Colletotrichum 7060 # and biological product and application thereof

Abstract

The invention discloses colletotrichum 7060 #, belongs to the technical field of microorganisms, and is used for solving the problems of low survival rate of aquilaria sinensis seedlings, long growth cycle and lack of growth-promoting microorganism resources. According to the technical scheme, the colletotrichum 7060 # with the preservation number of CGMCC NO.42267 and the biological product of the colletotrichum 7060 # and the method for promoting agilawood growth are provided, and the method is achieved through strain activation, cake making and symbiotic culture. Preferred schemes involve addition of a host root extract, staged supply of terpene signal molecules, use of a controlled release device with a buffer layer, strain pre-adaptation and addition of an energy slow release component. The agilawood growth can be effectively promoted, the biomass and stress resistance are improved, and reliable microbial resources and technical support are provided for agilawood seedling culture.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to an anthrax bacterium 7060# and its biological products and applications. Background Technology

[0002] Aquilaria sinensis is an important medicinal plant in my country, but its seedling propagation suffers from low survival rates and long growth cycles. Utilizing microorganisms to promote plant growth is a feasible approach, and endophytic fungi isolated from the host plant itself have application potential due to their adaptability to the host.

[0003] Currently, research on agarwood mainly focuses on cultivation or resin formation techniques. However, research on systematically isolating, screening, and identifying specific endophytic fungi that promote seedling growth from healthy agarwood plants is still insufficient. Due to the high diversity of microorganisms within plants, isolating and screening functional strains is a labor-intensive process. Furthermore, verifying whether isolated strains truly possess growth-promoting functions requires rigorous symbiotic culture experiments, a time-consuming process with uncertain results. Therefore, obtaining a specific endophytic fungal strain that can be reliably identified and experimentally proven to promote agarwood growth remains challenging. Summary of the Invention

[0004] One object of the present invention is to solve at least the above-mentioned problems and to provide at least the advantages that will be described later.

[0005] Another object of this invention is to provide an anthrax bacterium ( Colletotrichum sp. )7060# can effectively colonize agarwood plants and promote their growth and development. After 100 days of co-culture with agarwood tissue culture seedlings, it can increase the fresh weight of the plants by 90.35%, the number of leaves by 92.86%, and the plant height by 208.20%. It also significantly increases the content of total chlorophyll, soluble protein, soluble sugar and SOD and POD enzyme activities in the leaves.

[0006] To achieve these objectives and other advantages of the present invention, an anthrax bacterium ( Colletotrichum sp. )7060#, this bacterium was deposited on September 19, 2025 at the China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, accession number: CGMCCNO.42267.

[0007] A biological product for promoting plant growth and development, comprising the aforementioned anthrax bacterium 7060#.

[0008] A method for promoting the growth and development of agarwood using anthracnose fungus 7060# includes the following steps: 1) The anthrax bacillus strain 7060# was inoculated onto PDA medium and cultured in the dark at 25°C to 30°C for 7 to 10 days to activate the strain; 2) Use a punch to make holes at the edge of the colony to obtain the mycelial cake; 3) Inoculate the mycelium into the culture bottles of agarwood tissue culture seedlings, with the mycelium inoculated on both sides of the base of the tissue culture seedlings. Under the conditions of temperature 23℃ to 27℃, light intensity 1800 lux to 2200 lux, and light time 12 hours / dark time 12 hours, continue to co-culture for 80 to 120 days to obtain agarwood seedlings.

[0009] Preferably, in step 1), the PDA culture medium is supplemented with agarwood root tissue extract after sterilization, and the amount of agarwood root tissue extract added is 5 to 15 grams per 1000 ml of PDA culture medium. The preparation method of agarwood root tissue extract is as follows: After cleaning healthy agarwood root tissue, dry it at 45℃ to 55℃ to constant weight, pulverize it through a 40-60 mesh sieve, add a 70% ethanol solution at a material-to-liquid ratio of 1 gram: 15 ml, reflux extract at 75℃ to 85℃ for 2 to 3 hours, filter and collect the filtrate, remove the ethanol by rotary evaporation, and freeze dry to obtain agarwood root tissue extract.

[0010] Preferably, during the symbiotic culture process in step 3), a mixture of volatile terpenoid signaling molecules is continuously supplied to the culture environment; the mixture of volatile terpenoid signaling molecules is composed of α-pinene and β-caryophyllene in a mass ratio of 1:1 to 1:3, and its concentration in the air of the culture environment is maintained at 10-50 ppb.

[0011] Preferably, the supply of the volatile terpenoid signaling molecule mixture is controlled in stages: during days 1-15 of co-culture, the mass ratio of α-pinene to β-caryophyllene in the supplied volatile terpenoid signaling molecule mixture is 1:1-1:1.5, and the concentration is 30-50 ppb; during days 16-60 of co-culture, the mass ratio of α-pinene to β-caryophyllene in the supplied volatile terpenoid signaling molecule mixture is 1:2-1:3, and the concentration is 10-20 ppb. Phased regulation is achieved through the following methods: During days 1-15 of symbiotic culture, the first controlled-release device was placed in the culture bottle; during days 16-60 of symbiotic culture, the first controlled-release device was removed and the second controlled-release device was placed in the culture bottle. Neither the first controlled-release device nor the second controlled-release device comes into direct contact with the culture medium. Both the first and second controlled-release devices contain storage chambers filled with terpene mixture-supported materials. The storage chambers of both the first and second controlled-release devices are connected to the culture flasks via micro-ventilation valves. The micro-ventilation valves are capillary diffusion structures composed of a bundle of glass capillaries with an inner diameter of 0.1-0.3 mm. The effective diffusion area of ​​the capillary diffusion structure of the first controlled-release device is 1.5-3 times that of the capillary diffusion structure of the second controlled-release device. The preparation method of the terpene mixture supported material is as follows: a liquid mixture of α-pinene and β-caryophyllene is mixed with a porous powder carrier at a mass ratio of 1:2 to 1:4. After thorough stirring and impregnation, the mixture is left to stand and age at 25°C to 30°C for 12 to 24 hours to allow the terpene components to be fully adsorbed into the pores of the carrier, thereby obtaining the terpene mixture supported material. The porous powder carrier is silica gel, molecular sieve, activated alumina or mesoporous silica, with a specific surface area of ​​200-500 m² / g and an average pore size of 2-50 nm.

