Trichoderma longibrachiatum YP7 and application thereof

Abstract

The invention discloses trichoderma longibrachiatum YP7 and application thereof, and relates to the technical field of microorganisms, the trichoderma longibrachiatum YP7 is preserved in China General Microbiological Culture Collection Center on November 13, 2024, the classification name of the trichoderma longibrachiatum YP7 is Trichoderma longibrachiatum YP7, and the preservation number of the trichoderma longibrachiatum YP7 is CGMCC NO.41627. The trichoderma longibrachiatum YP7 is named as Trichoderma longibrachiatum YP7. The trichoderma longibrachiatum YP7 is combined with a bactericide to be used for preventing and treating corn sheath rot. According to the invention, the bactericide and the trichoderma longibrachiatum YP7 are combined for use, so that the control effect of the bactericide and the trichoderma longibrachiatum YP7 is obviously better than that of the bactericide and the trichoderma longibrachiatum YP7 which are independently used, and the bactericide and pesticide combination has a synergistic effect.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and particularly relates to a Trichoderma longibrachiatum YP7 and its application. Background Art

[0002] Maize sheath rot is a disease that has occurred relatively commonly in maize production in recent years, seriously affecting the yield and quality of maize. At present, the control measures for maize sheath rot include planting disease-resistant varieties, agricultural control, chemical control, biological control, etc.

[0003] Chemical control is the main control method for maize sheath rot in current production. Fungicides such as difenoconazole, pyraclostrobin, prochloraz, and carbendazim have obvious control effects and can effectively inhibit the infection of maize sheath rot pathogens on maize leaf sheaths. However, chemical control measures have certain drawbacks, such as environmental pollution and the problem of easily causing pathogens to develop drug resistance.

[0004] Using Trichoderma agents to control plant diseases will neither pollute the environment nor easily cause pathogens to develop drug resistance. However, the disease prevention mechanism is single, the control effect is easily affected by adverse environments, and the existing Trichoderma agents currently have not very ideal control effects on maize sheath rot.

[0005] In the integrated control of maize sheath rot, when chemical control and biological control are adopted simultaneously, the biocontrol strains are extremely susceptible to chemical fungicides and cannot fully play their biocontrol roles.

[0006] Based on this, in order to effectively control maize sheath rot in Shanxi Province, it is very meaningful to find new Trichoderma and develop a disease prevention and control method for jointly using biocontrol Trichoderma and suitable fungicides to control maize sheath rot. Summary of the Invention

[0007] In view of the above problems, the present invention provides a Trichoderma longibrachiatum YP7 and its application, which solves the problems of environmental pollution caused by chemical fungicides and the easy induction of drug resistance in pathogens in the prior art, as well as the problems such as the single disease prevention mechanism of Trichoderma agents and the susceptibility of the control effect to adverse environments and chemical fungicides.

[0008] In order to achieve the above object, the technical solutions adopted by the present invention are as follows: The present invention provides a Trichoderma longibrachiatum YP7, which was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on November 13, 2024, and the taxonomic name is Trichoderma longibrachiatum YP7, and the deposit number is CGMCC NO.41627.

[0009] The present invention also provides an application of Trichoderma longibrachiatum YP7, and the Trichoderma longibrachiatum YP7 is used in combination with a fungicide to control maize sheath rot.

[0010] Furthermore, the bactericide is fludioxonil, prochloraz, difenoconazole, pyraclostrobin, hymexazol or carbendazim.

[0011] The present invention also provides an application of Trichoderma longibrachiatum YP7, and the Trichoderma longibrachiatum YP7 is used for inhibiting the growth of plant pathogenic bacteria.

[0012] Furthermore, the pathogenic bacteria are Fusarium verticillioides ( Fusarium verticillioides ), Fusarium proliferatum ( Fusarium proliferatum ).

[0013] Compared with the prior art, the beneficial effects of the present invention are as follows: In the present invention, by applying the isolated and screened Trichoderma longibrachiatum YP7 strain to the prevention and control of maize sheath rot in Shanxi Province, the native Trichoderma has strong adaptability in local application and is more likely to play a role. By combining the bactericide with Trichoderma longibrachiatum YP7, the control effect is significantly better than that of the bactericide and Trichoderma used alone, indicating that the combination of the bacteria and the drug has a synergistic effect. The biological bactericide with the YP7 strain as the active ingredient is pollution-free, does not cause environmental pollution, and can also reduce the usage amount of chemical bactericides. In addition, in agricultural production, the combination of Trichoderma longibrachiatum YP7 and chemical bactericides has a synergistic effect; when Trichoderma longibrachiatum YP7 and chemical bactericides are used simultaneously or successively, Trichoderma longibrachiatum YP7 will not be affected by chemical bactericides, ensuring its control effect on diseases. Description of the Drawings

