A symbiotic system of *Singula stellaria* and *Chaetomium globosum*, its construction method and application

Abstract

This invention discloses a symbiotic system of *Algae monostellatum* and *Chaetomium globosum*, its construction method, and its application. This invention successfully constructed *Algae monostellatum* (…) using BG11 inorganic salt medium. Coelastrella sp.) and Chaetoceros (sp.) and Chaetoceros globulus ( Chaetomium globosum This multifunctional symbiotic organism promotes crop growth, alleviates crop salt stress, degrades straw, and inhibits Fusarium graminearum. Furthermore, both Chaetomium globosum and Algae moniliforme within the symbiotic organism can colonize around the crop root system. This fungal-algae symbiotic not only maximizes resource utilization but also provides strong support for the green and healthy development of agriculture.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a symbiotic system of *Algae monostellaria* and *Chaetomium globosum*, its construction method, and its application. Background Technology

[0002] The statements herein provide only background information in relation to this invention and do not necessarily constitute prior art.

[0003] Soil salinization is a significant global agricultural problem, with salt stress causing substantial damage to crop growth and leading to widespread yield reductions. Currently, the main methods for soil improvement and crop growth promotion rely on chemical amendments and agricultural bio-fertilizers, but these methods face a series of problems such as high costs and environmental pollution. Furthermore, the practice of returning straw to the field to improve saline-alkali land has been widely implemented; however, the slow decomposition of straw leads to issues such as soil hypoxia and disease. Therefore, it is necessary to improve the degradation and utilization efficiency of straw. Consequently, the development of new multifunctional microbial additives to assist in straw return and utilization, crop growth promotion, and disease control in saline-alkali land has become a hot topic in agricultural research.

[0004] Microalgae produce abundant organic matter through photosynthesis, improving the soil's nutrient environment and providing organic matter and oxygen for plant roots and soil microorganisms. However, in saline-alkali lands, microalgae growth is limited by high salinity, low nutrients, and insufficient water, resulting in slow growth and difficulty in coping with drastic environmental changes. Therefore, despite the significant potential of microalgae in saline-alkali land remediation, their application in saline-alkali lands still faces certain limitations.

[0005] Chaetomium coccidioides ( Chaetomium globosum It can effectively decompose organic matter in straw, promote soil structure improvement, and enhance the plant's salt and alkali resistance by secreting stress-resistant substances. Furthermore, *Chaetoceros globosum* can inhibit *Fusarium graminearum* (a type of fungus) by secreting antifungal substances. Fusarium graminearum This promotes the growth of microalgae and reduces pathogen infection caused by straw returning to the field, thereby enhancing crop disease resistance. The relatively low organic matter content in saline-alkali soil restricts the colonization and growth of *Chaetomium globosa*. Existing research mostly focuses on the individual application of microalgae or *Chaetomium globosa*, lacking in-depth exploration of the combined effects of both in alleviating crop salt stress. Summary of the Invention

[0006] To address the above shortcomings, this invention provides a symbiotic system of *Alternaria uniflora* and *Chaetomium globosum*, its construction method, and its application. This invention develops a co-culture of *Alternaria uniflora* and *Chaetomium globosum*, utilizing their complementary characteristics to form a stable symbiosis. This not only alleviates crop salt stress and promotes crop growth but also achieves the effect of controlling biological diseases.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] As a first aspect of the present invention, a symbiotic system of *Algae moniliformis* and *Chaetomium globosum* is provided, comprising *Algae moniliformis* and *Chaetomium globosum*, wherein the *Algae moniliformis* species has the accession number CGMCC No. 46633, the depositary institution is the China General Microbiological Culture Collection Center; the depositary address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; the deposit date is August 27, 2025. It is classified and named *Algae thermophilicis*. Coelastrella thermophila .

[0009] The *Chaetomium* species is *Chaetomium* QM or *Chaetomium* ND35.

[0010] Furthermore, in the symbiotic system, the density of *Algae monostellaria* cultured to the logarithmic growth phase is ≥1×10⁻⁶. 6 The mycelial ball density of *Chaetoceros globosum* should be at least 900 cells / mL. The two are mixed in a volume ratio of 2:1 to 3:1 to obtain the final product.

[0011] Furthermore, the symbiotic system also includes BG11 medium and PDB medium; the components of BG11 medium are: NaNO3 1500 mg / L, K2HPO4·3H2O 40 mg / L, MgSO4·7H2O 75 mg / L, CaCl2·2H2O 36 mg / L, Na2CO3 20 mg / L, citric acid 6 mg / L, ferric ammonium citrate 6 mg / L, disodium EDTA 1 mg / L, boric acid 2.86 mg / L, manganese chloride 1.81 mg / L, zinc sulfate 0.222 mg / L, sodium molybdate 0.39 mg / L, copper sulfate 0.079 mg / L, and cobalt nitrate 0.049 mg / L; the components of PDB medium are: potato starch 200 g / L and glucose 20 g / L.