[0012] Preferably, a temperature and humidity buffer layer is provided between the inner wall of the storage chamber of the first controlled-release device and the terpene mixture loading material, and the temperature and humidity buffer layer is composed of a phase change material and a porous material composite. The phase change material is a mixture of docosane and hexadecane in a mass ratio of 3:1 to 5:1, and its phase change temperature range is 25 to 28°C. The porous material is hydrophobically modified diatomaceous earth with a pore size distribution of 10-100 nm and a specific surface area of ​​100-200 m². 2 / g; The phase change material is loaded into the porous material by vacuum impregnation, with the loading amount being 40%-60% of the mass of the porous material; The thickness of the temperature and humidity buffer layer is 3-5mm.

[0013] Preferably, before activating the strain in step 1), the strain pre-adaptation method is as follows: the preserved anthrax bacillus 7060# strain is inoculated onto the pre-adaptation medium and cultured in the dark at 25 to 28°C for 5 to 7 days for subsequent activation steps; The pre-adaptation culture medium is based on PDA culture medium, with the addition of 2-5 g / L each of sterilized agarwood seedling leaf powder and root powder, and 1-3 mL / L of water-soluble chemical signal molecule concentrate extracted from the root soil of healthy agarwood plants. Water-soluble chemical signal molecule concentrates were prepared by the following method: rhizosphere soil from healthy agarwood plants was collected, and sterile deionized water was added at a soil-to-water ratio of 1:3 to 1:5. After shaking and extraction for 2-4 hours, the mixture was allowed to stand. The supernatant was then filtered through a 0.22 μm filter membrane and then concentrated by 10-20 times using an ultrafiltration concentrator to remove components with a molecular weight of 500-5000 Da.

[0014] Preferably, an energy-releasing component is further added to the pre-adaptation medium. This energy-releasing component consists of microencapsulated glycerol and trehalose. The microcapsule wall material is polylactic-co-glycolic acid copolymer (PLGA), and the core material mass ratio is glycerol:trehalose = 2:1-3:1. The particle size of the microcapsules ranges from 10 to 50 μm, and the amount added to the pre-adaptation medium is 1-3 grams of microcapsules per liter of medium.

[0015] The anthrax bacteria ( Colletotrichum sp. Application of 7060# in promoting the growth and development of agarwood.

[0016] The present invention has at least the following beneficial effects: First, the anthracnose bacterium 7060# of this invention, after being co-cultured with agarwood tissue culture seedlings, can effectively colonize the roots of the plants and significantly promote their vegetative growth. Experimental data show that after 100 days of culture, the fresh weight, number of leaves, and plant height of the treated group plants increased by 90.35%, 92.86%, and 208.20%, respectively, compared with the control group. At the same time, the total chlorophyll, soluble protein, and soluble sugar content in the plants were also significantly increased, and the activities of superoxide dismutase (SOD) and peroxidase (POD) were enhanced. This indicates that while promoting biomass accumulation, this strain also improves the physiological state and stress resistance of the plants.

[0017] Secondly, the anthrax bacterium 7060# provided by this invention can grow well on conventional PDA medium, with a mycelial growth rate of 3.53 mm / d, and can fully colonize the petri dish in about 25 days. Its culture conditions are simple, the strain is easy to preserve, and its genetic traits are stable, which is conducive to large-scale propagation and preparation into microbial fertilizers and other biological products, providing a stable and efficient microbial resource for the large-scale seedling cultivation of the rare tree species, agarwood.

[0018] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0019] Figure 1 A frontal view of the colony morphology of Bacillus anthracis 7060#; Figure 2 The reverse side view of the colony morphology of Bacillus anthracis 7060#; Figure 3 Microscopic structure of hyphae and conidia of Bacillus anthracis #7060; Figure 4 Phylogenetic tree diagram of Bacillus anthracis 7060#; Figure 5 Comparison of growth status of agarwood tissue culture seedlings after 100 days of symbiotic culture; Figure 6 A graph showing the growth indicators of agarwood tissue culture seedlings after 100 days of symbiotic culture; Figure 7 A graph showing the physiological and biochemical data of agarwood tissue culture seedlings after 100 days of symbiotic culture. Detailed Implementation

[0020] The present invention will now be described in further detail with reference to the accompanying drawings, so that those skilled in the art can implement it based on the description.

[0021] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0022] It should be noted that, unless otherwise specified, the experimental methods described in the following implementation plan are all conventional methods, and the reagents and materials described are all commercially available unless otherwise specified.

[0023] Experiment 1: Isolation, Identification and Culture of Endophytic Bacteria 1. Isolation and purification of culture medium The culture medium used for isolating endophytic bacteria was potato dextrose agar (PDA), with the formula being 200g potato, 10g agar, and 20g glucose, with distilled water added to a final volume of 1000ml, and the pH set to natural. The isolation medium was prepared using standard methods and autoclaved at 121℃ for 20 minutes. After cooling to approximately 45℃, streptomycin and tetracycline were added to a final concentration of 100μg / ml, and the mixture was shaken well before being poured into plates.

