Application of cello-oligosaccharide in inducing plant PTI to promote rhizobium recruitment and symbiotic nodulation

Abstract

The invention provides application of cellooligosaccharide in inducing plant PTI to promote rhizobium recruitment and symbiotic nodulation, and belongs to the technical field of plant-microorganism interaction. The invention provides an application of cellooligosaccharide in inducing plant innate immunity, cellooligosaccharide with structure specificity is used for inducing PTI signals, and accurate targeting recruitment of rhizobium is carried out to improve the nodulation nitrogen fixation and growth performance of plants. The invention further provides a method for promoting plant growth, the compatibility state of plant growth and immunity is maintained under PTI activation only through PTI stimulation of the cello-oligosaccharide, and the nodulation efficiency and yield of crops are effectively improved by establishing a positive feedback model of PTI immune activation-microbial interaction-nutrition enhancement-growth promotion. According to the method, the in-situ symbiotic rhizobium can be accurately enriched without supplementing an exogenous microbial agent, and the method is an in-situ microorganism regulation and control method which is high in ecological adaptability and high in field generalizability.

Description

Technical Field

[0001] This invention relates to the field of plant-microbe interaction technology, specifically to the application of cellulosic oligosaccharides in inducing plant PTI to promote rhizobium recruitment and symbiotic nodulation. Background Technology

[0002] The rhizosphere is a crucial ecological interface for close interactions between plants and soil microorganisms. Rhizosphere microbial communities play a key role in plant nutrient absorption, growth and development, and disease and stress resistance. Particularly in legumes, symbiotic microorganisms such as rhizobia can establish root nodules to fix atmospheric nitrogen, forming an important biological basis for achieving green agricultural development and sustainable nitrogen utilization.

[0003] Existing plant-microbe interaction regulation strategies mainly rely on the application of exogenous microbial agents, such as rhizobium inoculants and growth-promoting fertilizers. However, in actual field applications, these agents often face problems such as low colonization efficiency, unstable interactions with plant hosts, and significant influence from soil environmental background, which limits their adaptability and promotion in various ecosystems.

[0004] In recent years, the mechanism by which plants regulate the rhizosphere microbiome through endogenous signaling has received increasing attention. Among these mechanisms, the "cry for help" mechanism has been proposed to describe the phenomenon in which plants actively recruit beneficial microorganisms by secreting metabolites through their roots when subjected to biotic or abiotic stresses or specific signal stimuli. This mechanism provides a theoretical basis for plants to achieve "autonomous regulation of the rhizosphere microbiome."

[0005] Studies have shown that plants can trigger immune (PTI) pathways by activating pattern recognition receptors, inducing root metabolic reprogramming and releasing specific compounds (such as flavonoids and organic acids) to form a distress signal, thereby attracting beneficial microorganisms such as growth-promoting bacteria and nitrogen-fixing bacteria to accumulate in the rhizosphere. This process enables plants to dominate the construction of rhizosphere ecological niches through their own immune system, thereby enhancing their response to nutrient stress or microbial invasion.

[0006] Furthermore, existing research largely focuses on sterile or simplified microbial settings, neglecting the regulatory role of complex native microbial communities in natural soil on the induction of immune signals. Therefore, there is an urgent need to develop a novel regulatory method that, under natural microecological conditions, induces low-level PTI responses in plants to guide them in autonomously releasing "recruitment signals" to recruit beneficial local microorganisms in situ, thereby enhancing plant nutrient absorption and nodulation nitrogen fixation capabilities. This would provide technical support for green agriculture and microbiome engineering. Summary of the Invention

[0007] This invention aims to overcome the problems of existing plant-microbe interaction regulation methods, such as reliance on exogenous microbial inoculation, low colonization efficiency, and poor synergy with plants. It provides the application of fiber oligosaccharides in inducing plant PTI to promote rhizobium recruitment and symbiotic nodulation. Based on the plant's endogenous immune system regulation ability, fiber oligosaccharides induce PTI signals to selectively enrich beneficial microorganisms such as rhizobia, thereby enhancing plant-microbe interactions. It has broad crop adaptability and application prospects.

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] This invention provides the application of fiber oligosaccharides in inducing innate immunity in plants.

