Biocontrol composite bacterial strain yk and application thereof

Abstract

This invention discloses a biocontrol compound microbial system YK and its application, relating to the field of microbial technology. The biocontrol compound microbial system YK consists of *Trichoderma harzianum* YP2 and *Bacillus subtilis* K14. *Trichoderma harzianum* YP2 was deposited on May 22, 2025, at the China General Microbiological Culture Collection Center, and is classified as follows: Trichoderma afroharzianum YP2, with accession number CGMCC NO. 41947; the described Bacillus subtilis K14 was deposited at the China General Microbiological Culture Collection Center on May 22, 2025, and classified as... Bacillus subtilis K14, with accession number CGMCC NO. 34540. The biocontrol compound microbial system YK described herein is used to control maize stalk rot. The biocontrol compound microbial system YK of this invention exhibits stable efficacy and strong antibacterial ability, opening up new avenues for the biological control of maize stalk rot in my country.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a biocontrol compound bacterial system YK and its applications. Background Technology

[0002] Corn stalk rot is a major disease affecting corn production in my country, severely reducing corn quality and yield. With the increasing adoption of straw return to the field, pathogens accumulate in the soil, leading to a worsening of corn stalk rot. Biological control of corn stalk rot has become a research hotspot, with Bacillus and Trichoderma being the most extensively studied. However, in practical applications, single biocontrol strains suffer from unstable efficacy, decreased inhibitory and disease-preventing abilities over time, and strong environmental dependence, significantly limiting their application scope and control effectiveness. Therefore, a stable and efficient control method is urgently needed to address this issue.

[0003] The application of compound biocontrol bacteria can provide more antagonistic mechanisms to a certain extent, increase the competitiveness of biocontrol bacteria, improve the antibacterial and disease-resistant capabilities and stability of biological control, and achieve high efficiency and long-lasting effects in production. However, there are currently few reports on the use of compound bacterial systems in the control of corn stalk rot. Summary of the Invention

[0004] To address the above-mentioned problems, this invention provides a biocontrol compound bacterial strain YK and its applications.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: This invention provides a biocontrol compound microbial system YK, which is composed of *Trichoderma harzianum* YP2 and *Bacillus subtilis* K14; the *Trichoderma harzianum* YP2 was deposited at the China General Microbiological Culture Collection Center on May 22, 2025, and is classified and named as follows: Trichoderma afroharzianum YP2, with accession number CGMCC NO. 41947; the described Bacillus subtilis K14 was deposited at the China General Microbiological Culture Collection Center on May 22, 2025, and classified as... Bacillus subtilis K14, with accession number CGMCC NO.34540.

[0006] This invention also provides an application of a biocontrol compound microbial system YK in the biological control of corn stalk rot, wherein the biocontrol compound microbial system YK is composed of Trichoderma harzianum YP2 and Bacillus subtilis K14.

[0007] This invention also provides the application of the biocontrol compound bacterial strain YK in the biological control of the pathogen of corn stalk rot.

[0008] Furthermore, the pathogen causing corn stalk rot is *Fusarium graminearum* (…). Fusarium graminearum Fusarium pseudoverticum ( Fusarium verticillioides ).

[0009] Furthermore, the inoculation time of the biocontrol compound strain YK is as follows: first inoculate with Trichoderma harzianum YP2, and then inoculate with Bacillus subtilis K14 60h later; the inoculation ratio of Trichoderma harzianum YP2 to Bacillus subtilis K14 is 1:3.

[0010] Furthermore, the optimal culture conditions for the compound bacterial strain YK to inhibit the pathogen of maize stalk rot are: 2% soluble starch, 0.2% ammonium sulfate, 0.3% magnesium sulfate, 0.001% KH2PO4, pH value of 5, liquid volume of 80mL, inoculum amount of 2%, and temperature of 30℃, with inhibition rates of 93.88% and 89.40%, respectively.

[0011] This invention also provides the application of a biocontrol compound bacterial strain YK in the preparation of a product for controlling corn stalk rot, wherein the product is a compound biological seed dressing agent YK.

[0012] Furthermore, the formulation of the compound biological seed coating agent YK is as follows: 2% polyvinyl alcohol, 0.5% kaolin, 0.1% sodium lignobenzenesulfonate, 0.01% sodium citrate, 0.04% glycerol, 0.3% fuchsin, and 97.05% compound microbial fermentation broth YK, wherein the effective spore count of Trichoderma reaches 7.58 × 10⁻⁶. 6 CFU / mL, with an effective viable bacterial count of 2.75 × 10⁻⁶. 8 The concentration of CFU / mL showed a potted control efficacy of 63.16% and a field control efficacy of 62.37% against corn stalk rot.

[0013] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention uses five biocontrol Bacillus strains and five biocontrol Trichoderma strains as research subjects, and *Fusarium graminearum* and *Fusarium verticillatum* as target bacteria for maize stalk rot. Through strain compatibility testing, screening of biocontrol strain combinations, and determination of inoculation time and ratio, a biocontrol compound microbial system YK (*Trichoderma harzianum* YP2 / *Bacillus subtilis* K14, inoculation time: YP2-60h-K14; inoculation ratio: 1:3) was constructed. Then, the optimal culture conditions for the compound microbial system YK were optimized through a combination of single-factor and orthogonal experiments, and a compound biological seed coating agent YK was prepared. The biocontrol compound microbial system YK of this invention exhibits stable efficacy and strong antibacterial ability, opening up new avenues for the biological control of maize stalk rot in my country and providing a reliable guarantee for the green and sustainable development of the maize industry. Attached Figure Description