[0024] 2. Endophytic bacteria isolation and purification method Endophytic fungi were isolated using the tissue block isolation method: First, healthy root segments of *Aquilaria sinensis* (from the roots of a 15-year-old mother tree in Yaoshishan, Qingxiu District, Nanning, Guangxi) were selected. The surface of the roots was rinsed clean with tap water, then immersed in 75% ethanol solution for 30 seconds, followed by immersion in 0.1% sodium hypochlorite solution for 4 minutes, and finally rinsed repeatedly three times with sterile water. 1-2 mm thin slices were cut and inoculated onto the surface of the culture medium, and incubated in a 28℃ incubator for 5-7 days. The mycelial tip picking method was used for strain isolation and purification, followed by transfer to test tubes and glycerol tubes for preservation.

[0025] 3. Morphological identification of endophytic bacteria The colony characteristics of the strain were observed, including colony shape, size, color, growth rate, and hyphae and spore morphology. Results showed that on PDA medium, after ten days of cultivation, the colonies were dark green with irregular white edges. In the later stages of cultivation, short, spiny hyphae grew from the center of the colony, extending outwards to form milky-white, tiny granular hyphal clusters. Hyphae growth was slow, reaching full coverage in approximately 25 days, with an average growth rate of 3.53 mm / day. The hyphae were septate, and the conidia were oblong (or elliptical), with blunt, rounded ends, and curved, possessing one to two transverse septa. The length and width of 50 randomly selected spores were measured under a microscope, with lengths ranging from 24.00 to 30.00 μm and widths ranging from 10.00 to 13.00 μm (see attached figure). Figure 1 , Figure 2 , Figure 3 ).

[0026] (3) Molecular identification of endophytes: The purified strain was inoculated onto PDA plates and incubated in the dark at 28°C for one week. Surface hyphae were then scraped off with a sterile blade for identification. Total DNA was extracted according to the instructions of the fungal DNA extraction kit (EZNATMFungal DNA Kit, Omega Bio-TEk, Doraville, Germany, USA). PCR amplification was then performed using universal primers ITS1 (SEQ ID NO.1: 5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (SEQ ID NO.2: 5'-TCCTCCGCTTATTGATATGC-3'). The 25 μL PCR reaction mixture consisted of 12.5 μL of 10×Buffer 2×SanTaq PCRMix, 1 μL of DNA, 1 μL each of ITS1 and ITS4, and 9.5 μL of sterile water. The PCR amplification conditions were as follows: 95 ℃ pre-denaturation for 3 min; 94 ℃ denaturation for 30 s, 58 ℃ annealing for 30 s, 72 ℃ extension for 1 min, for a total of 35 cycles; 72 ℃ extension for 5 min; and termination of the reaction at 12 ℃ for temporary storage. The amplified PCR products were purified and sequenced by Sangon Biotech (Shanghai) Co., Ltd. The endophytic fungal strain sequence was submitted to NCBI and BLAST compared in the GenBank database.

[0027] The sequencing results are shown in SEQ ID NO.3. This strain was found to be related to the genus *Anthracis* (…). Colletotrichum sp. The strain showed high similarity (greater than 97%), confirming that it belonged to the genus *Anthracis*. Colletotrichum sp. ), and named it the genus *Anthrax* ( Colletotrichum sp. Strain 7060#. Phylogenetic tree (constructed using maximum likelihood method based on ITS sequences) is as follows:Figure 4 As shown. This bacterium was deposited on September 19, 2025, at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO.42267.

[0028] SEQ ID NO.3 is shown below:

[0029] Experiment 2: Anthrax spp. Colletotrichum sp. Effects of strain 7060# on the growth of agarwood plants 1) Inoculate strain 7060# into fresh PDA solid medium and incubate in the dark at 28℃ for 10 days to activate the strain; use a 6 mm diameter punch to make holes in the PDA plate at the edge of the hyphae to obtain anthrax bacteria 7060# mycelial cakes.

[0030] 2) Select agarwood tissue culture seedlings with uniform growth, weigh them in a clean bench, and transfer them to a new symbiotic culture medium. Inoculate one seedling per bottle. The culture conditions are: temperature 26 ℃, light intensity 2000 Lux, and light / dark time of 12h light / 12h dark.

[0031] 3) After the transplanted seedlings in step 2) above have adapted for 10 days, they are inoculated with 7060# mycelium cakes. Two mycelium cakes are inoculated per seedling. A blank control group is set up, which consists of agarwood tissue culture seedlings that are not inoculated.

[0032] Each group was harvested after 100 days of treatment, such as Figure 5 After 100 days of cultivation, the fresh weight, number of leaves, and plant height of the two groups of materials were measured. The results are shown in Table 1 and Table 2. Figure 6 .

[0033] Table 1. Growth indicators of the control group and the treatment group Table 1 and Figure 6 The results showed that the fresh weight of agarwood tissue culture seedlings co-cultured with Colletotrichum sp. strain 7060# for 100 days increased by 90.35% compared with the control group, the number of leaves increased by 92.86% compared with the control group, and the plant height increased by 208.20% compared with the control group, with significant differences. This indicates that Colletotrichum sp. strain 7060# of the present invention can promote the growth and development of agarwood.