[0010] In a preferred embodiment of the present invention, the cellulose oligosaccharide comprises a linear oligosaccharide composed of β-1,4-D-glucose residues.

[0011] In a preferred embodiment of the present invention, the degree of polymerization of the linear oligosaccharide is 2 to 6.

[0012] In a preferred embodiment of the present invention, the plant includes a plant that interacts with rhizobia.

[0013] This invention also provides the application of fiber oligosaccharides in regulating rhizosphere microbial ecology by inducing plant innate immunity.

[0014] In a preferred embodiment of the present invention, the regulation of rhizosphere microbial ecology includes in situ recruitment of rhizobia.

[0015] The present invention also provides a method for regulating plant-microbe interactions based on plant PTI signaling induced by cellulosic oligosaccharides, comprising applying cellulosic oligosaccharides to plants, wherein the cellulosic oligosaccharides are linear oligosaccharides composed of β-1,4-D-glucose residues.

[0016] In a preferred embodiment of the present invention, the method of applying the oligosaccharide includes at least one of the following: foliar spraying, root irrigation, seed coating, or root zone application.

[0017] The present invention also provides a plant-microbe interaction enhancer composition comprising a cellulosic oligosaccharide and a supportive solution or buffer system; wherein the cellulosic oligosaccharide is a linear oligosaccharide composed of β-1,4-D-glucose residues.

[0018] The present invention also provides a method for enhancing the ability of plants to recruit beneficial microorganisms, comprising applying to the plant the above-mentioned plant-microorganism interaction enhancer composition or cellulosic sugar, wherein the cellulosic sugar is a linear oligosaccharide composed of β-1,4-D-glucose residues.

[0019] Beneficial effects: This invention provides the application of oligosaccharides in inducing plant innate immunity. Using oligosaccharides as a signal to induce plant innate immunity (PTI), it is the first time that PTI signals can be precisely induced by structurally specific oligosaccharides to target and recruit symbiotic microorganisms such as rhizobia, thereby significantly improving plant nodulation efficiency (including the colonization capacity and number of nodules of rhizobia), biological nitrogen fixation capacity, nitrogen utilization capacity and overall growth performance.

[0020] This invention also provides a method for promoting plant growth and nutrient absorption without relying on exogenous microbial agents. This method utilizes low-intensity PTI stimulation to activate immunity while maintaining a compatible state between plant growth and defense, constructing a positive feedback regulatory mechanism of "PTI activation - microbial interaction - nutrient enhancement - growth promotion." This mechanism significantly improves the colonization capacity of in-situ rhizobia in the plant rhizosphere, leading to a simultaneous increase in nodule number, nitrogen accumulation, and aboveground biomass.

[0021] The method described in this invention can reconstruct plant-microbe interaction functions without destroying the natural microbial community structure of the soil. It has the advantages of strong ecological adaptability, simple operation, no need for genetic modification, and high field scalability. It is suitable for green nitrogen fixation cultivation systems of legumes such as soybeans and alfalfa, as well as other crop planting systems that need to strengthen rhizosphere interaction capabilities. It has broad application prospects in ecological agriculture and microbiome engineering. Attached Figure Description

[0022] Figure 1 Figure 1 shows the results of immune-related experiments for different treatment groups. In the figure, a: reactive oxygen species burst results, b: relative expression level of PR1.

[0023] Figure 2 Figures showing the growth-related data for different treatment groups;

[0024] Figure 3 The graph shows the relative abundance of Rhizobiales in the rhizosphere of Arabidopsis thaliana under PTI activation induced by cellulosic oligosaccharides.

[0025] Figure 4 Figure a shows the results of rhizosphere microbial community shaping mediated by cellotetrasaccharide-mediated PTI response. Figure a: Marker bacterial groups significantly enriched in rhizosphere after cellotetrasaccharide activation of PTI; b: Cellotetrasaccharide treatment significantly increased the relative abundance of rhizobium (Rhizobium, etc.) in rhizosphere (p<0.05); c: Various potentially pathogenic fungal groups (such as Fusarium and Alternaria) were significantly inhibited after cellotetrasaccharide treatment. In Figures a and c, green text indicates groups with decreased relative abundance, and black text indicates groups with increased relative abundance.

