Bacteriostatic and growth-promoting microorganism and compound microbial inoculant thereof, preparation method and application

Abstract

The present application relates to a kind of bacteria inhibiting and growth promoting microorganism and its compound microbial inoculant, preparation method and application.The present application provides a kind of bacillus subtilis (Bacillus subtilis) YCBA0319, preservation number is CGMCC No.33006;A kind of trichoderma longibrachiatum (Trichoderma longibrachiatum) YCFA0522, preservation number is CGMCC No.41655.In the technical solution of the present application, through bacillus subtilis and trichoderma longibrachiatum and the compound microbial inoculant of its composition, through the synergistic effect of two strains, it has the promotion effect to soil health and apple yield, quality etc., compared with control, soil bulk density is reduced, organic matter and available nutrient are improved, and apple single fruit size, yield and quality are all improved.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a microbial compound agent consisting of Bacillus subtilis, Trichoderma longifolia, and antibacterial and growth-promoting agents, as well as its preparation method and application. Background Technology

[0002] In recent years, the overuse of pesticides and fertilizers has led to an imbalance in soil microbial communities, a decline in soil quality, and frequent crop diseases, seriously affecting the sustainable development of agricultural production. Microbial inoculants, as a green and environmentally friendly agricultural input, have shown broad application prospects. However, due to the diversity of farmland environments and the variability of climatic conditions, single inoculants often fail to achieve the expected results in practical applications. Therefore, microbial inoculants are usually used in the form of multi-strain compounds, that is, by uniformly mixing two or more microorganisms in a certain proportion and then propagating and culturing them in vitro, in order to fully utilize the synergistic effects and complementary advantages between different strains, thereby significantly enhancing the growth-promoting effect.

[0003] Apple root rot is one of the most widespread and damaging root diseases. It is primarily caused by fungi such as *Fusarium oxysporium*, which have a strong infectious capacity on weak apple plants, especially during the replanting of older orchards. Therefore, maintaining healthy plant growth and effectively suppressing the pathogen are crucial for controlling apple root rot. Summary of the Invention

[0004] The present invention aims to solve at least one of the technical problems existing in the prior art or related art.

[0005] Therefore, the purpose of this invention is to provide a microorganism that inhibits bacterial growth and promotes growth, its compound microbial agent, preparation method and application, which achieves significant prevention and control effects on a variety of crop diseases through the synergistic effect of two strains, while improving the physical and chemical properties of the soil and the micro-ecological environment.

[0006] To achieve the above objectives, the first aspect of the present invention provides a Bacillus subtilis strain YCBA0319, with accession number CGMCC No. 33006.

[0007] The second aspect of the present invention provides a long-branched Trichoderma longibrachiatum YCFA0522, with accession number CGMCC No. 41655.

[0008] The third aspect of the present invention provides a microbial compound agent for inhibiting bacteria and promoting growth, including Bacillus subtilis YCBA0319 as described in the first aspect of the present invention and Trichoderma longifolia YCBA0522 as described in the second aspect of the present invention.

[0009] In the above technical solution, preferably, the application form of the microbial compound agent includes at least one of mycelial fermentation broth, freeze-dried powder and spore suspension.

[0010] The fourth aspect of the present invention provides the application of Bacillus subtilis YCBA0319 or Trichoderma longifolia YCBA0522 or microbial compound inoculants in inhibiting apple root rot pathogens and / or promoting plant growth.

[0011] In the above technical solution, preferably, the growth promotion includes at least one of the following: reducing soil bulk density, increasing soil organic matter, increasing soil available nitrogen, increasing soil available phosphorus content, increasing soil available potassium, increasing apple yield, improving apple fruit morphology, and increasing the soluble solids content in apple fruit.

[0012] In the above technical solution, preferably, the application includes trenching or root irrigation.

