Trichoderma asperellum-bacillus synthetic bacterial consortium, construction method and application thereof

Abstract

The application provides a bacillus cereus, which is named as Bacillus cereus , is preserved in the Guangdong Microbial Culture Collection Center and has a preservation number of GDMCC NO: 66395. The application also provides a synthetic microbial flora containing the bacillus cereus and the trichoderma aculeatum (Trichoderma aculeatum) Trichoderma asperellum ) MM7. The synthetic microbial flora constructed by the bacillus cereus and the trichoderma aculeatum (Trichoderma aculeatum) Trichoderma asperellum ) MM7 has a better effect of promoting the growth of banana plants, can be used to prepare a mixed microbial agent or a microbial fertilizer, is beneficial to the improvement of economic benefits of a banana planting industry, and has a wide application space in green and sustainable development of the banana industry.

Description

Technical Field

[0001] This invention relates to the field of biology, specifically to a synthetic microbial community of Trichoderma echinosporum and Bacillus, its construction method, and its application. Background Technology

[0002] In the agricultural production system, the use of fertilizers and pesticides is a crucial link. For a long time, the unreasonable application of chemical fertilizers has placed a heavy burden on the environment. The excessive use of chemical fertilizers not only leads to soil compaction and acidification, reducing soil fertility, but also causes elements such as nitrogen and phosphorus in chemical fertilizers to flow into rivers and lakes with rainwater, causing eutrophication of water bodies and ecological disasters such as cyanobacterial blooms and fish and shrimp deaths. In addition, the overuse of pesticides has also led to increased pesticide resistance in pests, and pesticide residues can also pollute agricultural products and threaten human health. Therefore, in order to meet the needs of sustainable development, the use of fertilizers must strictly follow two principles: (1) ensure that no harmful substances remain in agricultural products to protect the health of consumers; (2) ensure that the use of fertilizers will not have a negative impact on crop growth and the ecological environment.

[0003] Bananas are an important dual-purpose crop, widely cultivated in tropical and subtropical regions, and a source of income for smallholder farmers in approximately 150 tropical and subtropical countries. Bananas are also a very popular fruit, consumed as a staple food in many countries, and bananas are the world's largest fresh fruit traded crop, ranking as the fourth largest food crop after rice, wheat, and corn. Bananas are a rich source of important plant nutrients, including vitamins and phenolic compounds, and possess high medicinal value and processing potential. With rising living standards and increasing health awareness, the demand for bananas is increasing year by year, and increasing banana production is key to improving the economic benefits of the banana industry. However, the negative consequences of excessive use of chemical fertilizers and high production costs in current agricultural production are becoming increasingly prominent, urgently requiring the creation of more sustainable agricultural systems. This places higher demands on methods for promoting banana growth, requiring measures to avoid environmental pollution caused by excessive pesticide and fertilizer use, prevent resource waste, and reduce production costs. An effective and feasible alternative is to develop environmentally friendly growth-promoting microbial agents, laying the foundation for the green and sustainable development of the banana industry. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a synthetic microbial community of Trichoderma echinosporum and its construction method and application.

[0005] The first aspect of this invention is to provide a Bacillus cereus, wherein the Bacillus cereus is named Bacillus cereus It is registered and preserved at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC NO: 66395.

[0006] A second aspect of the present invention is to provide a fermentation broth of Bacillus cereus as described in the first aspect of the present invention.

[0007] A third aspect of the present invention is to provide a synthetic microbial community containing *Bacillus cereus* and *Trichoderma echinosporum* as described in the first aspect of the present invention, wherein the *Trichoderma echinosporum* is named *Trichoderma echinosporum* (…). Trichoderma asperellum MM7 was deposited at the China Center for Type Culture Collection (CCTCC) on March 30, 2021, with accession number CCTCC NO: M2021302.

[0008] The method for constructing the synthetic microbial community includes the following steps: taking the fermentation broth of a strain containing Bacillus cereus and the fermentation broth of a strain containing Trichoderma echinosporum, respectively, inoculating them into PDB medium, and culturing them to obtain the culture.

[0009] A fourth aspect of the present invention is to provide a mixed microbial agent containing the synthetic microbial community described in the third aspect of the present invention.

[0010] The fifth aspect of the present invention is to provide a microbial fertilizer containing Bacillus cereus as described in the first aspect of the present invention, or a synthetic microbial community as described in the third aspect of the present invention, or a mixed microbial agent as described in the fourth aspect of the present invention.