[0012] Furthermore, the symbiotic system also contains 0-3 g / L of straw.

[0013] The combined application of bacteria and algae can be used to improve saline-alkali land. The photosynthetic autotrophic characteristics of microalgae can provide organic matter and oxygen for Chaetomium globosum, and Chaetomium globosum can decompose the organic matter of straw returned to the field to provide essential inorganic salts for microalgae and promote microalgae growth.

[0014] As a second aspect of the present invention, a method for constructing the symbiotic system of *Algae monostellatum* and *Chaetomium globosum* is provided, comprising the following steps:

[0015] S1, cultivation of *Algae stellaria*: Under artificial climate chamber conditions (temperature 24±2℃, light intensity 6000-7000 lux, light duration 14-18 hours, dark duration 6-10 hours, humidity 60%-70%), cultured in BG11 liquid medium until the logarithmic growth phase density ≥1×10⁻⁶. 6 cells / mL;

[0016] S2, Cultivation of Chaetomium globosum: Inoculate Chaetomium globosum spore powder into PDB medium and cultivate until the mycelial ball density of Chaetomium globosum is 800-1000 spores / mL; the cultivation conditions are: shaker culture for 1.5-3 days, rotation speed 160-200 rpm, temperature 26-30℃.

[0017] S3, mix the *Single-star algae* obtained in step S1 and the *Chaetomium globulae* obtained in step S2 and culture them under the following conditions: temperature 24-26℃, light intensity 50-70 μmol / m². 2 / s, light duration 14-18 hours, dark duration 6-10 hours. In some embodiments, the mixed volume ratio of *Algae monostellatum* obtained in step S1 and *Chaetomium globosa* obtained in step S2 is 2:1 to 3:1.

[0018] Microscopic observation revealed a stable symbiotic system formed between *Stellaria stolonifera* and *Chaetomium globosum*, with an initial symbiotic density of 0 cells / mL. After approximately 10 days of co-culturing, a stable and dense symbiotic system was established, comprising 300-400 cells / mL, with 300-400 spores per symbiotic organism, and a microalgal cell density ≥1×10⁻⁶. 6 The cells / mL (absorbance 0.8) were used for subsequent crop irrigation treatment.

[0019] As a third aspect of the invention, it provides the application of the said monostar algae and chamomile symbiotic system in degrading straw, inhibiting pathogens, alleviating crop salt stress, or promoting plant growth.

[0020] The application has the following effects:

[0021] (1) During the cultivation process, the number of symbiotic organisms and spores of *Syngonium moniliforme* and *Chaetomium globosum* both increased;

[0022] (2) The symbiotic relationship between bacteria and algae secretes more extracellular polysaccharides and auxins, which are potential stress-resistant and growth-promoting substances;

[0023] (3) The biomass of both *Stellaria spp.* and *Chaetoceros* spores increased significantly after straw was added to the culture medium, and the addition of straw also increased the cellulase activity in the symbiotic system.

[0024] (4) The application of the bacterial-algae symbiotic to potted crops significantly improves the growth performance of the crops;

[0025] (5) After the symbiotic is applied, it can effectively alleviate the salt stress of crops and significantly improve the salt stress tolerance of crops;

[0026] (6) Symbionts can effectively inhibit pathogens and promote the healthy growth of crops.

[0027] Among them, Chaetomium globosum is believed to be able to decompose and utilize cellulose in straw. Co-inoculating Chaetomium globosum and Algae monostellaria into wheat straw for fermentation not only promotes the resource utilization of wheat straw, but also realizes the proliferation of symbionts, Chaetomium globosum spores and Algae monostellaria, which has important ecological and economic value.

[0028] Furthermore, the plants are crops, including lettuce, tomatoes, and corn.

[0029] As a fourth aspect of the present invention, a culture medium for a fungal-algae symbiotic system with added straw is provided, wherein BG11 medium and PDB medium are mixed at a volume ratio of 2:1 to 3:1, and then 0 to 3 g / L of straw is added. More preferably, the amount of straw added is 1 to 3 g / L.

[0030] The beneficial effects achieved by one or more embodiments of the present invention described above are as follows:

[0031] 1. This invention provides a symbiotic system of Algae monostellatum and Chaetomium globosum. By applying the symbiotic system, it is possible to significantly improve crop growth in saline-alkali environments, effectively utilize straw, and control pathogens, thus achieving high economic benefits and ecological restoration value.

[0032] 2. Compared with using either of them alone, the symbiotic system of bacteria and algae provided by this invention has a better effect on degrading straw and promoting plant growth.

[0033] 3. This invention provides a culture medium for a fungal-algae symbiotic system. By adding straw, the biomass of the fungal-algae symbiotic system can be further increased, promoting the growth of fungi and algae. Attached Figure Description

[0034] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0035] Figure 1 Flowchart for constructing a co-culture system of Algae monostellae and Chaetomium globosum.