[0014] Figure 1The results of the bacterial compatibility test are shown in the figure. A: B2 / JN5; B: M28 / JN5; C: K14 / JN5; D: B31 / JN5; E: CY / JN5; F: B2 / CK1A; G: M28 / CK1A; H: K14 / CK1A; I: B31 / CK1A; J: CY / CK1A; K: B2 / PG1; L: M28 / PG1; M: K14 / PG1; N: B31 / PG1; O: CY / PG1; P: B2 / LZ1; Q: M28 / LZ1; R: K14 / LZ1; S: B31 / LZ1; T: CY / LZ1; U: B2 / YP2; V: M28 / YP2; W: YP2 / K14; X: B31 / YP2; Y: CY / YP2. Figure 2 This is a diagram illustrating the antifungal effect of the fungal-bacterial strain combination YP2 / K14 of this invention against *Hypericum cerevisiae*, the causal agent of corn stalk rot. In the diagram: A: Fg ( Fusarium graminearum B: K14-Fg; C: YP2-Fg; D: YP2 / K14-Fg; E: Fv ( Fusarium verticillioides Control group; F: K14-Fv; G: YP2-Fv; H: YP2 / K14-Fv; Figure 3 The study investigated the effects of the bacterial strain combination YP2 / K14 on the growth of maize seedlings, where A: plant height; B: stem diameter; C: leaf area; D: root length; E: aboveground fresh weight; and F: underground fresh weight. Figure 4 The results show the pot growth-promoting effect and pot control efficacy of strain combination YP2 / K14 against maize stalk rot. A: Pot growth-promoting effect of strain combination YP2 / K14 (from left to right in the figure: CK, strain YP2, strain K14, and strain combination YP2 / K14); B: Pot control efficacy of strain combination YP2 / K14 against maize stalk rot. Figure 5 The inoculation time and ratio of the strain combination YP2 / K14 showed the inhibitory effect on *Hymenocortis macrantha*, the causal agent of maize stalk rot. A: Inoculation time of the strain combination YP2 / K14 on *Hymenocortis macrantha*. In the figure, YK60h indicates inoculation with YP2 for 60 hours followed by inoculation with K14; KY60h indicates inoculation with K14 for 60 hours followed by inoculation with YP2. B: Inhibitory effect of different inoculation ratios of the strain combination YP2 / K14 on *Hymenocortis macrantha*. Figure 6 This is a morphological characteristic diagram of strain YP2, where A: characteristics of the strain cultured on a PDA plate (72h); B: morphological characteristics of the conidiophores of the strain; C: morphological characteristics of the conidia. Figure 7 The phylogenetic tree of strain YP2 was constructed based on the TEF1-α sequence; Figure 8 This is a morphological diagram of strain K14, where A: characteristics of the strain cultured on NA plates (48h); B: Gram staining of the strain. Figure 9 The phylogenetic tree of strain K14 was constructed based on the gyrA sequence; Figure 10 The effect of different culture media on the antibacterial effect of the compound bacterial strain YK; Figure 11 The effect of carbon source on the antibacterial effect of compound bacterial strain YK is shown in the figure, where A: type of carbon source; B: soluble starch content. Figure 12 The effect of nitrogen source on the antibacterial effect of compound bacterial strain YK is investigated, where A: type of nitrogen source; B: ammonium sulfate content; Figure 13 The study investigated the effect of inorganic salts on the antibacterial effect of the compound bacterial strain YK, where A represents the types of inorganic salts and B represents the magnesium sulfate content. Figure 14 The effects of temperature and liquid volume on the antibacterial effect of the compound bacterial strain YK are investigated, where A: temperature; B: liquid volume. Figure 15 The effects of inoculum size and pH on the antibacterial effect of the compound bacterial strain YK are investigated, where A: inoculum size; B: pH. Figure 16 The results of treatment with compound microbial strain YK to promote the growth of potted plants are shown in the following figures: A: Day 6; B: Day 12; C: Day 18; D: Day 24; E: Day 30.

[0015] Figure 17 The compound microbial strain YK promotes the growth of maize, where A: plant height; B: stem diameter; C: maximum leaf area; D: chlorophyll content. Figure 18 The results show the control efficacy of a compound biological seed dressing agent against corn stalk rot, where A represents potted plant control efficacy and B represents field control efficacy.

[0016] Note: The data in the table are mean ± standard error. Different lowercase letters indicate that the difference is significant at the 0.05 level. Detailed Implementation

[0017] To make the objectives and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention.

[0018] Unless otherwise specified, the instruments, reagents, and materials used in the following embodiments are all conventional instruments, reagents, and materials already available in the prior art and can be obtained through legitimate commercial channels. Unless otherwise specified, the experimental methods and detection methods used in the following embodiments are all conventional experimental methods and detection methods already available in the prior art.

[0019] In the following examples, all experimental data were statistically analyzed using Excel 2019 and SPSS 25.0 software. Duncan's multiple range method was used to compare the significance of differences between different treatments. P The range of the orthogonal experiment results was analyzed to determine the value of the result (<0.05).

[0020] Example 1: Isolation and Screening of Strains 1. Isolation and purification of strains Isolation and purification of maize endophytic strains: Healthy maize plants were collected, rinsed clean, and 2g of maize tissue was weighed, wrapped in sterile gauze, and placed in a laminar flow hood. The tissue was disinfected with 75% alcohol for 30 seconds, then immersed in 10% sodium hypochlorite solution for 3-5 minutes. After rinsing 3-4 times with sterile water, the tissue was dried and ground into a homogenate. 5mL of sterile water was added and allowed to stand for 15 minutes. The supernatant was diluted 10-fold and 100-fold, and 0.2mL of each was spread onto NA and PDA media, respectively. The mixture was repeated three times and incubated upside down at 28℃. Representative single colonies were picked, numbered, and stored at 4℃ for later use.

[0021] Isolation and purification of soil strains: Randomly weigh 5g of soil sample and add it to 100ml of sterile water. Shake at 37℃ and 180rpm for 1h. Take 100μL of the soil suspension supernatant and dilute it sequentially to 10... -3 10 -4 10 -5 10 -6 Take 100 μL of the sample and spread it evenly on NA and PDA plates, repeating the process three times. Incubate the plates upside down at 28°C. Pick representative single colonies, record their numbers, and store them at 4°C for later use.

[0022] Table 1 shows that 8 endophytic fungi were isolated from maize, and 2 fungi and 6 bacteria were isolated from soil.

[0023] Table 1. Results of strain isolation

[0024] 2. Effects of isolated strains on the mycelial growth of *Mycotoxin Bacillus subtilis* (maize stalk rot pathogen). After activating the isolated strains, they were inoculated onto PDA agar plates. The isolated strains and pathogens were inoculated at two points on the same horizontal line, 2.5 cm from the center of the plate, respectively. A control group was inoculated solely with *Cephalotaxus fortunei*. Each treatment was repeated three times. After the control group had fully colonized, the colony radius of *Cephalotaxus fortunei* was measured, and the inhibition rate was calculated.

