A compound inoculant containing Streptomyces longissima and Trichoderma guildrum and its application

Abstract

This invention discloses a compound microbial agent containing *Streptomyces longiflora* and *Trichoderma guiyuanensis*, wherein *Streptomyces longiflora* is *Streptomyces longiflora* A25 and *Trichoderma guiyuanensis* is *Trichoderma guiyuanensis* NJAU4742. This invention also discloses the application of the compound microbial agent in promoting cucumber growth and in increasing the yield and improving the fruit quality of seedless lychee. This invention uses *Streptomyces longiflora* A25 and *Trichoderma guiyuanensis* NJAU4742 isolated and screened from the rhizosphere soil of seedless lychee trees to prepare a compound microbial agent. When applied to cucumber pot experiments, it significantly promotes cucumber growth. When applied to seedless lychee field experiments, it significantly promotes the increase in seedless lychee yield and improves fruit quality. This compound microbial agent is of great significance to the sustainable development of the seedless lychee industry.

Description

Technical Field

[0001] This invention relates to the field of agricultural microbial technology, specifically to a compound microbial agent containing Streptomyces longissima and Trichoderma guizhouense and its application. Background Technology

[0002] Seedless lychees have high economic value due to their seedless nature, delicate flesh, and unique flavor. However, in actual production, seedless lychees generally face challenges such as difficulty in balancing yield and quality, and subpar fruit flavor. Traditional cultivation often relies on chemical fertilizers and plant growth regulators to promote plant growth and fruit development, but long-term use can easily lead to soil degradation, microbial community imbalance, decreased fruit flavor, and even environmental pollution and food safety issues. Therefore, developing green and efficient bio-promoting and quality-improving technologies is of great significance for the sustainable development of the seedless lychee industry.

[0003] Rhizosphere growth-promoting microorganisms have garnered significant attention in sustainable agriculture due to their functions in promoting growth, resisting disease, and improving the soil microecological environment. Common growth-promoting microorganisms include Bacillus and Trichoderma. However, existing single-strain microbial agents often have limited functions and cannot simultaneously meet the comprehensive needs of crop growth promotion and quality improvement. Compared to single-strain agents, compound microbial agents can exert a more comprehensive function through the synergistic effect of multiple strains. However, the interaction effects between different strains (such as antagonism or synergy) are not yet clear, limiting the development of compound microbial agents. Furthermore, existing compound microbial agents still suffer from poor strain compatibility and weak field colonization ability on seedless litchi. Summary of the Invention

[0004] The purpose of this invention is to provide a compound fungal agent containing Streptomyces longissima and Trichoderma guizhouense and its application, so as to overcome the shortcomings of the prior art.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] The first aspect of this invention provides a compound fungal agent containing *Streptomyces longissima* and *Trichoderma guildrum*, wherein the *Streptomyces longissima* is *Streptomyces longissima* A25, and its classification name is *Streptomyces longissima*. Streptomyces longisporus Latin name Streptomyces longisporus The specimen is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, on September 1, 2025, with accession number CGMCC NO.35794; the described *Trichoderma guiyuanensis* is *Trichoderma guiyuanensis* NJAU4742, and its classification name is *Trichoderma guiyuanensis*. Trichoderma guizhouense Latin name Trichoderma guizhouenseIt is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, on April 11, 2016, with accession number CGMCC NO.12166.

[0007] Furthermore, the compound microbial agent is prepared by the following steps:

[0008] (1) Inoculate Streptomyces A25 into the first liquid culture medium for liquid fermentation culture to obtain a culture broth; centrifuge the culture broth, discard the supernatant, resuspend the precipitate with sterile water, and adjust the cell concentration with sterile water to obtain A25 single-strain agent;

[0009] Trichoderma guiyuan NJAU4742 was inoculated into a second liquid culture medium for liquid fermentation to obtain a culture broth; the culture broth was filtered to remove mycelia to obtain a spore liquid; the spore liquid was centrifuged, the supernatant was discarded, the precipitate was resuspended in sterile water, and the spore concentration was adjusted with sterile water to obtain a single-strain NJAU4742 agent;

[0010] (2) Mix A25 single bacterial agent and NJAU4742 single bacterial agent evenly to obtain the compound bacterial agent.

[0011] Furthermore, in step (1), the cell concentration of the A25 single-strain agent is ≥1.0×10⁻⁶. 8 CFU / mL, spore concentration of NJAU4742 single-agent fungicide ≥0.5×10 8 / mL; in step (2), A25 single-strain agent and NJAU4742 single-strain agent are mixed evenly in equal volumes.