[0013] The fifth aspect of the present invention provides a method for culturing Bacillus subtilis as provided in the first aspect of the present invention, comprising the following steps:

[0014] Step S1, prepare the basic solid culture medium for Bacillus subtilis: mix 8g peptone, 2g beef extract, 4g sodium chloride, 16g agar powder and 900mL distilled water evenly, adjust the pH to 7.2, add 2g agar, and sterilize at 118℃ for 15 minutes;

[0015] Step S2, prepare the basic liquid culture medium for Bacillus subtilis: mix 8g peptone, 2g beef extract, 4g sodium chloride with 900mL distilled water, adjust the pH to 7.2, and sterilize at 118℃ for 15 minutes;

[0016] Step S3, Activation and Cultivation: Bacillus subtilis is inoculated into the basic solid culture medium prepared in step S1 for activation. Single colonies are selected and inoculated into the basic liquid culture medium prepared in step S2. The culture is carried out at 33℃ and 116r / min for 16 hours to obtain Bacillus subtilis fermentation broth.

[0017] The sixth aspect of the present invention provides a method for culturing Trichoderma longicornis as provided in the second aspect of the present invention, comprising the following steps:

[0018] Step S1, prepare Trichoderma longifolia basic solid culture medium: mix 10g glucose, 1g dipotassium hydrogen phosphate, 0.5g magnesium sulfate, 2g sodium nitrate, 15g agar with 1000mL distilled water, adjust the pH to 5.5, autoclave at 121℃ for 20 minutes, cool and pour into plates for later use.

[0019] Step S2, prepare Trichoderma longifolia basic liquid culture medium: mix 10g glucose, 3g sodium nitrate, 0.5g magnesium sulfate, 1g dipotassium hydrogen phosphate, 1g yeast extract with 1000mL distilled water, adjust the pH to 5.5, autoclave at 121℃ for 20 minutes, and cool for later use.

[0020] Step S3, strain activation: Inoculate Trichoderma longifolia into the basic solid culture medium prepared in step S1, and incubate in an incubator at 28℃ for 5-7 days until the colonies have grown sufficiently for use.

[0021] Step S4, liquid fermentation culture: Inoculate the activated Trichoderma longifolia spore liquid from step S3 into the basic liquid culture medium prepared in step S2, with an inoculation amount of 10% (v / v), and culture on a shaker at 28℃ and 150-200 r / min for 48-72 hours.

[0022] Step S5, Fermentation product collection: After the culture is completed, the fermentation broth is filtered through gauze or centrifuged at 5000 rpm for 10 minutes to separate the mycelium.

[0023] The seventh aspect of the present invention provides a method for preparing the microbial compound agent provided in the third aspect of the present invention, comprising the following steps:

[0024] Step S1, Preparation of Bacillus subtilis fermentation broth: According to step S3 in the fifth aspect of the technical solution of the present invention, Bacillus subtilis is cultured in liquid culture medium for 16 hours to obtain Bacillus subtilis fermentation broth;

[0025] Step S2, Preparation of Trichoderma longifolia fermentation broth: According to step S4 of the sixth aspect of the technical solution of the present invention, Trichoderma longifolia is cultured in liquid culture medium for 48-72 hours to obtain Trichoderma longifolia fermentation broth;

[0026] Step S3, determining the mixing ratio of microbial agents: The Bacillus subtilis fermentation broth prepared in step S1 and the Trichoderma longifolia fermentation broth prepared in step S2 are mixed at a mass ratio of 1:1 and stirred thoroughly to obtain a mixed microbial agent fermentation broth;

[0027] Step S4, Stabilization treatment of mixed microbial agent: Add a protectant (such as 5% lactose or skim milk powder) to the fermentation broth of mixed microbial agent from step S3, and prepare it into a powdered mixed microbial agent by spray drying or freeze drying;

[0028] Step S5, Mixed bacterial agent packaging: Dispense the powdered mixed bacterial agent prepared in step S4 into sterile aluminum foil bags, seal and store in a low temperature (4°C) environment for later use.

[0029] Compared with existing technologies, the advantages of the antibacterial and growth-promoting microorganisms, their compound microbial agents, preparation methods, and applications provided by this invention are as follows: Through the synergistic effect of Bacillus subtilis and Trichoderma longifolia and their compound microbial agents, soil health, apple yield, and quality are promoted. Compared with the control, soil bulk density is reduced, organic matter and available nutrients are increased, and the size, yield, and quality of individual apples are all improved. The Trichoderma longifolia of this invention has an antagonistic effect on Fusarium oxysporium in laboratory plates. As an environmentally friendly and pollution-free control method, it can control the occurrence of apple root rot and promote seedling growth. It is also of great significance for the prevention and control of larch twig blight.