[0011] The sixth aspect of the present invention is to provide the use of Bacillus cereus as described in the first aspect of the present invention, or the fermentation broth as described in the second aspect of the present invention, or the synthetic microbial community as described in the third aspect of the present invention, or the mixed microbial agent as described in the fourth aspect of the present invention, or the microbial fertilizer as described in the fifth aspect of the present invention in the preparation of formulations that promote banana growth and / or promote corn seed germination.

[0012] The seventh aspect of this invention is to provide the use of Bacillus cereus as described in the first aspect of this invention, or the fermentation broth as described in the second aspect of this invention, or the synthetic microbial community as described in the third aspect of this invention, or the mixed microbial agent as described in the fourth aspect of this invention, or the microbial fertilizer as described in the fifth aspect of this invention in the preparation of formulations that increase banana chlorophyll content, and / or increase banana plant height, and / or increase banana stem diameter, and / or increase banana leaf area, and / or increase banana fresh weight, and / or increase banana dry weight, and / or increase corn seed germination rate, and / or increase corn seed germination length.

[0013] The eighth aspect of the present invention is to provide the use of Bacillus cereus as described in the first aspect of the present invention, or the fermentation broth as described in the second aspect of the present invention, or the synthetic microbial community as described in the third aspect of the present invention, or the mixed microbial agent as described in the fourth aspect of the present invention in the preparation of biofertilizer.

[0014] The ninth aspect of the present invention is to provide the use of Bacillus cereus as described in the first aspect of the present invention, or the fermentation broth as described in the second aspect of the present invention, in the preparation of formulations that produce IAA and / or promote the growth of Trichoderma MM7.

[0015] The *Bacillus cereus* strain of this invention can produce IAA and has a good effect on promoting the growth of *Trichoderma emblica* MM7 and promoting plant growth. It can increase plant chlorophyll content, plant height, stem diameter, leaf area, fresh weight and dry weight to a moderate extent. Moreover, it is similar to *Trichoderma emblica* (… Trichoderma asperellum The synthetic microbial community constructed by MM7 has a better effect on promoting the growth of banana plants. It can significantly increase the chlorophyll content, plant height, stem diameter, leaf area, fresh weight and dry weight of plants. It can be used to prepare mixed microbial agents or microbial fertilizers, which is conducive to improving the economic benefits of banana planting industry. It has broad application space in the green and sustainable development of banana industry and has broad development and application prospects in the field of microbial agents or microbial fertilizers. Attached Figure Description

[0016] Figure 1 Phylogenetic tree of Bacillus cereus CSH-9.

[0017] Figure 2 The left image shows the inoculation of MM7 and the right image shows the inoculation of MM7+CSH-9 as a plate interaction experiment.

[0018] Figure 3 The number of spores after co-culturing MM7+CSH-9.

[0019] Figure 4 The effect of the MM7+CSH-9 synthetic microbial community on maize seed germination. A, Germination status of maize seeds under different treatments; B, Germination rate of maize seeds under different treatments; C, Germination length of maize seeds under different treatments.

[0020] Figure 5 The effects of different microbial fertilizer treatments on the growth of banana plants were investigated. CK was the treatment group (watering only); 9 was the treatment group T1 (CSH-9 applied alone); M7 was the treatment group T2 (MM7 applied alone); and 9+M7 was the treatment group T3 (CSH-9 and MM7 applied together).

[0021] The Bacillus cereus strain described in this invention (strain number CSH-9) is named Bacillus cereus It was deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on May 26, 2025, with accession number GDMCC NO: 66395. The deposit address is the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Detailed Implementation

[0022] The present invention will be further described below with reference to the accompanying drawings and specific embodiments to better understand the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0023] Example 1

[0024] Bacillus cereus ( Bacillus cereus CSH-9 was isolated from banana rhizosphere soil samples collected in Haikou, Hainan, China (110°10′ 41′′E, 20°04′52″N), and named CSH-9. Bacillus cereus It was deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on May 26, 2025, with accession number GDMCC NO: 66395. The deposit address is the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0025] The colony morphology of strain CSH-9 was observed. The strain was activated by inoculation onto LB medium and incubated at 37°C for 48 hours. After single colonies grew, the colony morphology was observed. The colonies of strain CSH-9 on LB medium were grayish-white, nearly round, relatively densely distributed, with a rough surface, clear but irregular edges, and some colonies showed a tendency to merge.