[0012] Furthermore, in step (1), the first liquid culture medium includes LB liquid culture medium; the second liquid culture medium includes PDB liquid culture medium.

[0013] Furthermore, in step (1), the liquid fermentation culture conditions for Streptomyces A25 are: temperature 28-30℃, rotation speed 170-200rpm, and fermentation culture time 1-2d; the liquid fermentation culture conditions for Trichoderma Guizhouensis NJAU4742 are: temperature 28-30℃, rotation speed 170-200rpm, and fermentation culture time 4-6d.

[0014] The second aspect of this invention provides the application of the above-mentioned compound microbial agent in promoting cucumber growth.

[0015] The third aspect of this invention provides the application of the above-mentioned compound microbial agent in promoting the increase of seedless lychee yield and improving fruit quality.

[0016] Furthermore, the increase in yield includes increases in single fruit weight, number of fruits per plant, and yield per plant.

[0017] Furthermore, the improvement in fruit quality includes an increase in the sugar-acid ratio and vitamin C content.

[0018] The beneficial effects of this invention are:

[0019] This invention isolated and screened a strain of *Streptomyces longissima* A25 from the rhizosphere soil of seedless lychee trees. *Streptomyces longissima* A25 and *Trichoderma guizhouense* NJAU4742 were formulated into a compound microbial agent. In pot experiments with cucumbers, this agent significantly promoted cucumber growth. In field trials with seedless lychees, it significantly increased yield and improved fruit quality. This compound microbial agent is of great significance for the sustainable development of the seedless lychee industry. Attached Figure Description

[0020] Figure 1 This is a colony morphology diagram of strain A25.

[0021] Figure 2 Phylogenetic tree diagram of strain A25 based on 16S rDNA sequence.

[0022] Figure 3 The production of auxin (IAA) by strains A25, NJAU4742, and A25+ strain NJAU4742.

[0023] Figure 4 The effects of different inoculants on the growth of potted cucumber seedlings.

[0024] Figure 5 The effects of different inoculants on the biomass-above-fresh weight of potted cucumber seedlings.

[0025] Figure 6 The effect of different inoculants on the biomass-above-ground dry weight of potted cucumber seedlings.

[0026] Figure 7 The effects of different inoculants on the biomass-underground fresh weight of potted cucumber seedlings.

[0027] Figure 8 The effect of different inoculants on the biomass-dry weight of underground parts of potted cucumber seedlings.

[0028] Figure 9 The effect of different inoculants on the biomass-plant height of potted cucumber seedlings.

[0029] Figure 10 The effects of NJAU4742+A25 compound microbial agent on the growth status of seedless litchi fruit in the field.

[0030] Figure 11 The effects of NJAU4742 inoculant on the growth status of seedless litchi fruit in the field.

[0031] Figure 12 The effect of control (CK) on the growth status of seedless litchi fruit in the field.

[0032] Figure 13 The effects of different microbial agents on the single fruit weight of seedless litchi in the field.

[0033] Figure 14 The effect of different microbial agents on the number of seedless litchi fruits (per tree) in the field.

[0034] Figure 15 The effects of different inoculants on the yield (per tree) of seedless litchi in the field.

[0035] Figure 16 The effects of different microbial agents on the quality of seedless litchi fruit in the field - soluble solids content.

[0036] Figure 17 The effects of different microbial agents on the quality and acidity of seedless litchi fruit in the field.

[0037] Figure 18 The effect of different microbial agents on the quality of seedless litchi fruit in the field - sugar-acid ratio.

[0038] Figure 19 The effects of different microbial agents on the quality and vitamin C content of seedless litchi fruits in the field.

[0039] Information on the preservation of biological materials

[0040] A25, classified as *Streptomyces longissima* Streptomyces longisporus Latin name Streptomyces longisporus The specimen is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The deposit date is September 1, 2025, and the accession number is CGMCC NO.35794.

[0041] NJAU4742, classified as *Trichoderma guiyuan*. Trichoderma guizhouense Latin name Trichoderma guizhouense It is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, on April 11, 2016, with accession number CGMCC NO.12166. Detailed Implementation

[0042] The following examples are provided to better understand the present invention, but do not limit the invention. Unless otherwise specified, the experimental methods in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent stores.

[0043] The culture medium formulations involved in the following examples are as follows:

[0044] LB liquid medium (1L): 10g tryptone, 5g yeast extract, 10g sodium chloride, bring the volume to 1L with deionized water, and sterilize at 115℃ for 30min.