[0030] Biological Preservation Instructions:

[0031] Bacillus subtilis YCBA0319 was deposited on December 9, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33006. The address of the depository is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0032] Trichoderma longibrachiatum YCFA0522 was deposited on December 9, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 41655. The address of the depository is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. Attached Figure Description

[0033] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0034] Figure 1 The following are plate images showing two pathogens causing apple rot: Wherein:

[0035] Figure 1 .1 shows a plate plot of Fusarium oxysporum; Figure 1 Figure 2 shows a plate diagram of Fusarium solani.

[0036] Figure 2 The diagram shows the antagonistic effect between strain Bacillus subtilis YCBA0319 and two pathogens causing apple rot.

[0037] Figure 2 Figure 1 shows the antagonistic effect of Fusarium oxysporum - YCBA0319; Figure 2 Figure 2 shows the antagonistic effect of Fusarium solani-YCBA0319.

[0038] Figure 3 The antagonistic effects of strain *Trichoderma longibrachiatum* YCFA0522 on two pathogens causing apple rot were studied, including:

[0039] Figure 3 Figure 1 shows the antagonistic effect of Fusarium oxysporum - YCFA0522; Figure 3 Figure 2 shows the antagonistic effect of Fusarium solani-YCFA0522.

[0040] Figure 4 Phylogenetic tree of strain Bacillus subtilis YCBA0319.

[0041] Figure 5 Phylogenetic tree of strain Trichoderma longibrachiatum YCFA0522. Detailed Implementation

[0042] To better understand the above-mentioned objectives, features, and advantages of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. It should be noted that, unless otherwise specified, the embodiments and features described in these embodiments can be combined with each other.

[0043] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and therefore the scope of protection of the invention is not limited to the specific embodiments disclosed below.

[0044] like Figure 4 As shown, Embodiment 1 of the present invention provides a Bacillus subtilis YCBA0319, with accession number CGMCC No. 33006.

[0045] Example 2 of this invention provides the application of Bacillus subtilis in inhibiting apple root rot pathogens and / or promoting plant growth.

[0046] In the above embodiments, preferably, the growth promotion includes at least one of the following: reducing soil bulk density, increasing soil organic matter, increasing soil available nitrogen, increasing soil available phosphorus content, increasing soil available potassium, increasing apple yield, improving apple fruit morphology, and increasing the soluble solids content in apple fruit.

[0047] In the above embodiments, preferably, the application takes the form of trenching or root drenching.

[0048] Example 3 of the present invention provides a method for culturing Bacillus subtilis, comprising the following steps:

[0049] Step S1, prepare the basic solid culture medium for Bacillus subtilis: mix 8g peptone, 2g beef extract, 4g sodium chloride, 16g agar powder and 900mL distilled water evenly, adjust the pH to 7.2, add 2g agar, and sterilize at 118℃ for 15 minutes;

[0050] Step S2, prepare the basic liquid culture medium for Bacillus subtilis: mix 8g peptone, 2g beef extract, 4g sodium chloride with 900mL distilled water, adjust the pH to 7.2, and sterilize at 118℃ for 15 minutes;

[0051] Step S3, Activation and Cultivation: Bacillus subtilis is inoculated into the basic solid culture medium prepared in step S1 for activation. Single colonies are selected and inoculated into the basic liquid culture medium prepared in step S2. The culture is carried out at 33℃ and 116r / min for 16 hours to obtain Bacillus subtilis fermentation broth.

[0052] In this embodiment, if used to prepare a compound microbial agent, the fermentation broth can be mixed with other microbial fermentation broths in a certain proportion (e.g., 1:1) to form the target microbial fermentation broth.

[0053] like Figure 5 As shown, Embodiment 4 of the present invention provides a Trichoderma longibrachiatum YCFA0522, with accession number CGMCC No.41655.

[0054] Example 5 of the present invention provides the application of Trichoderma longifolia in inhibiting apple root rot fungi and / or promoting plant growth.