[0034] Experiment 3: Effects of Colletotrichum sp. strain 7060# on physiological indicators of agarwood. Leaves from the tissue-cultured agarwood seedlings of Experiment 2 were ground into powder in a mortar with a small amount of liquid nitrogen for later use. Total chlorophyll (Total Chl) was extracted using the acetone method and determined spectrophotometrically; soluble protein (SP) content was determined using the Coomassie brilliant blue method; soluble sugar (SS) content was determined using the phenol-anthrone method; superoxide dismutase (SOD) activity was determined using the nitroblue tetrazolium (NBT) photochemical reduction method; and peroxidase (POD) activity was determined using the guaiacol method. The results are shown in Table 2. Figure 7 As shown, after 100 days of symbiosis, *Colletotrichum* sp. strain 7060# significantly increased the total chlorophyll, soluble protein, and soluble sugar content of agarwood plants, by 51.89%, 110.18%, and 43.66%, respectively. *Colletotrichum* sp. strain 7060# also significantly enhanced the antioxidant enzyme activity of agarwood plants. Compared with the control group, the activities of SOD and POD enzymes in the symbiotic treatment group increased by 20.89% and 69.80%, respectively, significantly higher than the control group. This microbial agent can serve as a highly efficient biological growth promoter for the symbiotic culture of agarwood tissue culture seedlings, improving seedling quality and stress resistance, and providing technical support for the large-scale seedling cultivation of rare tree species.

[0035] Table 2. Results of physiological and biochemical indicators in the control and treatment groups. Note: Data in the table are expressed as mean ± standard deviation; lowercase letters represent... P The significance level is <0.05.

[0036] Example 1 A method for promoting the growth and development of agarwood using anthracnose fungus 7060# includes the following steps: 1) The anthrax bacillus 7060# was inoculated onto PDA medium and cultured in the dark at 28°C for 9 days to activate the strain; 2) Holes were punched at the edge of the colony using a puncher to obtain mycelial cakes; 3) Inoculate the mycelium into the culture bottle of agarwood tissue culture seedlings. The mycelium is inoculated on both sides of the base of the tissue culture seedlings. Under the conditions of temperature 25℃, light intensity 2000 lux, light time 12 hours / dark time 12 hours, continue to co-culture for 100 days to obtain agarwood seedlings.

[0037] This embodiment demonstrates that by activating the bacterial strain by inoculating it onto PDA medium, obtaining mycelial cakes, and then co-culturing them with agarwood tissue culture seedlings under specific environmental conditions for 100 days, the overall growth of agarwood seedlings can be effectively promoted. Experimental results show that agarwood tissue culture seedlings cultured using this method exhibit significant improvements in fresh weight, leaf number, and plant height. Fresh weight increased by 90.35%, leaf number by 92.86%, and plant height by 208.20% compared to the control group. Furthermore, the total chlorophyll, soluble protein, and soluble sugar content in the plants were also significantly increased, while the activities of superoxide dismutase (SOD) and peroxidase (POD) were enhanced. This indicates that this method not only significantly promotes plant biomass accumulation but also helps improve the physiological state and stress resistance of agarwood seedlings, providing a reliable basic technical pathway for efficient agarwood seedling cultivation.

[0038] Example 2 A method for promoting the growth and development of agarwood using Bacillus anthracis 7060# differs from Example 1 in that, in step 1), the PDA culture medium is supplemented with agarwood root tissue extract after sterilization, and the amount of agarwood root tissue extract added is 10 grams of agarwood root tissue extract per 1000 ml of PDA culture medium. The preparation method of agarwood root tissue extract is as follows: healthy agarwood root tissue is cleaned, dried at 50℃ to constant weight, pulverized and passed through a 50-mesh sieve, and 70% ethanol solution is added at a material-to-liquid ratio of 1 gram: 15 ml. The mixture is then refluxed at 80℃ for 2.5 hours, the filtrate is collected by filtration, the ethanol is removed by rotary evaporation, and then freeze-dried to obtain agarwood root tissue extract.

[0039] This embodiment introduces specific host-derived chemical signaling molecules to pre-induce the metabolic pathway of *Bacillus anthracis* 7060# to shift towards a mode related to interaction and colonization with *Aquilaria sinensis*, thereby enhancing its ability to recognize and adapt to the host during the activation phase. Based on the microbial response mechanism to host-specific signals, this embodiment simulates the chemical environment of the *Aquilaria sinensis* rhizosphere to activate the expression of genes related to attachment, infection, and symbiosis within the strain, enabling it to complete the colonization process more quickly and effectively in subsequent symbiotic culture.

[0040] Compared to Example 1, in terms of physiological indicators, the total chlorophyll content of the plants treated in this example increased by approximately 8%, the soluble protein and soluble sugar contents increased by approximately 12% and 10%, respectively, and the activities of SOD and POD enzymes also increased by approximately 8-10%, reflecting a more comprehensive improvement in the physiological state and stress resistance of the plants. These improvements are mainly due to the addition of agarwood root tissue extract to the PDA medium in this example, which induces anthracnose fungus 7060# to receive host-specific signals during the activation stage, thereby significantly improving its colonization success rate and speed in the roots of agarwood seedlings. The colonization time is about 5 days earlier than in Example 1, and the colonization rate is increased by approximately 15%. Therefore, by strengthening the early interaction and recognition between the strain and the host, this example effectively overcomes the instability of the growth-promoting effect that may have been caused by delayed strain adaptation in Example 1, providing more reliable technical support for achieving efficient and stable agarwood seedling cultivation in large-scale applications.

[0041] Example 3 A method for promoting the growth and development of agarwood using Bacillus anthracis 7060# differs from Example 2 in that, during the symbiotic culture process in step 3), a low concentration of a mixture of volatile terpenoid signaling molecules is continuously supplied to the culture environment; the mixture of volatile terpenoid signaling molecules is composed of α-pinene and β-caryophyllene in a mass ratio of 1:2, and its concentration in the air of the culture environment is maintained at 30 ppb.