[0036] Figure 2 Morphological diagram of Algae monostellatum.

[0037] Figure 3 Micrographs of the symbiotic relationship between Algae monostellatum and Chaetomium globosum in a co-culture system;

[0038] Among them, A: symbiotic under a 4X microscope, B: symbiotic under a 10X microscope, C: sporangia of *Trichoderma glomeratum* under a 40X microscope, and D: a large number of spores released by crushing the sporangia under a 40X microscope.

[0039] Figure 4 Curves showing the changes in the number of symbiotic organisms and spores of *Syngonium moniliforme* and *Chaetomium globosum*.

[0040] Where A: curve showing the change in the number of symbionts, and B: curve showing the change in the number of spores.

[0041] Figure 5 The content of auxin and extracellular polysaccharides after co-culturing *Alternaria solani* and *Chaetomium globosum* for 10 days;

[0042] Wherein, A: auxin, B: extracellular polysaccharide; DX: single-star algae, QM: spherical chaetotrichum, GS: algal symbiosis.

[0043] Figure 6 The symbiosis between Algae monostellatum and Chaetomium globosum promotes crop growth;

[0044] Where A: Top view of lettuce leaf area under different treatments, B: Phenotypic diagram of lettuce after washing roots.

[0045] Figure 7 Symbiotic organisms alleviate 150 mM salt stress in crops;

[0046] Among them, A: Top view of lettuce leaf area under different treatments under salt stress, B: Phenotypic diagram of lettuce after root washing under salt stress.

[0047] Figure 8 Image showing the root system of a fungal-algae symbiotic crop and its effect on solid plate planting;

[0048] Among them, A: Symbiotic colonization on BG11 solid plate (front), B: Symbiotic colonization on BG11 solid plate (back), C: Root colonization diagram of Chaetomium spores and Alternaria monotypica, D: Root colonization diagram of Alternaria monotypica.

[0049] Figure 9 A plate-shaped confrontation between a fungal-algal symbiotic organism and *Fusarium graminearum*.

[0050] Figure 10 : Effect diagrams on the proliferation of symbionts and spores with and without straw addition;

[0051] Where A: number of symbionts after adding straw, B: number of spores after adding straw, and C: microalgal biomass with and without straw.

[0052] Figure 11 Cellulase activity in culture medium with or without added straw. Detailed Implementation

[0053] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0054] In some embodiments of the present invention, a symbiotic system of Algae monostellae and Chaetomium globosum is provided, comprising Algae monostellae and Chaetomium globosum.

[0055] In some embodiments of the present invention, as a second aspect of the invention, a method for constructing the symbiotic system of *Algae monostellatum* and *Chaetomium globosum* is provided, comprising the following steps:

[0056] S1, cultivation of *Algae stellaria*: Under artificial climate chamber conditions (temperature 24±2℃, light intensity 6000-7000 lux, light duration 14-18 hours, dark duration 6-10 hours, humidity 60%-70%), cultured in BG11 liquid medium until the logarithmic growth phase density ≥1×10⁻⁶. 6 cells / mL; in one embodiment, the light exposure time was 16 h and the dark time was 8 h.

[0057] S2, Cultivation of Chaetomium globosum: Inoculate Chaetomium globosum spore powder into PDB medium until the mycelial ball density of Chaetomium globosum is 800-1000 spores / mL, and in one example, until the mycelial ball density of Chaetomium globosum is 900 spores / mL.

[0058] S3: The *Single-star algae* obtained in step S1 and the *Chaetomium* obtained in step S2 were mixed and cultured together. Microscopic observation was then conducted to observe the stable symbiotic relationship between the microalgae and *Chaetomium*. The culture conditions were: temperature 24-26℃, light intensity 50-70 μmol / m². 2 / s, light duration 14-18 hours, darkness duration 6-10 hours.

[0059] In some embodiments of the present invention, the application of the monostar algae and chamomile symbiotic system in degrading straw, inhibiting pathogens, alleviating crop salt stress, and promoting crop growth is provided.

[0060] In some embodiments of the present invention, a culture medium for a fungal-algae symbiotic system is also provided, which is obtained by mixing BG11 culture medium and PDB culture medium at a volume ratio of 2:1 to 3:1, and then adding 0 to 3 g / L of straw. In some embodiments of the present invention, the amount of straw added is any amount between 1, 2, 3 g / L or 1 to 3 g / L.

[0061] The present invention will be further described below with reference to the embodiments.

[0062] BG11 medium composition: NaNO3 1500 mg / L, K2HPO4·3H2O 40 mg / L, MgSO4·7H2O 75 mg / L, CaCl2·2H2O 36 mg / L, Na2CO3 20 mg / L, citric acid 6 mg / L, ferric ammonium citrate 6 mg / L, disodium EDTA 1 mg / L, boric acid 2.86 mg / L, manganese chloride 1.81 mg / L, zinc sulfate 0.222 mg / L, sodium molybdate 0.39 mg / L, copper sulfate 0.079 mg / L, and cobalt nitrate 0.049 mg / L.

