Talaromyces rope and application of VOCs (Volatile Organic Compounds) generated by talaromyces rope in promoting tobacco growth

Abstract

The invention relates to talaromyces rope and application of VOCs (volatile organic compounds) produced by the talaromyces rope in promotion of tobacco growth, and belongs to the technical field of microorganisms. Carbon nano sol (CNS) is used for treating tobacco, a talaromyces rope F126 is separated and screened from rhizosphere soil of the tobacco, and the strain F126 and VOCs generated by the strain F126 can promote growth of the tobacco, so that application of chemical fertilizer in the tobacco is reduced, the ecological environment is further protected, and sustainable development of agriculture is promoted.

Description

Technical Field

[0001] This invention relates to a fungus called *Bacillus ligustus* and the application of its VOCs in promoting tobacco growth, belonging to the field of microbial technology. Background Technology

[0002] Tobacco (Nicotiana tabacum L.), belonging to the genus Nicotiana in the family Solanaceae, is an annual herbaceous plant. However, in most tobacco-growing areas, continuous cropping and the constant application of chemical fertilizers have led to slow tobacco growth, poor resistance, and a gradual decline in quality and yield.

[0003] To promote tobacco growth and ensure its yield and quality, the existing method is to apply large amounts of chemical fertilizers. However, excessive application of chemical fertilizers can lead to soil compaction, affecting the absorption of nutrients by tobacco roots. The excessive application of chemical fertilizers has also had certain impacts on the ecological environment, food security, resource utilization, and human health. To achieve sustainable agricultural development, reducing the amount of chemical fertilizers used and improving their utilization rate are urgent problems that need to be solved at this stage.

[0004] Utilizing natural plant symbiotic microorganisms to promote plant growth and related research has become a new direction for sustainable agricultural development. In the natural environment, plants and microorganisms establish a mutually beneficial symbiotic relationship during the infection process. These microorganisms can enhance the host plant's resistance to biotic and abiotic stresses, promote plant growth, and improve nutrient absorption. However, currently, the types of plant growth-promoting bacteria specifically targeting tobacco growth are limited, and it is still difficult to meet the needs for promoting rapid tobacco growth. Summary of the Invention

[0005] The first objective of this invention is to provide a rope-shaped basket-shaped bacterium to provide a plant growth-promoting bacterium with the function of promoting tobacco growth.

[0006] A second objective of this invention is to provide the application of the aforementioned *Bambusa cordiformis* in promoting tobacco growth.

[0007] A third objective of this invention is to provide the application of VOCs produced by the aforementioned *Bacillus ligustus* in promoting tobacco growth.

[0008] To achieve the first objective mentioned above, the technical solution adopted by this invention is as follows:

[0009] A fungus named *Talaromyces funiculosus* F126, with accession number CGMCC NO.41208.

[0010] This invention is groundbreaking. The obtained cordyceps fungus F126 was isolated from the rhizosphere soil of tobacco. Through synergistic effects with tobacco, this strain and the VOCs it produces effectively promote tobacco growth and have the potential to be developed into a highly efficient bio-fertilizer, protecting the ecological environment and promoting the sustainable development of agriculture.

[0011] To achieve the second objective mentioned above, the technical solution adopted by this invention is as follows:

[0012] The application of the aforementioned *Bambusa cordiformis* in promoting tobacco growth.

[0013] When *Bambusa fasciata* F126 and tobacco seedlings were co-cultured in petri dishes, the growth of tobacco seedlings in the experimental group was significantly better than that in the control group. The average fresh weight of tobacco plants increased by 233.3% compared with the control, and the root length and maximum leaf width were significantly higher than those in the control group (P<0.05), thus proving that *Bambusa fasciata* F126 can promote tobacco growth.

[0014] To achieve the third objective mentioned above, the technical solution adopted by this invention is as follows:

[0015] The application of VOCs produced by the aforementioned *Bacillus ligustus* in promoting tobacco growth.

[0016] When *Bacillus ligustus* F126 was co-cultured with tobacco seedlings without physical contact, the growth of tobacco seedlings in the experimental group was better than that in the control group. The average fresh weight of the plants increased by 108.3% compared with the control, and the root length, maximum leaf width, and maximum leaf length were all significantly higher than those of the control (P<0.05). This proves that the VOCs produced by *Bacillus ligustus* F126 can promote tobacco growth. Attached Figure Description

[0017] Figure 1 The image shows the morphology of a single colony obtained after separation and purification in Example 1 of this invention, where O is the front view of the obtained single colony and R is the back view of the obtained single colony.