[0014] Figure 1 shows the inhibitory effect of the isolated strain of the present invention on the mycelial growth of Fusarium verticillioides; in the figure, A: YP7; B: CK1A; C: GN1; D: JN5; E: YP8; F: YP7④; G: YP6; H: Fusarium verticillioides; Figure 2 shows the inhibitory effect of the non-volatile metabolites of the isolated strain of the present invention on the mycelial growth of Fusarium verticillioides; in the figure, A: YP7; B: CK1A; C: GN1; D: JN5; E: YP8; F: YP7④; G: YP6; H: Fusarium verticillioides; Figure 3 shows the inhibitory effect of the volatile metabolites of the isolated strain of the present invention on the mycelial growth of Fusarium verticillioides; in the figure, A: YP7; B: CK1A; C: GN1; D: JN5; E: YP8; F: Fusarium verticillioides; Figure 4 shows the field control effect of the antagonistic bacteria suspension of the present invention on maize stalk rot; in the figure, A: YP7 spraying; B: CK1A spraying; C: YP8 spraying; D: spraying control; E: YP7 irrigation; F: CK1A irrigation; G: YP8 irrigation; H: irrigation control; Figure 5 It is the morphological characteristic diagram of strain YP7 of the present invention; A: The culture characteristics of the strain on the PDA plate (72 h); B: The morphological characteristics of the conidiophores of the strain; C: The morphological characteristics of the conidia. Figure 6 It is the phylogenetic tree of strain YP7 constructed by the present invention based on the TEF1-α sequence. Figure 7 It is the effect of different concentrations of fungicides of the present invention on the mycelial growth of Fusarium verticillioides ( F. verticillioides ). In the figure, the concentrations of fludioxonil from top to bottom are CK, 5, 1, 0.1, 0.01, 0.001 μg·mL -1 , and the concentrations of hymexazol from top to bottom are CK, 150, 100, 50, 10, 1 μg·mL -1 , and the concentrations of prochloraz from top to bottom are CK, 1, 0.5, 0.05, 0.01, 0.001 μg·mL -1 , and the concentrations of difenoconazole from top to bottom are CK, 50, 5, 0.5, 0.05, 0.005 μg·mL -1 , and the concentrations of pyraclostrobin from top to bottom are CK, 20, 10, 1, 0.01, 0.001 μg·mL -1 , and the concentrations of carbendazim from top to bottom are CK, 10, 5, 1, 0.1, 0.01 μg·mL -1 ; among them, CK is the PDA medium. Figure 8 It is the effect of different concentrations of fungicides of the present invention on the mycelial growth of Fusarium proliferatum ( F. proliferatum ). In the figure, the concentrations of fludioxonil from top to bottom are CK, 10, 5, 1, 0.1, 0.01 μg·mL -1 , and the concentrations of hymexazol from top to bottom are CK, 150, 100, 50, 10, 1 μg·mL -1 , and the concentrations of prochloraz from top to bottom are CK, 1, 0.5, 0.05, 0.01, 0.001 μg·mL -1 , and the concentrations of difenoconazole from top to bottom are CK, 25, 5, 0.5, 0.05, 0.005 μg·mL -1 , and the concentrations of pyraclostrobin from top to bottom are CK, 20, 10, 1, 0.01, 0.001 μg·mL -1 , and the concentrations of carbendazim from top to bottom are CK, 25, 10, 5, 0.1, 0.01 μg·mL -1 ; among them, CK is the PDA medium. Figure 9These are the results of confrontation cultures (for 7 days) between three Trichoderma strains (Trichoderma atroviride CK1A, Trichoderma longibrachiatum YP7, and Trichoderma longibrachiatum YP8) of the present invention and Fusarium verticillioides; Figure 10 These are the results of confrontation cultures (for 7 days) between three Trichoderma strains (Trichoderma atroviride CK1A, Trichoderma longibrachiatum YP7, and Trichoderma longibrachiatum YP8) of the present invention and Fusarium proliferatum; Figure 11 This is the inhibition effect (for 5 days) of the combined use of Trichoderma longibrachiatum YP7 and carbendazim against Fusarium verticillioides in the present invention; In the figure, A: The combined use of Trichoderma longibrachiatum YP7 and carbendazim (A1 is the back of the colony, A2 is the front of the colony); B: Carbendazim used alone; T0: CK; T1: 0.001 μg·mL -1 ; T2: 0.005 μg·mL -1 ; T3: 0.01 μg·mL -1 ; T4: 0.05 μg·mL -1 ; T5: 0.1 μg·mL -1 ; Figure 12 This is the inhibition effect (for 5 days) of the combined use of Trichoderma longibrachiatum YP7 and hymexazol against Fusarium proliferatum in the present invention; A: The combined use of Trichoderma longibrachiatum YP7 and hymexazol (A1 is the back of the colony, A2 is the front of the colony); B: Hymexazol used alone; T0: CK; T1: 1 μg·mL -1 ; T2: 10 μg·mL -1 ; T3: 25 μg·mL -1 ; T4: 50 μg·mL -1 ; T5: 100 μg·mL -1 . Detailed implementation manners

[0015] In order to make the objectives and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0016] For the instruments, reagents, materials, etc. involved in the following embodiments, unless otherwise specified, they are all conventional instruments, reagents, materials, etc. existing in the prior art and can be obtained through regular commercial channels. For the experimental methods, detection methods, etc. involved in the following embodiments, unless otherwise specified, they are all conventional experimental methods, detection methods, etc. existing in the prior art.

[0017] Example 1 Isolation and primary screening of antagonistic strains 1. Test materials (1) Test samples Tested maize materials: The collection site was the maize planting area in Taoyuanbao, Taigu, Shanxi. The sampling method was to select healthy maize plants in the plots severely affected by maize stalk rot for the isolation of endophytes.

[0018] (2) Tested culture media The main culture media required for this experiment are shown in Table 1.

[0019] Table 1 Culture medium formula

[0020] (3) Tested strains Fusarium verticillioides causing maize stalk rot: The strain number is FV-1, provided by the Plant Pathology Laboratory of Shanxi Agricultural University. Fusarium verticillioides )

[0021] (4) Tested maize varieties Inbred line Ye 478 maize seeds, provided by the Plant Pathology Laboratory of Shanxi Agricultural University.

[0022] (5) Main instruments and equipment The main instruments required for this experiment are shown in Table 2.