[0055] In the above embodiments, preferably, the growth promotion includes at least one of the following: reducing soil bulk density, increasing soil organic matter, increasing soil available nitrogen, increasing soil available phosphorus content, increasing soil available potassium, increasing apple yield, improving apple fruit morphology, and increasing the soluble solids content in apple fruit.

[0056] In the above embodiments, preferably, the application takes the form of trenching or root drenching.

[0057] Example 6 of this invention provides a method for culturing Trichoderma longifolia, comprising the following steps:

[0058] Step S1, prepare Trichoderma longifolia basic solid culture medium: mix 10g glucose, 1g dipotassium hydrogen phosphate, 0.5g magnesium sulfate, 2g sodium nitrate, 15g agar with 1000mL distilled water, adjust the pH to 5.5, autoclave at 121℃ for 20 minutes, cool and pour into plates for later use.

[0059] Step S2, prepare Trichoderma longifolia basic liquid culture medium: mix 10g glucose, 3g sodium nitrate, 0.5g magnesium sulfate, 1g dipotassium hydrogen phosphate, 1g yeast extract with 1000mL distilled water, adjust the pH to 5.5, autoclave at 121℃ for 20 minutes, and cool for later use.

[0060] Step S3, strain activation: Inoculate Trichoderma longifolia into the basic solid culture medium prepared in step S1, and incubate in an incubator at 28℃ for 5-7 days until the colonies have grown sufficiently for use.

[0061] Step S4, liquid fermentation culture: Inoculate the activated Trichoderma longifolia spore liquid from step S3 into the basic liquid culture medium prepared in step S2, with an inoculation amount of 10% (v / v), and culture on a shaker at 28℃ and 150-200 r / min for 48-72 hours.

[0062] Step S5, Fermentation product collection: After the culture is completed, the fermentation broth is filtered through gauze or centrifuged at 5000 rpm for 10 minutes to separate the mycelium. The fermentation broth and mycelium can be applied to agriculture, industry or scientific research fields according to actual needs.

[0063] Example 7 of the present invention provides a microbial compound agent for inhibiting bacteria and promoting growth, including Bacillus subtilis YCBA0319 described in Example 1 of the present invention and Trichoderma longifolia YCBA0522 described in Example 3 of the present invention.

[0064] In the above embodiments, preferably, the application form of the microbial compound inoculant includes at least one of mycelial fermentation broth, freeze-dried powder, and spore suspension.

[0065] Example 8 of the present invention provides the application of a microbial compound agent that inhibits bacteria and promotes growth in the inhibition of apple root rot fungi and / or the promotion of plant growth.

[0066] Example 9 of this invention provides a method for preparing a microbial compound agent that inhibits bacteria and promotes growth, comprising the following steps:

[0067] Step S1, Preparation of Bacillus subtilis fermentation broth: According to step S3 in Example 3 of the present invention, Bacillus subtilis is cultured in liquid culture medium for 16 hours to obtain Bacillus subtilis fermentation broth;

[0068] Step S2, Preparation of Trichoderma longifolia fermentation broth: Following step S4 in Example 6 of this invention, Trichoderma longifolia is cultured in liquid culture medium for 48-72 hours to obtain Trichoderma longifolia fermentation broth;

[0069] Step S3, determining the mixing ratio of microbial agents: The Bacillus subtilis fermentation broth prepared in step S1 and the Trichoderma longifolia fermentation broth prepared in step S2 are mixed at a mass ratio of 1:1 and stirred thoroughly to obtain a mixed microbial agent fermentation broth;

[0070] Step S4, Stabilization treatment of mixed microbial agent: Add a protectant (such as 5% lactose or skim milk powder) to the fermentation broth of mixed microbial agent from step S3, and prepare it into a powdered mixed microbial agent by spray drying or freeze drying;

[0071] Step S5, Mixed bacterial agent packaging: Dispense the powdered mixed bacterial agent prepared in step S4 into sterile aluminum foil bags, seal and store in a low temperature (4°C) environment for later use.

[0072] In the above embodiments, preferably, the growth promotion includes at least one of the following: reducing soil bulk density, increasing soil organic matter, increasing soil available nitrogen, increasing soil available phosphorus content, increasing soil available potassium, increasing apple yield, improving apple fruit morphology, and increasing the soluble solids content in apple fruit.

