Plant growth-promoting synthetic flora, plant growth-promoting microbial agent and application of plant growth-promoting synthetic flora and plant growth-promoting microbial agent

Abstract

The invention discloses a plant growth-promoting synthetic flora, a plant growth-promoting microbial agent and application of the plant growth-promoting synthetic flora and the plant growth-promoting microbial agent, and the plant growth-promoting synthetic flora comprises strains Penicillium sp. PQxj3 and Penicillium bilaiae LS18. According to the invention, an integrated construction strategy is adopted, Penicillium sp. PQxj3 with a growth-promoting advantage is taken as a core functional strain, directional screening is carried out according to a regulation rule of Penicillium sp. PQxj3 on a soil microbial community structure, a homologous penicillium resource is obtained, Penicillium sp. PQxj3 is taken as a core strain, the Penicillium sp. PQxj3 and a plant growth-promoting synthetic flora constructed by the Penicillium sp. PQxj3 can be stably colonized in rhizosphere, and the growth-promoting effect of the Penicillium sp. PQxj3 is improved. The growth of tobacco roots, branches and leaves is obviously promoted, the quality and yield of tobacco are improved, and the method has important significance on green development of plants such as tobacco.

Description

Technical Field

[0001] This invention relates to the field of crop cultivation technology, and in particular to a plant growth-promoting synthetic microbial community, a plant growth-promoting microbial agent, and their applications. Background Technology

[0002] Plant growth-promoting bacteria (PGPM) are a class of beneficial bacteria or fungi that can directly promote plant growth, resist pests and diseases, and repair soil through various mechanisms, showing great promise for application. Some PGPMs primarily colonize the rhizosphere, and their physical, chemical, and biological characteristics are influenced by the growth and activity of plant roots; therefore, these are also known as rhizosphere growth-promoting bacteria (PGPR). However, most single-species PGPMs suffer from weak growth-promoting functions, poor colonization, and instability due to environmental conditions and interactions between microorganisms. Therefore, the application of single-microorganism-based inoculants remains somewhat limited and constrained.

[0003] Synthetic microbial communities (SynCom) are microbial communities based on ecological and synthetic biology theories. They involve artificially mixing two or more strains of microorganisms with known taxonomic positions and functional characteristics under specific conditions in a certain proportion. This results in highly efficient, multifunctional, controllable, easily preserved, and readily applicable microbial communities. Through rational design and precise division of labor, synthetic microbial communities promote the efficient biosynthesis of complex chemical substances that are difficult to obtain through monoculture, and have shown great application potential in the field of sustainable agricultural development in recent years. With the rapid development of microbiome, bioinformatics, and synthetic biology technologies, research on synthetic microbial communities has shifted from single microbial inoculants to the construction of complex communities, aiming to solve many challenges in agricultural production through microbial engineering.

[0004] Penicillium ( Penicillium These plants are widely found in nature and can produce auxin, thereby stimulating root development, increasing root surface area, improving water and fertilizer absorption efficiency, and secreting antibiotics to directly inhibit or kill rhizosphere pathogenic fungi and bacteria. Some strains have strong growth-promoting abilities. The inventors isolated and screened plant rhizosphere growth-promoting bacteria from the rhizosphere soil of healthy tobacco plants. Penicillium sp. PQxj3 (deposited at the China General Microbiological Culture Collection Center) can significantly increase the leaf area and plant height of tobacco leaves after treatment, thereby improving tobacco yield and quality. However, there is currently no known method to utilize this strain. Penicillium There are reports on the use of sp. PQxj3 and other Penicillium strains to construct synthetic microbial communities to promote tobacco growth. Summary of the Invention

[0005] In view of this, the present invention proposes a plant growth-promoting synthetic microbial community, a plant growth-promoting microbial agent, and the application of the plant growth-promoting synthetic microbial community and the plant growth-promoting microbial agent.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: The plant growth-promoting synthetic microbial community described in this invention includes strains Penicillium sp. PQxj3 and Penicillium bilaiae LS18, the Penicillium sp. PQxj3 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO: 40147 on March 17, 2022; Penicillium bilaiae LS18 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO: 42207, on September 18, 2025.