[0025] Inhibition rate (%) = (colon radius of control group - colony radius of treatment group) / (colon radius of control group - 0.3) × 100.

[0026] The effects of isolated strains on the mycelial growth of *Cephalotaxus fortunei* are shown in Table 2. The results indicated that different strains exhibited varying antagonistic effects against *Cephalotaxus fortunei*. Five fungal strains (YP2, JN5, PG1, CK1A, and LZ1) showed inhibition rates exceeding 80%, while three bacterial strains (M28, K14, and B2) showed inhibition rates exceeding 50%. To broaden the screening range of fungal-bacterial compatible strain combinations during the construction of the composite bacterial system, five biocontrol bacteria and five biocontrol fungi with good antibacterial effects were selected.

[0027] Table 2. Inhibitory effect of isolated strains on mycelial growth of *Mycotoxin Bacillus subtilis*.

[0028] Note: Data in the table are mean ± standard error. "-" indicates no data in this column. Lowercase letters after the data indicate significance between treatments. P <0.05), the rest are the same.

[0029] Example 2 Construction of a biocontrol compound microbial system 1. Test materials 1.1 Test strains and maize varieties Five biocontrol fungi were identified: YP2, JN5, PG1, CK1A, and LZ1.

[0030] Five biocontrol bacteria strains: B31, B2, M28, CYS, and K14.

[0031] The strains mentioned above were all obtained through previous screening by our research group and have a good inhibitory effect on maize stalk rot pathogens.

[0032] Corn stalk rot pathogen: Fusarium graminearum ( Fusarium graminearum ) and Fusarium verticillata ( Fusarium verticillioides ( ) as target bacteria.

[0033] All the strains mentioned above were isolated, identified, and preserved by the Key Laboratory of Plant Pathology, Shanxi Agricultural University.

[0034] The maize variety tested was the inbred line Ye 478, provided by the Key Laboratory of Plant Pathology, Shanxi Agricultural University.

[0035] 1.2 Test Culture Medium The culture media used in this experiment are shown in Table 3.

[0036] Table 3 Culture medium formulation

[0037] 1.3 Test Instruments The instruments required for this experiment are listed in Table 4.

[0038] Table 4. Main instruments and manufacturers used in the experiment

[0039] 2. Construction of the biocontrol compound microbial system YK 2.1 Compatibility determination of strain combinations The compatibility of five biocontrol fungi with five biocontrol bacteria was determined using the plate confrontation method to observe whether there was compatibility between the fungal and bacterial strains. A biocontrol fungal mycelium was inoculated in the center of a PDA plate, and a single colony of the biocontrol bacteria was spotted 2 cm from the center of the plate. Each treatment was repeated three times, and the plates were incubated at 28°C for 5 days. The presence of an inhibition zone between the biocontrol bacteria and the biocontrol fungi was observed.

[0040] The compatibility test results are shown in Table 5 and Figure 1 As shown, there was a significant antagonistic effect between biocontrol fungi and biocontrol bacteria. Only biocontrol fungi YP2 and biocontrol bacteria K14 showed no antagonistic effect, indicating good compatibility. A total of one compatible fungal / bacterial strain combination, YP2 / K14, was screened.

[0041] Table 5 Results of strain compatibility test

[0042] Note: "+" indicates no antagonistic effect, and "-" indicates antagonistic effect.

[0043] 2.2 Determination of the antibacterial effect of the strain combination YP2 / K14 Preparation of fungal spore suspension: Add a small amount of sterile water to a PDA plate containing Trichoderma cultured for 5 days, gently scrape the spores off the plate, and dilute the spore concentration to 1.0 × 10⁻⁶. 6 CFU / mL.

[0044] Preparation of bacterial seed culture: Use an inoculation loop to pick a single colony from the activated bacterial plate and inoculate it into 30 mL of LB liquid medium. Incubate at 28°C and 180 r / min in a shaker for 12 h to obtain the bacterial seed culture.

[0045] Preparation of fermentation broth for strain combination YP2 / K14: Take a concentration of 1.0 × 10⁻⁶... 6A fungal spore suspension of CFU / mL was inoculated into a 75mL PDB conical flask. After culturing for 48 hours, a 0.5% bacterial seed culture was added, and the flask was incubated in a shaker at 28℃ for 3 days to obtain the fermentation broth of the strain combination.

[0046] The antibacterial effect of the strain combination YP2 / K14 was determined using the plate confrontation method. A 6mm inoculum of *Cephalotaxus fortunei* was inoculated onto the center of a PDA plate. A 6mm sterile filter paper disc was placed 3.5cm away from the inoculum disc. 10μL of the strain combination fermentation broth was pipetted onto the filter paper disc. After the fermentation broth on the filter paper disc dried, the plate was incubated upside down in a 26℃ incubator for 5 days. The diameter of the pathogen colonies was measured using the cross-sectional method, and the average colony diameter and inhibition rate were calculated.

[0047] Inhibition rate (%) = (Coronavirus diameter of control group - Coronavirus diameter of treatment group) / (Coronavirus diameter of control group - 0.6) × 100.

[0048] The antibacterial test results of the strain combination YP2 / K14 are shown in Table 6 and Figure 2 It can be seen that the inhibition rate of strain combination YP2 / K14 against Fusarium graminearum reached 78.13%, which is significantly different from that of the two single strains. However, the inhibition rate against Fusarium verticillatum was 66.57%, which was lower than that of the single strain YP2 treatment, but there was no significant difference between the two.

[0049] Table 6. Antifungal effect of strain combination YP2 / K14 against *Hymenococcus faecium*, the causal agent of maize stalk rot.

[0050] 2.3 Pot control efficacy determination of strain combination YP2 / K14 Garden soil and substrate soil were sterilized at high temperature and then mixed in a 4:1 ratio. Fusarium graminearum propagated from corn culture medium was inoculated into the sterilized soil at a concentration of 5%, mixed thoroughly with sterile water, and placed in disposable flowerpots. Corn seeds were soaked for 6 hours in the fermentation broth of strain combination YP2 / K14 and the fermentation broth of two single strains, respectively, before being planted in the soil inoculated with Fusarium graminearum. The control group was soaked in sterile water for 6 hours. Each experiment was repeated three times. One month after planting, the growth indicators of the corn seedlings were measured, and the disease incidence of the corn seedlings was recorded. The disease grading criteria are shown in Table 7.

