Biocontrol composite bacterial system yc and application thereof

Abstract

This invention discloses a biocontrol compound microbial strain YC and its application, relating to the field of microbial technology. The biocontrol compound microbial strain YC is composed of *Trichoderma harzianum* YP2 and *Trichoderma viride* CK1A. *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 *Trichoderma viride* CK1A was deposited on May 22, 2025, at the China General Microbiological Culture Collection Center, and classified as... Trichoderma atroviride CK1A, with accession number CGMCC NO. 41948. The biocontrol compound microbial system YC described herein is used to control maize stalk rot. The biocontrol compound microbial system YC 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 YC 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 effectiveness. Therefore, a stable and efficient control method is urgently needed to alleviate this problem.

[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 YC 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 strain YC, which is composed of *Trichoderma harzianum* YP2 and *Trichoderma viride* CK1A. *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 *Trichoderma viride* CK1A was deposited on May 22, 2025, at the China General Microbiological Culture Collection Center, and classified as... Trichoderma atroviride CK1A, with accession number CGMCC NO.41948.

[0006] This invention also provides an application of a biocontrol compound microbial system YC in the biological control of corn stalk rot, wherein the biocontrol compound microbial system YC is composed of Trichoderma harzianum YP2 and Trichoderma viride CK1A.

[0007] This invention also provides the application of a biocontrol compound microbial strain YC in the biological control of pathogens causing corn stalk rot.

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

[0009] Furthermore, the inoculation time of the biocontrol compound strain YC is as follows: first inoculate Trichoderma CK1A, and then inoculate Trichoderma harzianum YP2 36 hours later; the inoculation ratio of Trichoderma harzianum YP2 to Trichoderma CK1A is 3:3.

[0010] Furthermore, the optimal fermentation conditions for the compound microbial strain YC to inhibit the pathogen of corn stalk rot are: 2% glucose, 0.5% peptone, 0.1% magnesium sulfate, 0.001% KH2PO4, pH value of 7, liquid volume of 80mL, inoculum amount of 4%, and temperature of 30℃.

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

[0012] Furthermore, the formulation of the compound biological seed coating agent is as follows: 1% sodium carboxymethyl cellulose, 0.25% kaolin, 0.4% sodium methylene dinaphthalene sulfonate, 0.01% potassium sorbate, 0.03% glycerol, 0.3% carmine, and 98.01% compound bacterial strain YC fermentation broth, wherein the effective spore count of Trichoderma reaches 7.58 × 10⁻⁶. 6 CFU / mL. The pot control efficacy against maize stalk rot reached 55.50%, and the field control efficacy reached 46.24%.

[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 YC (*Trichoderma harzianum* YP2 / *Trichoderma viride* CK1A, inoculation time: CK1A-36h-YP2; inoculation ratio: 3:3) was constructed. Then, through a combination of single-factor and orthogonal experiments, the optimal fermentation conditions of the compound microbial system YC were optimized, and a compound biological seed coating agent YC was prepared. The biocontrol compound microbial system YC of this invention exhibits stable efficacy, strong antibacterial ability, and growth-promoting properties, 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 1These are the results of strain compatibility testing. In the figure: A: JN5 / CK1A; B: PG1 / CK1A; C: YP2 / LZ1; D: YP2 / PG1; E: JN5 / YP2; F: PG1 / JN5; G: PG1 / LZ1; H: LZ1 / JN5; I: CK1A / YP2; J: CK1A / LZ1; Figure 2 The figure shows the antibacterial effect of the bacterial strain combinations on Fusarium graminearum. In the figure: A: YP2, CK1A, YP2 / CK1A; B: YP2, JN5, YP2 / JN5; C: YP2, PG1, YP2 / PG1; D: YP2, LZ1, YP2 / LZ1; E: JN5, PG1, JN5 / PG1; F: JN5, LZ1, JN5 / LZ1; G: JN5, CK1A, JN5 / CK1A; H: PG1, LZ1, PG1 / LZ1; I: PG1, CK1A, PG1 / CK1A; J: CK1A, LZ1, CK1A / LZ1. Figure 3 This is a graph showing the antibacterial effect of the bacterial strain combination against Fusarium graminearum. In the graph: A: Control; B: YP2; C: PG1; D: JN5; E: CK1A; F: LZ1; G: CK1A / YP2; H: PG1 / LZ1; I: LZ1 / JN5; J: CK1A / LZ1; K: CK1A / JN5; L: YP2 / JN5; M: YP2 / PG1; N: YP2 / LZ1; O: JN5 / PG1; P: CK1A / PG1; Figure 4 The figure shows the antibacterial effect of the bacterial strain combinations on Fusarium verticillatum. In the figure: A: YP2, CK1A, YP2 / CK1A; B: YP2, JN5, YP2 / JN5; C: YP2, PG1, YP2 / PG1; D: YP2, LZ1, YP2 / LZ1; E: JN5, PG1, JN5 / PG1; F: JN5, LZ1, JN5 / LZ1; G: JN5, CK1A, JN5 / CK1A; H: PG1, LZ1, PG1 / LZ1; I: PG1, CK1A, PG1 / CK1A; J: CK1A, LZ1, CK1A / LZ1. Figure 5 This is a graph showing the antibacterial effect of the bacterial strain combination against Fusarium verticillatum. In the graph: A: Control; B: YP2; C: PG1; D: JN5; E: CK1A; F: LZ1; G: CK1A / YP2; H: PG1 / LZ1; I: LZ1 / JN5; J: CK1A / LZ1; K: CK1A / JN5; L: YP2 / JN5; M: YP2 / PG1; N: YP2 / LZ1; O: JN5 / PG1; P: CK1A / PG1; Figure 6The figures show the growth-promoting effects of different strains on indoor potted plants. In the diagram: A: YP2 / CK1A; B: YP2 / JN5; C: YP2 / PG1; D: YP2 / LZ1; E: JN5 / PG1; F: JN5 / LZ1; G: JN5 / CK1A; H: PG1 / LZ1; I: PG1 / CK1A; J: CK1A / LZ1. Figure 7 The study investigated the effects of bacterial strain combinations on the growth of maize seedlings, including: A: plant height; B: stem diameter; C: maximum leaf area; D: root length; E: aboveground fresh weight; F: underground fresh weight. Figure 8 The efficacy of the bacterial strain combination against potted corn stalk rot; Figure 9 This is a potted plant control efficacy diagram of some bacterial strain combinations against maize stalk rot, where: A: CK1A / LZ1; B: CK1A / JN5; C: JN5 / PG1; D: YP2 / LZ1; E: YP2 / CK1A; F: CK; Figure 10 The inoculation time and ratio of the strain combination YP2 / CK1A have an inhibitory effect on *Cercospora maize* stalk rot. A: Inoculation time of the strain combination YP2 / CK1A has an inhibitory effect on *Cercospora maize* stalk rot. In the figure, YC12h indicates inoculation with YP2 for 12 hours followed by CK1A; CY12h indicates inoculation with CK1A for 12 hours followed by YP2. B: Inoculation ratio of the strain combination YP2 / CK1A has an inhibitory effect on *Cercospora maize* stalk rot. Figure 11 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 12 The phylogenetic tree of strain YP2 was constructed based on the TEF1-α sequence; Figure 13 This is a morphological characteristic diagram of strain CK1A, 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.