[0073] In the above embodiments, preferably, the application takes the form of trenching or root drenching.

[0074] The application method is radial, with four radial trenches dug in four directions under the tree canopy. The trenches are 100cm long, 40cm wide, and 30cm deep. After applying the compound microbial agent, mix it and cover it with soil.

[0075] Experimental example: Application effects in apple cultivation

[0076] Test site

[0077] The experiment was conducted in Xizhangbai Village, Beijing Township, Linyi County, Yuncheng City, Shanxi Province. The village falls within a continental semi-humid monsoon climate zone, at an altitude of 550m, with an average annual temperature of 13.2℃, a frost-free period of 205-212 days, an annual sunshine duration of 2293.4 hours, and an annual rainfall of approximately 550mm. The tested soil was loam.

[0078] Table 1. Physicochemical properties of soil at the test site

[0079]

[0080] Test materials

[0081] The tested apple variety was Fuji, with a plant spacing of 4m × 6m. The tested compound fertilizer was applied as a basal fertilizer (N:P₂O₅:K₂O = 16:16:16). The tested farmyard manure was well-rotted sheep manure (N:P₂O₅:K₂O ≥ 3%, organic matter ≥ 25%). The tested commercial organic fertilizer was also applied (N+P₂O₅+K₂O ≥ 5%, organic matter ≥ 45%).

[0082] Experimental Design

[0083] The experiment consisted of four treatment groups, with an inoculant application rate of 0 kg / hm². 2 (A), 15 kg / hm 2 (B) 30kg / hm 2 (C) 45 kg / hm 2 (D). The effectiveness of the microbial agent and the optimal application rate were verified by examining soil nutrient conditions, plant growth, and yield and quality. Apple trees with uniform growth were selected as test materials, with 5 trees per plot and 3 replicates. Except for the fertilization treatment, all other management practices were the same.

[0084] Application method

[0085] After apple harvest, the microbial agent is applied together with the base fertilizer. The base fertilizer application rate is 3000 kg / hm² of compound fertilizer. 2 +45t / hm of well-rotted sheep manure 2 Apply fertilizer in radial trenches. Dig four radial trenches at four points under the tree canopy, each trench 100cm long, 40cm wide, and 30cm deep. After applying the fertilizer, mix it and cover with soil. The microbial agent is a fermented liquid.

[0086] Results Analysis

[0087] 1. Impact on soil bulk density

[0088] Table 2. Effects of microbial inoculants on soil bulk density in orchards

[0089]

[0090] At a depth of 40-60 cm, there were no significant differences in soil bulk density among the treatment groups. At a depth of 20-40 cm, the bulk density of group A was 1.410±0.021 g / cm³, significantly higher than that of groups C (1.279±0.041 g / cm³) and D (1.224±0.012 g / cm³), while group B (1.355±0.053 g / cm³) was lower than that of group CK. This indicates that microbial inoculants can effectively reduce soil bulk density, especially in group D. At a depth of 0-20 cm, the bulk density of group CK was 1.468±0.023 g / cm³, significantly higher than that of groups C (1.291±0.044 g / cm³) and D (1.277±0.068 g / cm³), while group S1 (1.362±0.049 g / cm³) was lower than that of group A. This further demonstrates the effectiveness of microbial inoculants in reducing the bulk density of topsoil.

[0091] 2. Impact on soil organic matter

[0092] Table 3. Effects of microbial inoculants on orchard soil organic matter (g / kg)

[0093]

[0094] Soil samples from different depths showed little difference in organic matter content among the groups at a depth of 40-60 cm. Data for group A and treatment groups (B, C, and D) ranged from 5.72 to 5.84 g / kg, indicating that the microbial inoculant had a relatively small impact at this depth. However, the effect of the microbial inoculant began to appear at a depth of 20-40 cm. The organic matter content of groups C and D was significantly higher than that of group A, at 7.6 g / kg and 7.7 g / kg, respectively, indicating that the application of microbial inoculants at this depth effectively increased the soil's organic matter level. At a depth of 0-20 cm, the organic matter content of groups C and D reached 22.34 g / kg and 22.45 g / kg, respectively, significantly higher than the 19.34 g / kg of group A. This suggests that the application of microbial inoculants in the topsoil can significantly promote the accumulation of organic matter, possibly by enhancing soil microbial activity.