[0026] The physiological and biochemical characteristics of strain CSH-9 were analyzed according to the methods in the "Manual of Systematic Identification of Common Bacteria" and "Bergey's Manual of Bacterial Identification". The results are shown in Tables 1 and 2.

[0027] Table 1. Physiological and biochemical characteristics of strain CSH-9

[0028] Characteristics Results Hydrogen sulfide - Gelatin liquefaction - Cellulose decomposition - Nitrate reduction ＋ Siderophore + pH tolerance test 6-10 (optimum 7) NaCl tolerance test (%) Starch hydrolysis Phosphorus solubilization - Nitrogen fixation - Potassium solubilization - Carbon source utilization -

[0029] Note: "+" indicates a positive result; "-" indicates a negative result.

[0030] Table 2 Utilization of Single Carbon and Nitrogen Sources

[0031] Results Nitrogen source utilization Results Myo-inositol Salicin + Dextrin + Histidine + L-arabinose + Thiocyanate + L-rhamnose - Glutamic acid + D-galactose - Potassium nitrate + D-sorbitol - Valine - D-trehalose - L-asparagine + Xylan + Glycine + D-fructose + Tyrosine + D-turanose - Cysteine + Raffinose + Phenylalanine - Sucrose - Proline - Ribose + L-tyrosine + D-cellubiose - Figure 1 -

[0032] Note: "+" indicates good growth; "-" indicates weak growth.

[0033] The bacterial cells preserved in glycerol were reactivated using LB solid medium. A single colony was picked using an inoculation loop in a clean bench and inoculated into King B liquid medium containing 100 mg / L tryptophan. The culture was then incubated at 30°C and 160 r / min for 24 h with shaking. The IAA production activity of strain CSH-9 was then tested. The specific procedures are as follows:

[0034] (1) Initial screening

[0035] Pipette 1 mL of fermentation broth into a test tube, add 2 mL of Salksowski colorimetric solution (1 mL of 0.5 mol / L FeCl3 and 50 mL of 35% perchloric acid), wrap with aluminum foil, and place in the dark for 30 min. Observe whether it turns pink, and the deeper the red, the higher the IAA yield (Glickmann et al., 1995).

[0036] (2) Second screening

[0037] The culture conditions for the strain were the same as above. The fermentation broth was centrifuged at 8000 r / min for 10 min, and the supernatant was collected. Then, an equal volume of Salkowski colorimetric solution was added, and the process was repeated three times. After standing in the dark for 30 min to allow the color to develop, the OD530 value was measured, and the IAA content was calculated according to the standard curve.

[0038] Prepare the IAA standard curve: Weigh 0.01 g of analytical grade IAA on an analytical balance and add it to 100 ml of water to prepare a 100 µg / mL stock solution. Then, use a stepwise dilution method to obtain standard solutions with concentrations of 0, 10, 20, 30, 40, 50, 60, 80, and 100 mg / L.

[0039] The colorimetric reaction of IAA with Salksowsk reagent showed minimal color change during the reaction. Further screening and quantitative determination were performed, and the concentration of IAA produced by CSH-9 was calculated to be 98.14 mg / L by referring to the standard curve.

[0040] PCR amplification was performed using universal primers 27F (5ʹ-AGAG TTTG ATCC TGGC TCAG-3ʹ) and 1492R (5ʹ-TACG GCTACCTT GTTA CGAC TT-3ʹ). The PCR reaction system is shown in Table 3, and the PCR amplification conditions are shown in Table 4. After product purification, the gene sequence was determined. The determined 16S rRNA gene sequence was compared for homology with known 16S rRNA sequences stored in the public databases GenBank and EzBiocloud server (https: / / www.EzBiocloud.net / identify) (Kim et al., 2012). Twenty standard strains with high homology were selected, and a phylogenetic tree was constructed using the neighbor-joining method in MEGA version X software. Bacillus cereus The strain CSH-9 was identified as belonging to the genus Bacillus, and was compared with the standard strain. Reaction system ATCC 14579 (AE016877) is the most closely related, and based on the results of morphological, culture and physiological and biochemical characteristics, CSH-9 was further identified as Bacillus cereus.