[0026] Figure 5 Figure 1 shows the effect of cellotetrasaccharide-mediated PTI response on promoting plant growth and nitrogen metabolism in the context of natural microbial communities. Figure 2 shows: a) Comparison of the aboveground phenotype of Arabidopsis thaliana after cellotetrasaccharide treatment under two conditions: sterilized substrate and inoculation with natural soil microorganisms; b) Statistical analysis of flowering time (bolting time) of Arabidopsis thaliana; c) Changes in the expression level of genes related to flowering regulation; d) Activity of key enzymes related to nitrogen utilization.

[0027] Figure 6 The figure shows the results of the soybean-rhizobium system nodulation verification experiment. The results show that cellotetrasaccharide treatment promotes soybean growth and nodule development under nitrogen-free (N0) conditions. a: Comparison of aboveground phenotypes of soybean plants without inoculation or inoculated with rhizobium (USDA110) under N0 conditions after cellotetrasaccharide treatment. b: Root morphology and nodule formation under the corresponding treatments. c: Biomass statistics of aboveground and belowground parts, showing that cellotetrasaccharide treatment significantly increased plant biomass in the USDA110 inoculated group (p<0.05). d: Nodule development traits, including nodule number and fresh weight. Detailed Implementation

[0028] This invention provides the application of fiber oligosaccharides in inducing innate immunity in plants.

[0029] The cellulose oligosaccharides of this invention comprise polymers formed from β-1,4-D-glucose monomers, particularly linear oligosaccharides formed from β-1,4-D-glucose monomers, with a degree of polymerization of 2–6, and are named DP2, DP3, DP4, DP5, and DP6, respectively. Examples have verified that DP4 exhibits the best in-situ targeted recruitment effect on rhizobia. This invention utilizes the cellulose oligosaccharides to induce low-level pattern recognition receptor-triggered immune (PTI) responses in plants. Under PTI activation conditions, through plant root metabolite reprogramming and signal regulation, rhizosphere microorganisms promoting plant growth behavior, especially rhizobia, are selectively recruited. The above treatment is performed in a non-sterile matrix containing natural native microbial communities to maintain microbiome-mediated symbiotic construction, thereby enhancing the plant's ability to recognize and colonize beneficial microorganisms, promoting nitrogen absorption, nodulation, and growth.

[0030] The fiber oligosaccharides described in this invention can serve as PTI-inducing signals in various plant systems. These plants can be those capable of interacting with rhizobia, including legumes, herbaceous plants, dicotyledonous plants, as well as specific model plants and economic crops. The legumes include the following subfamilies and genera: Papilionoideae (Fabaceae), such as soybean, alfalfa, broad bean, pea, adzuki bean, cowpea, mung bean, common bean, hyacinth bean, pigeon pea, peanut, milkvetch, indigo, Dalbergia, kudzu, kudzu vine, astragalus gum, and sophora; Mimosoideae (Mimosa), such as mimosa, thorny bush, sophora, gum arabic, and copal; and Caesalpinioideae (Caesalpinioideae), such as sappanwood, soapberry, privet, red bean, and hemp. Any of these plants can interact with rhizobia or rhizosphere-promoting bacteria and is suitable for the PTI activation regulation strategy described in this invention. The model plants are representative plant models such as Arabidopsis thaliana or tomato. The agricultural crops mentioned include, but are not limited to, grasses such as rice, corn, wheat, sorghum, oats, foxtail grass, rye, sugarcane, reeds, bamboo, barnyard grass, cogongrass, jointed barnyard grass, and crabgrass.

[0031] This invention also provides the application of fiber oligosaccharides in regulating rhizosphere microbial ecology by inducing plant innate immunity.

[0032] The embodiments of this invention have confirmed that the aforementioned oligosaccharide can induce low-level ROS bursts and PR gene expression, characteristic of PTI (post-inflammatory hyperbacterial disease) basal immune responses in plants; and achieve microbial enrichment, particularly significant enrichment of plant growth-promoting rhizobia (PGPR) such as Rhizobiales in the rhizosphere, thereby increasing the number of root nodules, nodule size, nitrogenase activity, and plant biomass. The regulation of rhizosphere microbial ecology described in this invention includes in situ recruitment of rhizobia. When using the aforementioned oligosaccharide as a PTI-inducing signal, the oligosaccharide can be applied through various exogenous methods.