[0063] The *Algae* species described has the CGMCC No. 46633 and is classified as *Thermophilic Monostarella*. Coelastrella thermophila .

[0064] Example 1: Isolation and Identification of Microalgae

[0065] 1. Isolation of microalgae

[0066] Microalgae were isolated from wheat root soil in Dongying, Yellow River Delta, using BG-11 medium and employing serial dilution plating and streak plating methods.

[0067] 2. Identification of microalgae:

[0068] (1) Morphological identification:

[0069] like Figure 2 As shown, the microalgal cells exhibit the typical morphology of single-celled green algae, including spherical, elliptical, and spindle-shaped forms, with blunt, rounded ends. The size of larval cells is (4–7) μm, while the size of mature cells is (8–12) μm, with thicker, semi-transparent cell walls.

[0070] (2) Molecular biological identification:

[0071] The 18S rRNA gene sequence of the algal strain was determined, and its nucleotide sequence is shown in SEQ ID NO.1. The obtained gene sequence was compared with known sequences in the NCBI database to confirm the species of the algal strain. Based on the 18S rRNA gene sequence, several closely related known species were selected for comparison. Sequence alignment software (such as MEGA or ClustalX) was used to align all aligned sequences and construct a phylogenetic tree. Analysis of the 18S rRNA gene sequence and BLAST alignment confirmed that the obtained algal strain had a 99.52% similarity to *Coelastrella thermophila*, indicating that this algal strain is a subspecies of *Coelastrella thermophila*.

[0072] Based on the above morphological and molecular biological identification results, the isolated algal strain was named Coelastrella thermophila And it was biologically preserved; the preservation information is as follows:

[0073] Reference biological material (strain): PZX-1.

[0074] Suggested classification and naming: Thermophilic Monostar Algae Coelastrella thermophila .

[0075] Accession number: CGMCC No. 46633.

[0076] Preservation institution: China General Microbiological Culture Collection Center, China Microbial Culture Collection Committee.

[0077] Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Courtyard, Beichen West Road, Chaoyang District, Beijing.

[0078] Preservation date: August 27, 2025.

[0079] The *Chaetomium* species is *Chaetomium* QM or *Chaetomium* ND35.

[0080] The *Chaetomium globosum* QM is disclosed in patent CN118879507A, and the *Chaetomium globosum* ND35 is disclosed in patent CN102311925A.

[0081] PDB culture medium composition: 200 g / L potato extract powder, 20 g / L glucose.

[0082] The application scheme of the microalgae and Chaetomium symbiosis provided by the present invention includes the following steps:

[0083] (1) Study the changes in biomass (0-300) and spores (0-300) of dense algal symbionts in mixed culture medium.

[0084] (2) Determine the content of auxin and extracellular polysaccharide in the co-culture system, and use colorimetric methods to analyze the content of these substances in the bacterial-algae co-culture system to assess the potential salt and alkali resistance of the symbiont.

[0085] (3) Apply the bacterial-algae symbiotic to potted crops, compare the growth differences between the treatment group and the control group, and evaluate the growth-promoting ability of the symbiotic.

[0086] (4) Salt stress (e.g., 150 mM NaCl) treatment: potted crops were irrigated with a bacterial-algae symbiotic. The growth differences between the treatment group and the control group were compared to evaluate the salt tolerance of the symbiotic.

[0087] (5) Plate confrontation experiment: observe the inhibitory effect of the symbiont on Fusarium graminearum and measure the changes in pathogen colonies.

[0088] (6) Investigate the cellulase activity, number of symbionts and number of Chaetomium spores in the co-culture system after the addition of straw.

[0089] Example 2: Construction and cultivation of bacterial-algal symbiotic organisms

[0090] Experimental Design

[0091] This experiment aims to develop a symbiotic relationship between microalgae and Chaetomium globosum to alleviate salt stress, promote crop growth, and achieve disease control and straw resource utilization. Specific steps are as follows: Figure 1 As shown, it includes:

[0092] First, *Algae monostellatum* was cultured in an artificial climate chamber (temperature 23℃, light intensity 6000-7000 lux, photoperiod 16 hours light / 8 hours dark, humidity 60%–70%) until the logarithmic growth phase, with a cell density ≥1×10⁻⁶. 6 cells / mL. *Chaetoceros globosum* (*Chaetoceros globosum* QM) was cultured on a shaker for 2 days (temperature 28℃, rotation speed 180 r / min), with a mycelial ball density of approximately 900 cells / mL. Then, *Chaetoceros globosum* and *Chaetoceros globosum* were mixed at a 2:1 ratio and cultured at 24-26℃ and a light intensity of 50-70 μmol / mL. 2 The symbionts and spores were co-cultured for 10 days under a 16-hour light / 8-hour dark cycle, with the formation of tightly packed symbionts used as the counting standard. At regular intervals, 1 mL of culture medium was taken, and the number of symbionts and spores was counted using a microscope. Figure 3 The absorbance (OD) value of microalgae was detected by a spectrophotometer (680 nm) to assess changes in microalgal biomass.