[0051] Table 7 Grading Criteria for Corn Seedling Stalk Rot

[0052] The disease index and relative efficacy are calculated using the following formula: Disease index = ∑ (number of diseased plants at each level × representative value at each level) / (total number of plants surveyed × highest representative value) × 100; Relative efficacy (%) = (Control disease index - Treatment disease index) / Control disease index × 100; Growth promotion rate (%) = (Corn index after treatment - control corn index) / control corn index × 100.

[0053] The effects of the bacterial strain combination YP2 / K14 on maize seedling growth indicators were investigated in a pot experiment. The results are as follows: Figure 3 and Figure 4 As shown in Figure A, the strain combination YP2 / K14 treatment had a good growth-promoting effect on maize. The maize stalk diameter reached 4.74 mm, with a growth promotion rate of 28.80%, but there was no significant difference compared with the K14 treatment group. The maize root length and aboveground fresh weight were 23.56 cm and 2.89 g, respectively, with growth promotion rates of 39.61% and 59.67%, respectively, and there were significant differences compared with other treatment groups. The best growth promotion effect was on the fresh weight of the underground maize, with a growth promotion rate of 81.93%, but there was no significant difference compared with the K14 treatment group.

[0054] Depend on Figure 4 As shown in B, the control effect of the YP2 / K14 strain combination on maize stalk rot was significantly higher than that of other treatment groups, with a control effect of up to 78.96%.

[0055] Based on a comprehensive analysis of the antifungal effect of the bacterial strain combination YP2 / K14 against maize stalk rot pathogen, its growth-promoting effect on maize seedlings, and its control effect on maize stalk rot, the fungal-bacterial strain combination YP2 / K14 has high biocontrol potential. Therefore, the optimal inoculation time and inoculation ratio of this combination should be further determined.

[0056] 2.4 Determination of inoculation time for strain combination YP2 / K14 First, inoculate with seed culture of biocontrol bacteria K14, and then inoculate with spore suspension of biocontrol fungus YP2 (KY treatment) at intervals of 48h, 60h, 72h, 84h, and 96h. Alternatively, inoculate with spore suspension of biocontrol fungus YP2, and then inoculate with seed culture of biocontrol bacteria K14 (YK treatment) at intervals of 48h, 60h, 72h, 84h, and 96h. After inoculation, continue culturing in a shaker at 28℃ for a total of 5 days to obtain the combined fermentation broth of the bacterial strains. The prepared combined fermentation broth of the bacterial strains was subjected to a plate confrontation test according to the method in section 2.2, and its inhibition rate was calculated.

[0057] Fermentation broths prepared from the YP2 / K14 strain combination at different inoculation times were subjected to confrontation culture with *Cercospora cerevisiae*, the pathogen causing corn stalk rot. The results are as follows: Figure 5As shown in Figure A, the YK treatment group exhibited high inhibition rates against *Fusarium graminearum* at 48h and 60h, reaching 79.29% and 78.13%, respectively. While there was no significant difference between the two treatments, significant differences were observed compared to other treatment groups. The YK treatment group also showed high inhibition rates against *Fusarium verticillatum* at 60h and 72h, with inhibition rates of 74.36% and 75.12%, respectively. Again, there was no significant difference between the two treatments, but significant differences were observed compared to other treatment groups. Therefore, the optimal inoculation time for the YP2 / K14 strain combination is to first inoculate with strain YP2, followed by inoculation with K14 60h later.

[0058] 2.5 Determination of the inoculation ratio of strain combination YP2 / K14 YP2 spore suspension and K14 seed culture were inoculated into PDB medium at five ratios: 1:3, 2:3, 3:3, 3:2, and 3:1. The optimal inoculation time was determined according to section 2.4. The total inoculation amount was 1% for each strain. The cultures were incubated at 28°C and 180 rpm for 5 days. The prepared YP2 / K14 fermentation broth was then subjected to a plate confrontation test according to the method described in section 2.2, and its inhibition rate was calculated.

[0059] Based on the determined inoculation time of the bacterial strain combination, the antibacterial effect of fermentation broths prepared with different inoculation ratios on *Hypericum cuspidatum*, the pathogen causing corn stalk rot, was measured. The results are as follows: Figure 5 As shown in B, the inoculation ratio of strain YP2 / K14 at 1:3 showed the highest inhibition rate against *Fusarium graminearum*, at 74.70%, but there was no significant difference compared to the treatments with inoculation ratios of 3:3, 3:2, and 2:3, except for a significant difference compared to the treatment with an inoculation ratio of 3:1. The treatments with inoculation ratios of 1:3 and 2:3 showed higher inhibition rates against *Fusarium verticillatum*, at 72.41% and 72.33%, respectively, which were significantly different from the treatment with an inoculation ratio of 3:3. Therefore, the optimal inoculation ratio for strain YP2 / K14 is 1:3.

[0060] Based on the above results, a composite bacterial strain YK was constructed, consisting of strains YP2 and K14; the inoculation time was YP2-60h-K14; and the inoculation ratio was 1:3.

[0061] 3. Identification of strains Morphological identification of fungal strains: After culturing the tested strains on PDA medium at 28°C for 3 days, the changes in the strains were observed and recorded. After the strains produced a large number of sporulations, the morphology of the spores and the characteristics of the sporulation structures were observed under a microscope.

[0062] Morphological and physiological-biochemical identification of bacterial strains: Observe the characteristics of single colonies of each strain and Gram staining; perform physiological-biochemical identification of antagonistic bacteria in accordance with the methods of the "Manual of Systematic Identification of Common Bacteria" and "Bergey's Manual of Bacterial Identification".

[0063] Molecular biological identification of the strain: Genomic DNA was extracted from the strain using a genomic DNA kit (Beijing Solarbio Science & Technology Co., Ltd.) according to the manufacturer's instructions. PCR amplification of the corresponding strain was performed using the primer sequences and PCR reaction conditions listed in Table 8. The amplification system was as follows: 25 μL: 1 μL each of forward and reverse primers (10 μmol / L), 2 μL DNA template, 12.5 μL Master Mix, and 8.5 μL ddH2O. The PCR products were detected by 1% agarose gel electrophoresis. After observing that the amplified bands were bright and single, the samples were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing results were then analyzed using Blast software for sequence alignment, and a phylogenetic tree was constructed using MEGA5 software to determine the taxonomic position of the strain.