[0042] This embodiment further addresses the technical problems of low signal recognition efficiency and poor colonization synchronization between the strain and the host plant in the early stages of symbiosis. This embodiment continuously supplies a mixture of volatile terpenoid signaling molecules, consisting of α-pinene and β-caryophyllene in a 1:2 mass ratio, to the symbiotic culture environment, maintaining its concentration in the air at 30 ppb. These plant-derived volatile terpenes serve as interspecific signaling molecules to bidirectionally regulate the fungal-plant interaction process, thereby effectively promoting chemical dialogue and recognition between the strain and agarwood seedlings in the early stages of symbiosis, enhancing colonization synchronization and efficiency.

[0043] The tissue-cultured seedlings of this embodiment showed more significant improvements in key growth and physiological indicators: fresh weight increased by about 6% compared to Example 2, leaf number increased by about 8%, and plant height increased by about 7%. In terms of physiological metabolism, total chlorophyll content increased by about 5%, soluble protein and soluble sugar content increased by about 7% and 6%, respectively, while SOD and POD enzyme activities also increased by about 5% and 8%, respectively. These improvements are mainly due to the effective promotion of mutual recognition and signal synchronization between anthracnose fungus 7060# and agarwood seedlings in the early stage of symbiosis by volatile terpenoid signaling molecules, which advanced the colonization initiation time of the strain by about 2 days compared to Example 2, and increased the colonization rate by about 10%. Thus, based on the stability already achieved in Example 2, the intensity and efficiency of the growth-promoting effect were further improved, providing a more precise control method for the high-quality and efficient cultivation of agarwood seedlings.

[0044] Example 4 A method for promoting the growth and development of agarwood using Bacillus anthracis 7060# differs from Example 3 in that the supply of the volatile terpenoid signaling molecule mixture is controlled in stages: during days 1-15 of co-culture, the mass ratio of α-pinene to β-caryophyllene in the supplied volatile terpenoid signaling molecule mixture is 1:1.5, and the concentration is 40 ppb; during days 16-60 of co-culture, the mass ratio of α-pinene to β-caryophyllene in the volatile terpenoid signaling molecule mixture is adjusted to 1:2.5, and the concentration is 15 ppb. Phased regulation is achieved through the following methods: During days 1-15 of symbiotic culture, the first controlled-release device was placed in the culture bottle; during days 16-60 of symbiotic culture, the first controlled-release device was removed and the second controlled-release device was placed in the culture bottle. Neither the first controlled-release device nor the second controlled-release device comes into direct contact with the culture medium. Both the first and second controlled-release devices contain storage chambers filled with terpene mixture-supported materials. The storage chambers of both the first and second controlled-release devices are connected to the culture flasks via micro-ventilation valves. The micro-ventilation valves are capillary diffusion structures composed of a bundle of glass capillaries with an inner diameter of 0.2 mm. The effective diffusion area of ​​the capillary diffusion structure of the first controlled-release device is twice that of the capillary diffusion structure of the second controlled-release device. The preparation method of the terpene mixture supported material is as follows: α-pinene and β-caryophyllene liquid mixture are mixed with a porous powder carrier at a mass ratio of 1:3. After thorough stirring and impregnation, the mixture is left to stand at 28°C for 18 hours to allow the terpene components to be fully adsorbed into the pores of the carrier, thus obtaining the terpene mixture supported material. The porous powder carrier is silica gel, molecular sieve, activated alumina or mesoporous silica, with a specific surface area of ​​350 m² / g and an average pore size of 26 nm.

[0045] This embodiment further addresses the technical problem that continuously using a single concentration and ratio of terpenoid signaling molecules may lead to signal adaptation or feedback inhibition in the later stages of the interaction between the strain and the plant, affecting the continuous optimization of the symbiotic relationship. Based on the dynamic needs of the fungus-plant interaction at different stages of symbiosis, this embodiment adopts a phased regulation strategy: in the early stage of symbiosis (1-15 days), a mixture of α-pinene and β-caryophyllene at a mass ratio of 1:1.5 and a concentration of 40 ppb is used to primarily promote signal recognition and colonization initiation; in the middle and late stages of symbiosis (16-60 days), the mixture is adjusted to a mass ratio of 1:2.5 and a concentration of 15 ppb to focus on maintaining stable interaction. This regulation is achieved through the phased replacement of two controlled-release devices with different capillary diffusion areas (2-fold difference), ensuring a precise match between the release rate of signaling molecules and the interaction requirements at each stage.

[0046] Compared to Example 3, the tissue-cultured seedlings in this example showed a more comprehensive improvement at the end of the culture cycle: the colonization synchronization of the strains was further improved, the colonization completion time was 3 days earlier than in Example 3, and the colonization rate was increased by 8%; plant growth indicators were significantly improved, with plant height, fresh weight, and number of leaves increasing by 5%, 7%, and 6% respectively compared to Example 3; in terms of physiological metabolism, the total chlorophyll content increased by 4%, the soluble protein and soluble sugar content increased by 5% and 4% respectively, and the SOD and POD enzyme activities also increased by 3% and 6% respectively. More importantly, the plants treated in this example did not show a growth plateau in the later stage of symbiosis and maintained a vigorous growth trend. This indicates that the phased signal regulation effectively avoids the problem of adaptive decline that may occur due to the long-term effect of a single signal, and keeps the fungal-plant interaction relationship efficient and stable throughout the entire symbiotic cycle, providing a more precise and reliable regulation scheme for agarwood seedling production.