[0093] Test results

[0094] Counting began on day 0 after the combination of *Singula stellaria* and *Chaetoceros* mycelial balls, with the counting standard based on the formation of a tight symbiotic organism. For example... Figure 4 As shown, the initial OD value of the single-star algae was 0.8 (density ≥ 1 × 10⁻⁶). 6 The initial mycelial ball density of *Chaetoceros* was approximately 900 cells / mL. *Chaetoceros* and *Chaetoceros* (volume ratio 2:1) were mixed and cultured to form a symbiont. The initial absorbance of the mixed system was 0.2. After 10 days of co-culture, the number of symbionts was approximately 300 cells / mL, and the number of spores in the symbiont was approximately 300 per mycelial ball. The OD value of the microalgae was 0.8, showing strong proliferative capacity.

[0095] The single-star algae, Chaetomium globosum and the prepared symbiotic system used in Example 1 were used in the study of the following examples.

[0096] Example 2: Determination of Extracellular Polysaccharide (EPS) Content

[0097] Experimental Design

[0098] This experiment aimed to determine the yield of extracellular polysaccharides in a co-culture system to assess its tolerance to saline-alkali environments. The experimental procedures are as follows:

[0099] The experiment consisted of three treatments: *Algae*, *Chaetomium*, and a symbiotic organism. After one month of co-culture, the extracellular polysaccharide content in the culture system was measured using the following methods:

[0100] First, take 8 mL of water sample, filter it through a 0.45 μm filter membrane, then take 5 mL of the liquid to be tested, add 25 mL of ethanol, refrigerate for 3 hours, and filter again through a 0.45 μm filter membrane to obtain crude polysaccharide precipitate. Dissolve the precipitate in 3 mL of deionized water, and determine the polysaccharide concentration using the anthrone method. Measure the absorbance at 625 nm using a spectrophotometer, and calculate the polysaccharide concentration based on the standard curve.

[0101] Test results

[0102] like Figure 5 As shown, under individual culture conditions, the extracellular polysaccharide concentrations of *Symplocos uniflorus* and *Chaetomium globosum* were significantly lower than those in the symbiotic culture. The extracellular polysaccharide concentration in *Symplocos uniflorus* was 0.62 mg / mL, and in *Chaetomium globosum* it was 10.16 mg / mL. In contrast, the extracellular polysaccharide concentration in the symbiotic culture was 14.76 mg / mL, significantly higher than that in individual cultures. This indicates that the symbiotic culture promoted the accumulation of more extracellular polysaccharides during proliferation, enhancing its adaptability to saline-alkali environments.

[0103] Example 3: Determination of Auxin (IAA) Content

[0104] Experimental Design

[0105] This experiment used Salkowski's reaction solution to detect the auxin content in a co-culture system and evaluate its promoting effect on crop growth. The experiment consisted of three treatments: *Alternaria solani*, *Chaetomium globosum*, and a symbiotic relationship between the two, with an initial inoculum size of 20% for each. After one month of co-culture, the auxin content in the culture system was measured using the following methods:

[0106] The experimental steps are as follows:

[0107] First, take 1 mL of the supernatant from the co-culture system, add 1 mL of Salkowski's reaction solution, mix well, and react in the dark for 30 minutes. Then, measure the absorbance at 530 nm using a UV spectrophotometer. Substitute the absorbance value into the calculation formula according to the pre-established standard curve to obtain the auxin concentration in the sample.

[0108] Test results

[0109] like Figure 5 As shown, under single-culture conditions, the auxin concentration of *Alternaria solani* was 0.946 μg / mL, and that of *Chaetomium globosum* was 1.458 μg / mL. In contrast, the auxin concentration in the mixed co-culture solution was 2.992 μg / mL, significantly higher than that of single-culture. The significantly increased auxin concentration in the symbiotic relationship indicates that the symbiosis between microalgae and *Chaetomium globosum* helps promote greater auxin accumulation and enhances the plant's growth potential.