[0007] The beneficial effects are as follows: This invention uses the growth-promoting strain PQxj3 as the core strain, and through the analysis of the regulatory law of PQxj3 on the soil microbial community structure, strain LS18 is selected by directional screening. The plant growth-promoting synthetic microbial community constructed by PQxj3 and LS18 can stably colonize in the rhizosphere. The two strains synergistically enhance each other, promoting the growth of tobacco roots and branches and leaves, which is of great significance for realizing the green development of tobacco and other plants.

[0008] More preferably, the plant growth-promoting synthetic microbial community of the present invention also includes bacterial strains. Penicillium bilaiae LS25, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO: 42208, was deposited on September 18, 2025. The beneficial effects are: the synthetic microbial community of this invention is constructed from three species of Penicillium, including PQxj3, LS18, and LS25, which can further promote the growth of tobacco roots, branches, and leaves, and improve the quality of tobacco leaves.

[0009] This invention also proposes a plant growth-promoting microbial inoculant, which is a compound microbial inoculant comprising: Penicillium sp. PQxj3 and Penicillium bilaiae Plant growth-promoting synthetic microbial communities constructed by LS18, or Penicillium sp. PQxj3、 Penicillium bilaiae LS18 and Penicillium raperi The plant growth-promoting synthetic microbial community constructed by LS25 can be added to the rhizosphere soil of plants to promote plant growth and improve quality.

[0010] The present invention also provides the application of plant growth-promoting synthetic microbial communities or plant growth-promoting microbial agents in promoting plant growth, which can significantly promote the root growth and branch and leaf growth of plants such as tobacco.

[0011] Strategies for synthesizing microbial communities can be categorized into "top-down," "bottom-up," and an integrated construction strategy that combines both approaches. The "top-down" approach uses indigenous microbial strains from natural microbial communities to construct the synthetic community, focusing on a detailed description of the community's structure and dynamic changes. The "bottom-up" approach, on the other hand, primarily relies on combining single species, designing strains with well-defined functions, and using gene editing techniques to construct the community, thereby obtaining the desired synthetic microbial community. When solving practical application problems and designing and constructing synthetic microbial communities, a reasonable integration of these two strategies is necessary—that is, an integrated construction strategy.

[0012] This invention employs an "integrated construction" strategy, using Penicillium fungi with growth-promoting advantages (… Penicillium sp. PQxj3) is the core functional strain, through Penicillium Sp. PQxj3 was used to conduct targeted screening of the regulatory patterns of soil microbial community structure, obtaining homologous Penicillium resources. Penicillium Using sp. PQxj3 as the core strain, the plant growth-promoting synthetic flora constructed by combining it with the homologous Penicillium can stably colonize in the rhizosphere, significantly promoting the growth of tobacco roots and leaves, improving the quality and yield of tobacco, and is of great significance for achieving the green development of tobacco and other plants. Attached Figure Description

[0013] Figure 1 This is the phylogenetic tree of strain LS25 in Example 1 of the present invention.

[0014] Figure 2 This is the phylogenetic evolutionary tree of strain LS20 in Example 1 of the present invention.

[0015] Figure 3 This is the phylogenetic evolutionary tree of strain LS18 in Example 1 of the present invention.

[0016] Figure 4 This is the effect of the inoculant corresponding to each strain of the present invention on the growth of tobacco (20 days after cultivation).

[0017] Figure 5 This refers to the effects of five groups of tobacco fresh weight, root weight, and root length in Example 2 of the present invention.

[0018] Figure 6 This is the result of plate confrontation of the four strains in Example 3 of this implementation.

[0019] Figure 7This is one of the effects of synthetic microbial communities on tobacco growth in Example 4 of the present invention.

[0020] Figure 8 This is the second example of the effect of synthetic microbial communities on tobacco growth in Example 4 of the present invention.

[0021] Figure 9 This describes the effect of the synthetic microbial community on the fresh weight, root weight, and root length of tobacco in Example 4 of this invention. Detailed Implementation

[0022] The present invention will be further described in detail below with reference to specific embodiments. The embodiments given are only for illustrating the present invention and are not intended to limit the scope of the present invention.