[0064] Table 8. PCR primers and reaction conditions for different genes of the strain.

[0065] 3.1 Morphological identification of strain YP2 Morphological characteristics of strain YP2 as follows Figure 6 As shown. YP2 grows extremely rapidly on PDA plates, completely covering the plate after 2 days of cultivation at 28℃. The mycelium is abundant, initially appearing as white cottony fibers, producing conidia from the center. The surface is green and has no obvious special odor. The conidiophores are pyramidal, with branches at acute or right angles to the main axis. The main axis branches multiple times, with the ends often having whorled 3 phialides. The base is swollen, and the tip is pointed, resembling a flask or ampoule. Conidia are borne at the tips of the phialides, appearing as green oval or round shapes with a smooth surface, averaging (4.25 × 3.00) μm in size. Based on its morphological characteristics, strain YP2 was preliminarily identified as *Trichoderma harzianum* (African *Trichoderma*). Trichoderma afroharzianum ).

[0066] 3.2 Molecular biological identification of strain YP2 PCR amplification of strain YP2 was performed using the universal fungal primer ITS and the Trichoderma-specific primer TEF1-α. The sequenced sequences were then compared with BLAST data on NCBI. The results showed that the amplified sequence of strain YP2 had the highest similarity to *Trichoderma harzianum*, clustering with it in a single line with a support rate of 95%. Figure 7 Based on morphological identification, strain YP2 was identified as *Trichoderma harzianum* (African Trichoderma). Trichoderma afroharzianum ).

[0067] 3.3 Morphological and physiological-biochemical identification of strain K14 Morphological characteristics of strain K14 as follows Figure 8As shown. K14 appears milky white and opaque on NA medium, with single, nearly circular colonies. The colony edges are irregular, the surface is slightly raised, and it is moist and smooth. The bacteria are solitary, short rods, and Gram-positive. The physiological and biochemical characteristics of strain K14 are shown in Table 9. Based on the morphological observation and physiological and biochemical identification of the strain, strain K14 was preliminarily identified as Bacillus subtilis (…). Bacillus subtilis ).

[0068] Table 9 Physiological and biochemical characteristics of strain K14

[0069] Note: + indicates a positive reaction; - indicates a negative reaction.

[0070] 3.4 Molecular biological identification of strain K14 PCR amplification of strain K14 was performed using the universal bacterial primer 27F / 1492R and the Bacillus-specific primer gyrA. The sequenced sequences were then compared with BLAST data on NCBI. The results showed that the amplified sequence of strain K14 had the highest similarity to Bacillus subtilis. Phylogenetic analysis revealed that K14 and Bacillus subtilis were most closely related, clustering together in one clade with 100% support. Figure 9 Based on morphological and physiological-biochemical identification, strain K14 was identified as Bacillus subtilis (…). Bacillus subtilis ).

[0071] Example 3: Optimization of culture conditions for the biocontrol compound bacterial strain YK 1. Test materials 1.1 Test strains and complex bacterial strains Example 2 constructed a composite bacterial strain YK, with the following strain composition: Trichoderma harzianum YP2 ( T. afroharzianum ) and Bacillus subtilis K14 ( B. subtilis ); Vaccination time: YP2-60h-K14; Vaccination ratio: 1:3.

[0072] Corn stalk rot pathogen: Fusarium graminearum ( F. graminearum Fusarium pseudoverticum ( F. verticillioides ).

[0073] 1.2 Test Culture Medium The names and components of the seven culture media used in the experiment are shown in Table 10.

[0074] Table 10 Culture media used in the experiment

[0075] 1.3 Test Instruments The main instruments used in the experiment are shown in Table 11.

[0076] Table 11. Main instruments and manufacturers used in the experiment

[0077] 2. Test methods 2.1 Optimization of Fermentation Nutrient Conditions for the Biocontrol Compound Microbial Strain YK Based on the experimental results of the previous nutrient conditions, the subsequent experiments were carried out sequentially according to the various nutrient conditions listed in Table 12 to optimize the culture conditions of the compound bacterial strain YK. For each treatment, strains YP2 and K14 were added sequentially according to the screening range in the table and the culture method of the compound bacterial strain in Example 2. The strains were then placed in a shaker at 28°C and 180 rpm for 5 days. Then, a plate confrontation test was conducted with maize stalk rot pathogen to screen out the nutrient conditions with better antibacterial effect. The antibacterial test method was the same as 2.2 in Example 2.

[0078] Table 12 Screening of nutrient conditions for fermentation of the YK compound microbial strain

[0079] The effects of different culture media on the antibacterial effect of the YK compound bacterial fermentation broth, such as Figure 10 As shown in the figure, the fermentation broths of Bilay, Cazpek-Dox, and Armstrong media exhibited good antibacterial effects against *Fusarium graminearum*, with inhibition rates of 83.79%, 86.17%, and 84.11%, respectively, showing significant differences compared to the PDB medium treatment group. The fermentation broth of Armstrong medium showed the best antibacterial effect against *Fusarium verticillatum*, with an inhibition rate of 83.33%, significantly different from the other treatment groups. Based on comprehensive analysis, Armstrong medium was selected as the optimal medium for further culture optimization.

[0080] Depend on Figure 11 A indicates that when glucose and soluble starch were used as carbon sources, the YK fermentation broth of the compound microbial strain showed good antibacterial effects against *Fusarium graminearum*, with inhibition rates of 85.97% and 85.66%, respectively, which were significantly different from the maltose and lactose treatment groups. When soluble starch was used as the carbon source, the YK fermentation broth of the compound microbial strain showed the best antibacterial effect against *Fusarium verticillatum*, with an inhibition rate of 86.14%, which was significantly different from the other four carbon sources. Therefore, soluble starch was selected as the optimal carbon source. Figure 11As shown in Figure B, when 2% soluble starch was used as the carbon source, the YK fermentation broth of the compound microbial system exhibited the best antibacterial effect against *Fusarium graminearum*, with an inhibition rate of 90%; followed by 1% soluble starch, with an inhibition rate of 89.02%. Significant differences were observed between this and the 0% and 3% treatment groups. When 3% soluble starch was used as the carbon source, the YK fermentation broth of the compound microbial system showed the best antibacterial effect against *Fusarium verticillatum*, with an inhibition rate of 89.19%, followed by 1% soluble starch, with an inhibition rate of 89.06%. Based on comprehensive analysis, 1% soluble starch was selected as the optimal carbon source addition amount.