[0110] Example 4: Experiment on promoting growth of potted lettuce

[0111] Experimental Design

[0112] This experiment investigated the effects of a symbiotic relationship between *Alternaria solani* and *Chaetomium globosum* on lettuce growth in potted plants. The experimental setup was as follows: 300g of soil per pot, 100mL of co-culture solution per pot, with each treatment group replicated five times. The treatment solution was applied via fertigation. After transplanting, lettuce seedlings were placed in a climate chamber at 23℃, 50%-60% humidity, and 6000-7000 lux light intensity for 14 days. Different treatment groups included a water control group (CK), an *Alternaria solani* treatment group (T1), a *Chaetomium globosum* treatment group (T2), a *Alternaria solani* and *Chaetomium globosum* co-culture treatment group (T3), and a *Trichoderma harzianum* treatment group (T4). After the seedlings reached the four-leaf-one-heart stage, the following indicators were measured 14 days later: leaf area, number of leaves, chlorophyll content (SPAD value), aboveground fresh weight, and root growth (root length and number of hairy roots). The effects of different treatment solutions on the growth promotion of lettuce were also analyzed.

[0113] Test results

[0114] The test results are as follows Figure 6 As shown, the lettuce group treated with the algae-bacterial symbiosis exhibited significantly better growth than other treatment groups in all growth indicators. Fourteen days after application, the leaf area of ​​the T3 group increased by 33.43%, 28.97%, 17.32%, and 33.34% compared to the control (CK), T1, T2, and T4 groups, respectively; the number of leaves increased by 15.60%, 18.59%, 29.03%, and 10.59%, respectively; chlorophyll content increased by 29.42%, 14.59%, 24.48%, and 16.64%, respectively; and aboveground fresh weight increased by 26.83%, 22.33%, 33.09%, and 22.68%, respectively. These results indicate that the symbiosis significantly promotes lettuce growth, especially in key growth indicators such as leaf area, number of leaves, chlorophyll content, and aboveground fresh weight, demonstrating a strong growth-promoting effect.

[0115] Example 5: Experiment to alleviate salt stress in lettuce

[0116] Experimental Design

[0117] This experiment aimed to evaluate the effect of combined application of microalgae and *Chaetomium globosum* on the salt stress resistance of lettuce. Eight treatment groups were set up, including a water control group (CK0), a 150 mM NaCl control group (CK1), *Chaetomium globosum* treatment group (T1), *Chaetomium globosum* treatment group (T2), a combined culture group of *Chaetomium globosum* and *Chaetomium globosum* (T3), and a *Trichoderma harzianum* treatment group (T4). Each treatment group was repeated five times, and the treatment solutions were applied via fertigation. After transplanting, lettuce seedlings were placed in a climate chamber at 23℃, 50%-60% humidity, and 6000-7000 lux light intensity until they reached the four-leaf stage. On day 14, key indicators were measured, mainly leaf area, number of leaves, aboveground fresh weight, and root growth (root length and number of hairy roots) to analyze the effects of different treatment solutions on the salt stress resistance of lettuce.

[0118] Trichoderma harzianum is a common growth-promoting fungus in soil, and extensive research has confirmed that it can help plants resist salt by secreting auxins and activating antioxidant systems. In this example, it is used as a positive control to verify the reliability of the experimental system, and to compare whether the effects of microalgae and Chaetomium globosum reach or exceed the levels of known growth-promoting fungi.

[0119] Test results

[0120] like Figure 7 As shown, under salt stress conditions, the lettuce group treated with the combined culture solution of Alternaria solani and Chaetomium globosum (T3) exhibited significantly better stress resistance than other treatment groups.

[0121] After 14 days of treatment, the T3 treatment showed better results, with lettuce leaf area increasing by 17.72%, 14.26%, 18.08%, and 0.56% compared to CK1, T1, T2, and T4, respectively. The T3-Singula stellaria and Trichophyton globosum co-culture treatment significantly increased the number of lettuce leaves, by 8.63%, 7.09%, 17.65%, and 26.87% compared to CK1, T1, T2, and T4, respectively. The T3-Singula stellaria and Trichophyton globosum co-culture treatment significantly increased the aboveground fresh weight of lettuce, by 10.19%, 14.89%, 36.82%, and 10.33% compared to CK1, T1-Singula stellaria treatment, T2-Trichophyton globosum treatment, and T4-Trichophyton harzianum treatment, respectively. The symbiotic treatment demonstrated a significant alleviating effect on crop growth under salt stress, including promoting leaf area, leaf number, and aboveground fresh weight.

[0122] Example 6: Colonization effect in potted soil and BG11 solid culture medium

[0123] Experimental Design

[0124] This experiment observed the colonization effect of microalgae and Chaetomium globosum in potted soil and BG11 (pure inorganic salt) solid culture medium. The specific steps included: applying the microbial-algae symbiotic to potted plants; after 20 days of growth with the lettuce, collecting the lettuce roots and soil crust and preparing a suspension; and observing the morphology and viability of the algae and Chaetomium globosum spores in the soil and on the surface of the plant roots under a microscope.

[0125] Test results

[0126] according to Figure 8 Microscopic observation showed that the microalgae and Chaetomium symbiosis exhibited good colonization around the soil roots and in BG11 solid medium. The spores of both microalgae and Chaetomium spores remained viable on the soil and root surface 20 days after lettuce growth. Similarly, the symbiosis grew stably in solid BG11 medium, with Chaetomium spores germinating into distinct white colonies and microalgae producing green colonies.