[0095] 3. Impact on soil alkaline nitrogen

[0096] Table 4. Effects of microbial inoculants on alkaline nitrogen availability in orchard soil (mg / kg)

[0097]

[0098] At all depths, there were no significant differences in available nitrogen among the groups, but the application of microbial agents in the 0-20 cm depth may have a potentially positive effect, with S2 and S3 showing increases of 18.0% and 18.1% compared to the control, respectively.

[0099] 4. Impact on available phosphorus in soil

[0100] Table 5. Effects of microbial inoculants on available phosphorus in orchard soil (mg / kg)

[0101]

[0102] The differences among all treatment groups at a depth of 20-60 cm were not statistically significant, indicating that the microbial inoculant had no significant effect on available phosphorus at this depth. At a depth of 0-20 cm, the available phosphorus content in group A was 64.77 mg / kg, significantly lower than that in groups C and D. In the topsoil (0-20 cm), the application of a certain amount of microbial inoculant significantly increased the available phosphorus content.

[0103] 5. Impact on available potassium in soil

[0104] Table 6. Effects of microbial inoculants on available potassium in orchard soil (mg / kg)

[0105]

[0106] At a depth of 20-60 cm, the available potassium content in all treatment groups was not significantly different from that in the control (CK). At a depth of 0-20 cm, the available potassium content in the CK group was significantly lower than that in the other treatment groups. S2 (363.69 mg / kg) and S3 (369.67 mg / kg) showed the greatest increase. These results indicate that microbial inoculants significantly increased the available potassium content in the topsoil (0-20 cm).

[0107] 6. The effects of microbial inoculants on the leaves of orchard fruit trees

[0108] Table 7 Effects of microbial inoculants on orchard leaves.

[0109]

[0110] The differences between the groups were not statistically significant (P>0.05), indicating that the application of microbial agents did not have a significant impact on the physiological characteristics of fruit tree leaves.

[0111] 7. Effects of microbial inoculants on fruit morphology

[0112] Table 8 Effects of microbial inoculants on apple morphology

[0113]

[0114] The data show that the transverse and longitudinal diameters of fruit treated with C were significantly higher than those treated with CK, while other treatments showed no significant differences from CK. However, there were no significant differences in fruit shape index among all treatments, therefore it can be concluded that C can increase the overall size of apples.

[0115] 8. Impact on quality

[0116] Table 9. Effects of microbial inoculants on the internal quality of apples

[0117]

[0118] There were no significant differences in firmness, titratable acidity, and solids-acid ratio among the groups, indicating that the microbial agent had a limited impact on apple ripeness and acidity. The significantly increased soluble solids content of group C may mean that, under similar acidity conditions, the soluble solids content of apples in this group was relatively high, thus enhancing the flavor and texture of the fruit.

[0119] 9. The impact of microbial inoculants on apple yield

[0120] Table 10. Effects of microbial inoculants on apple yield

[0121]

[0122] Data shows that group C exhibited a significant advantage in both single fruit weight and yield, with a single fruit weight reaching 271.62g and a yield of 67.8t / hm². Although groups B and D showed some improvement in single fruit weight and yield, they were still lower than group C.

[0123] In this experiment, treatment C performed best, with a single fruit weight of 271.62 g and a yield of 67.8 t / hm², significantly superior to other treatment groups. This treatment effectively increased soil organic matter and available phosphorus content, improved soil structure, reduced bulk density, and thus enhanced soil moisture and nutrient retention capacity, while avoiding the negative effects of excessive application, achieving a good balance. In conclusion, the application of microbial inoculants significantly improved apple yield and quality, and the recommended application rate is 30 kg / hm².

[0124] In summary, this compound microbial agent is a highly efficient biocontrol and growth-promoting strain for apples. The strain is simple to cultivate, has a significant biocontrol effect, is environmentally friendly and pollution-free, and is not prone to developing drug resistance.