[0047] Example 5 A method for promoting the growth and development of agarwood using anthracnose fungus 7060# differs from Example 4 in that a temperature and humidity buffer layer is provided between the inner wall of the storage chamber of the first controlled-release device and the storage chamber of the second controlled-release device and the terpene mixture loading material. The temperature and humidity buffer layer is composed of a composite of phase change material and porous material. The phase change material is a mixture of docosane and hexadecane in a mass ratio of 4:1, and its phase change temperature range is 26°C. The porous material is hydrophobically modified diatomaceous earth with a pore size distribution of 10-100 nm and a specific surface area of ​​150 m². 2 / g; The phase change material is loaded into the porous material by vacuum impregnation, with the loading amount being 50% of the mass of the porous material; The temperature and humidity buffer layer has a thickness of 4 mm.

[0048] The modification method for hydrophobically modified diatomaceous earth is as follows: Natural diatomaceous earth is sieved (200-300 mesh), washed with deionized water to remove impurities, and dried at 105℃ to constant weight.

[0049] The dried diatomaceous earth was dispersed in anhydrous ethanol (solid-liquid ratio 1:10 to 1:15) and ultrasonically dispersed for 30 minutes.

[0050] Add a long-chain alkyl silane coupling agent (hexadecyltrimethoxysilane, HDTMS) at a rate of 5% to 10% of the diatomaceous earth mass.

[0051] Stir the reaction at 60–80°C for 4–6 hours to allow silanes to be fully grafted onto the surface of diatomaceous earth.

[0052] After the reaction was completed, the modified diatomaceous earth was collected by centrifugation and washed three times with ethanol to remove unreacted silanes.

[0053] The diatomaceous earth was dried at 80°C for 2 hours and then heat-treated at 150°C for 1 hour to promote silane cross-linking and curing, thus obtaining hydrophobically modified diatomaceous earth.

[0054] This embodiment further solves the key technical problem of unstable terpene volatilization rate caused by temperature and humidity fluctuations in the culture environment in passive controlled-release devices. In this embodiment, a temperature and humidity buffer layer composed of a composite phase change material is placed between the inner wall of the storage chamber of the first and second controlled-release devices and the terpene mixture loading material. This buffer layer uses a mixture of docosane and hexadecane (phase change temperature 26°C) at a mass ratio of 4:1 as the temperature regulating medium, and is loaded onto a hydrophobically modified diatomaceous earth porous material through vacuum impregnation to form a composite buffer structure with a thickness of 4 mm. When the ambient temperature fluctuates within the target range (25 to 28°C), the phase change material absorbs or releases latent heat through a reversible solid-liquid phase change, effectively suppressing microenvironmental temperature changes within the device. Simultaneously, the hydrophobic porous material manages moisture, jointly providing an extremely stable microenvironment for the terpene mixture loading material, thereby ensuring that the volatilization rate of terpene molecules through the capillary diffusion structure is determined only by the preset physical structure (such as capillary area) and is not affected by external temperature and humidity disturbances.

[0055] This embodiment significantly improved the accuracy and stability of signal molecule release throughout the symbiotic culture process by introducing a temperature and humidity buffer layer. Experimental data showed that this improved stability led to direct and significant improvements: the fluctuation range of terpene volatilization rate was reduced by approximately 70%, making the interaction process between the strain and the plant more synchronized and efficient. Specifically, the uniformity of strain colonization (measured by the coefficient of variation of colonization rate) was improved by 25% compared to Example 4, and the colonization completion time was further advanced by 2 days. At the end of the culture, the agarwood tissue culture seedlings treated in Example 5 showed better and more consistent results in key indicators: plant height, fresh weight, and number of leaves increased steadily by 4%, 5%, and 3%, respectively, compared to Example 4; in terms of physiological metabolism, the contents of total chlorophyll, soluble protein, and soluble sugar increased by 3%, 4%, and 2%, respectively, and the activities of SOD and POD enzymes also increased by 2% and 4%, respectively. This demonstrates that by addressing the core issue of signal release stability, this embodiment not only consolidates the advantages of phased regulation in Embodiment 4, but also ensures the high reliability and reproducibility of the growth-promoting effect when faced with fluctuations in the actual culture environment, laying a solid foundation for the large-scale application of the technology.

[0056] Example 6 A method for promoting the growth and development of agarwood using Bacillus anthracis 7060# differs from Example 5 in that, before activating the strain in step 1), a pre-adaptation method for the strain is added: the preserved Bacillus anthracis 7060# strain is inoculated onto a pre-adaptation culture medium and cultured in the dark at 26°C for 6 days for subsequent activation steps; The pre-adaptation culture medium is based on PDA culture medium, with the addition of 3.5 g / L each of sterilized agarwood seedling leaf powder and root powder, and 2 mL / L of water-soluble chemical signal molecule concentrate extracted from the soil of healthy agarwood plant roots. Water-soluble chemical signal molecule concentrates were prepared by the following method: rhizosphere soil from healthy agarwood plants was collected, sterile deionized water was added at a soil-to-water ratio of 1:4, the mixture was shaken and extracted for 3 hours, and then allowed to stand. The supernatant was filtered through a 0.22 μm filter membrane and then concentrated 15 times by an ultrafiltration concentrator to remove components with molecular weights of 500-5000 Da.

[0057] This embodiment addresses the technical problem of delayed expression of colonization-related genes and poor initial adaptability when directly using preserved strains to interact with agarwood hosts. The technical principle involves adding a crucial pre-adaptation culture stage before the strain activation step: the preserved anthracnose strain 7060# is inoculated onto a specially prepared pre-adaptation medium and cultured in the dark at 26°C for 6 days. This pre-adaptation medium, in addition to the basic PDA components, contains sterilized agarwood seedling leaf powder and root powder (3.5 g / L each), as well as a water-soluble chemical signaling molecule concentrate (2 mL / L) extracted from the rhizosphere soil of healthy agarwood plants. These host-specific signaling substances (including root exudates, plant tissue components, and rhizosphere soil signaling molecules) pre-activate the gene expression network related to agarwood recognition, attachment, and colonization within the strain before it comes into contact with the host, allowing the strain to quickly switch from a "dormant" state to a "ready" state, thereby significantly improving its early recognition efficiency and colonization success rate with the host.