[0041] Table 3. PCR reaction system for Bacillus cereus CSH-9 16S rRNA sequence

[0042] Volume Template DNA 1 μL 2x Taq PCR MasterMix 12.5 μL Upstream primer 27F 0.5 μL Downstream primer 1492R 0.5 μL 10.5 μL ddH2O Total volume 25 μL Name

[0043] Table 4. PCR amplification reaction conditions for Bacillus cereus CSH-9 16S rRNA sequence

[0044]

[0045] Example 2: Synthetic flora of Trichoderma echinosporum and Bacillus subtilis

[0046] 1. Experimental Materials

[0047] 1.1 Test Instruments and Equipment

[0048] The main instruments and equipment required for this experiment are shown in Table 5.

[0049] Table 5 Instruments and Equipment

[0050] Model Manufacturer Biochemical incubator SPX-150 Beijing Hengrui Tianchuang Electromechanical Equipment Co., Ltd. pH meter Delta 320 Mettler-Toledo Instruments Constant temperature water bath HHS-11-2 Hangzhou Hui'er Instrument Co., Ltd. High-pressure steam sterilization pot HVE-2510 HIRAYAMA, Japan PCR amplifier TAdvanced 96 SG Biometra, Germany Gel imaging analysis system UVP EC3 UVP, USA Horizontal electrophoresis apparatus HR / 022 Beijing Hengrui Tianchuang Electromechanical Equipment Co., Ltd. Scanning electron microscope Sigma VP Zeiss, Germany Electric heating air drying oven DHG-9140A Shanghai Yiheng Scientific Instruments Co., Ltd. Single-person single-side horizontal clean bench SW-CF-1F Suzhou Suji Purification Equipment Co., Ltd. Tabletop refrigerated centrifuge ST16R Thermo, Germany Laser confocal laser microscope ZEISS Axio Scope A1 Carl Zeiss, Germany Culture medium name

[0051] 1.2 Main Culture Medium

[0052] The main culture media used in this study are shown in Table 6.

[0053] Table 6 Main Culture Media and Formulations

[0054] Culture medium formula Potato glucose agar (PDB) Potato 200.0 g, glucose 20.0 g, water 1000 mL. LB medium Yeast powder 5 g, tryptone 10 g, NaCl 10 g, agar 20 g, water 1000 mL, pH 7.2~7.5 Strain number

[0055] 1.3 Banana seedlings tested

[0056] The banana tissue culture seedlings were all healthy Brazilian banana seedlings with relatively uniform growth and at the 3-4 leaf stage, provided by the Danzhou Tissue Culture Center of the Chinese Academy of Tropical Agricultural Sciences.

[0057] 1.4 Test Soil

[0058] The soil used in the experiment was taken from a banana plantation in Danzhou City, Hainan Province (109°54'66"E, 19°44′63"N), and the soil type was red soil.

[0059] 1.5 Information on the tested functional strains

[0060] The bacterial strains tested in this experiment are shown in Table 7.

[0061] Table 7 Information on the tested bacterial strains

[0062] Species Classification Function Source CSH-9 Bacillus cereus Bacteria Ability to reduce nitrate; growth promotion Isolated from banana rhizosphere soil MM7 Trichoderma asperellum Fungus Predation of banana wilt; growth promotion Isolated from banana rhizosphere soil ​

[0063] Trichoderma acicularis ( Trichoderma asperellum Trichoderma MM7 was deposited at the China Center for Type Culture Collection (CCTCC) on March 30, 2021, with accession number CCTCC NO: M2021302, located at Wuhan University, Wuhan, Hubei Province, China. It was isolated from banana rhizosphere soil. For the mycological characteristics of Trichoderma MM7, please refer to Chinese Patent Application No. 202111570689.8, entitled "A Trichoderma MM7 that preys on banana wilt pathogens and its application".

[0064] 2. Experimental Methods and Results

[0065] 2.1 Construction of Synthetic Microbial Community

[0066] Prepare PDA medium and sterilize it at 121 °C for 20 min. Streak a line down the center of a PDA plate, then inoculate CSH-9 at the center of the line. After incubating at 28 °C for 2 days, inoculate MM7 at the center of the plate. For the control group, inoculate MM7 alone onto sterilized PDA medium. Incubate upside down at 28 °C for 5 days, observing the growth of each strain to confirm whether the functional strains can coexist.

[0067] like Figure 2 As shown, after cultivation, it was found that CSH-9 can coexist well with MM7, with no antagonism between them, and the symbiosis is strong, making it suitable for constructing a stable functional microbial synthetic community.