[0015] Figure 14 The phylogenetic tree of strain CK1A was constructed based on the TEF1-α sequence; Figure 15 The effect of different culture media on the antibacterial effect of the compound bacterial strain YC; Figure 16 The study focuses on the effect of carbon sources on the antibacterial effect of the compound bacterial strain YC, where A represents the type of carbon source and B represents the glucose content. Figure 17 The effect of nitrogen source on the antibacterial effect of compound bacterial strain YC is investigated, where A: type of nitrogen source; B: peptone content. Figure 18 The study investigated the effect of inorganic salts on the antibacterial effect of the compound bacterial strain YC, where A represents the types of inorganic salts and B represents the magnesium sulfate content. Figure 19 The effects of temperature and liquid volume on the antibacterial effect of the compound bacterial strain YC are investigated, where A represents temperature and B represents liquid volume. Figure 20 The effects of inoculum size and pH on the antibacterial effect of the compound bacterial strain YC are investigated, where A: inoculum size; B: pH. Figure 21 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 <0.05), range analysis and variance analysis were performed on the orthogonal experiment results.

[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 the 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 *Mycorrhiza cerevisiae*, the causal agent of maize stalk rot, are shown in Table 2. The results indicated that different strains exhibited varying antagonistic effects against *Mycorrhiza cerevisiae*. Five fungal strains (YP2, JN5, PG1, CK1A, and LZ1) showed inhibition rates exceeding 80%, while two bacterial strains (M28 and K14) showed inhibition rates exceeding 53%. Five biocontrol fungi with good antibacterial effects were selected for constructing the composite bacterial system.

[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 biocontrol compound microbial system 1. Test materials 1.1 Test strains and maize varieties Five biocontrol fungi—YP2, JN5, PG1, CK1A, and LZ1—were obtained through preliminary screening by our research group and showed good inhibitory effects against maize stalk rot pathogens.

[0030] Corn stalk rot pathogen: Fusarium graminearum ( Fusarium graminearum Fusarium pseudoverticum ( Fusarium verticillioides ( ) as target bacteria.

[0031] All the strains mentioned above were isolated, identified, and preserved by the Key Laboratory of Plant Pathology, Shanxi Agricultural University.

[0032] The maize variety tested was the inbred line Ye 478, provided by the Key Laboratory of Plant Pathology, Shanxi Agricultural University.

[0033] 1.2 Test Culture Medium The culture media used in this experiment are shown in Table 3.

[0034] Table 3 Culture medium formulation

[0035] 1.3 Test Instruments The instruments required for this experiment are listed in Table 4.

[0036] Table 4. Main instruments and manufacturers used in the experiment

[0037] 2. Construction of the biocontrol compound microbial system YC 2.1 Compatibility determination of strains The compatibility of five biocontrol fungi was determined by pairwise comparison using the plate confrontation method. Six-mm biocontrol fungal mycelial discs were inoculated at both ends of a PDA plate, with a 4cm distance between the two discs. Each treatment was repeated three times. The plates were incubated at 28℃ for 5 days, and the presence of an inhibition zone between the two fungi and any changes in the hyphae at the colony edges were observed.