[0033] The present invention also provides a method for regulating plant-microbe interactions based on plant PTI signaling induced by cellulosic oligosaccharides, comprising applying cellulosic oligosaccharides to plants, wherein the cellulosic oligosaccharides are linear oligosaccharides composed of β-1,4-D-glucose residues.

[0034] The application method described in this invention can be foliar spraying, root irrigation, seed coating, or root zone application. One embodiment uses foliar spraying as an example. When applying the oligosaccharide topically, the concentration of the oligosaccharide is 1–200 μM, for example, within the range of 10–100 μM, with 50 μM being particularly effective. In one embodiment, the solvent for the oligosaccharide solution is distilled water.

[0035] The present invention also provides an enhancer composition for enhancing plant-microbe interactions, comprising a cellulose oligosaccharide and a supportive solution or buffer system.

[0036] The cellulose oligosaccharide described in this invention is an oligosaccharide linked by β-1,4-D-glucose groups, with a degree of polymerization (DP) of 2 to 6. In one embodiment, DP4 (cellotetrasaccharide) was shown to have the best overall effect. It can act as an inducing signaling molecule of the plant's innate immune system (PTI), promoting the recognition and recruitment of beneficial microorganisms (such as rhizobia and growth-promoting bacteria) by plant roots.

[0037] The supporting solution is used to stabilize and carry the oligosaccharides, facilitating their transport and absorption in the plant rhizosphere. This supporting solution may include water, alcohols, sugar alcohols, bio-release polymers, surfactants (such as Tween-20), etc., preferably distilled or sterile water. The buffer system is used to adjust the pH stability of the oligosaccharide system, preventing oligosaccharide degradation and maintaining its biological activity. The buffer may include one or more of the following: phosphate buffer (PBS, pH 5.8–7.2); Tris-HCl buffer (pH 6.0–8.0); MES buffer (suitable for weakly acidic stability); other agriculturally available buffer systems.

[0038] The plant-microbe interaction enhancer described in this invention may also contain surfactants, spreading agents, or nutrient cofactors to better induce the basic PTI response and promote rhizobium customization, thereby improving plant nodulation efficiency, nodule number, nitrogenase activity, and biomass accumulation.

[0039] The present invention also provides a method for enhancing the ability of plants to recruit beneficial microorganisms, comprising applying to the plant the above-mentioned plant-microorganism interaction enhancer composition or cellulosic sugar, wherein the cellulosic sugar is a linear oligosaccharide composed of β-1,4-D-glucose residues.

[0040] This invention induces a low-level PTI (Pattern-Triggered Immunity) response in plants, thereby avoiding the typical "growth-defense" trade-off while selectively enriching beneficial rhizosphere microorganisms, particularly rhizobium groups within growth-promoting bacteria (PGPR). The recruited PGPR enhance plant nutrient acquisition and growth performance through multiple mechanisms, including promoting nitrogen uptake and nitrogen fixation. Particularly in legumes, it significantly increases nodule number and biological nitrogen fixation levels, forming a positive feedback loop of "immune activation - microbial interaction - nutrient enhancement - growth promotion."

[0041] The immune-microbiome synergistic regulation mechanism described in this invention relies on the integrity of the natural soil microbial community. Under conditions where a native microbial community is present (non-sterile environment), plants exhibit more significant positive physiological responses, such as increased biomass accumulation, enhanced nitrogen uptake, and earlier growth stages. Therefore, the method described in this invention is preferentially applied to non-sterile substrate conditions containing a natural microbial background to maximize the benefits of plant-microbe interactions.