[0023] Table 2 Main instruments used in the experiment and their manufacturers

[0024] 2. Experimental methods and results 2.1 Isolation, purification and preservation of strains (1) Isolation, purification and preservation of maize endophytic strains Collect healthy maize plants in the areas severely affected by maize stalk rot. Wash the maize tissues with tap water, weigh 2 g, wrap them with sterile gauze and put them into the ultra-clean workbench. Then put the gauze and maize tissues into 75% alcohol for 30 s. After taking them out, transfer them to 10% sodium hypochlorite solution and soak for 3 - 5 min for surface disinfection of maize tissues. Rinse with sterile water 3 - 4 times; after air-drying, grind them into a homogenate, add 5 mL of sterile water and let it stand for 15 min. Dilute the supernatant of the homogenate 10 times and 100 times respectively, and take 0.2 mL of each and spread it on NA and PDA culture media. Set 3 replicates for each treatment and incubate in an incubator at 28 °C under constant temperature.

[0025] Pick representative single colonies with different colors, morphologies and textures from the NA and PDA plates of different treatments, record the numbers; inoculate the purified strains into the slant culture medium and store them at 4 °C for standby.

[0026] As can be seen from Table 3, a total of 29 endophytic strains were isolated from maize plants by the dilution coating method, including 15 bacteria and 14 fungi.

[0027] Table 3 Strain isolation results

[0028] Note: The prefix YP represents the strains isolated from corn leaves; CK1A and GN represent the strains isolated from corn roots; JN represents the strains isolated from corn stems.

[0029] 2.2 Effects of isolated strains on the mycelial growth of Fusarium verticillioides After activating the isolated and preserved strains, inoculate them on the PDA medium plate. The isolated strains and Fusarium verticillioides are respectively inoculated at two points on the same horizontal line 2.5 cm away from the center of the plate. Using the single inoculation of the pathogen of corn stalk rot as the control, and each treatment is repeated 3 times. After the CK just fills the plate, measure the colony radius of the pathogen of corn stalk rot and calculate the inhibition rate according to formula (1).

[0030] Inhibition rate (%) = (colony radius of the pathogen in the control group - colony radius of the pathogen in the treatment group) / colony radius of the pathogen in the control group × 100% (1) The effects of the isolated strains on the mycelial growth of Fusarium verticillioides are shown in Table 4. The results show that: A total of 7 fungal strains with better antagonistic effects against Fusarium verticillioides are initially screened out, and they are all fungi. When the antagonistic strains confront the pathogen for 10 days, the colony radius of the pathogen is significantly lower than that of the control, indicating that all antagonistic strains can significantly inhibit the growth of the pathogen mycelium.

[0031] The inhibition rate of the antagonistic fungi against Fusarium verticillioides ranges from 78.53% to 86.23%, and among them, the inhibition rate of the antagonistic fungus YP7 against Fusarium verticillioides is the highest.

[0032] All the antagonistic fungi have obvious contact with the mycelia of Fusarium verticillioides at 3 days after inoculation. Such as Figure 1 , after the antagonistic fungi come into contact with the pathogen mycelia, they invade and cover the colony of Fusarium verticillioides, resulting in sparse pathogen colonies and stopped growth.

[0033] Table 4 Inhibitory effects of isolated strains on the mycelial growth of Fusarium verticillioides

[0034] Note: The data in the table are mean ± standard error, "-" indicates no data in this column, and the lowercase letters after the data indicate the significance of differences between treatments (P < 0.05). The same applies to the rest of the tables.

[0035] 2.3 Effects of non-volatile metabolites of antagonistic strains on the mycelial growth of Fusarium verticillioides The glass paper method is used to evaluate the effects of non-volatile metabolites of antagonistic fungi on the mycelial growth of the pathogen, and the results are shown in Table 5. The results show that: All treatments have significant inhibitory effects on the mycelial growth of the pathogen.

[0036] After 5 days of treatment with 7 antagonistic fungi, the colony diameter of Fusarium verticillioides was 30.14 mm to 67.94 mm, and the inhibition effect ranged from 24.51% to 66.51%. Among them, YP7 had a better inhibition effect.

[0037] The effects of non-volatile metabolites of the antagonistic bacteria on the mycelial growth of Fusarium verticillioides were as follows Figure 2 . Obvious dissolution phenomena occurred in the pathogen mycelia. After 5 days of treatment with most of the antagonistic bacteria, the pathogen mycelia were relatively sparse, with weak growth and uneven colony edges.

[0038] The inhibitory effects of the non-volatile metabolites of the antagonistic fungi YP7④ and YP6 on the mycelial growth of the pathogen were relatively weak. Therefore, the other 5 antagonistic bacteria were selected for subsequent experiments.

[0039] Table 5 Inhibitory effects of non-volatile metabolites of antagonistic bacterial strains on the mycelial growth of maize stalk rot pathogen

[0040] 2.4 Effects of volatile metabolites of antagonistic bacterial strains on the mycelial growth of Fusarium verticillioides The plate counter method was used to evaluate the effects of volatile metabolites of antagonistic bacteria on the mycelial growth of Fusarium verticillioides. The results are shown in Table 6 and Figure 3 as follows. The results showed that when the antagonistic bacterial strains and the pathogen were co-cultured for 5 days, the colony diameter of the pathogen was significantly lower than that of the control, indicating that the volatile metabolites of all antagonistic bacterial strains had a significant inhibitory effect on the pathogen.

[0041] When the antagonistic fungi and Fusarium verticillioides were co-cultured for 5 days, the colony diameter of Fusarium verticillioides ranged from 54.58 mm to 74.40 mm, and the inhibition rate ranged from 17.33% to 39.36%. The inhibition rate of YP7 was 39.36%.