[0125] Figure 1 .1 and Figure 2 Figure 1 shows the plate diagram of Fusarium oxysporum and the antagonistic effect of strain YCBA0319 on Fusarium oxysporum. It can be seen that after 10 days of confrontation culture, strain YCBA0319 has a good inhibitory effect on Fusarium oxysporum and there is a clear inhibition zone between it and Fusarium oxysporum.

[0126] Figure 1 .1 and Figure 3Figure 1 shows the plate plot of *Fusarium oxysporum* and the antagonistic effect of strain YCFA0522 against *Fusarium oxysporum*. It can be seen that after 10 days of confrontation culture, strain YCFA0522 has a good inhibitory effect on *Fusarium oxysporum*, with an antagonism index of grade III, and a clear inhibition zone between it and *Fusarium oxysporum*. (The *Trichoderma* antagonism index grading method is as follows: Grade I: *Trichoderma* mycelial coverage 100%; Grade II: *Trichoderma* mycelial coverage > 2 / 3; Grade III: 1 / 3 < *Trichoderma* mycelial coverage < 2 / 3; Grade IV: *Trichoderma* mycelial coverage < 1 / 3; Grade V: pathogenic fungal mycelial coverage 100%.)

[0127] Figure 1 .2 and Figure 2 Figure 2 shows the plate diagram of Fusarium solani and the antagonistic effect of strain YCBA0319 on Fusarium solani. It can be seen that after 10 days of confrontation culture, strain YCBA0319 has a certain inhibitory effect on Fusarium solani.

[0128] Figure 1 .2 and Figure 3 Figure 2 shows the plate plots of *Fusarium solani* and the antagonistic effect of strain YCFA0522 against *Fusarium solani*. It can be seen that after 10 days of confrontation culture, strain YCFA0522 has a good inhibitory effect on *Fusarium solani*, with an antagonism index of grade II, showing a clear inhibition zone between grade II and III. (The antagonism index grading method for *Trichoderma* is as follows: Grade I: *Trichoderma* mycelial coverage 100%; Grade II: *Trichoderma* mycelial coverage > 2 / 3; Grade III: 1 / 3 < *Trichoderma* mycelial coverage < 2 / 3; Grade IV: *Trichoderma* mycelial coverage < 1 / 3; Grade V: pathogenic fungal mycelial coverage 100%.)

[0129] In the description of this specification, the terms "one embodiment," "some embodiments," "specific embodiment," etc., refer to a specific feature, structure, material, or characteristic described in connection with that embodiment or example, which is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0130] 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 Bacillus subtilis YCBA0319, with preservation number CGMCCNo.33006; the Bacillus subtilis YCBA0319 has antagonistic activity against both Fusarium oxysporum and Fusarium solani, pathogens of apple root rot, and after 10 days of confrontation culture, a clear inhibition zone is formed between it and both Fusarium oxysporum and Fusarium solani.

2. A *Trichoderma longibrachiatum* fungus, YCFA0522, with accession number CGMCCNo.41655; the *Trichoderma longibrachiatum* fungus YCFA0522 exhibits antagonistic activity against both *Fusarium oxysporum* and *Fusarium solani*, pathogens of apple root rot. After 10 days of confrontation culture, the antagonism index against *Fusarium oxysporum* is grade III and the antagonism index against *Fusarium solani* is grade II, and a clear inhibition zone is formed between it and the pathogens.

3. A microbial compound agent for inhibiting bacteria and promoting growth, characterized in that, It includes Bacillus subtilis YCBA0319 as described in claim 1 and Trichoderma longifolia YCBA0522 as described in claim 2.

4. The antibacterial and growth-promoting microbial compound agent according to claim 3, characterized in that, The application forms of the microbial compound agent include at least one of mycelial fermentation broth, freeze-dried powder and spore suspension, and the mass ratio of the fermentation broth of Bacillus subtilis YCBA0319 to the fermentation broth of Trichoderma longifolia YCFA0522 is 1:

1.

5. The application of a microbial compound agent of Bacillus subtilis YCBA0319 as described in claim 1, or Trichoderma longifolia YCBA0522 as described in claim 2, or the antibacterial and growth-promoting agent as described in claim 3 or 4 in inhibiting apple root rot pathogens and / or promoting plant growth; The inhibition of apple root rot fungi is the inhibition of Fusarium oxysporum and / or Fusarium solani; Plant growth promotion includes at least one of the following: reducing soil bulk density, increasing soil organic matter, increasing soil available nitrogen, increasing soil available phosphorus content, increasing soil available potassium, increasing apple yield, increasing the transverse and longitudinal diameter of apple fruits, and increasing the soluble solids content of apple fruits.