[0038] Five biocontrol fungi were subjected to pairwise plate confrontation experiments, and the results are shown in Table 5. Figure 1 As shown, there is no obvious antagonism among the biocontrol fungi YP2, JN5, PG1, CK1A, and LZ1, and there are a total of 10 compatible strain combinations.

[0039] Table 5 Results of strain compatibility test

[0040] Note: "+" indicates no antagonistic effect.

[0041] 2.2 Initial screening of strain combinations in biocontrol compound bacterial systems 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.

[0042] Preparation of fermentation broth for the strain combination: 1.0 × 10⁻⁶ spore suspensions of the two fungal spores constituting the strain combination were prepared. 6 CFU / mL was inoculated into a conical flask containing 0.5% to 75mL PDB at a 1:1 ratio. After inoculation, the flask was placed in a shaker at 28°C and fermented for 5 days to obtain the fermentation broth of the strain combination.

[0043] Determination of the antibacterial effect of bacterial strain combinations: Ten bacterial strain combinations were cultured against *C. corn stalk rot* using the plate confrontation method. A 6mm *C. corn stalk rot* inoculum dish was inoculated 3cm from the center of the plate. A 6mm sterile filter paper disc was placed on the other side, 3cm from the center of the plate. 10μL of the bacterial strain combination fermentation broth was pipetted onto the filter paper disc. After the fermentation broth on the filter paper disc dried, the plates were incubated upside down in a 26℃ incubator for 5 days. The diameter of the pathogen colonies was measured using the cross-cross method, and the average colony diameter and inhibition rate were calculated.

[0044] Inhibition rate (%) = (Coronavirus diameter of control group - Coronavirus diameter of treatment group) / (Coronavirus diameter of control group - 0.6) × 100.

[0045] The results of confrontation culture between the strain combination and Fusarium graminearum are as follows: Figure 2 , Figure 3 As shown, the six strain combinations (YP2 / CK1A, JN5 / LZ1, JN5 / CK1A, PG1 / LZ1, PG1 / CK1A, CK1A / LZ1) showed better antibacterial effects against Fusarium graminearum than single strains. Only two strain combinations, YP2 / CK1A and JN5 / LZ1, showed significant differences compared to single strains, with inhibition rates of 82.03% and 79.46%, respectively.

[0046] The results of confrontation culture between the strain combination and Fusarium verticillatum are as follows: Figure 4 , Figure 5 As shown, the four strain combinations (YP2 / CK1A, PG1 / LZ1, PG1 / CK1A, and CK1A / LZ1) showed better antibacterial effects against Fusarium verticillatum than single strains. Only three strain combinations, YP2 / CK1A, PG1 / LZ1, and CK1A / LZ1, showed significant differences compared to single strains, with inhibition rates of 78.67%, 74.15%, and 75.35%, respectively.

[0047] 2.3 Secondary screening of strain combinations in biocontrol compound bacterial systems The secondary screening of strain combinations was conducted using a pot experiment to assess their control efficacy. Garden soil and substrate soil were sterilized at a 4:1 ratio. Fusarium graminearum propagated from corn culture medium was inoculated at 5% into the sterilized soil, 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 was recorded. The disease grading criteria are shown in Table 6.

[0048] Table 6. Grading Criteria for Corn Seedling Stalk Rot

[0049] 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.

[0050] A pot experiment was conducted on a combination of well-compatible bacterial strains to investigate their effects on maize seedling growth indicators. The results are as follows: Figure 6 and Figure 7 As shown.

[0051] Depend on Figure 7 As shown in Figure A, the maize plant height treated with the six strain combinations PG1 / LZ1, YP2 / CK1A, CK1A / JN5, CK1A / PG1, YP2 / LZ1, and JN5 / LZ1 was significantly different from that of the control (CK). The strain combination PG1 / LZ1 showed significant differences from both individual strains and from all other strain combinations except YP2 / CK1A, with a plant height of 53.53 cm and a growth promotion rate of 56.93%. The strain combination YP2 / CK1A also showed significant differences from other strain combinations except PG1 / LZ1, but not from the individual strain CK1A.

[0052] Depend on Figure 7As shown in B, the corn stalk diameter treated with the six fungal strain combinations YP2 / CK1A, PG1 / LZ1, YP2 / PG1, CK1A / JN5, CK1A / PG1, and YP2 / LZ1 was significantly different from that of the control group (CK). Among them, five strain combinations YP2 / CK1A, CK1A / PG1, PG1 / LZ1, YP2 / PG1, and CK1A / JN5 were significantly different from the two single strains. The stalk diameters of strain combinations YP2 / CK1A, CK1A / PG1, and PG1 / LZ1 were 4.36 mm, 4.46 mm, and 4.47 mm, respectively, with growth promotion rates of 30.54%, 33.53%, and 33.83%, respectively.

[0053] Depend on Figure 7 As shown in C, the maximum leaf area of ​​maize treated with the two strain combinations PG1 / LZ1 and YP2 / CK1A was significantly different from that of the control (CK) and significantly higher than that of other strain combinations. Significant differences also existed between these treatments and those of individual strains. The maximum leaf area was 47.22 cm². 2 57.88cm 2 The fertility rate can reach 76.46% and 116.29%.