[0042] To further illustrate the present invention, the application of the fiber oligosaccharide provided by the present invention in inducing plant PTI to promote rhizobium recruitment and symbiotic nodulation is described in detail below with reference to embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0043] Example 1: Cellotetrasaccharide-induced immune response and rhizosphere remodeling experiment in Arabidopsis thaliana

[0044] 1. Materials and plant growth conditions

[0045] The cellulose oligosaccharides used in this embodiment include cellobiose (DP2, molecular formula C). 12 H 22 O 11 (Molecular weight 342.30), cellotriose (DP3, C) 18 H 32 O 16 (Molecular weight 504.45), cellotetrasaccharide (DP4, C 24 H 42 O 21 (Molecular weight 666.59), Fiber pentasaccharide (DP5, C) 30 H 52 O 26 (molecular weight 828.73) and cellohexasaccharide (DP6, C 36 H 62 O 31 All of the above-mentioned cellulose oligosaccharides (molecular weight 990.90) were purchased from Megazyme (Bray, Ireland) and had a purity of ≥90%. These oligosaccharides are composed of β-1,4-linked D-glucose units and have good water solubility.

[0046] The experimental plant was wild-type Arabidopsis thaliana (Col-0). Seeds were surface-sterilized sequentially with 70% ethanol and 3% sodium hypochlorite, and then vernalized for 3 days at 4°C in the dark. The substrate consisted of a 1:1:1 volume ratio of peat moss, vermiculite, and perlite, packaged in 8×8×12.5cm square containers, moistened from the bottom. 12–16 seeds were sown per container, arranged in a 4×4 pattern with a row spacing of approximately 2.5cm. Seeds were covered with film to promote germination; after true leaves emerged, the film was removed and seedlings were thinned, ultimately retaining two uniformly shaped seedlings per container. Cultivation conditions included an artificial greenhouse with full-spectrum white light and a light intensity of 200 μmol / m². 2 / s, temperature 25℃ (day) / 20℃ (night), humidity 40%, CO2 at natural concentration, supplemented with modified Hogrange nutrient solution 1-2 times per week. All treatments began when the plants were 22 days old. Cellobiose to cellohexaose were dissolved in sterile distilled water to a final concentration of 50 μM; the control group used sterile water without cellooligosaccharides. The solution was applied by foliar spraying 3 hours after the start of light exposure, once every 3 days, for 10 consecutive days.

[0047] 2. Rhizosphere microbiome transplantation treatment

[0048] Freshly collected farmland soil (approximately 50g from the rhizosphere of wheat in the field; source: Institute of Agricultural Environment and Sustainable Development, Chinese Academy of Agricultural Sciences, Shunyi, Beijing; 40°09′65.15″N, 116°92′33.91″E) was vigorously mixed with 200mL of sterile water for 60 seconds. The resulting soil suspension was used to inoculate 16-day-old Arabidopsis thaliana pot substrate, with 1mL added to each pot to form the "infected group". The same batch of suspension was sterilized by high-pressure steam at 121℃ and then cooled, and added in the same way to form the "sterile group", which served as the control.

[0049] One week after inoculation with soil microorganisms (plant age 23 days), foliar spraying was performed on four groups: "with microorganisms + cellotetrasaccharide", "with microorganisms + control", "sterile + cellotetrasaccharide", and "sterile + control". Spraying was done every 3 days for a total of 4 applications. Spraying was performed 3 hours after the plants were exposed to sunlight, and the amount sprayed was just enough to moisten the leaves to the point of slight dripping. Before spraying, sterile absorbent cotton was used to cover the surface of the potting soil to prevent direct contact between the liquid and the root zone.

[0050] Two days after treatment, the plants and rhizosphere soil were harvested. During collection, the plants were uprooted, and the compacted soil near the roots was placed in a 50 mL centrifuge tube containing 20 mL of PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM phosphate buffer, pH 7.3-7.5). The tube was incubated at 30°C and 200 rpm for 20 min, after which the root tissue was discarded. The tube was then centrifuged at 1000 rpm for 20 min, the supernatant was discarded, and the precipitate (rhizosphere soil sample) was retained. This sample was immediately frozen in liquid nitrogen and stored at -80°C until subsequent analysis.

[0051] The entire experimental process was conducted under aseptic conditions as possible to avoid environmental microbial contamination.