[0042] Table 6 Inhibitory effects of volatile metabolites of antagonistic bacterial strains on the mycelial growth of Fusarium verticillioides

[0043] 2.5 Effects of antagonistic bacterial strains on the spore germination of Fusarium verticillioides (1) Effects of the spore suspension of antagonistic bacteria on the spore germination of Fusarium verticillioides 5 mL of sterile water was added to the PDA plate of the antagonistic bacteria cultured for 7 days and shaken well to fully disperse the conidia of the antagonistic bacteria in the sterile water, obtaining the spore suspension of the antagonistic bacteria. The concentration of the spore suspension was counted using a hemocytometer, and the concentration of the spore suspension was adjusted to 1×10 5 CFU·mL -1Reserve. Prepare the spore suspension of *Fusarium verticillioides* using the same method as above, and adjust the concentration of the spore suspension to 1×10 5 CFU·mL -1 Reserve.

[0044] The effect of the spore suspension of the antagonistic strain on the spore germination of *Fusarium verticillioides* was determined by the slide spore germination method. Add 1 mL of the pathogen spore suspension to a sterilized 2 mL centrifuge tube, add the spore suspension of the antagonistic strain according to a volume ratio of 1:1. Use the volume ratio of the pathogen spore suspension and sterile water of 1:1 as the control, and set 3 replicates for the experiment. Pipette 60 μL of the mixed spore suspension and drop it on a sterilized concavity slide. Place the concavity slide in a petri dish lined with moist filter paper, put it in an incubator and culture at 28 °C. Microscopic examination was carried out at 12 h to observe the spore germination situation. A spore was recorded as a germinated spore when the length of the spore germ tube exceeded 1 / 2 of the spore diameter.

[0045] The effect of the spore suspension of the antagonistic strain on the spore germination of *Fusarium verticillioides* is shown in Table 7. The results of the spore suspension of the antagonistic strain on the spore germination of *Fusarium verticillioides* showed that when treated with the spore suspension of the antagonistic strain for 12 h, there were significant differences in the spore germination rate of *Fusarium verticillioides* compared with the control. After treatment with the antagonistic strain, the spore germination rate of *Fusarium verticillioides* ranged from 42.31% to 55.46%, and the inhibition rate ranged from 25.12% to 42.88%.

[0046] Table 7 Inhibitory effect of the spore suspension of the antagonistic strain on the spore germination of the pathogen

[0047] (2) Effect of the fermentation broth of the antagonistic strain on the spore germination of *Fusarium verticillioides* Take 2 mL of the prepared spore suspension of the antagonistic strain and add it to a 250 mL Erlenmeyer flask containing 100 mL of PDB medium. Culture it under dark shaking conditions at 28 °C and 180 r / min for 7 d to obtain the fermentation broth of the antagonistic strain. Prepare the spore suspension of *Fusarium verticillioides* using the same method as in 2.5 (1).

[0048] The effect of the fermentation broth of the antagonistic strain on the spore germination of *Fusarium verticillioides* was determined by the slide spore germination method. Add 1 mL of the pathogen spore suspension to a sterilized 2 mL centrifuge tube, add the fermentation broth of the antagonistic strain according to a volume ratio of 1:1. Use the volume ratio of the pathogen spore suspension and sterile water of 1:1 as the control, and set 3 replicates for the experiment. Pipette 60 μL of the mixed liquid and drop it on a sterilized concavity slide. Place the concavity slide in a petri dish lined with moist filter paper, put it in an incubator and culture at 28 °C. Microscopic examination was carried out at 12 h to observe the spore germination situation. A spore was recorded as a germinated spore when the length of the spore germ tube exceeded 1 / 2 of the spore diameter.

[0049] The effects of the antagonistic bacterium fermentation broth on the spore germination of Fusarium verticillioides are shown in Table 8. The results of the antagonistic bacterium fermentation broth on the spore germination of Fusarium verticillioides indicate that after treatment with the antagonistic fungal fermentation broth, the spore germination rate of Fusarium verticillioides ranges from 15.19% to 56.58%, and the inhibition rate ranges from 30.41% to 81.32%. Among them, the inhibition rate of the YP7 fermentation broth on the spore germination of Fusarium verticillioides reaches 81.32%.

[0050] Table 8 Inhibitory effects of the antagonistic bacterium fermentation broth on the spore germination of pathogenic bacteria

[0051] Based on the comprehensive experimental results of 2.2, 2.3, 2.4, and 2.5, it is found that the antagonistic strains YP7 and CK1A have certain inhibitory effects on Fusarium verticillioides in all aspects. Although the inhibitory effect of strain YP8 on the spore germination of pathogenic bacteria is poor, its growth rate is fast and it has strong spatial competition potential. Therefore, YP7, CK1A, and YP8 are selected for subsequent experiments.

[0052] 2.6 Field control efficacy of antagonistic strains against maize stalk rot Maize sowing and field management: Maize was planted in Taoyuanbao, Taigu District, Jinzhong City, Shanxi Province in early May 2022. The planting interval was 40 cm in row spacing and 25 cm in plant spacing. Two disinfected maize seeds were planted in each hole. The field management measures for each treatment and the control were kept consistent.

[0053] Preparation of inoculum: After multiplying the maize stalk rot pathogen with maize tassels, a spore suspension of 1×10 6 CFU·mL -1 was prepared, and the prepared spore suspension was placed in a low-temperature storage box and taken to the field for standby.

[0054] Field experiment treatment: This experiment adopted a single-factor randomized block design, and the distribution of each treatment in the field was arranged in a randomized block. After the female flowers of maize plants bloomed for 7 - 10 days, the maize stalk rot pathogen was inoculated by the stem injection method. Treatment 1: One week after inoculating the maize stalk rot pathogen, 200 mL of the antagonistic bacterium spore suspension was irrigated to the roots of each plant every week for 3 consecutive weeks; Treatment 2: One week after inoculating the maize stalk rot pathogen, 200 mL of the antagonistic bacterium spore suspension was sprayed at the base of the maize stem of each plant every week for 3 consecutive weeks; Blank control: Sterile water was used to replace the antagonistic bacterium spore suspension for spraying and root irrigation treatments respectively.

