: there were highly significant differences among treatments (F(3,56) = 618.2, p<0.001). Stages 2-3: there were highly significant differences among treatments (F(3,56) = 110, p<0.001). Stages 4-5: there were highly significant differences among treatments (F(3,56) = 11348, p<0.001). This indicates that different treatments had highly significant effects on the development of *Cymbidium goeringii*. MS treatment had a certain promoting effect on germination (38.55% ± 2.46), but the promoting effect on seedling development in the later stage (stages 4-5) was significantly reduced (2.70% ± 0.43). OMA treatment was significantly lower than other groups in all stages, indicating that it had no effect on germination or seedling development. The OMA+Tulasnella colaspora treatment showed performance between MS and OMA in the early stages, and its promoting effect on the later stages was only comparable to MS, indicating that the addition of Tulasnella colaspora had limited effect on germination or seedling growth. Notably, the OMA+ZSP3_1 treatment showed outstanding performance in stages 1-5 (93.10% ± 0.70) and stages 4-5 (90.75% ± 0.62), indicating that ZSP3_1 not only effectively promoted seed germination but also significantly promoted the transformation of Cymbidium goeringii to later developmental stages. In summary, ZSP3_1 exhibited the strongest development-promoting effect among the treatment combinations.

[0052] 4. Verification of fungal infection

[0053] To determine the success of mycorrhizal symbiosis under ZSP3-1 treatment, fungal infection was observed in *Cymbidium goeringii* seedlings and roots after 60 and 90 days of co-cultivation using trypan blue staining. Five seedlings and roots were randomly selected. The materials were treated with 10% KOH at 90°C for 30 min for clearing, followed by bleaching with alkaline hydrogen peroxide (0.5% NH4OH and 0.5% H2O2 v / v). The solution was then acidified with 1% HCl for 30 min to neutralize the alkalinity. The acidified materials were then stained in 0.05% trypan blue acetic acid glycerol solution at 37°C for 30 min, and finally destained with acetic acid glycerol solution. The staining was observed and photographed under a microscope. The results showed that numerous mycelial clusters were present at the base of the seedlings after 60 days and in the roots after 90 days. Figure 4 This indicates that ZSP3-1 has successfully colonized, and that Cymbidium goeringii obtains nutrients from ZSP3-1 through mycelial decomposition.

[0054] To further verify the mycorrhizal fungal specificity of the tested strain ZSP3-1, the aforementioned OMA was used as the symbiotic medium to conduct plate symbiotic germination of seeds from 24 species of orchids belonging to 17 genera, including *Renantheracoccinea*, *Renanthera citrina*, *Galeola faberi*, *Galeola lindleyana*, *Malaxis monophyllos*, *Lecanorchis japonica*, *Lecanorchis nigricans*, *Aphyllorchis montana*, *Dendrobium officinale*, *Bletilla striata*, *Calanthe discolor*, *Cymbidium mannii*, *Cymbidium tracyanum*, *Aerides falcata*, *Cymbidium ensifolium*, *Cymbidium faberi*, and *Cymbidium goeringii*. *Phaius columnaris*, *Neofinetia falcata*, *Arundina graminifolia*, *Platanthera japonica*, *Rhynchostylis retusa*, *Liparis nervosa*, and *Zeuxine strateumatica*. Seed counts in stages 2-5 after 60 days. Results showed that ZSP3-1 had no growth-promoting effect on most other orchid species, but exhibited the strongest promoting effect on *Zeuxine strateumatica*. Figure 6 This confirms that it has high host specificity, rather than being a broad-spectrum promoting bacterium.

[0055] Soil-based cultivation:

[0056] A 1:1 ratio of coconut coir to sawdust was used as the fungal culture medium. ZSP3-1 was inoculated into the medium and cultured at 28℃ for 10 days. Soil from the lawn where Cymbidium faberi grew was collected. 30% of the sterilized original soil was mixed with the fungal culture medium as the seed germination substrate. After the seeds were sterilized and counted under a microscope, they were evenly sown on the surface of the substrate, covered with plastic wrap to keep them moist, and placed in a greenhouse for cultivation. Seed germination and seedling development were observed regularly. The biomass of the seedlings was counted at 90, 180, 270 and 360 days (flowering period).

[0057] The results showed that ZSP3-1 significantly promoted seedling growth and produced protocorms and seedlings of Cymbidium goeringii. Figure 5 This indicates that ZSP3-1 can provide full support during the seedling stage under soil ecological conditions.

[0058] Ten Cymbidium plants were randomly selected at 90, 180, 270 and 360 days, respectively. The soil on the root surface was washed off, the surface moisture was dried, and the fresh weight of each plant was measured as biomass. Figure 7 The results showed that when Cymbidium goeringii was in symbiosis with ZSP3-1, the biomass in the later stage was significantly higher than that in the earlier stage. That is, as the number of days increased, its biomass continued to accumulate, and there was no obvious growth stagnation or bottleneck period in its life cycle. After one year, 50.4% of the plants reached the flowering state, indicating that ZSP3-1 not only promotes the germination of Cymbidium goeringii seeds, but also promotes the growth and development of the plants.

[0059] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A type of chalcobacillus ( Ceratobasidium strain (sp.), characterized in that, The *Castrodinium* strain has the accession number CCTCC NO: M20252398 at the China Center for Type Culture Collection, and the accession date is October 30, 2025.

2. The application of the *Acer basidiomyces* strain according to claim 1 in promoting seed germination, protocorm formation, seedling growth, root development, aboveground growth, and flower bud differentiation and flowering of *Cymbidium goeringii*.

3. The application according to claim 2, characterized in that, The application involves symbiotic culture of the *Cyclocarya paliurus* strain described in claim 1 with *Cyclocarya lanceolata* seeds.

4. A method for promoting seed germination in orchidaceae plants, characterized in that, Includes the following steps: (1) Preparation of culture medium; (2) The chalcogenide strain according to claim 1 is inoculated into the culture medium; (3) Perform surface disinfection treatment on the seeds of Cymbidium goeringii; (4) Place the sterilized Cymbidium goeringii seeds on the culture medium containing the Cymbidium spp. for symbiotic culture.

5. The method according to claim 4, characterized in that, The culture medium is OMA medium, MS medium, or soil-based culture medium containing a substrate.

6. The method according to claim 5, characterized in that, The soil-based substrate is a combination of coconut coir and sawdust.

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