[0068] 2.2 Co-culture of synthetic microbial communities

[0069] Seed culture: Fresh colonies of CSH-9 and MM7 were picked from the plates and inoculated into LB and PDB liquid media, respectively. CSH-9 was cultured overnight at 37°C and 180 rpm / min on a shaker, while MM7 was cultured for 3 days at 28°C and 180 rpm / min on a shaker.

[0070] Fresh seed cultures of CSH-9 and MM7 were inoculated into PDB liquid medium at a 1:1 ratio and co-cultured, with separately cultured MM7 serving as a control. The cultures were incubated at 28°C and 180 rpm / min in a shaker. On days 3 and 5, the number of spores in the treated MM7 cells was counted under a microscope using a hemocytometer.

[0071] like Figure 3 As shown, co-culturing CSH-9 with MM7 significantly increased the spore count of MM7, and this promoting effect persisted on both day 3 and day 5. In the MM7 treatment group, the spore count was approximately 40 spores / mL on day 3 and approximately 85 spores / mL on day 5; while in the MM7 + CSH-9 treatment group, the spore count was approximately 90 spores / mL on day 3 and approximately 250 spores / mL on day 5. This indicates that the addition of CSH-9 significantly promotes the growth of MM7.

[0072] 2.3 Preparation of Synthetic Microbial Community

[0073] Raw materials for preparation: potatoes, glucose, and inoculants (as shown in Table 8).

[0074] A single CSH-9 colony was picked and inoculated into KingB liquid medium containing 100 mg / L tryptophan, and cultured in a shaker at 30℃ and 160 rpm for 24 h to obtain CSH-9 fermentation broth.

[0075] MM7 bacterial cake was inoculated into PDB medium and cultured in a shaker at 30℃ and 160 rpm for 24 h to obtain MM7 fermentation broth.

[0076] Prepare PDB liquid culture medium, dispense it into 250mL Erlenmeyer flasks, sterilize at 121℃ for 20 minutes, and after cooling, inoculate fresh test strains at a rate of 1% (V / V) of the culture medium volume according to the strain combination in Table 6. Incubate at 28℃ on a shaker at 180rpm / min for 5-7 days to prepare synthetic bacterial groups.

[0077] Table 8. Microbial Strain Combinations

[0078] Bacterial colony number Bacterial strain combination Effective viable bacterial number ratio of inoculated bacteria CK No bacteria added / T1 MM7 100% T2 CSH-9 100% T3 MM7+CSH-9 50%；50%

[0079] 2.4 Experiment on the promotion of seed germination by synthetic microbial communities

[0080] This experiment used maize seeds as material to evaluate the ability of synthetic microbial communities to promote seed germination. The maize seeds were purchased from an agricultural supply store in Chengmai, Haikou, China. After washing and sterilizing, the maize seeds were placed on filter paper in petri dishes. The fermentation broths T1, T2, and T3 were diluted 10-fold with sterile water, and then the sterilized maize seeds were treated using the following method (each treatment group contained 30 seeds, with 3 replicates): the glass petri dishes containing maize seeds were incubated at 26±1℃, and 3 mL of each treatment solution was sprayed onto the filter paper daily to keep the seeds moist. Physiological changes in the seeds were recorded. The entire experiment was conducted under controlled conditions using a completely randomized design.

[0081] To further explore the potential of synthetic microbial communities to promote seed germination, the germination rate and sprout length of maize seeds were recorded after 7 days. The results are as follows: Figure 4 As shown, the germination rate of maize seeds in the T3 treatment group was 88.89%, significantly higher than that in the T2 treatment group (50.00%), T1 treatment group (55.56%), and control group (62.84%). Figure 4 A, 4B). And the budding length also showed similar differences ( Figure 4 C). The average germination length of maize seeds in the T3 treatment group was 12.20 cm, significantly higher than that in the T2 treatment group (4.88 cm), T1 treatment group (6.25 cm), and control group (8.27 cm); while the average germination length of maize seeds in the control group was significantly higher than that in the T1 and T2 treatment groups. In conclusion, the synthetic microbial community plays an important role in promoting maize seed germination.