[0055] Investigation of the disease situation of maize plants: One month after inoculating the maize stalk rot pathogen, random samples were taken from each treatment. According to the disease grading standard in Table 9, the longitudinal stem dissection method was used to count the incidence of maize stalk rot. The disease index and relative control efficacy were calculated according to the following formulas (2) and (3): Disease index = ∑(number of diseased plants at each level × representative value at each level) / (total number of plants surveyed × representative value of the highest level) × 100; (2) Relative control efficacy (%) = (control disease index - treatment disease index) / control disease index × 100. (3) Table 9 Disease grading standard of corn stalk rot at maturity

[0056] The field control efficacy results of antagonistic strains against corn stalk rot are as Figure 4 shown in Table 10. The results of the antagonistic bacteria spray treatment showed that the disease index of the sterile water control was 68.15%, and the disease indices of all antagonistic bacteria treatments were significantly lower than that of the control, indicating that the spray treatment with the antagonistic spore suspension had a certain control effect on field corn stalk rot. Among them, the control efficacy of the spray treatment of YP7 was 33.71%.

[0057] The results of the antagonistic bacteria root irrigation treatment showed that the disease index of the sterile water control was 68.89%, and the disease indices of all treatments were significantly lower than that of the control, indicating that the root irrigation treatment with the antagonistic spore suspension also had a certain control effect on field corn stalk rot. Among them, the control efficacy of CK1A reached 37.64%.

[0058] Table 10 Field control efficacy of antagonistic spore suspension spray and root irrigation treatments against corn stalk rot

[0059] 2.7 Identification of biocontrol strains Based on the above screening results of biocontrol strains, it can be seen that strains YP7 and CK1A have better comprehensive inhibitory effects on Fusarium verticillioides; in addition, YP-8 also has a certain inhibitory effect on Fusarium verticillioides. Although the effect is not significant, its growth rate is faster, and it is more likely to form spatial competition and nutrient competition with the pathogen. Therefore, strains YP7, CK1A, and YP-8 were identified and used for subsequent experiments.

[0060] (1)Morphological identification of biocontrol strains Fungal morphological identification: After culturing the test strains on PDA medium in an incubator at 28°C for 3 days, observe and record the changes in the culture characteristics of the strains. After the fungi produce a large amount of spores, observe the morphology of the spores and the characteristics of the spore-bearing structures under a microscope.

[0061] The morphological characteristics of strain YP7 are as Figure 5As shown in the figure. YP7 grows relatively fast on the PDA plate. When cultured at 28 °C for 2 days, the mycelial diameter is 65 - 70 mm. The mycelia are sparse, initially white and felt-like, closely spreading flat on the medium, and sporulating outward with the mycelial cake as the center. The plate turns dark green after 3 - 5 days; the conidiophores are at right angles or acute angles to the main axis, the main axis branches multiple times, the phialides are mostly solitary or in whorls of 2, swollen at the base, and pointed at the top, flask-shaped. The conidia are borne at the top of the phialides, oval, with an average size of (4.58 × 6.95) μm. According to its morphological characteristics, the strain YP7 was identified as Trichoderma longibrachiatum ( Trichoderma longibrachiatum ).

[0062] (2)Molecular biological identification of biocontrol strains Extraction of strain DNA: The fungal genomic DNA kit (Beijing Solarbio Science & Technology Co., Ltd.) was used to extract the fungal genomic 18S rDNA according to the instructions.

[0063] PCR amplification: The 18S rDNA gene of the antagonistic fungus was amplified with ITS1 / ITS4, and the TEF1-α fragment was amplified with EF1-728F / TEF1LLErev. The amplification system was 25 μL: 1 μL of each upstream and downstream primer (10 μmol / L), 2 μL of DNA template, 12.5 μL of MasterMix, and 8.5 μL of ddH 2 O. The primer sequences and PCR reaction conditions are shown in Table 11, and the primer sequences are as shown in SEQ ID NO: 1 - 2.

[0064] Gel electrophoresis detection: The PCR products were detected by 1% agarose gel electrophoresis. After observing that the amplified bands were bright and single, they were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0065] Sequence alignment: After obtaining the sequencing results, log in to NCBI and use the Blast software for alignment and analysis.

[0066] Constructing a phylogenetic tree: Use the MEGA5 software to analyze and construct a phylogenetic tree to determine the taxonomic status of the strain.

[0067] Table 11 PCR primers and reaction conditions corresponding to different genes of the strain

[0068] After PCR amplification and sequencing of strain YP7 using the fungal universal primer ITS and the Trichoderma-specific primer TEF1-α, the sequences were obtained. The sequences were Blast-aligned on NCBI and found that the amplified sequences of strain YP7 had the highest similarity rate with Trichoderma longibrachiatum. The phylogenetic tree showed that YP7 and Trichoderma longibrachiatum clustered into one branch, with a support rate of 98% ( Figure 6 ). Combining morphological identification, the strain YP7 was identified as Trichoderma longibrachiatumTrichoderma longibrachiatum ).

[0069] The remaining 2 antagonistic strains were identified by the above morphological identification method and molecular biology identification method. The results showed that: strain YP8 was Trichoderma longibrachiatum ( Trichoderma longibrachiatum ), and strain CK1A was Trichoderma atroviride ( Trichoderma atrovirid ).