[0045] LB solid medium (1L): 10g tryptone, 5g yeast extract, 10g sodium chloride, deionized water to a final volume of 1L, 15g agar powder, sterilized at 115℃ for 30min.

[0046] 1 / 10 LB solid culture medium (1L): 1g tryptone, 0.5g yeast extract, 1g sodium chloride, deionized water to a final volume of 1L, 15g agar powder, sterilize at 115℃ for 30min.

[0047] PDB liquid culture medium (1L): 200g peeled potatoes cut into 1cm pieces 3 For the potato, add 800mL of water to a pot and bring it to a boil. Add the potato and cook for 15-20 minutes until the water becomes viscous. Add 20g of glucose to a beaker, place two layers of gauze at the mouth of the beaker, pour the viscous potato liquid through the gauze, and then add water to make up to 1L. Sterilize at 115℃ for 30 minutes.

[0048] PDA solid culture medium (1L): 200g peeled potatoes cut into 1cm pieces 3 For the potato mixture, add 800mL of water to a pot and bring it to a boil. Add the potato mixture and cook for 15-20 minutes until the water becomes viscous. Add 20g of glucose to a beaker, place two layers of gauze over the mouth of the beaker, and pour the viscous potato mixture through the gauze. Add water to bring the volume to 1L. Add 20g of agar powder and sterilize at 115℃ for 30 minutes.

[0049] Example 1

[0050] Isolation, screening and identification of functional strains

[0051] 1. Isolation of Functional Strains: Strains were isolated from the rhizosphere soil of healthy litchi trees in the Yuanyanghong seedless litchi orchard in Bailian Town, Chengmai County, Hainan Province. 5g of rhizosphere soil from healthy litchi trees was weighed and placed in an Erlenmeyer flask containing 45mL of sterile water (4-6 glass beads). The flask was shaken at 30℃ and 170rpm for 30min, followed by serial dilutions to obtain soil suspensions of different dilution gradients. 100μL of each dilution was spread onto 1 / 10LB solid medium plates and incubated at 30℃ for 2 days. After colonies appeared, differentially expressed colonies were selected and purified on 1 / 10LB solid medium plates at 30℃. The purified strains were inoculated onto LB solid medium plates and incubated at 30℃ until distinct single colonies formed. The plates were then stored at 4℃ for later use. The purified single colonies were preserved in glycerol tubes and stored at 80℃. A total of 32 strains were obtained.

[0052] 2. Determination of the strain's IAA production capacity:

[0053] (1) Thirty-two strains were inoculated into LB liquid medium and cultured at 30℃ and 170 rpm for 2 days to obtain culture medium. 2 mL of culture medium was centrifuged at 5000 rpm for 10 min, the supernatant was removed, and the precipitate was resuspended in 1 mL of sterile water to obtain strain suspension. The concentration of the strain suspension was adjusted to OD using sterile water. 600 ≈1.0. The strain suspension after concentration adjustment was added to LB liquid medium containing 200 mg / L tryptophan at an inoculum rate of 1 v / v%, and cultured in the dark at 30 °C and 170 rpm for 2 days to obtain the bacterial culture.

[0054] (2) Then, take 100µL of the bacterial culture from step (1) and drop it onto a white ceramic plate. At the same time, add an equal volume of colorimetric solution (weigh 1g FeCl3·6H2O, dissolve it in 21.485mL of concentrated H2SO4, slowly drop it into distilled water to dilute, and then make up to 50mL with distilled water to obtain the colorimetric solution; the same applies below). Use a mixture of 100µL of uninoculated LB liquid medium containing 200mg / L tryptophan and an equal volume of colorimetric solution as a control. After the white ceramic plate has been left to stand at room temperature and in the dark for 30min, observe it. If the color turns pink, it indicates that it can secrete IAA. The darker the color, the greater the secretion intensity. If it does not change color, it indicates that it cannot secrete IAA.

[0055] (3) For strains capable of secreting IAA (all 32 strains were found to have IAA secretion ability as determined in step (2)), quantitative determination of IAA production was performed. 1 mL of the bacterial culture from step (1) was centrifuged at 5000 rpm for 10 min, the supernatant was collected, an equal volume of colorimetric solution was added, and the mixture was allowed to stand at room temperature in the dark for 30 min. Three replicates were set up for each strain, and its OD was measured. 530The IAA content per unit volume of bacterial culture was calculated by referring to the standard curve. The standard curve was obtained as follows: (a) IAA standard solutions of varying concentrations (0, 3, 6, 12, 24, 48 ng / mL) were prepared using sterile water; (b) each IAA standard solution was mixed with an equal volume of colorimetric solution and allowed to stand at room temperature in the dark for 30 min; (c) OD was measured. 530 Value; (d) OD with IAA concentration on the x-axis. 530 A linear regression was performed on the ordinate to obtain the standard curve. The results are shown in Table 1.