[0054] Depend on Figure 7 As shown in D, the single strain LZ1 treatment resulted in the best root length effect in maize, with a root length of 26.06 cm and a growth promotion rate of 54.48%. The strain combination YP2 / CK1A treatment also showed good root length effect in maize, with significant differences compared to CK and single strains YP2 and CK1A, with a root length of 25.42 cm and a growth promotion rate of 50.68%.

[0055] Depend on Figure 7 As shown in E, the aboveground fresh weight of maize treated with the three strain combinations PG1 / LZ1, YP2 / CK1A, and CK1A / PG1 was higher, showing significant differences from CK and from the single strains. The aboveground fresh weights were 3.83 g, 3.77 g, and 3.25 g, respectively, with growth promotion rates of 111.60%, 108.29%, and 79.56%.

[0056] Depend on Figure 7 As shown in F, the fresh weight of the underground part of maize treated with the strain combination YP2 / CK1A was significantly different from that of other strain combination treatment groups, and also significantly different from that of single strains YP2 and CK1A. The fresh weight of the underground part reached 1.43g, and the growth promotion rate was 72.28%.

[0057] The efficacy of strain combinations in potted control of maize stalk rot is as follows: Figure 8 , Figure 9As shown, the strain combination YP2 / CK1A showed the best control effect against maize stalk rot, reaching 66.67%, which was significantly different from other combinations except for strain combination YP2 / LZ1, and also significantly different from the control effect of single strains, effectively improving the disease control ability.

[0058] Based on the antibacterial effect of the strain combination against the causal agent of maize stalk rot, its potted control effect on maize stalk rot, and its growth-promoting effect on maize, the strain combination YP2 / CK1A was selected through comprehensive analysis, and its inoculation time and ratio were further determined.

[0059] 2.4 Determination of inoculation time for strain combination YP2 / CK1A The inoculation time for the strain combination YP2 / CK1A selected in section 2.3 was determined. A concentration of 1×10⁻⁶ was used. 6 CFU / mL of strain YP2 (or CK1A) spore suspension was inoculated into the culture medium and cultured in a shaker at 28℃ and 180 rpm. CK1A (or YP2) spore suspension was inoculated at 0h, 12h, 24h, 36h, and 48h post-inoculation, and cultured in a shaker at 28℃ for a total of 5 days to obtain the combined fermentation broth. The antibacterial effect of the combined fermentation broth was determined using the plate confrontation method described in section 2.2, and its inhibition rate was calculated.

[0060] Fermentation broths prepared from the YP2 / CK1A strain combination at different inoculation times were subjected to confrontation culture with *Cercospora macrantha*, the causal agent of maize stalk rot. The results are as follows: Figure 10 As shown in Figure A, the CY 24h and CY 36h treatment groups showed good antibacterial effects against *Fusarium graminearum*, with inhibition rates of 81.07% and 82.54%, respectively. However, there was no significant difference between these groups and the 0h treatment group (inhibition rate 82.03%), but significant differences were observed between these groups and other treatments. The CY 36h, CY 24h, and YC 24h treatment groups showed high inhibition rates against *Fusarium verticillatum*, with inhibition rates of 78.15%, 77.20%, and 77.49%, respectively. These rates were significantly different from the 0h treatment group and significantly different from other treatment groups except for the YC 48h treatment group. Based on the analysis of the antibacterial effects and significant differences against the two pathogens, it was determined that inoculating CK1A for 36 hours followed by inoculating YP2 was the optimal inoculation time for the strain combination YP2 / CK1A.

[0061] 2.5 Determination of the inoculation ratio of strain combination YP2 / CK1A Take concentrations of 1×10 6Spore suspensions of strains YP2 and CK1A at CFU / mL were inoculated into PDB liquid medium at five ratios (1:3, 2:3, 3:3, 3:2, and 3:1) according to the optimal inoculation time selected in section 2.4, with a total inoculation volume of 1%. Each group was replicated in triplicate and cultured at 28°C and 180 rpm for 5 days. The antibacterial effect of the prepared strain combination fermentation broth was determined using the plate confrontation method described in section 2.2, and the inhibition rate was calculated.

[0062] The antifungal effects of fermentation broths of strain YP2 / CK1A prepared with different inoculation ratios on *Hypericum micranthum*, the causal agent of maize stalk rot, are as follows: Figure 10 As shown in B, the strain combination with an inoculation ratio of 3:3 showed the best antibacterial effect against *Fusarium graminearum*, with an inhibition rate of 82.54%, which was significantly different from the other four inoculation ratios. Inoculation ratios of 1:3, 2:3, 3:3, and 3:1 showed good antibacterial effects against *Fusarium verticillatum*, with no significant differences among these ratios, but a significant difference compared to the 3:2 ratio; the highest inhibition rate (80.71%) was achieved at an inoculation ratio of 2:3. Based on the analysis of the antibacterial effects and significant differences of the strain combinations with different inoculation ratios, the optimal inoculation ratio for the strain combination YP2 / CK1A was determined to be 3:3.

[0063] Based on the above results, a composite bacterial strain YC was constructed, consisting of strain YP2 and strain CK1A; the inoculation time was CK1A - 36h - YP2; and the inoculation ratio was 3:3.