[0070] Example 2 Inhibitory effect of the combination of bacteria and drugs on maize sheath rot pathogens (Fusarium verticillioides, Fusarium proliferatum) 1. Test materials (1) Test strains Biocontrol bacteria: Trichoderma longibrachiatum YP7 ( Trichoderma longibrachiatum ), Trichoderma atroviride CK1A ( Trichoderma atroviride ), Trichoderma longibrachiatum YP8 ( Trichoderma longibrachiatum ) (all provided by the Key Laboratory of Plant Pathology, Shanxi Agricultural University).

[0071] Maize sheath rot pathogens: Fusarium verticillioides ( Fusarium verticillioides ) strain number TG-3.1, Fusarium proliferatum ( Fusarium proliferatum ) strain number WM-2.2.

[0072] (2) Test agents The agents used in this test are shown in Table 12.

[0073] Table 12 Test agents

[0074] (3) Test media Potato dextrose agar medium (PDA): 200 g of potatoes, 20 g of glucose, 20 g of agar, 1000 mL of distilled water.

[0075] Carboxymethyl cellulose sodium medium (CMC-Na): 20 g of CMC-Na, Na 2 HPO 4 2.5 g, KH 2 PO 4 1.5 g, 2.5 g of peptone, 20 g of agar, made up to 1000 mL with deionized water.

[0076] (4) Test maize varieties Inbred line Ye 478 maize seeds, provided by the Plant Pathology Laboratory of Shanxi Agricultural University.

[0077] (5) Test instruments The main instruments used in the test are shown in Table 13.

[0078] Table 13 Main instruments used in the test and manufacturers

[0079] 2. Test Methods and Results 2.1 Inhibitory Effect of Fungicides on Fusarium spp. Causing Sheath Rot of Maize The inhibitory effects of 6 fungicides on Fusarium spp. (Fusarium verticillioides and Fusarium proliferatum) causing sheath rot of maize were studied by the mycelial growth rate method. First, the stock solutions of 6 fungicides were prepared respectively, and then diluted with sterile water into 5 concentration gradients. The medicated plates were made with V fungicide:V medium = 1:9. The sterile water in equal amount was used to replace the fungicide as the blank control, and each concentration treatment was repeated 3 times. After culturing at 25 °C for 7 d, the colony diameter was measured by the cross method, and the inhibition rate / % was calculated according to formula (4).

[0080] Inhibition rate (%) = (radius of pathogen colony in the control group - radius of pathogen colony in the treatment group) / radius of pathogen colony in the control group × 100%. (4) (1) Inhibitory Effect of Fungicides on Fusarium verticillioides The results showed that: all the 6 fungicides had inhibitory effects on the mycelial growth of Fusarium verticillioides, and the inhibition rate increased with the increase of concentration. There were significant differences in the inhibitory effects among different fungicides ( Figure 7 ). Among the 6 chemical fungicides, the best inhibitory effects were obtained with fludioxonil and prochloraz, and the EC 50 were 0.0039 μg·mL -1 and 0.0082 μg·mL -1 respectively; followed by pyraclostrobin, difenoconazole and carbendazim, and the EC 50 were 0.0125 μg·mL -1 , 0.0418 μg·mL -1 and 0.0997 μg·mL -1 respectively; while the inhibitory effect of hymexazol was relatively poor, and its EC 50 was as high as 4.7388 μg·mL -1 . It indicated that Fusarium verticillioides was more sensitive to fludioxonil and prochloraz, and less sensitive to hymexazol (Table 14).

[0081] Table 14 Inhibitory Effects of 6 Fungicides on Fusarium verticillioides

[0082] (2) Inhibitory Effect of Fungicides on Fusarium proliferatum The virulence determination results of 6 fungicides on Fusarium proliferatum are shown in Table 15. There were significant differences in the inhibitory effects among different fungicides ( Figure 8 ). Among the 6 tested fungicides, the EC 50The values from large to small are hymexazol, difenoconazole, carbendazim, pyraclostrobin, fludioxonil, and prochloraz, and their EC 50 values are 5.4850 μg·mL -1 , 0.3459 μg·mL -1 , 0.3162 μg·mL -1 , 0.1914 μg·mL -1 , 0.0609 μg·mL -1 , 0.0095 μg·mL -1 . Fludioxonil and prochloraz have strong inhibitory effects on Fusarium proliferatum, and their EC 50 values are both less than 0.1.

[0083] Table 15 Inhibitory effects of 6 fungicides on Fusarium proliferatum ,

[0084] 2.2 Inhibitory effects of Trichoderma on Fusarium graminearum The plate confrontation culture method was used to screen biocontrol strains against Fusarium graminearum (Fusarium verticillioides, Fusarium proliferatum). On PDA plates, a biocontrol Trichoderma strain and a pathogen disc (Φ = 0.6 cm, with a 5 cm distance between the two discs) were inoculated simultaneously. Plates without inoculating the biocontrol Trichoderma strain were used as controls, and each treatment was repeated 3 times. Incubate at a constant temperature of 25 °C for 7 days, measure the colony radius, and calculate the inhibition rate / % according to formula (4).

[0085] (1) Inhibitory effects of Trichoderma on Fusarium verticillioides The plate confrontation culture method was used to compare the inhibitory effects of 3 Trichoderma strains on the mycelial growth of Fusarium verticillioides on PDA medium. The antibacterial effects of each strain are as Figure 9 shown in and Table 16. All 3 biocontrol Trichoderma strains can inhibit the growth of Fusarium verticillioides. Trichoderma longibrachiatum YP7 has a good antibacterial effect on the mycelial growth of Fusarium verticillioides, with an inhibition rate of 72.26%, which is significantly higher than the other 2 treatments; while the inhibition rates of Trichoderma longibrachiatum YP8 and Trichoderma atroviride CK1A on Fusarium verticillioides are not significantly different, being 68.21% and 68.54% respectively. It shows that Trichoderma longibrachiatum YP7 has the best inhibitory effect on the mycelial growth of Fusarium verticillioides.