[0081] The effect of nitrogen source on the antibacterial effect of YK fermentation broth of compound bacteria, such as Figure 12 As shown. From Figure 12 As shown in Figure A, when ammonium sulfate was used as the nitrogen source, the YK fermentation broth of the compound bacterial strain exhibited the best antibacterial effect against *Fusarium graminearum*, with an inhibition rate of 88.00%, which was significantly different from the potassium nitrate and peptone treatment groups. When ammonium nitrate was used as the nitrogen source, the YK fermentation broth of the compound bacterial strain showed the best antibacterial effect against *Fusarium verticillatum*, with an inhibition rate of 85.14%, which was significantly different from the peptone treatment; ammonium sulfate was the second best, with an inhibition rate of 82.76%, which was not significantly different from other treatments. Based on comprehensive analysis, ammonium sulfate was selected as the optimal nitrogen source. Figure 12 As shown in B, when 0.2% ammonium sulfate was used as the nitrogen source, the YK fermentation broth of the compound bacteria system showed the best antibacterial effect against Fusarium graminearum, with an inhibition rate of 87.60%, which was significantly different from the treatment group without nitrogen source. When 0.3% ammonium sulfate was used as the nitrogen source, the YK fermentation broth of the compound bacteria system showed the best antibacterial effect against Fusarium verticillata, with an inhibition rate of 89.08%, which was significantly different from the other treatment groups. Therefore, 0.3% ammonium sulfate was selected as the optimal amount of nitrogen source to be added.

[0082] The effect of inorganic salts on the antibacterial effect of YK fermentation broth of compound bacteria, such as Figure 13 As shown. From Figure 13 As shown in Figure A, magnesium sulfate had the best inhibitory effect on *Fusarium graminearum* and *Fusarium verticillatum* in the fermentation broth of the YK composite strain, with inhibition rates of 87.24% and 81.97%, respectively, which were significantly different from other treatment groups. Therefore, magnesium sulfate was selected as the optimal inorganic salt. Figure 13 As shown in B, 0.3% magnesium sulfate had the best effect on inhibiting Fusarium graminearum and Fusarium verticillatum in the fermentation broth of the YK compound bacteria, with inhibition rates of 88.51% and 88.50%, respectively, which were significantly different from the groups without magnesium sulfate or with 0.1% magnesium sulfate. Therefore, 0.3% magnesium sulfate was selected as the optimal amount of inorganic salt to be added.

[0083] 2.2 Optimization of fermentation culture conditions for the biocontrol compound strain YK Following the culture conditions listed in Table 13, and based on the experimental results of the previous culture conditions, subsequent experiments were conducted sequentially to optimize the culture conditions of the compound bacterial strain YK. Other specific experimental methods are the same as described in 2.1.

[0084] Table 13 Screening of culture conditions for YK compound bacterial strain fermentation

[0085] The effect of temperature on the antibacterial effect of the YK fermentation broth of the compound bacterial strain, such as Figure 14 As shown in Figure A, the YK fermentation broth of the compound microbial strain exhibited good antibacterial effect against *Fusarium graminearum* at 30℃, with an inhibition rate of 86.27%, which was significantly different from the 20℃ and 25℃ treatment groups. The YK fermentation broth of the compound microbial strain showed good antibacterial effect against *Fusarium verticillatum* at 28℃ and 30℃, with inhibition rates of 84.70% and 84.22%, respectively, which were significantly different from the 20℃, 25℃, and 35℃ treatment groups. Based on comprehensive analysis, 30℃ was selected as the optimal culture temperature.

[0086] The effect of liquid volume on the antibacterial effect of the YK compound bacterial strain fermentation broth is as follows: Figure 14 As shown in Figure B, at a liquid volume of 30 mL, the YK fermentation broth of the compound microbial strain exhibited an inhibition rate of 88.18% against Fusarium graminearum. This inhibition rate gradually decreased with increasing liquid volume. At a liquid volume of 80 mL, the YK fermentation broth showed the best antibacterial effect against Fusarium verticillatum, with an inhibition rate of 87.00%, which was significantly different from other treatment groups. Based on comprehensive analysis, 80 mL was selected as the optimal liquid volume.

[0087] The effect of inoculum size on the antibacterial effect of the YK compound bacterial strain fermentation broth, as follows: Figure 15 As shown in Figure A, at a 2% inoculum size, the YK fermentation broth of the compound microbial strain exhibited a high inhibitory effect against *Fusarium graminearum*, with an inhibition rate of 88.80%. At 2% and 3% inoculum sizes, the YK fermentation broth showed good inhibitory effects against *Fusarium verticillatum*, with inhibition rates of 83.95% and 83.94%, respectively, showing significant differences compared to the 4% and 5% inoculum treatment groups. Based on comprehensive analysis, a 2% inoculum size was selected as the optimal inoculum size.

[0088] The effect of pH on the antibacterial effect of the YK fermentation broth of the compound bacteria is as follows: Figure 15 As shown in Figure B, the YK fermentation broth of the compound microbial strain exhibited better antibacterial effects against *Fusarium graminearum* at pH 5 and 7, with inhibition rates of 87.40% and 87.24%, respectively, showing significant differences compared to the other three pH treatment groups. At pH 3, the YK fermentation broth of the compound microbial strain showed the best antibacterial effect against *Fusarium verticillatum*, with an inhibition rate of 87.70%, showing a significant difference compared to the other treatment groups; at pH 5, the inhibition rate was 84.25%, showing a significant difference compared to the treatment groups with pH values ​​of 7, 9, and 11. Based on comprehensive analysis, pH 5 was selected as the optimal pH.