[0052] 2.1 Analysis of the basic immune response of Arabidopsis thaliana PTI induced by oligosaccharides with different degrees of polymerization: To verify the structural specificity differences of the oligosaccharides described in this invention in inducing plant PTI (pattern recognition receptor triggered immunity), the reactive oxygen species (ROS) burst was detected by luminol chemiluminescence assay, and the relative expression level of the classical disease resistance gene PR1 was detected by qRT-PCR.

[0053] The primer sequences used are as follows:

[0054] Forward(SEQ ID No.1):5'-GGTGACTTGTCTGGCGTCTC-3',

[0055] Reverse (SEQ ID No. 2): 5'-ACTTTGGCACATCCGAGTCT-3'. RNA extraction and reverse transcription were performed according to standard operating procedures. qPCR data were normalized to the internal reference gene Actin.

[0056] The results show ( Figure 1 In (a), at a treatment concentration of 20 μM, only DP4 (cellotetrasaccharide) and DP6 (cellohexasaccharide) could induce a significant transient reactive oxygen species burst response, exhibiting the typical early immune response of PTI; no significant ROS signal was observed in the DP2 and DP3 treatment groups. Figure 1 Figure b shows that DP3 to DP6 can all induce significant upregulation of the PR1 gene (p<0.01), with DP6 inducing the highest expression level, followed by DP4, both significantly higher than the control group and the DP2 group. DP2 did not induce significant upregulation. In summary, oligosaccharides with a degree of polymerization ≥3 can induce a typical PTI immune response in plants, with DP4 and DP6 showing more significant effects in ROS burst and PR1 expression. The results indicate that the oligosaccharides used in this invention have the ability to induce plant PTI responses with structural specificity, among which DP4 and DP6 are the oligomers with the strongest immunomodulatory activity.

[0057] 2.2 Aboveground biomass determination: The aboveground biomass (fresh weight) of Arabidopsis thaliana in sterilized soil and soil with preserved natural microbial communities was compared by comparing the activation of plant PTI immune responses by fiber oligosaccharides with different degrees of polymerization over a certain period.

[0058] The results are as follows Figure 2 As shown, in the "sterile group" where the natural microbial community was preserved, all oligosaccharides of different degrees of polymerization increased the aboveground biomass of Arabidopsis thaliana to varying degrees, with DP4 (cellotetrasaccharide) showing the most significant effect, demonstrating a growth-promoting effect under PTI activation. DP2 and DP3 showed increased biomass with increasing concentration, while DP6 exhibited growth inhibition at high concentrations (100 μM), showing a decreasing trend in biomass. In contrast, the increase in biomass was not significant in the sterile treatment group, suggesting that the plant growth-promoting effect of oligosaccharides depends on the presence of rhizosphere microorganisms.

[0059] 2.3 Rhizosphere microbial community structure determination: To analyze whether PTI induced by different degrees of polymerization of cellulosic oligosaccharides remodels the plant rhizosphere microbiome structure, rhizosphere soil samples were collected from the infected group, and high-throughput sequencing was performed using 16S rRNA and ITS amplified regions.

[0060] 1) Bacterial sequencing was performed using primers 338F (SEQ ID No. 3: 5'-ACTCCTACGGGAGGCAGCAG-3') and 806R (SEQ ID No. 4: 5'-GGACTACHVGGGTWTCTAAT-3') to amplify the V3-V4 region of bacterial 16S rRNA;

[0061] 2) Fungal sequencing used primers ITS1F (SEQ ID No. 5: 5'-CTTGGTCATTTAGAGGAAGTAA-3') and ITS2R (SEQ ID No. 6: 5'-TGCGTTCTTCATCGATGC-3') to amplify the ITS1-ITS2 region.

[0062] PCR products were screened for fragments approximately 460 bp in length by agarose gel electrophoresis, quantified using a Qubit 2.0 fluorometer, diluted to 1 ng / μl, and sent to Allwegene (Beijing) for sequencing using the Illumina MiSeq platform (PE250). Raw data have been uploaded to the NCBI SRA database, accession number PRJNA803271. Sequencing data underwent bioinformatics analysis.