[0056] Table 1

[0057] strain name IAA content (ng / mL) A25 72.32 A34 70.51 B13 65.13 A16 62.73 A31 61.13 D5 60.13 A48 60.95 D4 59.33 D20 57.99 C3 56.17 B1 55.70 B7 55.57 C11 55.57 C9 55.50 C4 55.33 C19 55.19 A5 54.93 A46 54.37 A35 54.24 D8 53.75 A10 52.74 B14 52.22 D28 52.13 B12 52.06 B3 50.51 B2 50.04 A9 49.67 D9 48.66 A12 47.64 C2 43.67 A14 40.74 D47 35.22

[0058] The results showed that strain A25 had the strongest IAA production capacity. Therefore, strain A25 and strain NJAU4742 (i.e., *Trichoderma guiyuan* NJAU4742, deposited at the China General Microbiological Culture Collection Center (CGMCC) on April 11, 2016, with accession number CGMCC NO.12166) were selected to prepare a compound microbial agent for subsequent experiments. Strain A34 is involved in another patent.

[0059] 3. Identification of strain A25:

[0060] (1) Morphological identification: The A25 glycerol strain was streaked onto LB solid medium for activation inoculation, and then incubated at 30℃ for 2 days. Single colonies were then observed. Figure 1 As shown, the colony surface is milky yellow, loose in texture, dry and opaque; the edge of the colony is irregularly wavy, extending outward with fine radial hyphae.

[0061] (2) Molecular biological identification: DNA was extracted from strain A25, and the 16S rDNA gene in the genome of strain A25 was amplified by PCR using universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3', as shown in SEQ ID NO.1) and 1492R (5'-TACGGYTACCTTGTTACGACTT-3', as shown in SEQ ID NO.2). The PCR product was sent to a biotechnology company for sequencing. The obtained 16S rDNA gene sequence of strain A25 (as shown in SEQ ID NO.3) was compared with the GenBank database using the NCBI BLAST tool for homology comparison, and a phylogenetic tree was constructed using MEGA 11.0 software, as shown in SEQ ID NO.2. Figure 2 As shown, strain A25 is located in Streptomyces longisporus On the branch, with S. longisporusISP 5166 has the highest homology, reaching 100%.

[0062] Based on the colony morphology characteristics of strain A25 and the phylogenetic tree analysis results constructed from the 16S rRNA gene sequence, strain A25 was identified as *Streptomyces longissima*. Streptomyces longisporus Strain A25 has been deposited at the China General Microbiological Culture Collection Center (CGMCC) on September 1, 2025, with accession number CGMCC NO.35794.

[0063] Example 2

[0064] Quantitative determination of IAA production performance of strains A25, NJAU4742, and A25+ strain NJAU4742:

[0065] (1) Inoculate strain A25 into PDB liquid medium and culture at 30℃ and 170 rpm for 2 days to obtain the culture solution. Centrifuge the culture solution at 5000 rpm for 10 min, remove the supernatant, and resuspend the precipitate in sterile water to prepare a concentration of OD. 600 A25 suspension with a density of approximately 1.0.

[0066] Strain strain NJAU4742 was inoculated into PDB liquid medium and cultured at 28℃ and 170 rpm for 5 days to obtain the culture medium. The culture medium was centrifuged at 5000 rpm for 10 min, the supernatant was removed, and the precipitate was resuspended in sterile water to prepare a concentration of OD100. 600 NJAU4742 suspension ≈1.0.

[0067] To observe the IAA yield of individual strains, A25 suspension was inoculated at 1 v / v% in PDB liquid medium containing 200 mg / L tryptophan and cultured in the dark at 30°C with shaking at 170 rpm for 2 days to obtain A25 bacterial culture; NJAU4742 suspension was inoculated at 1 v / v% in PDB liquid medium containing 200 mg / L tryptophan and cultured in the dark at 28°C with shaking at 170 rpm for 5 days to obtain NJAU4742 bacterial culture.

[0068] To observe the IAA yield of the composite strain, A25 suspension and NJAU4742 suspension were mixed at a volume ratio of 1:1 to obtain a mixed suspension. The mixed suspension was inoculated into PDB liquid medium containing 200 mg / L tryptophan at an inoculation rate of 1 v / v% and cultured in the dark at 28°C and 170 rpm for 5 days to obtain the composite bacterial culture.