[0089] 2.3 Orthogonal experimental design for fermentation conditions of the YK complex bacterial strain The factor levels of the orthogonal experimental design for the compound microbial strain YK are shown in Table 14, and the orthogonal experimental combinations are shown in Table 15. The optimal fermentation conditions for the biocontrol compound microbial strain YK were screened using the inhibitory effect of the YK fermentation broth on the pathogen of maize stalk rot as the evaluation index.

[0090] Table 14 Factor Level Table for Optimizing Fermentation Conditions of Compound Microbial Strain YK

[0091] Table 15. Four-factor, three-level orthogonal table for optimizing fermentation conditions of the YK complex microbial strain.

[0092] Based on the determination of the optimal culture medium, optimal nutrient conditions, and optimal culture conditions, an orthogonal optimization experiment was conducted to study the effects of soluble starch content, ammonium sulfate content, magnesium sulfate content, and inoculum amount on the antibacterial effect of the YK compound bacterial fermentation broth.

[0093] The antibacterial effect of the YK compound microbial strain after orthogonal optimization of fermentation conditions is shown in Table 16. Range analysis of the results on the inhibition of *Fusarium graminearum* is shown in Table 17. The range R values ​​of the four factors differed. The influence of each factor on the inhibition of *Fusarium graminearum* growth by the YK compound microbial strain fermentation broth was reflected by the range R value. A larger R value indicates a greater influence of the factor on the inhibition rate, and vice versa. Table 17 shows that RB > RD > RA > RC, meaning that ammonium sulfate content has the greatest impact on the inhibition of *Fusarium graminearum* by the YK compound microbial strain, followed by inoculum size, soluble starch content, and magnesium sulfate content.

[0094] Based on a comprehensive analysis of the orthogonal experimental results, the optimal combination for the compound bacterial strain YK to inhibit Fusarium graminearum was determined to be A3B1C2D2, which contains 2% soluble starch, 0.2% ammonium sulfate, 0.3% magnesium sulfate, and 2% inoculum. This combination showed the highest inhibition rate against Fusarium graminearum.

[0095] Table 16 Antibacterial effect of YK compound bacterial strain after orthogonal optimization of fermentation conditions

[0096] Table 17 Range analysis of the inhibition of Fusarium graminearum by the YK compound bacterial strain after orthogonal optimization of fermentation conditions.

[0097] After orthogonal optimization of the fermentation conditions of the compound bacterial strain YK, range analysis was performed on the antibacterial effect of Fusarium verticillatum. The results are shown in Table 18. RC > RD > RA > RB, indicating that magnesium sulfate content has the greatest impact on the inhibition of Fusarium verticillatum by the compound bacterial strain YK, followed by inoculum size, soluble starch content, and ammonium sulfate content.

[0098] Based on a comprehensive analysis of the orthogonal experimental results, the optimal combination for the compound bacterial strain YK to inhibit Fusarium verticillatum was determined to be A2B1C2D1, which contains 1% soluble starch, 0.2% ammonium sulfate, 0.3% magnesium sulfate, and 1% inoculum. This combination showed the highest inhibition rate against Fusarium verticillatum.

[0099] Table 18 Range analysis of the inhibition of Fusarium verticillatum by the YK compound bacterial strain after orthogonal optimization of fermentation conditions.

[0100] Based on the above experimental results, the optimal fermentation conditions for the compound strain YK to inhibit corn stalk rot pathogens are: 2% soluble starch, 0.2% ammonium sulfate, 0.3% magnesium sulfate, 0.001% KH2PO4, pH value of 5, liquid volume of 80mL, inoculum amount of 2%, and temperature of 30℃, with inhibition rates of 93.88% and 89.40%, respectively.

[0101] Example 4: Application Effects of Compound Microbial Strains YK and Compound Biological Seed Coating Agent 1. Test Methods 1.1 Soil collection and treatment for potted plants In the maize experimental field of Shanxi Agricultural University in Taigu District, Shanxi Province, the topsoil (0 to 20 cm) of maize-grown plots for 10 consecutive years was collected using the five-point sampling method. The soil was mixed evenly, dried naturally at room temperature, and then sieved through a sieve with a diameter of 2 mm.

[0102] 1.2 Pretreatment of Corn Seeds Disinfect corn seeds in a 2% NaClO solution for 2 minutes, then rinse them three times with sterile water. Place two layers of sterile filter paper in a 15cm diameter sterile petri dish, moisten the filter paper with sterile water, place the disinfected seeds into the petri dish, and incubate at 28℃ for 2 days to germinate.

[0103] 1.3 Preparation of YK Fermentation Broth from Compound Microbial Culture YP2 and K14 were inoculated into the optimized culture medium of Example 3 at an inoculation time of YP2-60h-K14 and an inoculation ratio of 1:3. The mixture was then placed in a 30°C shaking incubator and cultured for 5 days to obtain the YK composite bacterial fermentation broth.

[0104] 1.4 Pot Experiment Design The continuously cropped soil was moistened with sterile water to a moisture content of 60% as the control group (A1). The prepared YK compound microbial fermentation broth was diluted 50 times with sterile water, and an equal amount of the diluted fermentation broth was mixed with soil as the treatment group (A2), as shown in Table 19. 1000g of soil was placed in 30×20cm flowerpots. Germinated and uniformly grown corn seeds from section 1.2 were selected and planted in the treated soil, with 6 plants per pot. Each treatment was repeated 5 times, and the flowerpots were placed in a greenhouse for normal cultivation. Plant height, stem diameter, leaf area, and chlorophyll content of the corn plants were measured at 6, 12, 18, 24, and 30 days after sowing. At 30 days, the corn plants were removed from the pots, and their fresh weight and root length were measured.

[0105] Table 19 Potted Plant Soil Treatment Methods

[0106] 2. Effects of the compound microbial strain YK on the growth of maize seedlings The effects of treating continuously cropped soil with the YK microbial strain on maize seedling growth indicators are as follows: Figure 16 , Figure 17 As shown in Table 20.

[0107] Both the A2 treatment group and the A1 control group showed a near-linear growth in maize plant height. At day 12, the plant heights of the control and treatment groups were similar. From day 12 onwards, the increase in plant height in the A2 treatment group was greater than that in the A1 control group, but there was no significant difference between the two. At day 30, the plant heights were 36.29 cm and 32.76 cm, respectively, with a growth promotion rate of 10.77%. Overall, the maize stem diameter showed an increasing trend, and the stem diameter of the A2 treatment group was consistently higher than that of the control group. Significant differences in stem diameter were found between the control and treatment groups at days 18 and 24. At day 30, the stem diameter of the A2 treatment group was still higher than that of the control group, but there was no significant difference between the two, with stem diameters of 3.82 mm and 3.38 mm, respectively, and a growth promotion rate of 13.02%.