[0064] 3. Identification of strains Morphological identification of fungal strains: After the tested strains were cultured on PDA medium in an incubator at 28°C, colony changes 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.

[0065] 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 7. 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 amplification products were detected by 1% agarose gel electrophoresis. After observing that the amplification bands were bright and single, the samples were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing results were analyzed by sequence alignment using Blast software, and a phylogenetic tree was constructed using MEGA5 software to determine the taxonomic position of the strain.

[0066] Table 7. PCR primers and reaction conditions for different genes of the strain.

[0067] 3.1 Morphological identification of strain YP2 Morphological characteristics of strain YP2 as follows Figure 11 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 ).

[0068] 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 12 Based on morphological identification, strain YP2 was identified as *Trichoderma harzianum* (African *Trichoderma*). Trichoderma afroharzianum ).

[0069] 3.3 Morphological identification of strain CK1A Morphological characteristics of strain CK1A as follows Figure 13 As shown. CK1A grows rapidly on PDA plates. After 2 days of cultivation at 28℃, the hyphae diameter is 50-60 mm. The hyphae are initially abundant and appear as white cottony fibers, expanding outwards from the center of the fungal disc to produce conidia, with obvious conidial concentric rings. The conidiophores are perpendicular or acute to the main axis, which branches multiple times. The phialpiniophores are usually whorled in groups of 2-3, with a swollen base and a pointed, ampoule-like tip. Conidia are borne at the tip of the phialpiniophore, round or oval, with an average size of (4.77 × 5.35) μm. Based on its morphological characteristics, the strain was identified as *Trichoderma viride* (…). Trichoderma atroviride ).

[0070] 3.4 Molecular biological identification of strain CK1A PCR amplification of strain CK1A 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 CK1A had the highest similarity to *Trichoderma viride*. Phylogenetic analysis revealed that strain CK1A and *Trichoderma viride* clustered together in one clade with a support rate of 99%. Figure 14 Based on morphological identification, strain CK1A was identified as *Trichoderma viride*. Trichoderma atroviride ).

[0071] Example 3: Optimization of fermentation conditions for the biocontrol compound strain YC 1. Test materials 1.1 Test strains and complex bacterial systems Example 2 constructed a composite bacterial strain YC, strain combination: Trichoderma harzianum YP2 ( T. afroharzianum ) / Dark green Trichoderma CK1A ( T. atroviride ); Vaccination time: CK1A-36h-YP2; Vaccination ratio: 3:3.

[0072] Corn stalk rot pathogen: Fusarium graminearum ( F. graminearum Fusarium pseudoverticum ( F. verticillioides All of the above strains were isolated, identified, and preserved by the Key Laboratory of Plant Pathology, Shanxi Agricultural University.

[0073] 1.2 Test Culture Medium The names and components of the six culture media used in the experiment are shown in Table 8.

[0074] Table 8. Culture media used in the experiment

[0075] 1.3 Test Instruments The main instruments used in the experiment are shown in Table 9.

[0076] Table 9. Main instruments and manufacturers used in the experiment

[0077] 2. Test methods 2.1 Screening of Nutrient Conditions for Fermentation of Biocontrol Compound Strain YC Based on the experimental results of the previous nutrient conditions, the subsequent experiments were carried out sequentially according to the nutrient conditions listed in Table 10 to optimize the culture conditions of the compound bacterial strain YC. For each treatment, strains CK1A and YP2 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℃ 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 10. Screening of nutrient conditions for YC fermentation by compound microbial strains

[0079] (1) Effect of culture medium type on the antibacterial effect of compound bacterial strain The effects of different culture media on the antibacterial effect of the YC fermentation broth of the compound bacterial strain, such as Figure 15 As shown in the figure, different culture media exhibited varying antibacterial effects against the two maize stalk rot pathogens. The figure reveals that the fermentation broths from Armstrong and PDB media showed better antibacterial effects against *Fusarium graminearum*, with inhibition rates of 82.70% and 79.98%, respectively, showing significant differences compared to the other three media. Armstrong medium showed the best antibacterial effect against *Fusarium verticillatum* after fermentation, with an inhibition rate of 80.02%, effectively improving the antibacterial effect of the YC compound strain against *Fusarium verticillatum*. The difference between Armstrong medium and PDB, Bilay, and Czapek-Dox media was significant, but not significant compared to Richead medium. Based on comprehensive analysis, Armstrong medium was selected as the optimal culture medium for subsequent nutrient condition screening.

[0080] (2) Effect of carbon source on the antibacterial effect of compound bacterial strain YC Depend on Figure 16 As shown in Figure A, when maltose and glucose were used as carbon sources, the YC fermentation broth of the compound microbial strain exhibited better antibacterial effects against Fusarium graminearum, with inhibition rates of 79.94% and 79.05%, respectively, showing significant differences compared to the treatments using soluble starch and lactose. Lactose showed the best antibacterial effect against Fusarium verticillatum in the YC fermentation broth, with an inhibition rate of 82.90%, which was significantly different from other treatments. Glucose was the second best, with an inhibition rate of 81.44%, which did not show significant differences compared to other treatments. Based on comprehensive analysis, glucose was selected as the optimal carbon source for further analysis of its addition amount.