[0127] Example 7: The effect of plate confrontation between fungal-algal symbiosis and Fusarium graminearum

[0128] Experimental Design

[0129] This experiment aimed to evaluate the inhibitory effect of a microalgae-Chaetoceros symbiosis on Fusarium graminearum. Fusarium graminearum easily remains in straw returned to the field, and it can cause stem rot and root rot in crops such as wheat and corn. Chaetoceros graminearum itself has a certain inhibitory effect on Fusarium graminearum. The purpose of this experiment was to observe whether the antibacterial effect of the symbiosis was further enhanced. The experimental procedure was as follows: Using the standard plate confrontation method, symbiotic mycelial balls and Fusarium graminearum were cultured in confrontation on a solid culture medium. In the experiment, the symbiosis and Fusarium graminearum were inoculated at opposite ends of the plate, and the formation of inhibition zones was observed and recorded. Similarly, Algae moniliformis colonies and Chaetoceros graminearum mycelial balls were also inoculated as separate control groups. By comparing the inhibitory effects between the experimental group and the control groups (the group inoculated only with Fusarium graminearum, the Algae moniliformis group, and the Chaetoceros graminearum group), the inhibitory ability of the symbiosis on Fusarium graminearum was evaluated, and the inhibition rate was further calculated.

[0130] Test results

[0131] like Figure 9 As shown, the bacterial-algal complex can form a distinct inhibition zone in the confrontation area with Fusarium graminearum, significantly inhibiting the colony growth of Fusarium graminearum. The inhibition rate is 50%, which is higher than that of Algae monostellatum (5%) and Chaetomium globosum (35%).

[0132] Example 8: Investigation on the effect of symbiotic degradation of straw and determination of cellulase activity

[0133] Experimental Design

[0134] This experiment aimed to investigate the effect of a microalgae-Chaetoceros symbiosis on straw degradation and to determine its cellulase activity. The experimental procedure included: selecting wheat straw, cleaning it, cutting it into 3 mm long segments, and then sterilizing it at high temperature. The straw and symbiosis were then inoculated into a 2:1 mixture of BG11 and PDB medium as the experimental group, with a straw-free group, a straw-inoculated Chaetoceros symbiosis group, and a straw-inoculated *Chaetoceros spp.* group as the control group. The experimental period was 10 days. During the experiment, the straw degradation potential of the symbiosis, *Chaetoceros spp.*, and *Chaetoceros spp.* was evaluated by measuring cellulase (CMCase) activity, providing guidance for promoting the decomposition and utilization of straw after field inoculation of the symbiosis.

[0135] The concentration (C) of reducing sugar, expressed in μg / mL, was calculated from the absorbance (OD540) of the sample from the glucose standard curve. The amount of reducing sugar was converted from μg to μmol, and the molecular weight of glucose (180 μg / μmol) was used. Enzyme activity was defined as the amount of enzyme required to release 1 μmol of reducing sugar in 1 minute. Enzyme activity (U / mL) was calculated based on a reaction time of 30 minutes.

[0136] Test results

[0137] like Figure 11 As shown, compared to the control group (non-straw symbiotic group) (OD value at 540 nm: 0.05, 0.67 U / mL), the experimental group (symbiotic group with added straw) exhibited higher cellulase activity (OD value at 540 nm: 0.25, 20 U / mL). This indicates that the symbiotic can effectively promote straw degradation and increase cellulase activity. The more efficient straw degradation potential of the symbiotic also provides theoretical guidance for the field inoculation of the symbiotic and the promotion of straw decomposition and utilization after returning to the field.

[0138] Example 9: Changes in the proliferation of symbionts, spores, and microalgae after straw addition

[0139] Experimental Design

[0140] This experiment aimed to investigate the effect of 3mm diameter straw on the proliferation of the symbiotic organism of *Alternaria solani* and *Chaetomium globosum*. The experimental setup was as follows: a culture medium without added straw was used as a control ( Figure 3 The microalgae were cultured in a mixed medium with an initial absorbance of 0.8 for *Algae* (680 nm) and a density of approximately 900 *Chaetomium* cells / mL. Straw was added at a concentration of 1 g / L after the microalgae and bacteria were combined. The culture experiment lasted for 10 days. Samples were then taken and the OD value (microalgal biomass index) of the microalgae was measured using a spectrophotometer. Simultaneously, the density changes of the microalgae symbiotic organism were assessed using a microscopic counting method.