[0082] 2.5 Potted Plant Experiment

[0083] 2.5.1 Banana Cultivation

[0084] The pot experiment was conducted in April-May 2025 at the Institute of Tropical Biological Sciences and Biotechnology, Chinese Academy of Tropical Agricultural Sciences. Greenhouse conditions included 25℃, 75% humidity, and natural light. Healthy soil from banana plantations in Danzhou City, Hainan Province, was collected and sieved through a 20-mesh sieve. Uniformly grown banana seedlings with 3-4 leaves were selected, rinsed with sterile water, and planted in plastic pots containing 1400 g of soil, with 30 seedlings per treatment. The experiment included four treatment groups: CK: watering only; T1: MM7 alone; T2: CSH-9 alone; T3: a combination of CSH-9 and MM7. Every 7 days, 100 mL of soil diluted 10 times was applied to each treatment. All other management practices remained consistent throughout the experiment. Samples were taken two months after treatment.

[0085] 2.5.2 Measurement of Physiological Indicators of Banana Seedlings

[0086] Physiological parameters of banana seedlings after transplanting, including chlorophyll content, plant height, stem diameter, leaf area, fresh weight, and dry weight, were determined according to the method of Chen et al. (2018).

[0087] (1) Determination of chlorophyll content in banana seedlings

[0088] The chlorophyll content was measured using a SPAD-502 portable chlorophyll meter. The second unfolded leaf at the top of the banana seedling was selected, and the chlorophyll content at the bottom, middle, and upper edges of both sides of the leaf was measured.

[0089] The chlorophyll content in leaves is closely related to the healthy growth of banana plants because it is directly related to photosynthesis. When banana plants are diseased, the synthesis of chlorophyll in the leaves is interfered with, or even destroyed, reducing the efficiency of photosynthesis and inhibiting plant growth and development. The experimental results of the effects of different microbial treatments on the chlorophyll content of banana seedling leaves are shown in Table 9. Compared with the control (CK), the chlorophyll content of treatments T1, T2, and T3 all showed significant differences, with the T3 treatment showing the most significant difference, reaching 48.94 mg / g, which was 1.11 times that of T1 and T2. The results indicate that CSH-9 and MM7 can significantly increase the chlorophyll content of banana leaves, and their synthetic microbial communities are more conducive to increasing the chlorophyll content of banana leaves. This suggests that CSH-9 and MM7 can effectively increase leaf chlorophyll content, enhance the efficiency of photosynthesis, and promote healthy plant growth.

[0090] Table 9. Effects of different microbial communities on chlorophyll content and leaf area of ​​bananas.

[0091] Treatment Chlorophyll content (mg / g) Leaf area (cm 2 / plant)]]> CK 40.92 ± 0.88c 239.26 ± 4.82b T1 44.12 ± 0.79b 428.78 ± 10.18a T2 44.18 ± 0.67b 412.52 ± 8.58a T3 48.94 ± 0.92a 461.01 ± 10.19a

[0092] Note: Different lowercase letters after each column indicate significant differences (P < 0.05) between different treatments.

[0093] (2) Leaf area of ​​banana seedlings

[0094] The leaf area of ​​banana seedlings was obtained using the formula proposed by Chen Zhijie (2018). The third unfolded leaf at the top of the banana seedling was selected, and the formula was: leaf length × leaf width × 0.75. The leaf length is the distance from the leaf base to the leaf tip, and the leaf width is the distance between the two sides of the widest part of the leaf.

[0095] Leaf area is an important trait in plant growth, directly affecting photosynthetic efficiency and playing a crucial role in plant physiological functions, ecological adaptation, and agricultural production. Table 9 shows that the leaf areas of treatments T1, T2, and T3 were significantly higher than the control (CK), with treatment T3 having the largest leaf area at 461.01 cm². 2Comprehensive analysis showed that both T1 and T2 treatments significantly increased chlorophyll content and leaf area in plants, with comparable effects; however, T3 treatment showed the best results in increasing chlorophyll content and leaf area, significantly outperforming the other treatments. This suggests that the two treatments may have a synergistic effect and have great potential in promoting plant growth.

[0096] (3) The stem of the banana seedling is thick

[0097] The diameter of the banana seedling stem was measured using a vernier caliper with a minimum scale of 0.01, at a point 3 cm from the potting substrate.