[0018] Figure 2 This is the phylogenetic tree constructed based on the ITS gene sequence in Example 1 of the present invention;

[0019] Figure 3 The image shows the phenotypic pattern of strain F126 of *Bacillus ligustus* on MS plates and tobacco seedlings in Example 2 of this invention, where CK is the control group and F126 is the experimental group.

[0020] Figure 4 The image shows the phenotypic pattern of strain F126 of *Bacillus ligustus* co-cultured with tobacco seedlings in a 6-well culture plate in Example 2 of this invention, where CK is the control group and F126 is the experimental group. Detailed Implementation

[0021] Nanomaterials are a special type of material with dimensions ranging from 0.1 to 100 nm. When a substance reaches the nanoscale, its physical or chemical properties undergo a sudden and qualitative leap, exhibiting unique characteristics. They are now widely used in agriculture and life sciences, possessing significant ecological, economic, and scientific research value. Carbon nanosols (CNS) are a novel type of highly hydrophilic carbon-based nanomaterial that has shown great application potential in regulating plant growth, promoting nutrient absorption by crops, increasing biomass accumulation, and remediating soil. Furthermore, they can indirectly regulate plant growth or physiological characteristics by improving the soil microbiome (e.g., by enriching beneficial microorganisms). Studies have shown that applying appropriate concentrations of CNS under various environments, including suspension cell culture, hydroponics, and field cultivation, can promote the growth of crops such as tobacco, Arabidopsis thaliana, and rice.

[0022] This invention utilizes CNS treatment of tobacco and screens out a fungus strain F126 from the tobacco rhizosphere soil. When F126 is co-cultured with tobacco seedlings in petri dishes and without physical contact, it is found that strain F126 and the VOCs (volatile organic compounds) it produces can promote tobacco growth.

[0023] The carbon nanoparticles used in this invention were prepared by Academician Xie's team. For details, please refer to the published literature (Highlyhydrophilic carbon nanopaticles: uptake mechanism by mammalian and plant cells; RSC Advances; Issur 61, 2018).

[0024] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto; unless otherwise specified, all reagents, instruments and other items used in the embodiments are commercially available products.

[0025] The following is a brief introduction to some of the biological materials, reagents, and equipment involved in the examples:

[0026] Biomaterials:

[0027] Tobacco: China Tobacco 100;

[0028] Tobacco seeds: K326.

[0029] Culture medium:

[0030] PDA medium: 6 g / L potato extract powder, 20 g / L glucose, 20 g / L agar, pH 5.6, add distilled water to 1000 mL, autoclave at 115℃ for 20 min;

[0031] MS agar medium: MS basal medium 4.43 g / L, sucrose 30 g / L, agar 7 g / L, pH 5.8, add distilled water to 1000 mL, autoclave at 121℃ for 20 min;

[0032] 1 / 2MS agar medium: MS basal medium 2.21 g / L, sucrose 30 g / L, agar 7 g / L, pH 5.8, add distilled water to 1000 mL, autoclave at 121℃ for 20 min.

[0033] Experimental reagents:

[0034] EZNA Fungal DNA Extraction Kit: Omega Biotek Inc., Doraville, GA, USA.

[0035] Experimental materials:

[0036] 6-well cell culture plate: Falcon, NJ, USA.

[0037] The implementation process of the present invention will be described in detail below with reference to specific embodiments.

[0038] I. Specific Embodiments of a Rope-shaped Basket Fungus of the Present Invention

[0039] Example 1

[0040] A strain of *Talaromyces funiculosus* F126, accession number: CGMCCNO.41208; accession date: April 24, 2024; depositary institution: China General Microbiological Culture Collection Center (CGMCC); depositary address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0041] The isolation, screening, and identification process of this strain are described below:

[0042] 1. Isolation and screening of *Basilella fragilis* F126

[0043] This experiment describes the isolation and screening process of *Bacteroides fragilis* F126. The specific procedures are as follows:

[0044] Using the tobacco (Nicotiana tabacum L.) variety Zhongyan 100 as experimental material, one-month-old tobacco seedlings were transplanted to an experimental field in Jiaxian County, Henan Province, China after germination. Three different concentrations of CNS (CK: 0 g / L, i.e., with the same volume of purified water added; T1: 600 mg / L; T2: 1200 mg / L) were used to treat the tobacco. Surrounding soil (BS), rhizosphere soil (RS), root surface soil (RP), and roots (R) were collected on days 5, 10, and 25 after CNS treatment. These samples were used to analyze the samples using primers ITS1 F (5'-CTTGGTCATTTAGAGGAAGTAA-3', as shown in SEQ ID No. 2) and ITS2 (5'-CTTGGTCATTTAGAGGAAGTAA-3', as shown in SEQ ID No. 2).

[0045] Amplicon sequencing of the ITS1 region of fungi (as shown in SEQ ID No. 3) revealed that CNS significantly increased the abundance of various fungi compared to the control, and these fungi may have plant growth-promoting potential.

[0046] Potential growth-promoting fungi were isolated using the infinite dilution method. 1 g of CNS-treated rhizosphere soil was placed in a 2 mL centrifuge tube, finely disrupted and homogenized using sterile glass beads, and serially diluted with sterile phosphate buffer (5.0 mL) at dilutions ranging from 10 to 150 μL. -4 and 10 -7 Samples were plated on PDA (potato dextrose agar) plates containing streptomycin. Different fungi were selected based on morphological characteristics such as colony color and edge shape, and purified by streaking continuously on PDA plates. Forty-four culturable fungi were isolated and subjected to plate growth promotion tests. Fungus F126 was found to have a significant growth-promoting effect on tobacco. Comparison with amplicon sequencing data showed that strain F126 was a fungus that was significantly enriched after CNS treatment. Strain F126 was preserved in 40% glycerol solution at -80°C and subcultured on PDA plates for further research.

[0047] 2. Identification of *Basilaria cordifolia* F126

[0048] This experiment describes the identification process of *Basilella fragilis* F126. The specific procedures are as follows:

[0049] Morphological identification: F126 strain was inoculated onto MS plates and incubated at 25°C. Colonies initially appeared white, gradually turning grayish-green in the center after one week. Colony diameter was 1–2 cm, with a rough surface, regularly circular borders, and a raised center. The reverse side was yellow in the center and white at the edges, without any raised areas. Figure 1Microscopically, the hyphae are septate, and their color gradually darkens with increasing culture time. Conidiophores occur on hyphal ropes or aerial hyphae, are smooth and transparent, and are shaped like brooms. The conidiophore stems are septate, and the conidia are typically elliptical with smooth single cell walls.

[0050] Molecular biological identification: DNA was extracted from strain F126 using the EZNA fungal DNA extraction kit. Using this DNA as a template, primers ITS1F and ITS2 were selected for PCR amplification of the ITS1 region. The amplification program was: 94℃ pre-denaturation for 2 min, 30 cycles (94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 45 s), and a final extension at 72℃ for 10 min. Sequencing was then performed, and the sequencing results (as shown in SEQ ID No. 1) were compared with the UNITE database. Phylogenetic analysis was performed using MEGAX software, and a phylogenetic tree was constructed using the neighbor-joining method. Figure 2 The results showed that strain F126 and Talamoyces funiculosus belong to the same branch, with a similarity of 97.93%.

[0051] Based on the morphological and molecular biological identification results, the strain was identified as *Talaromyces funiculosus*, named *Talaromyces funiculosus* F126, and deposited at the China General Microbiological Culture Collection Center on April 24, 2024, with accession number CGMCC NO.41208.

[0052] II. Specific Examples of the Application of the Rosaceae bacterium and its VOCs in Promoting Tobacco Growth

[0053] Example 2

[0054] In this embodiment, the selected fungus F126 was co-cultured with tobacco seedlings in a petri dish, or co-cultured without physical contact. It was found that strain F126 and the VOCs it produces can promote the growth of tobacco plants.