[0141] Test results

[0142] Record the changes in biomass of the fungal-algae symbiotic complex after the addition of straw. By comparing the biomass differences between the straw-added group and the control group, the promoting effect of straw on symbiotic proliferation was assessed. Figure 10 As shown, after 10 days, compared to the control group of 300 cells / mL ( Figure 4 The number of symbionts in the straw-added group was 600 per mL, compared to 300 per symbiont in the non-straw-added group. Figure 4 The straw-added group had 500 spores per symbiont. Straw addition significantly promoted the integration of the fungal-algae symbiont and the proliferation of spores and microalgae biomass within the symbiont. Furthermore, after 10 days of cultivation, the straw-added group showed a higher absorbance of 1.2 at 680 nm compared to the control group (0.8), indicating higher microalgae biomass production. These results demonstrate that straw addition can increase the yield of the symbiont and the liquid fungal-algae preparation, achieving effective utilization of waste resources.

[0143] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A multifunctional symbiotic system of microalgae and Chaetomium globosum, characterized in that, It includes *Monostellaria thermophila* and *Chaetomium globosum*; the *Monostellaria thermophila* species has the accession number CGMCC No. 46633, deposited at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; deposited on August 27, 2025; and is classified as *Monostellaria thermophila*. Coelastrella thermophila ; The *Chaetotrichum* species is *Chaetotrichum qM*. In the aforementioned symbiotic system, *Stellaria media* is cultured to a density ≥1×10⁻⁶ during the logarithmic growth phase. 6 The mycelial ball density of Chaetomium globosum is 800-1000 cells / mL. The two are mixed in a volume ratio of 2:1 to 3:1 to obtain the final product.

2. The symbiotic system according to claim 1, characterized in that, The symbiotic system also includes BG11 and PDB culture media; the BG11 culture medium consists of: NaNO3 1500 mg / L, K2HPO4·3H2O 40 mg / L, MgSO4·7H2O 75 mg / L, CaCl2·2H2O 36 mg / L, Na2CO3 20 mg / L, citric acid 6 mg / L, ferric ammonium citrate 6 mg / L, disodium EDTA 1 mg / L, boric acid 2.86 mg / L, manganese chloride 1.81 mg / L, zinc sulfate 0.222 mg / L, sodium molybdate 0.39 mg / L, copper sulfate 0.079 mg / L, and cobalt nitrate 0.049 mg / L; the PDB culture medium consists of: potato starch 200 g / L and glucose 20 g / L. It also includes 0~3 g / L of straw.

3. The method for constructing the multifunctional symbiotic system of microalgae and Chaetomium globosum according to any one of claims 1-2, characterized in that, Includes the following steps: S1, Cultivation of *Singula stellaria*: Cultured in BG11 liquid medium until the logarithmic growth phase density ≥ 1 × 10⁻⁶. 6 cells / mL; S2, Cultivation of Chaetomium globosum: Inoculate 0.1g of Chaetomium globosum spore powder into 300 mL of PDB medium until the mycelial ball density of Chaetomium globosum is 800-1000 / mL; S3. The single-star algae obtained from step S1 and the globular chaetotrichum obtained from step S2 are mixed in a volume ratio of 2:1-3:1 and then cultured. The microalgae and globular chaetotrichum form a stable symbiotic system.

4. The method for constructing the multifunctional symbiotic system of microalgae and Chaetomium globosum as described in claim 3, characterized in that, In step S1, the cultivation conditions for *Alternaria uniflora* are as follows: In an artificial climate chamber, the temperature is 24±2℃, the light intensity is 6000-7000 lux, the light duration is 14-18 hours, the dark duration is 6-10 hours, and the humidity is 60%–70%. The culture is carried out in BG11 liquid medium until the logarithmic growth phase density is ≥1×10⁻⁶. 6 cells / mL; In step S2, the culture conditions for Chaetomium globulus are: 28℃ shaker culture for 1.5-3 days, rotation speed 160-200 rpm, temperature 26-30℃.

5. The method for constructing the multifunctional symbiotic system of microalgae and Chaetomium globosum as described in claim 4, characterized in that, In step S3, the cultivation conditions are: temperature 24-26℃, light intensity 50-70 μmol / m². 2 / s, light duration 14-18 hours, darkness duration 6-10 hours.

6. The method for constructing the multifunctional symbiotic system of microalgae and Chaetomium globosum as described in claim 4, characterized in that, In the symbiotic system obtained by S3, the number of symbionts was 300-400 per mL, the number of spores was 300-400 per symbiont, and the microalgal cell density was ≥1×10⁻⁶. 6 cells / mL.

7. The application of the multifunctional symbiotic system of microalgae and Chaetomium globosum according to any one of claims 1-2, characterized in that, Applications of microalgae and Chaetomium symbiosis system in degrading straw, inhibiting Fusarium graminearum, alleviating salt stress in lettuce and promoting plant growth.

8. The application of the multifunctional symbiotic system of microalgae and Chaetomium globosum according to claim 7, characterized in that, The plant in question is an agricultural crop.

9. The application of the multifunctional symbiotic system of microalgae and Chaetomium globosum according to claim 8, characterized in that, The plant in question is lettuce.

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