[0069] (2) Take 1 mL of A25 bacterial solution, NJAU4742 bacterial solution or compound bacterial solution, centrifuge at 5000 rpm for 10 min, take the supernatant, add an equal volume of colorimetric solution, and let stand at room temperature in the dark for 30 min. Set up 3 replicates for each treatment and measure its OD. 530 Value. Calculate the IAA content per unit volume of bacterial culture by referring to the standard curve (same as step 2(3) in Example 1). Figure 3 As shown, the test results indicate that the combined strain (strain A25 + strain NJAU4742) has a higher IAA production capacity than either single strain (strain A25 or strain NJAU4742).

[0070] Example 3

[0071] Cucumber pot experiment to determine the growth-promoting performance of compound microbial agents

[0072] 1. Preparation of microbial agents

[0073] Preparation of A25 single-strain agent: Strain A25 was inoculated into LB liquid medium and cultured at 30℃ and 170 rpm with shaking for 2 days to obtain the culture broth. The culture broth was centrifuged at 5000 rpm for 10 min, the supernatant was discarded, the precipitate was resuspended in sterile water, and the bacterial concentration was adjusted to 1.0 × 10⁻⁶ cells / mL with sterile water. 8 CFU / mL (plate count) indicates the A25 single-strain agent.

[0074] Preparation of NJAU4742 single-strain agent: Strain NJAU4742 was inoculated into PDB liquid medium and cultured at 28℃ and 170 rpm for 5 days with shaking to obtain a culture solution. The culture solution was filtered through four layers of gauze to remove mycelia, yielding a spore solution. The spore solution was centrifuged at 5000 rpm for 10 min, the supernatant was discarded, the precipitate was resuspended in sterile water, and the spore concentration was adjusted to 0.5 × 10⁻⁶ with sterile water. 8 The count of cells / mL (platelet count) is the value of NJAU4742 single-strain agent.

[0075] Preparation of NJAU4742+A25 compound microbial agent: The bacterial cell concentration was 1.0 × 10⁻⁶. 8 The concentration of A25 single-strain agent (CFU / mL) and spore concentration is 0.5 × 10⁻⁶. 8 Mix NJAU4742 single bacterial agent (number / mL) at a volume ratio of 1:1 to obtain NJAU4742+A25 compound bacterial agent.

[0076] 2. Germination

[0077] Select plump, uniformly sized, dark brown cucumber seeds of variety Jin Chun No. 4. First, soak them in sterile water for 1 minute, then disinfect them in 3 w / v% sodium hypochlorite for 5 minutes, followed by disinfection in 70 v / v% ethanol for 1 minute. Finally, wash them 2-3 times with sterile water. Use tweezers to place the cucumber seeds into a glass petri dish containing moistened filter paper. Place the dish in an incubator at 25℃ and 70% relative humidity for 2 days. Once white sprouts appear, transfer them to seedling trays.

[0078] 3. Seedling raising

[0079] First, sterilize the seedling substrate (by mixing peat moss, perlite, and vermiculite in a volume ratio of 3:1:1) twice at 121℃ for 20 minutes each time, then dry it. Next, spread the dried seedling substrate throughout the entire seedling tray (the seedling tray is 54cm long, 28cm wide, and has 5×10 holes). Use tweezers to plant the germinated cucumber seeds into the seedling tray (1 seed per hole), then water with sterilized water. Finally, cultivate the seedlings in a light-controlled room at a temperature of 25℃, a relative humidity of 60%, a light cycle of 16h light / 8h darkness, and a light intensity of 3000-4000LX. Water with sterile water every day during the seedling period. Transplant the cucumber seedlings after they have grown two tender leaves.

[0080] 4. Transplanting and inoculation

[0081] The experimental soil used for potted plants was a mixture of soil and vermiculite from the Yuanyanghong seedless lychee orchard in Bailian Town, Chengmai County, Hainan Province, at a mass ratio of 96:4. The pots used had a diameter of 10cm, a height of 8.5cm, and a bottom diameter of 7cm. Each pot contained 500g of experimental soil. Uniformly growing cucumber seedlings were transplanted into each pot, one seedling per pot. The plants were cultivated in a light-controlled room at 25℃, relative humidity of 60%, a light cycle of 16h light / 8h darkness, and a light intensity of 3000-4000 LX. During cultivation, the soil was watered with sterile water every 2 days to keep it moist but not waterlogged. After 7 days of cultivation, each pot was inoculated (by slowly watering along the root zone of the cucumber plant) with 10mL of the soil. A25 single-strain agent (A25 single-strain agent treatment, denoted as A25), NJAU4742 single-strain agent (NJAU4742 single-strain agent treatment, denoted as NJAU4742), or NJAU4742+A25 compound agent (NJAU4742+A25 compound agent treatment, denoted as NJAU4742+A25) were used for continued cultivation under the same conditions as before. 10 mL of sterile water was used as a control (CK treatment, denoted as CK). Each treatment was set up with 4 replicates. After 30 days of cultivation, the growth of cucumber plants was photographed and recorded, and their aboveground fresh weight, aboveground dry weight, underground fresh weight, underground dry weight, and plant height were measured.