[0063] The results are as follows Figure 3As shown, activation of PTI with cellooligosaccharides of different degrees of polymerization (50 μM) significantly altered the structure of the Arabidopsis rhizosphere microbiome. Particularly in the DP4 (cellotetrasaccharide) group, the relative abundance of Rhizobiales significantly increased, indicating a trend of targeted recruitment of symbiotic nitrogen-fixing bacteria. In contrast, the DP2, DP3, DP5, and DP6 groups failed to effectively induce rhizobium enrichment.

[0064] 2.4 Analysis of rhizosphere microbiome characteristics shaped by cellotetrasaccharide-mediated PTI: The LefSe analysis method was used to screen for biomarkers of differentially expressed microorganisms between the cellotetrasaccharide-treated group and the control group.

[0065] The results are as follows Figure 4 As shown, cellotetrasaccharide-activated PTI significantly enriched several typical PGPR groups, particularly those in the orders Rhizobiales and Burkholderiales; simultaneously, pathogen-related groups such as Acetobacterales and Dothideomycetes fungi in the phylum Ascomycota were significantly inhibited. These results indicate that cellotetrasaccharide-induced PTI not only recruits growth-promoting bacteria but also effectively inhibits potential pathogenic microorganisms, achieving bidirectional regulation of the rhizosphere microecology and contributing to improved plant growth potential and soil health.

[0066] 2.5 Verification of the effect of cellotetrasaccharide-mediated PTI response on plant growth and nitrogen metabolism in a natural microbial community setting: Arabidopsis thaliana treatment was compared in sterile substrates and substrates inoculated with natural microorganisms to evaluate its phenotypic, developmental, molecular, and metabolic responses. The primer sequences used are as follows:

[0067] Forward(SEQ ID No.7):5'-CTGAGGCAATGATGTTCG-3',

[0068] Reverse (SEQ ID No. 8): 5'-TTGGGTTGTGACTTGTTCC-3'. RNA extraction and reverse transcription were performed according to standard operating procedures. qPCR data were normalized to the internal reference gene Actin.

[0069] The results are as follows Figure 5As shown: Aboveground phenotypic observations revealed that in the "infected group" inoculated with natural microbial communities, cellotetrasaccharide treatment significantly enhanced the growth status of Arabidopsis thaliana, manifested as darker green leaves, increased leaf area, and increased plant height, exhibiting a stronger growth-promoting effect compared to the sterile group. Statistics on Arabidopsis thaliana flowering time (bolus time) showed that cellotetrasaccharide treatment significantly accelerated the flowering process of the inoculated group (p<0.05), indicating that PTI activation can accelerate the transition of plants to reproductive growth. qRT-PCR was used to detect the expression levels of key flowering regulatory genes, revealing that the expression level of the DUF1216 gene was significantly upregulated in the cellotetrasaccharide-treated group, revealing its molecular-level flowering-promoting effect. Biochemical detection of nitrogen utilization-related enzyme activities showed that cellotetrasaccharide treatment significantly increased the activity of the key enzyme nitrite reductase (NiR), reflecting enhanced nitrogen utilization capacity in plants. In summary, cellotetrasaccharide-induced plant PTI responses exhibit significant growth-promoting and development-promoting functions in the context of natural microbial communities, particularly accelerating plant entry into the reproductive stage and enhancing nitrogen metabolism. This effect depends on the presence of the microbiome, suggesting that the application of this invention should preferably be carried out in non-sterile, natural soil ecosystems.

[0070] Example 2: Greenhouse verification of the regulation of rhizobium accumulation and nodulation efficiency by cellotetrasaccharides in soybeans

[0071] To verify the regulatory effect of cellotetraose-mediated plant PTI (Pattern-Triggered Immunity) response on nodulation and nitrogen fixation efficiency in the legume-rhizobium symbiotic system, a soybean-rhizobium nodulation verification system was constructed under greenhouse conditions to explore whether PTI activation promotes the symbiotic nitrogen fixation process.