[0055] The specific operating method is as follows:

[0056] 1. Growth-promoting effect of *Basilaria cordifolia* F126 on tobacco

[0057] This experiment verified the growth-promoting effect of *Bacillus ligustus* F126 on tobacco. The specific procedures are as follows:

[0058] Strain strain F126 was inoculated onto a PDA plate and incubated at 25°C until spores were produced. A spore suspension was then prepared, and 10 μL (1×10⁻⁶) of the spore suspension was taken. 5Conidia per mL were inoculated into the center of 1 / 2 MS agar medium. The control group was sterile water. Each experimental group was replicated in 3 parts. Tobacco seeds (K326) were surface-sterilized with 10% sodium hypochlorite (NaClO) for 10 min, then rinsed 3 times with sterile water. The sterilized tobacco seeds were inoculated onto 1 / 2 MS agar medium. When the seeds grew to the stage of 4 true leaves, they were transferred to 1 / 2 MS medium inoculated with strain F126. Five plants were evenly distributed on each medium. The culture dishes were then placed in the growth chamber and the growth conditions were controlled at 21℃, with light culture for 16 h / d and dark culture for 8 h / d for 10 days. After the culture, the growth indicators of the tobacco plants were measured, including fresh weight, plant height, root length, maximum leaf width and number of leaves.

[0059] The formula for calculating the fresh weight growth rate is as follows:

[0060] Fresh weight growth rate (%) = (Fresh weight of experimental group - Fresh weight of control group) / Fresh weight of control group × 100 (1)

[0061] Table 1. Growth-promoting effects of *Basilaria cordifolia* F126 on tobacco.

[0062]

[0063] By observing the growth of tobacco plants on plates, it can be found that *Basilaria cordifolia* F126 can effectively promote the growth of tobacco. Figure 3 The average fresh weight of the plants increased by 233.3% compared with the control, and the root length and maximum leaf width were significantly higher than those of the control group (P<0.05). The plant height and number of leaves were not significantly different from those of the control group (Table 1).

[0064] 2. The growth-promoting effect of VOCs produced by *Basilaria cordifolia* F126 on tobacco.

[0065] This experiment verified the growth-promoting effect of VOCs produced by *Bacillus floribunda* F126 on tobacco. The specific procedures are as follows:

[0066] Strain strain F126 was inoculated onto PDA plates and incubated at 25°C until spores were produced. A spore suspension was then prepared, and 10 μL (1×10⁻⁶) of the spore suspension was taken. 5Conidia (1 / mL) were inoculated into two wells of a 6-well cell culture plate. Each well of the 6-well cell culture plate had a septum in the middle to prevent physical contact between the fungus F126 and the tobacco seedlings. The control group was sterile water. Each experimental group was replicated three times. Tobacco seeds (K326) were surface-sterilized with 10% sodium hypochlorite (NaClO) for 10 min, then rinsed three times with sterile water. The sterilized tobacco seeds were inoculated onto 1 / 2 MS agar medium. When the seeds grew to the stage of 4 true leaves, they were transferred to the remaining 4 wells of the 6-well cell culture plate inoculated with strain F126. The culture plate was sealed and placed in a growth chamber (the experimental group and the control group were sealed separately and placed in the same environment). The growth conditions were controlled at 21℃, light culture for 16 h / d, dark culture for 8 h / d, and continued for 10 days. After the end of the growth, the growth indicators of the tobacco plants were measured, including fresh weight, root length, maximum leaf width, maximum leaf length, and number of leaves.

[0067] The formula for calculating the fresh weight growth rate is shown in equation (1).

[0068] Table 2. Effects of VOCs produced by *Basilaria cordifolia* F126 on tobacco growth.

[0069]

[0070] By observing the growth of tobacco plants on culture plates, it can be found that the VOCs produced by *Bacillus ligustus* F126 can promote tobacco growth. Figure 4 The average fresh weight of the plants increased by 108.3% compared with the control, and the root length, maximum leaf width and maximum leaf length were significantly higher than those of the control (P<0.05). The number of leaves was not significantly different from that of the control group (Table 2).

[0071] Based on the above experiments, it is clear that *Bacillus ligustus* and its VOCs can promote the growth of tobacco plants. In actual tobacco cultivation, it can be prepared into microbial fertilizer using conventional methods to further promote tobacco growth.

Claims

1. A rope-shaped basket-like fungus, characterized by: The strain was *Talaromyces funiculosus* F126, with accession number CGMCC NO.41208.

2. The application of the *Bambusa cordifolia* as described in claim 1 in promoting tobacco growth.

3. The application of VOCs produced by the fungus *Bacillus ligustus* as described in claim 1 in promoting tobacco growth.

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