[0098] As shown in Table 10, stem diameter increased to some extent in all treatments with different microbial communities. Compared with the control (CK), the stem diameters of treatments T1, T2, and T3 were significantly different, with the T3 treatment showing the largest stem diameter at 21.83 mm, significantly higher than the other treatments. Therefore, treatments T1 and T2 significantly increased stem diameter, and their effects were comparable; however, treatment T3 showed the best effect in increasing stem diameter, indicating that CSH-9 and MM7, especially their synthetic microbial communities, have a good growth-promoting effect and can significantly increase banana stem diameter, with treatment T3 showing the most significant effect.

[0099] (4) Fresh weight and dry weight of banana seedlings

[0100] Wash the banana seedlings with clean water, air dry them on newspaper, and measure their fresh weight. Place the weighed banana seedlings in an oven and dry them at 80℃ for 3 days, then remove them and weigh them again.

[0101] As shown in Table 10, the dry and fresh weights of banana plants remained consistent across all treatment groups. Compared to the control (CK), the fresh and dry weights of treatments T1, T2, and T3 all showed significant differences, with treatment T3 exhibiting the highest values ​​at 128.18 g and 26.63 g, respectively, significantly higher than the other treatments and representing increases of 50.64% and 77.30% compared to the control. This indicates that CSH-9 and MM7, especially their synthetic flora, can significantly improve banana biomass and are suitable for subsequent production.

[0102] (5) Plant height of banana seedlings

[0103] The height of a banana seedling is measured using a ruler with a minimum graduation of 0.1 cm, starting from the surface of the substrate in the pot and ending at the junction of the petiole and pseudostem of the top leaf.

[0104] Table 10 shows that there were significant differences in the plant height of banana seedlings under different microbial treatments, and all were significantly higher than those under the sterile fertilizer treatment. The plant height of banana seedlings was highest under the T3 treatment, significantly higher than other treatments, reaching 20.40 cm, an increase of 64.38% compared to the control. This indicates that CSH-9 and MM7, especially the synthetic microbial community treatments of the two, have the best growth-promoting effect on bananas.

[0105] Table 10 Effects of different microbial communities on banana growth

[0106] Treatment Plant height (cm) Stem diameter (mm) Fresh weight (g·plant -1 )]]> Dry weight (g·plant -1 )]]> CK 12.41 ± 0.28d 14.15 ± 0.28c 85.09 ± 2.34c 15.02 ± 0.94c T1 17.24 ± 0.26c 18.16 ± 0.43b 99.06 ± 1.01b 19.35 ± 0.37b T2 18.83 ± 0.26b 18.73 ± 0.36b 106.15 ± 2.67b 17.58 ± 0.40b T3 20.40 ± 0.36a 21.83 ± 0.35a 128.18 ± 3.31a 26.63 ± 0.37a

[0107] Note: Different lowercase letters after each column indicate significant differences (P < 0.05) between different treatments.

[0108] The specific embodiments of the present invention have been described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions to this invention are also within the scope of the present invention. Therefore, all equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered within the scope of the present invention.

Claims

1. The application of synthetic microbial communities, mixed microbial agents, or microbial fertilizers in the preparation of formulations that promote banana growth and / or corn seed germination, wherein the synthetic microbial communities, mixed microbial agents, or microbial fertilizers all contain Bacillus cereus and Trichoderma echinococcus, wherein Bacillus cereus (… Bacillus cereus The accession number is GDMCC NO: 66395; Trichoderma echinosporum ( Trichoderma asperellum The accession number is CCTCC NO: M 2021302.

2. The application of synthetic microbial communities, mixed microbial agents, or microbial fertilizers in the preparation of formulations that increase banana chlorophyll content, and / or banana plant height, and / or banana stem diameter, and / or banana leaf area, and / or banana fresh weight, and / or banana dry weight, and / or corn seed germination rate, and / or corn seed germination length, wherein the synthetic microbial communities, mixed microbial agents, or microbial fertilizers all contain Bacillus cereus and Trichoderma echinococcus, wherein Bacillus cereus (… Bacillus cereus The accession number is GDMCC NO: 66395; Trichoderma echinosporum ( Trichoderma asperellum The accession number is CCTCC NO: M 2021302.

3. The use of Bacillus cereus or fermentation broth containing Bacillus cereus in the preparation of formulations that increase the number of Trichoderma spores, wherein the Trichoderma spores is named Trichoderma spores ( Trichoderma asperellum MM7, was deposited at the China Center for Type Culture Collection on March 30, 2021, with accession number CCTCC NO: M 2021302; the described Bacillus cereus ( Bacillus cereus The accession number is GDMCC NO: 66395.

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