[0082] 5. Indicator Measurement

[0083] Carefully remove the cucumber plant whole, avoiding root damage. Gently rinse the soil surface of the roots with tap water, then rinse once with sterile water. After blotting off excess moisture with filter paper, perform the following tests:

[0084] (a) Plant height

[0085] Use a ruler to measure the vertical height from the base of the cucumber plant (soil surface position) to the growing point at the top of the cucumber plant, in cm.

[0086] (b) Fresh weight of aboveground parts and fresh weight of underground parts

[0087] Cut the cucumber plant at the root neck, separating the above-ground part (stem + leaves) and the underground part. Immediately weigh them using an electronic balance to obtain the fresh weight of the above-ground part and the fresh weight of the underground part, in grams.

[0088] (c) Dry weight of aboveground and belowground parts

[0089] The above-ground and underground parts, which have been weighed fresh, are placed separately into paper envelopes and dried in an oven at 65°C until constant weight. After cooling to room temperature, they are weighed to obtain the dry weight of the above-ground and underground parts, in grams.

[0090] 6. Test Results

[0091] The results are as follows Figure 4 and Figure 5-9 As shown, the cucumber plants treated with the NJAU4742+A25 compound microbial agent exhibited the best performance in terms of plant height, aboveground fresh weight, underground fresh weight, aboveground dry weight, and underground dry weight, significantly exceeding the control treatment and each single-agent treatment. This demonstrates that it can significantly increase the biomass of cucumber plants. In contrast, the cucumber plants treated with the A25 single-agent agent showed no significant differences in any of the indicators compared to the control treatment. Except for the cucumber plants treated with the NJAU4742 single-agent agent, whose plant height was significantly higher than the control treatment, other indicators showed no significant differences compared to the control treatment.

[0092] The NJAU4742+A25 compound microbial agent promotes the growth of the aboveground parts and roots of crops through the synergistic effect of strains NJAU4742 and A25, laying a material foundation for high and stable crop yields.

[0093] Example 4

[0094] Field trials of seedless litchi to determine the growth-promoting properties of compound microbial agents

[0095] 1. Preparation of microbial agents

[0096] The preparation of NJAU4742 single-strain agent and NJAU4742+A25 compound-strain agent is the same as step 1 in Example 3. NJAU4742 single-strain agent and rice husk are mixed evenly at a volume ratio of 1:1 to obtain an NJAU4742 single-strain agent-rice husk mixture, which is set aside for later use. NJAU4742+A25 compound-strain agent is mixed evenly with rice husk at a volume ratio of 1:1 to obtain an NJAU4742+A25 compound-strain agent-rice husk mixture, which is also set aside for later use.

[0097] 2. Experimental Design and Treatment Setup

[0098] A field trial was conducted in the Yuanyanghong seedless lychee orchard in Bailian Town, Chengmai County, Hainan Province. The test material was 8-year-old healthy Hainan seedless lychee (commercial name: Yuanyanghong seedless lychee). The planting density of the experimental orchard was 50 trees / mu, and the area occupied by each tree was approximately 13.33m².

[0099] The experiment included three treatments: CK (traditional farmer fertilization), NJAU4742, and NJAU4742+A25. Each treatment was replicated three times, with each replicate consisting of one seedless litchi tree. Tree vigor and fruit yield were kept consistent across treatments to ensure fairness and accuracy.

[0100] 3. Field fertilization management

[0101] (1) Base fertilizer: In June 2024 (after fruit harvest), the NJAU4742 treatment and the NJAU4742+A25 treatment were given a compound microbial fertilizer of Trichoderma harzianum and Bacillus belye (produced by Jiangsu Sinong High Technology Co., Ltd., fertilizer registration certificate number is Microbial Fertilizer (2024) Approval No. (13853)) as base fertilizer. The application method was hole application. Two fertilizer holes with a depth of 20cm and a diameter of 25cm were dug evenly inside the drip line of the tree canopy. The amount applied per tree was 30kg, that is, 15kg per hole. The soil was covered in time after application. The CK treatment was treated with organic fertilizer produced by Taicang Lvfeng Agricultural Resources Development Co., Ltd. (registration certificate number is Su Nongfei (2012) Approval No. 0636) in the same way. The amount applied per tree was 15kg, that is, 7.5kg per hole.