[0081] The effect of glucose content on the antibacterial effect of the compound bacterial strain YC is as follows: Figure 16 As shown in Figure B, the 2% glucose treatment exhibited the highest antibacterial effect against *Fusarium graminearum*, with an inhibition rate of 83.62%, which was significantly different from other treatment groups. The 2% glucose treatment also showed the highest antibacterial effect against *Fusarium verticillatum*, with an inhibition rate of 75.39%, which was significantly different from other treatment groups except for the 1% glucose treatment. Therefore, 2% glucose was selected as the optimal carbon source addition.

[0082] (3) Effect of nitrogen source on the antibacterial effect of compound bacterial strain The effect of nitrogen source on the antibacterial effect of YC fermentation broth of compound bacteria, such as Figure 17 As shown. By Figure 17As shown in Figure A, when peptone was used as the nitrogen source, the fermentation broth of the YC compound microbial strain exhibited the best antibacterial effect against *Fusarium graminearum*, with an inhibition rate of 90.58%, which was significantly different from other treatment groups. When ammonium sulfate was used as the nitrogen source, the YC compound microbial strain showed the best inhibitory effect against *Fusarium verticillatum*, with an inhibition rate of 80.47%, which was significantly different from the ammonium nitrate, potassium nitrate, and urea treatment groups. When peptone was used as the nitrogen source, the inhibition rate reached 79.32%, showing a significant difference only from the ammonium nitrate treatment. Based on comprehensive analysis, peptone was selected as the optimal nitrogen source for screening the optimal addition amount.

[0083] Depend on Figure 17 As shown in Figure B, 0.1% peptone as the nitrogen source exhibited the best antibacterial effect against *Fusarium graminearum*, with an inhibition rate of 86.39%, significantly different from the 0.3% and 0.4% treatments. 0.3% peptone showed the best antibacterial effect against *Fusarium verticillatum*, with an inhibition rate of 83.01%, significantly different from the 0% and 0.4% peptone treatments. The treatments with 0.1% and 0.2% peptone were the next most effective, with inhibition rates of 81.31% and 81.37%, respectively, showing no significant difference from the other treatments. Based on comprehensive analysis, 0.1% peptone was selected as the optimal nitrogen source addition.

[0084] (4) Effect of inorganic salts on the antibacterial effect of the compound bacterial strain The effect of inorganic salts on the antibacterial effect of the YC fermentation broth of the compound bacterial strain, such as Figure 18 As shown. From Figure 18 As shown in Figure A, magnesium sulfate and manganese sulfate had the best inhibitory effect on *Fusarium graminearum* in the fermentation broth of the YC composite microbial strain, with inhibition rates of 89.41% and 88.94%, respectively, showing significant differences compared to other treatment groups. Furthermore, magnesium sulfate and manganese sulfate had the best inhibitory effect on *Fusarium verticillatum* in the YC composite microbial strain, with inhibition rates of 79.30% and 79.11%, respectively, also showing significant differences compared to other treatment groups. Therefore, magnesium sulfate was selected as the optimal inorganic salt for addition.

[0085] from Figure 18 As shown in B, the 0.2% magnesium sulfate treatment group had the highest antibacterial effect against Fusarium graminearum and Fusarium verticillatum, with inhibition rates of 84.55% and 76.13%, respectively, which were significantly different from other treatment groups. Therefore, 0.2% magnesium sulfate was selected as the optimal amount of inorganic salt to be added.

[0086] 2.2 Screening of fermentation culture conditions for the biocontrol compound strain YC Following the culture conditions listed in Table 11, 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 YC. Other specific experimental methods are the same as described in 2.1.

[0087] Table 11 Screening of culture conditions for YC fermentation of compound microbial strain

[0088] (1) Effect of fermentation temperature on the antibacterial effect of the compound strain YC The effect of temperature on the antibacterial effect of the compound bacterial strain YC is as follows: Figure 19 As shown in Figure A, at 30℃, the YC fermentation broth of the compound microbial strain showed the best antibacterial effect against Fusarium graminearum, with an inhibition rate of 82.77%, which was significantly different from other treatment groups. At 28℃, the YC fermentation broth of the compound microbial strain showed the best antibacterial effect against Fusarium verticillatum, with an inhibition rate of 83.72%, which was significantly different from other treatment groups. The 30℃ treatment group was the second best, with an inhibition rate of 80.82%, which was also significantly different from other treatment groups. Based on comprehensive analysis, 30℃ was selected as the optimal culture temperature.

[0089] (2) Effect of liquid volume on the antibacterial effect of the compound bacterial strain The effect of liquid volume on the antibacterial effect of the compound bacterial strain YC is as follows: Figure 19 As shown in Figure B, at a volume of 30 mL, the YC fermentation broth of the compound microbial strain exhibited a high inhibitory effect against *Fusarium graminearum*, with an inhibition rate of 80.47%, showing a significant difference only compared to the 150 mL treatment group. At a volume of 80 mL, the YC fermentation broth of the compound microbial strain showed the best inhibitory effect against *Fusarium verticillatum*, with an inhibition rate of 82.04%, showing a significant difference compared to the 50 mL, 110 mL, and 150 mL treatment groups. At a volume of 30 mL, the YC fermentation broth of the compound microbial strain showed a good inhibitory effect against *Fusarium verticillatum*, showing a significant difference compared to the 50 mL and 110 mL treatment groups. Based on comprehensive analysis, 80 mL was selected as the optimal volume for further optimization.