[0086] Table 16 Growth inhibition of mycelia of Fusarium verticillioides by 3 Trichoderma strains

[0087] Note: Different lowercase letters indicate significant differences among treatments (P < 0.05).

[0088] (2) Inhibitory effects of Trichoderma on Fusarium proliferatum The antibacterial effect of Trichoderma on Fusarium proliferatum was determined by the plate confrontation method as follows Figure 10 , and the inhibition rates are shown in Table 17. The order of the inhibition rates of the three Trichoderma strains on Fusarium proliferatum from high to low is: Trichoderma longibrachiatum YP7, Trichoderma longibrachiatum YP8, Trichoderma atroviride CK1A, and their inhibition rates are 68.88%, 65.32%, and 62.03% respectively. The inhibition rate of Trichoderma longibrachiatum YP7 is significantly higher than that of the other two treatments. It shows that Trichoderma longibrachiatum YP7 has the best inhibitory effect on the mycelial growth of Fusarium proliferatum.

[0089] Table 17 Growth inhibition of mycelia of three Trichoderma strains on Fusarium proliferatum

[0090] Note: Different lowercase letters indicate significant differences among treatments (P<0.05).

[0091] 2.3 Determination of the compatibility between Trichoderma and fungicides Taking the ratio of the EC 50 of the fungicide on Trichoderma to the EC 50 of the fungicide on the pathogen of maize sheath rot disease > 1 as the standard for screening fungicides that can be used in combination with Trichoderma, calculate the EC 50 ratio according to formula (5). The method for determining the inhibitory effect of the fungicide on Trichoderma is the same as that in 2.1.

[0092] EC 50 ratio = EC 50 value of the fungicide on Trichoderma / EC 50 value of the fungicide on the pathogen (5) (1) Results of the determination of the compatibility between Trichoderma and fungicides in inhibiting Fusarium verticillioides Select Trichoderma atroviride CK1A, Trichoderma longibrachiatum YP7 and Trichoderma longibrachiatum YP8 for compatibility determination with 6 fungicides. The inhibitory effects of the 6 fungicides on the 3 Trichoderma strains are different, and the inhibitory effects of the same fungicide on Fusarium verticillioides (No. TG-3.1) and Trichoderma also vary. The EC 50 of the 6 fungicides on strain CK1A ranges from 0.0538 to 43.9030 μg·mL -1 , all less than 45 μg·mL -1 ; The order of EC 50 from small to large is fludioxonil, carbendazim, prochloraz, difenoconazole, pyraclostrobin, hymexazol. The order of EC 50 from small to large of the 6 fungicides on strain YP7 and strain YP8 is fludioxonil, prochloraz, carbendazim, difenoconazole, pyraclostrobin, hymexazol. The EC 50 on strain YP7 ranges from 0.0119 to 64.0221 μg·mL-1 , the EC of strain YP8 50 is between 0.0125 and 57.2008 μg·mL -1 , the EC 50 is all less than 65 μg·mL -1 .

[0093] Analyze the EC of fungicides against Trichoderma 50 and the EC of this fungicide against Fusarium verticillioides 50 ratio. It is found that the inhibitory effect of 6 fungicides on Fusarium verticillioides is always greater than that on 3 Trichoderma strains. The EC 50 ratio is all greater than 1, indicating that the 6 fungicides are compatible with 3 Trichoderma strains, and the 6 fungicides can be used in combination with 3 Trichoderma strains. Among them, the EC 50 ratio of pyraclostrobin is relatively high, and the EC 50 ratios against strain YP7, strain YP8 and strain CK1A are 2920.80, 1279.66 and 1144.83 respectively (Table 18).

[0094] Table 18 Inhibitory effects of 6 fungicides on 3 Trichoderma strains and Fusarium verticillioides

[0095] (2) Compatibility determination results of Trichoderma and fungicides in inhibiting Fusarium proliferatum There are also differences in the inhibitory effects of 6 fungicides on Fusarium proliferatum (No. WM-2.2) and 3 Trichoderma strains. Analyze the EC of fungicides against Trichoderma 50 and the EC of this fungicide against Fusarium proliferatum 50 ratio. It is found that the inhibitory effect of 4 fungicides, pyraclostrobin, prochloraz, difenoconazole and hymexazol, on Fusarium proliferatum is always greater than that on 3 Trichoderma strains. The EC 50 ratio is all greater than 1, indicating that these 4 fungicides are compatible with 3 Trichoderma strains, and the 4 fungicides can be used in combination with 3 Trichoderma strains; the EC 50 ratio of carbendazim and Trichoderma longibrachiatum YP7, Trichoderma longibrachiatum YP8 > 1, indicating that it is suitable for combined use, while the EC 50 ratio of carbendazim and Trichoderma atroviride CK1A is 0.55, indicating that it is not suitable for combined use; the inhibitory effect of fludioxonil on Fusarium proliferatum is less than that on 3 Trichoderma strains, and the EC 50 ratio is all less than 1, indicating that fludioxonil is not suitable for combined use with Trichoderma (Table 19).

[0096] Table 19 Inhibitory effects of 6 fungicides on 3 Trichoderma strains and Fusarium proliferatum ​​​​​​​​​

[0097] 2.4 Inhibitory effect of the combined use of Trichoderma and fungicides on Fusarium verticillioides The inhibitory effect of the combined use of the fungus and the drug was studied by the confrontation culture method on a drug-containing medium. Select fungicides with good compatibility with the biocontrol Trichoderma to prepare drug-containing medium plates. On the drug-containing plates, inoculate Trichoderma and pathogen discs (Φ = 0.6 cm, and the two discs are 5 cm apart) at the same time. Use the plates inoculated with only Trichoderma and only the pathogen as controls respectively, and use the PDA plate with an equal amount of sterile water instead of the fungicide as the blank control. Each treatment is repeated 3 times. After 7 days, measure the colony radius of the control pathogen, the control Trichoderma, as well as the colony radius of the pathogen and Trichoderma in each treatment respectively, and calculate the inhibition rate (%) according to formula (6).