[0108] The maximum leaf area of ​​maize plants showed a linear increase throughout the treatment, with the A2 treatment group consistently having a higher maximum leaf area than the control group. Significant differences were observed between the control and treatment groups at days 6, 12, and 18, but no significant differences were found at days 24 and 30. The maximum leaf area of ​​maize on day 30 was 22.19 cm². 2 17.39cm 2The growth promotion rate was 27.60%. The chlorophyll content in maize leaves showed a trend of first increasing and then decreasing, but overall, the A2 treatment group was higher than the control group. On days 6, 24, and 30, the chlorophyll content in maize leaves of the A2 treatment group was significantly higher than that of the control group, while there was no significant difference between the two groups on days 12 and 18. Table 20 shows that on day 30, the fresh weight of the A2 treatment group was significantly higher than that of the control group, with fresh weights of 1.95 g / plant and 1.52 g / plant, respectively, and a growth promotion rate of 28.29%. The root length of maize in the A2 treatment group was significantly higher than that in the control group, with root lengths of 24.65 cm and 22.43 cm, respectively, and a growth promotion rate of 2.90%.

[0109] Table 20 Fresh weight and root length of maize plants on day 30 after treatment with the YK compound microbial strain.

[0110] 3. Application effects of compound biological seed coating agents The optimized compound microbial strain YK was used to develop a biological seed coating agent. The types and dosages of various adjuvants were screened, and the quality of the seed coating agent was tested. Then, pot experiments and field trials were conducted to verify its application effect. The resulting compound biological seed coating agent formula was: 2% polyvinyl alcohol, 0.5% kaolin, 0.1% sodium lignin benzenesulfonate, 0.01% sodium citrate, 0.04% glycerol, 0.3% fuchsin, and 97.05% fermentation broth of the compound microbial strain YK. The effective spore count of Trichoderma reached 7.58 × 10⁻⁶. 6 CFU / mL, with an effective viable bacterial count of 2.75 × 10⁻⁶. 8 CFU / mL.

[0111] The application method for this seed coating agent is as follows: Select corn seeds of uniform size and soak them in 55℃ warm water for 15 minutes. After soaking, let the seeds air dry under natural conditions. Prepare the seed coating agent according to the formula of each adjuvant concentration. Weigh the seed coating agent and seeds at a ratio of 1:10 and coat the seeds until the seed surface is uniformly colored. After drying, the seeds are ready for planting.

[0112] The results showed that the compound biological seed dressing agent had a good growth-promoting effect on maize, with a plant height promotion rate of 9.26%, stem diameter promotion rate of 34.65%, maximum leaf area promotion rate of 117.20%, root length promotion rate of 16.49%, aboveground fresh weight promotion rate of 60.40%, and underground fresh weight promotion rate of 64.71%. The pot control efficacy against maize stalk rot reached 63.16%. The field yield of maize was 884.73 kg / mu, and the field control efficacy against maize stalk rot reached 62.37%. Figure 18 ).

[0113] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A biocontrol compound bacterial strain YK, characterized in that: The biocontrol compound strain YK consists of *Trichoderma harzianum* YP2 and *Bacillus subtilis* K14; *Trichoderma harzianum* YP2 was deposited at the China General Microbiological Culture Collection Center on May 22, 2025, and classified as... Trichoderma afroharzianum YP2, with accession number CGMCC NO. 41947; the described Bacillus subtilis K14 was deposited at the China General Microbiological Culture Collection Center on May 22, 2025, and classified as... Bacillus subtilis K14, with accession number CGMCC NO.34540.

2. The application of the biocontrol compound bacterial strain YK as described in claim 1 in the biological control of maize stalk rot, characterized in that: The biocontrol compound strain YK consists of *Trichoderma harzianum* YP2 and *Bacillus subtilis* K14; *Trichoderma harzianum* YP2 was deposited at the China General Microbiological Culture Collection Center on May 22, 2025, and classified as... Trichoderma afroharzianum YP2, with accession number CGMCC NO. 41947; the described Bacillus subtilis K14 was deposited at the China General Microbiological Culture Collection Center on May 22, 2025, and classified as... Bacillus subtilis K14, with accession number CGMCC NO. 34540.

3. The application of the biocontrol compound microbial system YK as described in claim 1 in the biological control of maize stalk rot pathogens.

4. The application according to claim 3, characterized in that: The pathogen causing corn stalk rot is Fusarium graminearum ( ). Fusarium graminearum Fusarium pseudoverticum ( Fusarium verticillioides ).

5. The application according to claim 3, characterized in that: The inoculation time of the biocontrol compound strain YK is as follows: first inoculate with Trichoderma harzianum YP2, and then inoculate with Bacillus subtilis K14 60h later; the inoculation ratio of Trichoderma harzianum YP2 to Bacillus subtilis K14 is 1:

3.

6. The application according to claim 3, characterized in that: The optimal culture conditions for the compound bacterial strain YK to inhibit the pathogen of maize stalk rot are: 2% soluble starch, 0.2% ammonium sulfate, 0.3% magnesium sulfate, 0.001% KH2PO4, pH 5, liquid volume 80mL, inoculum 2%, and temperature 30℃.

7. The application of the biocontrol compound microbial strain YK as described in claim 1 in the preparation of products for controlling maize stalk rot, characterized in that: The product is a compound biological seed coating agent YK.

8. The application according to claim 7, characterized in that: The formulation of the compound biological seed coating agent YK is as follows: 2% polyvinyl alcohol, 0.5% kaolin, 0.1% sodium lignin benzenesulfonate, 0.01% sodium citrate, 0.04% glycerol, 0.3% fuchsin, and 97.05% compound microbial strain YK fermentation broth, wherein the effective spore count of Trichoderma reaches 7.58 × 10⁻⁶. 6 CFU / mL, with an effective viable bacterial count of 2.75 × 10⁻⁶. 8 CFU / mL.

Images