[0023] It should be noted that, unless otherwise specified, the experimental methods in the following examples are all conventional methods; and the materials and reagents used in the following examples are all commercially available unless otherwise specified.

[0024] It should be noted that the tobacco seeds used in the specific embodiments of this invention are the Yunyan 87 variety, which is widely planted in Xinyang. To investigate the effect of synthetic microorganisms on tobacco, the seeds were sterilized before sowing: they were soaked in 0.1% mercuric chloride solution for 5 minutes, and then rinsed with sterile distilled water 3 times or more; then they were soaked in a sodium hypochlorite solution with an available chlorine concentration of 2.5% for 15 minutes, and then rinsed with sterile distilled water 3 times to obtain sterile tobacco seeds.

[0025] It should be noted that the PDA culture medium in this invention is a conventional culture medium. During preparation, agar can be added for sterilization to form a solid plate medium, which can then be dispensed into test tubes to form slant culture medium for culturing four strains (for cryopreservation). The bran solid culture medium in this invention is prepared from bran and sterile distilled water. Specifically: Bran solid culture medium (500 mL Erlenmeyer flask): Weigh 45 g of fine bran, add 35 mL of distilled water, control the moisture content of the bran to 80%, and sterilize at 121℃ for 20 min.

[0026] This invention uses MS medium as a substrate to obtain tobacco seedlings, which includes: MS powder (Murashige & Skoog Basal Medium with Vitamins, Phyto Technology Laboratories) 9.4 g / L, sucrose 10 g / L, pH adjusted to 5.8, agar powder 10 g / L, added to deionized water, and autoclaved at 115°C for 30 min.

[0027] This invention is based on Penicilliumsp. PQxj3 (abbreviated as PQxj3) is the core strain. This strain is a Penicillium that has a significant growth-promoting effect on tobacco. It is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC NO: 40147, and the deposit date is March 17, 2022.

[0028] The present invention Penicillium bilaiae LS18 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO: 42207, and the deposit date is September 18, 2025. Penicillium raperi LS25 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO: 42208, on September 18, 2025.

[0029] Example 1: Isolation, screening, and identification of strains LS18, LS20, and LS25 of the present invention. The first step is to screen tobacco-promoting molds from the soil. Molds from tobacco soil in Xinyang were co-cultured with tobacco on agar plates. Based on the tobacco fresh weight ratio (fresh weight ratio = (strain fresh weight - blank fresh weight) / blank fresh weight) and the number of fibrous roots, tobacco-promoting molds were screened. The co-culture of molds and tobacco seedlings included the following: Sterile tobacco seeds were sown in MS medium-cultured petri dishes, sealed with sealing film, and then cultured in the dark at 4℃ for 1 day. They were then placed in 28℃ and cultured under light for 12 hours, followed by 12 hours in the dark, for a total of 14 days to obtain sterile tobacco seedlings. The sterile tobacco seedlings were then transplanted into PDA petri dishes and cultured for another 7 days. Mold mycelium was inoculated at a distance of 1 mm from the middle of the tobacco root system (mold samples were taken from PDA plates by punching holes, with a depth of 1 cm). The plants were cultured at 28℃ for a total of 14 days. Tobacco-promoting molds were selected based on the fresh weight of the tobacco and the number of fibrous roots. The second step is to filter out those that match... Penicillium sp. PQxj3, a member of the same genus *Penicillium tumefaciens*. The whole genome DNA of each mold selected in the first step was extracted using the CTAB method. The ITS-specific sequence of each mold was amplified by PCR using the universal primers ITS1 and ITS4 to obtain the ITS gene fragment of each strain. The sequence was then sent to Beijing Qingke Biotechnology Co., Ltd. The obtained ITS gene fragments were compared with the NCBI strain database to construct a phylogenetic tree of the strains. Based on the comparison results from the NCBI bacterial strain database and the constructed phylogenetic tree, three strains were identified as being related to... Penicillium Strains belonging to the same genus as sp. PQxj3 (all of which are Penicillium genus) were named as follows: Penicillium sp. LS25 (abbreviated as LS25), Penicillium sp.LS20 (abbreviated as LS20) Penicillium sp. LS18 (abbreviated as LS18), see Table 1. The phylogenetic trees of LS25, LS20, and LS18 are shown below. Figures 1-3 The sequences of the ITS gene fragments of LS25, LS20 and LS18 are shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3, respectively.