[0102] (2) Topdressing:

[0103] (a) Flower-promoting fertilizer: applied in October 2024 (flower bud differentiation period). All treatments were given 15-15-15 NPK compound fertilizer (produced by Hubei Dayukou Chemical Co., Ltd., total nutrients (N-P2O5-K2O) ≥45%; the same below). The CK treatment was 0.7 kg per plant, i.e., 0.35 kg per hole. The NJAU4742 and NJAU4742+A25 treatments were 1 kg per plant, i.e., 0.5 kg per hole. The application method was the same as the basal fertilizer application.

[0104] (b) Fruit-preserving fertilizer: applied in March 2025 (young fruit stage). For the CK treatment, apply 15-15-15 NPK compound fertilizer at a rate of 0.93 kg per plant (0.465 kg per hole). For the NJAU-4742 and NJAU-4742+A25 treatments, apply 1 kg per plant (0.5 kg per hole). Simultaneously, for the NJAU-4742 treatment, apply NJAU fertilizer one week after the fruit-preserving fertilizer application. For the NJAU4742 single-strain agent-rice husk mixture, apply 2.0 kg of the NJAU4742 single-strain agent-rice husk mixture per plant, which is 1.0 kg per hole. For the NJAU4742+A25 treatment, apply the NJAU4742+A25 compound microbial agent-rice husk mixture as a top dressing one week after the fruit-setting fertilizer application, applying 2.0 kg of the NJAU4742+A25 compound microbial agent-rice husk mixture per plant, which is 1.0 kg per hole. The application method is the same as for basal fertilizer application.

[0105] 4. Field management

[0106] During the experiment, irrigation, weeding, pruning and other field agronomic practices were kept consistent across all treatments. Pest and disease control was strictly implemented in accordance with local pollution-free production standards. No additional microbial agents or bio-organic fertilizers were applied throughout the process to eliminate interfering factors and ensure the validity of the experimental results.

[0107] 5. Indicator Measurement

[0108] Lychee fruits were collected during the ripening period to determine their single fruit weight, fruit quantity, yield, soluble solids, acidity, sugar content, and vitamin C. Specifically, samples were collected on June 4, 2025, at the ripening stage of seedless lychees. Healthy fruits free from disease, pests, and mechanical damage, and of uniform ripeness, were randomly collected from five locations on each tree's canopy (east, south, west, north, and center). Fifty fruits were collected from each tree. The samples were placed in an insulated box with ice packs for refrigeration and transported back to the laboratory within 4 hours. The indicators (except for fruit quantity and yield) were measured within 24 hours. The methods for measuring each indicator are as follows:

[0109] Single fruit weight: 30 fruits were randomly selected for each treatment, i.e., 10 fruits were randomly selected for each replicate. The fresh weight of each fruit was weighed using an electronic balance with an accuracy of 0.01g, and the single fruit weight was recorded and the average value was calculated. Unit: kg / fruit.

[0110] Fruit count (per tree): After all mature fruits were harvested from each experimental tree, they were counted manually one by one to determine the total number of fruits per tree. Unit: fruits / tree.

[0111] Yield (per plant): Calculated by multiplying the average weight of a single fruit by the number of fruits per plant. Unit: kg / plant.

[0112] Soluble solids content (TSS): Determined according to NY / T 2637-2014 "Determination of soluble solids content in fruits and vegetables by refractometer method". 30 fruits were randomly selected for each treatment, i.e., 10 fruits were randomly selected for each replicate.

[0113] Acidity was determined by titration (TA): The acid-base indicator titration method was used in accordance with the "National Food Safety Standard GB 12456-2021 Determination of Total Acid in Food". 30 fruits were randomly selected for each treatment, i.e., 10 fruits were randomly selected for each replicate.

[0114] Sugar-acid ratio: The sugar-acid ratio is calculated by dividing the soluble solids content of the same fruit sample by the titratable acidity.

[0115] Vitamin C (Vc): The content of reduced ascorbic acid (AsA) / vitamin C was determined according to the Suzhou Greens Biotechnology Co., Ltd. Reduced Ascorbic Acid (AsA) / Vitamin C Content Kit (Catalog No.: G0201W48). 30 fruits were randomly selected for each treatment, i.e., 10 fruits were randomly selected for each replicate.