6. The antibacterial and growth-promoting microbial compound agent according to claim 5, characterized in that, The application methods include trenching or root irrigation, with an application rate of 30 kg / hm².

7. A method for culturing Bacillus subtilis according to claim 1, characterized in that, Includes the following steps: Step S1, prepare the basic solid culture medium for Bacillus subtilis: mix 8g peptone, 2g beef extract, 4g sodium chloride, 16g agar powder and 900mL distilled water evenly, adjust the pH to 7.2, add 2g agar, and sterilize at 118℃ for 15 minutes; Step S2, prepare the basic liquid culture medium for Bacillus subtilis: mix 8g peptone, 2g beef extract, 4g sodium chloride with 900mL distilled water, adjust the pH to 7.2, and sterilize at 118℃ for 15 minutes; Step S3, Activation and Cultivation: Bacillus subtilis is inoculated into the basic solid culture medium prepared in step S1 for activation. Single colonies are selected and inoculated into the basic liquid culture medium prepared in step S2. The culture is carried out at 33℃ and 116r / min for 16 hours to obtain Bacillus subtilis fermentation broth.

8. A method for culturing Trichoderma longifolia as described in claim 2, characterized in that, Includes the following steps: Step S1, prepare Trichoderma longifolia basic solid culture medium: mix 10g glucose, 1g dipotassium hydrogen phosphate, 0.5g magnesium sulfate, 2g sodium nitrate, 15g agar with 1000mL distilled water, adjust the pH to 5.5, autoclave at 121℃ for 20 minutes, cool and pour into plates for later use. Step S2, prepare Trichoderma longifolia basic liquid culture medium: mix 10g glucose, 3g sodium nitrate, 0.5g magnesium sulfate, 1g dipotassium hydrogen phosphate, 1g yeast extract with 1000mL distilled water, adjust the pH to 5.5, autoclave at 121℃ for 20 minutes, and cool for later use. Step S3, strain activation: Inoculate Trichoderma longifolia into the basic solid culture medium prepared in step S1, and incubate in an incubator at 28℃ for 5-7 days until the colonies have grown sufficiently for use. Step S4, liquid fermentation culture: Inoculate the activated Trichoderma longifolia spore liquid from step S3 into the basic liquid culture medium prepared in step S2, with an inoculation amount of 10% (v / v), and culture on a shaker at 28℃ and 150-200 r / min for 48-72 hours. Step S5, Fermentation product collection: After the culture is completed, the fermentation broth is filtered through gauze or centrifuged at 5000 rpm for 10 minutes to separate the mycelium.

9. A method for preparing the microbial compound inoculant according to claim 3, characterized in that, Includes the following steps: Step S1, Preparation of Bacillus subtilis fermentation broth: According to step S3 in claim 7, Bacillus subtilis is cultured in liquid culture medium for 16 hours to obtain Bacillus subtilis fermentation broth; Step S2, Preparation of Trichoderma longifolia fermentation broth: According to step S4 in claim 8, Trichoderma longifolia is cultured in liquid culture medium for 48-72 hours to obtain Trichoderma longifolia fermentation broth; Step S3, determining the mixing ratio of microbial agents: The Bacillus subtilis fermentation broth prepared in step S1 and the Trichoderma longifolia fermentation broth prepared in step S2 are mixed at a mass ratio of 1:1 and stirred thoroughly to obtain a mixed microbial agent fermentation broth; Step S4, Stabilization treatment of mixed microbial agent: Add a protectant to the fermentation broth of mixed microbial agent from step S3. The protectant is 5% lactose or skim milk powder. After spray drying or freeze drying, it is prepared into a powdered mixed microbial agent. Step S5, Mixed bacterial agent packaging: The powdered mixed bacterial agent prepared in step S4 is packaged into sterile aluminum foil bags, sealed and stored at a low temperature of 4°C for later use.

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