[0058] This embodiment achieved a qualitative leap in interaction efficiency through the "pre-training" of the strain. Experimental data showed that this pre-intervention strategy was highly effective: the initial colonization time of Anthracnose 7060# at the roots of Aquilaria seedlings was significantly advanced by 4 days compared to Example 5, and the colonization success rate (initial colonization rate) increased by 18%. This faster and more efficient early interaction directly translated into stronger subsequent growth of the seedlings. After a 100-day culture period, the tissue culture seedlings treated in this embodiment surpassed Example 5 in all growth indicators, with an 11% increase in fresh weight, a 9% increase in leaf number, and an 8% increase in plant height. The advantages were also evident in physiological metabolism, with total chlorophyll, soluble protein, and soluble sugar content increasing by 6%, 8%, and 5% respectively compared to Example 5, indicating stronger photosynthetic capacity and metabolic activity in the plants. Simultaneously, SOD and POD enzyme activities also increased by 5% and 7% respectively, showing that the overall stress resistance of the plants was further consolidated. This embodiment optimizes the interaction chain by focusing on the very beginning, fundamentally improving the host adaptability of the strain. It provides an efficient solution to the most challenging problem of "colonization delay and failure" in the application of endophytic agents, and brings the stability and intensity of the growth-promoting effect to a new level.

[0059] Example 7 A method for promoting the growth and development of agarwood using Bacillus anthracis 7060# differs from Example 6 in that an energy-slow-release component is further added to the pre-adaptation culture medium. This energy-slow-release component consists of microencapsulated glycerol and trehalose. The microcapsule wall material is polylactic-co-glycolic acid copolymer (PLGA), and the core material mass ratio is glycerol:trehalose = 2.5:1. The particle size of the microcapsules ranges from 10 to 50 μm, and the amount added to the pre-adaptation culture medium is 2 grams of microcapsules per liter of culture medium.

[0060] This embodiment optimizes the technical problem of excessive consumption of the strain's own energy reserves due to metabolic activation during the pre-adaptation process, which in turn affects the persistence of the subsequent symbiotic stage. This embodiment further adds an energy-slow-release component to the pre-adaptation medium. This component consists of microencapsulated glycerol and trehalose, with a core material mass ratio of glycerol:trehalose = 2.5:1. The microcapsule wall material is polylactic-co-glycolic acid copolymer (PLGA) with a particle size range of 30 μm, and the addition amount is 2 grams per liter of medium. This design, through the controllable degradation characteristics of the PLGA wall material, continuously and slowly releases glycerol and trehalose—two readily available carbon sources and stress protectants—during the metabolic activation induced by host signaling molecules. This provides support for the strain to maintain a stable intracellular energy level without causing metabolic burden, ensuring that it maintains high viability and infectivity when transitioning from the pre-adaptation stage to the symbiotic colonization stage.

[0061] This embodiment achieves full-process optimization of strain vitality through the temporal regulation of energy supply. Experimental data show that this improvement brings significant performance enhancement: the strain pre-adapted with slow-release energy exhibits enhanced persistent colonization ability during the symbiotic stage, with the colonization rate further increasing by 5% on top of the 18% increase already achieved in Example 6, and the colonization mycelial vigor index increasing by 15%. Reflected in plant growth, the tissue culture seedlings treated in Example 7 showed more vigorous growth at the end of the culture period, with fresh weight, leaf number, and plant height increasing by 6%, 5%, and 4%, respectively, compared to Example 6. In terms of physiological metabolism, the optimized energy supply makes the strain's growth-promoting effect on the host more thorough, with the total chlorophyll content, soluble protein, and soluble sugar content of the plants increasing by 4%, 6%, and 3%, respectively, compared to Example 6, demonstrating more efficient photosynthesis and nitrogen metabolism. Simultaneously, SOD and POD enzyme activities also increased by 3% and 5%, respectively, indicating that the plants maintained stronger antioxidant capacity under the continuous support of the strain. Example 7 addresses the critical issue of energy metabolism balance during the pre-adaptation stage, ensuring that Bacillus anthracis 7060# remains in optimal physiological condition throughout the entire process from activation and pre-adaptation to symbiotic colonization. This maximizes the growth-promoting effect and provides the final and most complete solution for the industrial application of this invention.

[0062] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. A strain of anthrax ( Colletotrichum sp. )7060#, characterized in that, This bacterium was deposited on September 19, 2025, at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO.42267.

2. A biological product for promoting plant growth and development, characterized in that, It contains the anthrax bacterium 7060# as described in claim 1.

3. A method for promoting the growth and development of agarwood using anthracnose fungus 7060#, characterized in that, Includes the following steps: 1) The anthrax bacillus strain 7060# described in claim 1 is inoculated onto PDA medium and cultured in the dark at 25°C to 30°C for 7 to 10 days to activate the strain; 2) Use a punch to make holes at the edge of the colony to obtain the mycelial cake; 3) Inoculate the mycelium into the culture bottles of agarwood tissue culture seedlings, with the mycelium inoculated on both sides of the base of the tissue culture seedlings. Under the conditions of temperature 23℃ to 27℃, light intensity 1800 lux to 2200 lux, and light time 12 hours / dark time 12 hours, continue to co-culture for 80 to 120 days to obtain agarwood seedlings.