[0072] 1. Experimental Materials and Conditions

[0073] The experimental plant material was soybean variety WS82, and the inoculated strain was the slow-growing soybean rhizobium *Bradyrhizobium japonicum* USDA110. The experiment used a completely vermiculite sterile substrate. During the seedling stage, no nutrient solution was applied; only deionized water (ddH2O) was used to maintain water supply, creating a complete nitrogen starvation (NO) condition to test the plant's dependence on symbiotic nitrogen fixation by the rhizobium. Cellotetrasaccharide was purchased from Megazyme, with a purity ≥90%, and dissolved in sterile water to prepare a 50 μM solution, which was prepared and used immediately. The inoculated strain was the slow-growing soybean rhizobium *Bradyrhizobium japonicum* USDA110. The inoculation amount was 1 mL of bacterial solution per plant in the rhizosphere, with a bacterial concentration of approximately 1 × 10⁻⁶. 8CFU / mL. Rhizosphere inoculation was performed 7 days after sowing, with no watering for 24 hours after inoculation to promote rhizobium colonization. The bacterial culture was incubated in YEM liquid medium at 28°C with shaking for 72 hours, and the culture in the logarithmic growth phase was used.

[0074] 2. Grouping settings and processing methods

[0075] The experiment was set up with the following four treatment groups:

[0076] Water treatment group (no cellotetrasaccharide, no rhizobium inoculation);

[0077] Cellotetraose group (treated with 50 μM cellotetraose, without inoculation with rhizobia);

[0078] Rhizobium group (inoculated with USDA110 rhizobium, without cellotetrasaccharide);

[0079] Cellotetraose + Rhizobium group (cellotetrasaccharide treatment + rhizobium inoculation).

[0080] Each group had 3 replicates, with each replicate containing 10 plants. Treatment began on the 15th day after sowing, with foliar application of cellotetrasaccharides every 3 days for a total of 4 treatments. The amount of spray was just enough to moisten the leaves, and all spraying was carried out 3 hours after the start of light exposure.

[0081] The results are as follows Figure 6 As shown: Under conditions without rhizobium inoculation, there was no significant difference in aboveground and belowground biomass of soybeans between the water treatment group and the cellotetrasaccharide group, indicating that PTI activation itself does not directly cause growth enhancement in the absence of symbiotic bacteria. Under conditions with rhizobium inoculation, cellotetrasaccharide treatment significantly increased the number of soybean root nodules (+19%), root nodule fresh weight (+37%), and aboveground biomass (+23%), showing enhanced symbiotic efficiency and improved plant nutritional status. This example demonstrates that in the soybean-rhizobium symbiotic system, cellotetrasaccharide can significantly promote the recruitment and nodulation process of rhizobia by activating the PTI reaction, thereby improving symbiotic nitrogen fixation efficiency and plant biomass, confirming the potential of this invention for application in leguminous crops to enhance symbiotic nitrogen fixation capacity.

[0082] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Use of cello-oligosaccharide in inducing plant innate immunity.

2. Use according to claim 1, characterized in that, The cello-oligosaccharide comprises linear oligosaccharide consisting of β-1, 4-D-glucose residues.

3. Use according to claim 2, characterized in that, The linear oligosaccharide has a degree of polymerization of 2-6.

4. The use according to claim 1, characterized in that, The plant comprises a plant interacting with rhizobium.

5. Use of cello-oligosaccharide in regulating rhizosphere microbial ecology by inducing plant innate immunity.

6. Use according to claim 5, characterized in that, The regulating rhizosphere microbial ecology comprises in-situ recruitment of rhizobium.

7. A method of inducing plant PTI signals based on a fibrous oligosaccharide to regulate plant-microbe interactions, characterized in that, The method comprises applying cello-oligosaccharide to the plant, the cello-oligosaccharide being linear oligosaccharide consisting of β-1, 4-D-glucose residues.

8. The method of claim 7, wherein, The method of applying the cello-oligosaccharide comprises at least one of foliar spraying, root irrigation, seed coating, and root zone irrigation.

9. A plant-microbe interaction enhancer composition, characterized in that, The method comprises applying cello-oligosaccharide to the plant, the cello-oligosaccharide being linear oligosaccharide consisting of β-1, 4-D-glucose residues.

10. A method of enhancing the ability of a plant to recruit beneficial microorganisms, comprising, The method comprises applying cello-oligosaccharide to the plant, the cello-oligosaccharide being linear oligosaccharide consisting of β-1, 4-D-glucose residues.

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