[0030] Table 1. Identification results of three Penicillium strains Example 2: Identification of the growth-promoting ability of the four strains in Example 1 of this invention. The four strains in Example 1 of this invention were verified by pot culture, specifically including the following: The first step is to sterilize the tobacco seeds. Using sterile tweezers or sterile toothpicks, the sterile tobacco seeds are sown in MS medium culture dishes and sealed with sealing film. The seed culture dishes are cultured in a dark environment at 4℃ for 1 day. Then they are placed at 28℃ and cultured in a cycle of 12 h light + 12 h dark for 14 days to obtain sterile tobacco seedlings. The second step involves inoculating the strains cultured on PDA plates (using a punch to obtain 1cm diameter mycelial cakes) onto bran solid culture medium and culturing them at 28℃ for 7 days. During the culture process, the initial static incubation allows for the extensive growth of mycelium, followed by appropriate shaking to ensure even distribution of the mycelium. Finally, the culture is statically incubated again until sufficient mycelium and spores are produced, thus obtaining the corresponding microbial inoculum for each strain. The third step is to transplant the sterile tobacco seedlings cultivated in the first step and continue to cultivate them in the cultivation room (28℃). The transplanting substrate is made by mixing water, nutrient soil and vermiculite in a ratio of 3:2:1. After the tobacco plants had grown 4-6 leaves, select plants with uniform growth and randomly divide them into five groups. One group served as a blank control group without any microbial inoculant. For the other four groups, the roots of each tobacco plant were treated with 1g of inoculant, with each group receiving one specific inoculant. The tobacco plants were then cultured for another 20 days, and their growth was observed. The results are shown in the table below. Figure 4 On day 20 after treatment, each group of tobacco seedlings was washed, and the fresh weight, root weight, and root length of the tobacco were recorded. The results are shown in [the table below]. Figure 5 .

[0031] Depend on Figure 4It was observed that all tobacco plants grew normally, and the tobacco plants treated with the four Penicillium strains showed no signs of disease or poor growth. Furthermore, on day 20 after treatment, the tobacco plants treated with the four Penicillium strains exhibited vigorous growth, with greener and larger leaves, showing significantly better growth than the control group. The results indicate that the four Penicillium strains used in this invention do not affect tobacco growth and are harmless to tobacco. Figure 5 It was found that on day 20 after cultivation, the fresh weight and root weight of tobacco seedlings treated with the four Penicillium strains were superior to the blank control. Strain PQxj3 showed a very significant promoting effect, with the fresh weight being 1.3 times that of the blank control group and the root weight being 1.8 times that of the blank control group. The results indicate that all four Penicillium strains can effectively promote tobacco seedling growth, with a particularly prominent effect on promoting tobacco growth (fresh weight).

[0032] Example 3: Antagonism among the four strains of the present invention To rule out antagonistic interactions among the four strains, a plate confrontation experiment was conducted. The four strains were randomly paired, and each pair was inoculated onto the same PDA plate at a distance of 1 cm (each pair was repeated three times). The plates were incubated at 28°C for 6 days, and the boundary region of each pair was observed. The results are shown below. Figure 6 .Depend on Figure 6 It can be seen that there are no obvious antagonistic lines between the four Penicillium strains and no growth inhibition or death was observed, indicating that there is no mutual inhibition among the four Penicillium strains, and they can be used to construct synthetic microbial communities.