[0090] (3) Effect of inoculum size on the antibacterial effect of the compound bacterial strain The effect of inoculum size on the antibacterial effect of the compound strain YC is as follows: Figure 20 As shown in Figure A, at inoculum levels of 3% and 4%, the YC fermentation broth of the compound microbial strain exhibited high antibacterial effects against *Fusarium graminearum*, with inhibition rates of 81.84% and 81.54%, respectively, showing significant differences compared to other treatment groups. At inoculum levels of 1%, 2%, and 4%, the YC fermentation broth of the compound microbial strain showed good antibacterial effects against *Fusarium verticillatum*, with inhibition rates of 82.69%, 82.93%, and 82.56%, respectively, also showing significant differences compared to other treatment groups. Based on comprehensive analysis, 4% was selected as the optimal inoculum level.

[0091] (4) Effect of pH on the antibacterial effect of the compound bacterial strain The effect of pH on the antibacterial effect of the YC fermentation broth of the compound bacteria strain, such as Figure 20As shown in B, the YC fermentation broth of the compound bacteria strain exhibited the best antibacterial effect against Fusarium graminearum and Fusarium verticillatum at pH 7, with inhibition rates of 83.97% and 78.20%, respectively, showing significant differences compared to other treatment groups. Therefore, pH 7 was selected as the optimal pH value.

[0092] 2.4 Orthogonal experimental design for fermentation conditions of the biocontrol compound strain YC Based on the single-factor experiment, L9(3) was adopted. 4 An orthogonal experimental design was used, selecting four factors: carbon source content, nitrogen source content, inorganic salt content, and inoculum size. Each factor had three levels, and a four-factor, three-level orthogonal experiment was conducted. The levels of each factor are shown in Table 12. The orthogonal experimental combinations were designed using SPSS software, as shown in Table 13. The antibacterial effect of the YC compound microbial fermentation broth on *Cyperus rotundus* was used as the evaluation index to optimize the fermentation conditions of the YC compound microbial strain.

[0093] Table 12 Factor Level Table for Optimizing Fermentation Conditions of YC Compound Microbial Culture

[0094] Table 13. Four-factor, three-level orthogonal table for optimizing fermentation conditions of the YC complex microbial strain.

[0095] 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 glucose content, peptone content, magnesium sulfate content, and inoculum size on the antibacterial effect of the YC composite bacterial fermentation broth.

[0096] The antibacterial effect of the YC compound microbial strain after orthogonal optimization of fermentation conditions is shown in Table 14. Range analysis of the results on the inhibition of *Fusarium graminearum* is shown in Table 15. The range R values ​​of the four factors differed. The degree of influence of each factor on the inhibition of *Fusarium graminearum* growth by the YC compound microbial strain fermentation broth is reflected by the range R value; a larger R value indicates a greater influence of that factor on the inhibition rate, and vice versa. Table 15 shows that RC > RD > RA > RB, meaning that magnesium sulfate content has the greatest impact on the inhibition of *Fusarium graminearum* by the YC compound microbial strain, followed by inoculum size, glucose content, and peptone content.

[0097] Based on a comprehensive analysis of the orthogonal experimental results, the optimal combination for the compound bacterial strain YC to inhibit Fusarium graminearum was determined to be A2B1C1D1, which consists of 2% glucose, 0.5% peptone, 0.1% magnesium sulfate, and 3% inoculum. This combination resulted in the highest inhibition rate against Fusarium graminearum.

[0098] Table 14 Antibacterial effect of YC compound bacterial strain after orthogonal optimization of fermentation conditions

[0099] Table 15 Range analysis of the inhibition of Fusarium graminearum by the YC compound bacterial strain after orthogonal optimization of fermentation conditions.

[0100] After orthogonal optimization of the fermentation conditions of the YC compound microbial strain, range analysis was performed on the results of the inhibition of Fusarium verticillatum, as shown in Table 16. The range R values ​​of the four factors differed, and the influence of each factor on the inhibition of Fusarium verticillatum growth by the YC fermentation broth was reflected by the range R value. A larger R value indicates a greater influence of that factor on the inhibition rate. In Table 16, RA > RB > RC > RD, meaning that glucose content had the greatest impact on the inhibition of Fusarium verticillatum by the YC compound microbial strain, followed by peptone content, magnesium sulfate content, and inoculum size.

[0101] Based on a comprehensive analysis of the orthogonal experimental results, the optimal combination for the compound bacterial strain YC to inhibit Fusarium oxysporum was determined to be A2B1C1D2, which consists of 2% glucose, 0.5% peptone, 0.1% magnesium sulfate, and 4% inoculum. This combination resulted in the highest inhibition rate against Fusarium oxysporum.

[0102] Table 16 Range analysis of the inhibition of Fusarium verticillatum by the YC fermentation conditions after orthogonal optimization.