[0116] 6. Test Results

[0117] The results are as follows Figure 10-12 and Figure 13-19 As shown in the figure, compared with the NJAU4742 and CK treatments, the NJAU4742+A25 treatment significantly promoted the increase in seedless litchi yield and fruit quality. In terms of yield performance, the single fruit weight (0.03505 kg / fruit) of the NJAU4742+A25 treatment increased by 15.52% compared to the CK treatment and by 7.28% compared to the NJAU4742 treatment. The number of fruits per tree (909 fruits / tree) and the yield per tree (approximately 31.86 kg / tree) were also significantly higher than those of the NJAU4742 and CK treatments, achieving simultaneous increases in single fruit weight, number of fruits per tree, and yield per tree. Regarding the main fruit quality indicators, the sugar-acid ratio (95.64) and vitamin C content (0.41 mg / g) of the NJAU4742+A25 treatment were significantly better than those of the NJAU4742 and CK treatments, which not only enhanced the flavor harmony of seedless litchi fruit and reduced its astringency but also improved its nutritional quality. In summary, the NJAU4742+A25 treatment, compared with the NJAU4742 treatment and the CK treatment, can better simultaneously improve the yield performance and fruit quality of seedless litchi.

[0118] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A compound fungal agent containing *Streptomyces longissima* and *Trichoderma guizhouense*, characterized in that, The *Streptomyces longissima* species is *Streptomyces longissima* A25, which is classified as *Streptomyces longissima*. Streptomyces longisporus The specimen is deposited at the China General Microbiological Culture Collection Center (CGMCC) on September 1, 2025, with accession number CGMCC NO.35794; the described *Trichoderma guiyuanensis* is *Trichoderma guiyuanensis* NJAU4742, and its classification name is *Trichoderma guiyuanensis*. Trichoderma guizhouense It is deposited at the China General Microbiological Culture Collection Center (CGMCC) on April 11, 2016, with accession number CGMCC NO.12166.

2. The compound fungal agent containing *Streptomyces longissima* and *Trichoderma guizhouense* according to claim 1, characterized in that, The compound microbial agent is prepared by the following steps: (1) Inoculate Streptomyces A25 into the first liquid culture medium for liquid fermentation culture to obtain a culture broth; centrifuge the culture broth, discard the supernatant, resuspend the precipitate with sterile water, and adjust the cell concentration with sterile water to obtain A25 single-strain agent; Trichoderma guiyuan NJAU4742 was inoculated into a second liquid culture medium for liquid fermentation to obtain a culture broth; the culture broth was filtered to remove mycelia to obtain a spore liquid; the spore liquid was centrifuged, the supernatant was discarded, the precipitate was resuspended in sterile water, and the spore concentration was adjusted with sterile water to obtain a single-strain NJAU4742 agent; (2) Mix A25 single bacterial agent and NJAU4742 single bacterial agent evenly to obtain the compound bacterial agent.

3. The compound fungal agent containing *Streptomyces longissima* and *Trichoderma guizhouense* according to claim 2, characterized in that, In step (1), the concentration of A25 single-strain agent is ≥1.0×10⁻⁶. 8 CFU / mL, spore concentration of NJAU4742 single-agent fungicide ≥0.5×10 8 / mL; in step (2), A25 single-strain agent and NJAU4742 single-strain agent are mixed evenly in equal volumes.

4. A compound fungal agent containing *Streptomyces longissima* and *Trichoderma guizhouense* according to claim 2 or 3, characterized in that, In step (1), the first liquid culture medium includes LB liquid culture medium; the second liquid culture medium includes PDB liquid culture medium.

5. A compound fungal agent containing *Streptomyces longissima* and *Trichoderma guizhouense* according to claim 2 or 3, characterized in that, In step (1), the liquid fermentation culture conditions for Streptomyces A25 are: temperature 28-30℃, rotation speed 170-200rpm, and fermentation culture time 1-2d; the liquid fermentation culture conditions for Trichoderma Guizhouensis NJAU4742 are: temperature 28-30℃, rotation speed 170-200rpm, and fermentation culture time 4-6d.

6. The application of the compound microbial agent according to any one of claims 1-5 in promoting cucumber growth.

7. The application of the compound microbial agent according to any one of claims 1-5 in promoting the increase of seedless lychee yield and improving fruit quality.

8. The application according to claim 7, characterized in that, The yield increase includes improvements in single fruit weight, number of fruits per plant, and yield per plant.

9. The application according to claim 7, characterized in that, The improvement in fruit quality includes an increase in the sugar-acid ratio and vitamin C content.

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