[0033] Example 4: Construction of the synthetic microbial community of the present invention There was no mutual inhibition among the four Penicillium strains. Randomly combining the four Penicillium strains yielded 11 synthetic groups, namely PQxj3+LS20, PQxj3+LS25, PQxj3+LS18, PQxj3+LS18+LS20, PQxj3+LS18+LS25, PQxj3+LS20+LS25, LS18+LS25, LS20+LS25, LS18+LS20, LS20+LS18+LS25, and PQxj3+LS20+LS25+LS18. Each synthetic microbial community was cultured to obtain microbial agents. Taking the PQxj3+LS20 compound microbial agent as an example: strains PQxj3 and LS20 cultured on PDA plates were punched with a 1 cm punch, and 1 cm mycelial cakes were inoculated into bran solid culture medium and cultured at 28℃ for 7 days. During the culture process, static incubation in the early stage allowed for a large amount of mycelial growth, followed by appropriate shaking to ensure even distribution of the mycelium. Finally, static incubation was performed again until sufficient mycelium and spores were produced. After separate culture, they were mixed to obtain the compound microbial agents corresponding to PQxj3 and LS20. Similarly, other combined microbial community compound microbial agents can be obtained. Sterile tobacco seedlings were obtained according to the cultivation method in Example 2. The sterile tobacco seedlings were transplanted into pots for cultivation. After transplanting, they were cultivated in a cultivation room (28°C) until the tobacco plants grew 4-6 leaves. Tobacco plants with uniform growth were randomly divided into thirteen groups. One group served as a blank control group without any microbial inoculant, and another group served as a control group with PQxj3 inoculant. For the other groups, 1 g of inoculant was added to the roots of each tobacco plant, with one inoculant per group. The tobacco plants were then cultured for 20 days, and their growth was observed. The results are shown in […]. Figure 7-8 On day 20 after treatment, each group of tobacco seedlings was washed, and the fresh weight of the tobacco and the root weight were recorded. The results are shown in [the table below]. Figure 9 .

[0034] Depend on Figure 7-8 It can be seen that the tobacco in each group grew normally, without any problems such as stunted growth, wilting, or disease. The tobacco in all 11 compound microbial agent treatment groups showed no adverse phenomena such as disease or slowed growth. The results indicate that the 11 compound microbial agents did not affect the normal growth of tobacco and were harmless to it. Furthermore, from... Figure 7-8 It can be seen that on day 20 after treatment, compared with the blank control, the tobacco of the PQxj3+LS25+LS18 combination grew vigorously, with greener and larger leaves, and the tobacco growth was significantly better than the blank control and other synthetic microbial combinations.

[0035] Depend on Figure 9 It was found that on day 20 after treatment, except for the PQxj3+LS25 treatment group, the fresh weight and root weight of tobacco seedlings treated with the other synthetic microbial groups were significantly higher than those of the blank control group. Among them, the fresh weight of tobacco seedlings treated with the PQxj3+LS25+LS18 group was the best, significantly higher than other combinations. The fresh weight reached 1.63 times that of the blank control, and the dry weight reached 1.6 times that of the blank control, demonstrating a significant ability to promote tobacco growth in the early stage of tobacco growth. Therefore, the synthetic microbial group constructed by PQxj3+LS25+LS18 is the optimal microbial group in this invention.

[0036] In summary, the invention employs an "integrated construction" strategy, using Penicillium fungi with growth-promoting advantages (… Penicillium sp.PQxj3) is the core functional strain, and through analysis Penicillium Based on the analysis of the regulatory mechanism of sp. PQxj3 on soil microbial community structure, targeted screening was carried out to obtain homologous Penicillium resources. Using PQxj3, Penicillium and core strains, a plant growth-promoting synthetic microbial community was constructed with homologous Penicillium. The synthetic microbial community can stably colonize in the rhizosphere, significantly promoting the growth of plant roots and branches, improving plant quality and yield, which is of great significance for realizing the green development of tobacco and other plants.

Claims

1. A plant growth-promoting synthetic microbial community, characterized in that: Including strains Penicillium sp. PQxj3 and Penicillium bilaiae LS18, the Penicillium sp. PQxj3 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO: 40147 on March 17, 2022; Penicillium bilaiae LS18 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO: 42207 on October 28, 2025.

2. The plant growth-promoting synthetic microbial community according to claim 1, characterized in that: Also includes strains Penicillium bilaiae LS25 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO: 42208 on October 28, 2025.

3. A plant growth-promoting microbial agent, characterized in that: Includes the plant growth-promoting synthetic microbial community as described in claim 1 or 2.

4. The application of the plant growth-promoting synthetic microbial community according to any one of claims 1-2 or the plant growth-promoting microbial agent according to claim 3 in promoting plant growth.

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