[0103] Based on the above experimental results, the optimal fermentation conditions for the compound strain YC to inhibit corn stalk rot pathogens are: 2% glucose, 0.5% peptone, 0.1% magnesium sulfate, 0.001% KH2PO4, pH value of 7, liquid volume of 80mL, inoculum amount of 4%, and temperature of 30℃, with inhibition rates of 90.73% and 86.51%, respectively.

[0104] Example 4: Application Effect of Compound Biological Seed Coating Agent The optimized compound microbial strain YC was used to develop a biological seed coating agent. A combination of single-factor and orthogonal experiments was used to screen the types and dosages of film-forming agents, stabilizers, dispersants, preservatives, antifreeze agents, and warning colors. The seed coating agent underwent quality testing, and its application effect was verified through pot and field trials. The resulting compound biological seed coating agent formula is: 1% sodium carboxymethyl cellulose, 0.25% kaolin, 0.4% sodium methylene dinaphthalene sulfonate, 0.01% potassium sorbate, 0.03% glycerol, 0.3% carmine, and 98.01% YC fermentation broth. The effective spore count of *Trichoderma* reached 7.58 × 10⁻⁶. 6 The CFU / mL concentration meets all national standards.

[0105] The method of using 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. 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.

[0106] The application effect of the compound biological seed dressing agent showed that it had a good growth-promoting effect on maize, with a plant height promotion rate of 24.21%, stem diameter promotion rate of 67.48%, maximum leaf area promotion rate of 36.68%, root length promotion rate of 85.38%, aboveground fresh weight promotion rate of 257.72%, and underground fresh weight promotion rate of 229.41%. The potted plant control efficacy against maize stalk rot reached 55.50%; the field yield of maize was 743.98 kg / mu, and the field control efficacy against maize stalk rot reached 46.24% (e.g., ...). Figure 21 (As shown).

[0107] 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 microbial strain YC, characterized in that: The biocontrol composite strain YC is composed of Trichoderma harzianum YP2 and T. atroviride CK1A; the Trichoderma harzianum YP2 is preserved in the China General Microbiological Culture Collection Center on May 22, 2025, and is classified as YP2, with the preservation number of CGMCC NO. 41947; the T. atroviride CK1A is preserved in the China General Microbiological Culture Collection Center on May 22, 2025, and is classified as CK1A, with the preservation number of CGMCC NO. 41948. Trichoderma afroharzianum The biocontrol composite strain YC is composed of Trichoderma harzianum YP2 and T. atroviride CK1A; the Trichoderma harzianum YP2 is preserved in the China General Microbiological Culture Collection Center on May 22, 2025, and is classified as YP2, with the preservation number of CGMCC NO. 41947; the T. atroviride CK1A is preserved in the China General Microbiological Culture Collection Center on May 22, 2025, and is classified as CK1A, with the preservation number of CGMCC NO. 41948. Trichoderma atroviride The biocontrol composite strain YC is composed of Trichoderma harzianum YP2 and 2. The application of the biocontrol compound microbial strain YC as described in claim 1 in the biological control of maize stalk rot, characterized in that: The biocontrol composite bacterial system YC is composed of Trichoderma harzianum YP2 and T. atroviride CK1A; the Trichoderma harzianum YP2 was preserved in the China General Microbiological Culture Collection Center on May 22, 2025, and was classified as YP2, with a preservation number of CGMCC NO. 41947; the T. atroviride CK1A was preserved in the China General Microbiological Culture Collection Center on May 22, 2025, and was classified as CK1A, with a preservation number of CGMCC NO. 41948. Trichoderma afroharzianum The biocontrol composite bacterial system YC is composed of Trichoderma harzianum YP2 and T. atroviride CK1A; the Trichoderma harzianum YP2 was preserved in the China General Microbiological Culture Collection Center on May 22, 2025, and was classified as YP2, with a preservation number of CGMCC NO. 41947; the T. atroviride CK1A was preserved in the China General Microbiological Culture Collection Center on May 22, 2025, and was classified as CK1A, with a preservation number of CGMCC NO. 41948. Trichoderma atroviride The biocontrol composite bacterial system YC is composed of Trichoderma harzianum YP2 3. The application of the biocontrol compound microbial system YC 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 fungus 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 YC is as follows: first inoculate Trichoderma viride CK1A, and then inoculate Trichoderma harzianum YP2 36 hours later; the inoculation ratio of Trichoderma harzianum YP2 to Trichoderma viride CK1A is 3:

3.

6. The application according to claim 3, characterized in that: The optimal fermentation conditions for the compound microbial strain YC to inhibit corn stalk rot fungus are: 2% glucose, 0.5% peptone, 0.1% magnesium sulfate, 0.001% KH2PO4, pH 7, liquid volume 80mL, inoculum size 4%, and temperature 30℃.

7. The application of the biocontrol compound microbial strain YC 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.

8. The application according to claim 7, characterized in that: The formula of the complex biological seed coating agent is: 1% sodium carboxymethyl cellulose, 0.25% kaolin, 0.4% sodium methylene bisnaphthalene sulfonate, 0.01% potassium sorbate, 0.03% glycerol, 0.3% cochineal, 98.01% complex bacterial strain YC fermentation broth, wherein the effective spore number of Trichoderma reaches 7.58 x 10 6 CFU / mL.

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