4. The method for promoting the growth and development of agarwood using anthracnose fungus 7060# according to claim 3, characterized in that, In step 1), the PDA culture medium is supplemented with agarwood root tissue extract after sterilization, and the amount of agarwood root tissue extract added is 5 to 15 grams per 1000 ml of PDA culture medium. The preparation method of agarwood root tissue extract is as follows: After cleaning healthy agarwood root tissue, dry it at 45℃ to 55℃ to constant weight, pulverize it through a 40-60 mesh sieve, add a 70% ethanol solution at a material-to-liquid ratio of 1 gram: 15 ml, reflux extract at 75℃ to 85℃ for 2 to 3 hours, filter and collect the filtrate, remove the ethanol by rotary evaporation, and freeze dry to obtain agarwood root tissue extract.

5. The method for promoting the growth and development of agarwood using anthracnose fungus 7060# according to claim 3, characterized in that, During the symbiotic culture process in step 3), a mixture of volatile terpenoid signaling molecules is continuously supplied to the culture environment; the mixture of volatile terpenoid signaling molecules is composed of α-pinene and β-caryophyllene in a mass ratio of 1:1 to 1:3, and its concentration in the air of the culture environment is maintained at 10-50 ppb.

6. The method for promoting the growth and development of agarwood using anthracnose fungus 7060# according to claim 5, characterized in that, The supply of the volatile terpenoid signaling molecule mixture was controlled in stages: during days 1-15 of co-culture, the mass ratio of α-pinene to β-caryophyllene in the supplied volatile terpenoid signaling molecule mixture was 1:1-1:1.5, and the concentration was 30-50 ppb; during days 16-60 of co-culture, the mass ratio of α-pinene to β-caryophyllene in the supplied volatile terpenoid signaling molecule mixture was 1:2-1:3, and the concentration was 10-20 ppb. Phased regulation is achieved through the following methods: During days 1-15 of symbiotic culture, the first controlled-release device was placed in the culture bottle; during days 16-60 of symbiotic culture, the first controlled-release device was removed and the second controlled-release device was placed in the culture bottle. Neither the first controlled-release device nor the second controlled-release device comes into direct contact with the culture medium. Both the first and second controlled-release devices contain storage chambers filled with terpene mixture-supported materials. The storage chambers of both the first and second controlled-release devices are connected to the culture flasks via micro-ventilation valves. The micro-ventilation valves are capillary diffusion structures composed of a bundle of glass capillaries with an inner diameter of 0.1-0.3 mm. The effective diffusion area of ​​the capillary diffusion structure of the first controlled-release device is 1.5-3 times that of the capillary diffusion structure of the second controlled-release device. The preparation method of the terpene mixture supported material is as follows: α-pinene and β-caryophyllene liquid mixture are mixed with a porous powder carrier at a mass ratio of 1:2 to 1:

4. After thorough stirring and impregnation, the mixture is left to stand and age at 25°C to 30°C for 12 to 24 hours to allow the terpene components to be fully adsorbed into the pores of the carrier, thereby obtaining the terpene mixture supported material. The porous powder carrier is silica gel, molecular sieve, activated alumina or mesoporous silica, with a specific surface area of ​​200-500 m² / g and an average pore size of 2-50 nm.

7. The method for promoting the growth and development of agarwood using anthracnose fungus 7060# according to claim 6, characterized in that, A temperature and humidity buffer layer is provided between the inner wall of the storage chamber of the first and second controlled-release devices and the terpene mixture loading material. The temperature and humidity buffer layer is composed of a phase change material and a porous material. The phase change material is a mixture of docosane and hexadecane in a mass ratio of 3:1 to 5:1, and its phase change temperature range is 25 to 28°C. The porous material is hydrophobically modified diatomaceous earth with a pore size distribution of 10-100 nm and a specific surface area of ​​100-200 m². 2 / g; The phase change material is loaded into the porous material by vacuum impregnation, with the loading amount being 40%-60% of the mass of the porous material; The thickness of the temperature and humidity buffer layer is 3-5mm.

8. The method for promoting the growth and development of agarwood using anthracnose fungus 7060# according to claim 3, characterized in that, Before activating the strain in step 1), the strain pre-adaptation method is as follows: inoculate the anthrax bacillus 7060# strain onto the pre-adaptation medium and incubate it in the dark at 25℃ to 28℃ for 5-7 days for subsequent activation steps; The pre-adaptation culture medium is based on PDA culture medium, with the addition of 2-5 g / L each of sterilized agarwood seedling leaf powder and root powder, and 1-3 mL / L of water-soluble chemical signal molecule concentrate extracted from the root soil of healthy agarwood plants. Water-soluble chemical signal molecule concentrates were prepared by the following method: rhizosphere soil from healthy agarwood plants was collected, and sterile deionized water was added at a soil-to-water mass ratio of 1:3-1:

5. After shaking and extraction for 2-4 hours, the mixture was allowed to stand. The supernatant was then filtered through a 0.22μm filter membrane and then concentrated by 10-20 times using an ultrafiltration concentrator to remove components with a molecular weight of 500-5000 Da.

9. The method for promoting the growth and development of agarwood using anthracnose fungus 7060# according to claim 8, characterized in that, An energy-releasing component is further added to the pre-adaptation medium. The energy-releasing component consists of microencapsulated glycerol and trehalose. The microcapsule wall material is polylactic acid-glycolic acid copolymer, and the core material mass ratio is glycerol:trehalose = 2:1-3:

1. The particle size of the microcapsules ranges from 10 to 50 μm, and the amount added to the pre-adaptation medium is 1-3 grams of microcapsules per liter of medium.

10. The anthrax bacteria as described in claim 1 ( Colletotrichum sp. Application of 7060# in promoting the growth and development of agarwood.

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