[0098] Inhibition rate = (colony radius of the control pathogen - colony radius of the confronting pathogen) / (colony radius of the control pathogen) × 100% (6) The evaluation of the combined synergistic effect of the biocontrol Trichoderma and the fungicide refers to the test method in 2.3, and calculate the synergy coefficient according to formula (7).

[0099] Synergy coefficient (S) = (R Bt - R BCK ) / (R Tt - R TCK ) (7) Where R TCK and R BCK represent the colony radii of the control Trichoderma and the pathogen respectively; R Tt and R Bt represent the opposite-facing colony radii of Trichoderma and the pathogen in confrontation respectively. If S ≥ 1.5, it indicates that the combined use of Trichoderma and the fungicide has a synergistic effect; 1 ≤ S < 1.5 indicates that the combined use of Trichoderma and the fungicide has an additive effect; S < 1 indicates that the combined use of Trichoderma and the fungicide has an antagonistic effect.

[0100] (1) Inhibitory effect of the combined use of Trichoderma and fungicides on Fusarium verticillioides The inhibitory effect of the combined use of Trichoderma and fungicides on Fusarium verticillioides, a pathogen of maize sheath rot, was evaluated by the synergy coefficient method ( Figure 11 ). The inhibitory rates of the 6 fungicides combined with Trichoderma on Fusarium verticillioides were all greater than those when used alone, indicating that there is a certain synergistic effect after the combined use of the fungus and the drug (Table 20).

[0101] After 6 fungicides were used in combination with Trichoderma atroviride CK1A, the number of treatments with a synergy coefficient S < 1 against Fusarium verticillioides accounted for 33.33% of the total, indicating that the combination of the fungus and the fungicide in these treatments had a certain antagonistic effect; the number of treatments with 1 ≤ S < 1.5 accounted for 66.67% of the total, indicating that the combination of the fungus and the fungicide in these treatments had an additive effect; there were no treatments with S ≥ 1.5, indicating that there was no synergistic effect after the 6 fungicides were used in combination with Trichoderma atroviride CK1A, that is, Trichoderma atroviride CK1A was not suitable for use in combination with fungicides.

[0102] After fludioxonil was used in combination with Trichoderma longibrachiatum YP7 and Trichoderma longibrachiatum YP8 respectively, the number of treatments with a synergy coefficient S < 1 against Fusarium verticillioides accounted for 30% of the total, indicating that the combination of the fungus and the fungicide in these treatments had a certain antagonistic effect; the treatments with 1 ≤ S < 1.5 accounted for 70% of the total, indicating that there was an additive effect after the combination of the fungus and the fungicide; there were no treatments with S ≥ 1.5, indicating that there was no synergistic effect after the combination of the fungus and the fungicide. In summary, fludioxonil was not suitable for use in combination with Trichoderma longibrachiatum YP7 and Trichoderma longibrachiatum YP8.

[0103] After prochloraz was used in combination with strain YP7 and strain YP8 respectively, its synergy coefficient changed with the change of the fungicide concentration. The synergy coefficients of the combination of prochloraz at three concentrations of 0.001 μg·mL -1 , 0.005 μg·mL -1 and 0.01 μg·mL -1 against Fusarium verticillioides were 1 ≤ S < 1.5, indicating that the combination of the fungus and the fungicide at these 3 concentrations had an additive effect; the S of the combination of prochloraz at 0.05 μg·mL -1 with strain YP7 and YP8 was ≥ 1.5, indicating that this treatment had a synergistic effect; while the S of the combination of prochloraz at 0.1 μg·mL -1 was < 1, indicating that there was an antagonistic effect between prochloraz and the biocontrol strain. Therefore, 0.05 μg·mL -1 prochloraz was suitable for use in combination with strain YP7 and strain YP8.

[0104] After carbendazim was used in combination with strain YP7 and strain YP8 respectively, the synergy coefficient 1 ≤ S < 1.5 against Fusarium verticillioides accounted for 80% of the total number of treatments, indicating that the combination of the fungus and the fungicide in these treatments had an additive effect; the combination of carbendazim at 0.01 μg·mL -1 and 0.05 μg·mL -1 with strain YP7 had a synergy coefficient S ≥ 1.5, accounting for 20% of the total number of treatments, indicating a synergistic effect, and the synergy coefficient of 0.01 μg·mL -1 carbendazim with strain YP7 was as high as 1.61.

[0105] 0.1 μg·mL -1 and 1 μg·mL-1 After pyraclostrobin was used in combination with strain YP7 and strain YP8 respectively, the synergy coefficient S of Fusarium verticillioides was ≥ 1.5. Among them, 1 μg·mL -1 The synergy coefficient of pyraclostrobin and Trichoderma longibrachiatum YP7 was as high as 1.62, indicating that 0.1 μg·mL -1 and 1 μg·mL -1 Pyraclostrobin is suitable for use in combination with strain YP7 and strain YP8.

[0106] Table 20 Synergistic effects of the combination of Trichoderma and fungicides on the inhibition of Fusarium verticillioides , ,

[0107] (2) Inhibitory effect of the combination of Trichoderma and fungicides on Fusarium proliferatum The inhibitory effect of the combination of Trichoderma and fungicides on Fusarium proliferatum, the causative agent of maize sheath rot, was evaluated by the synergy coefficient method ( Figure 12 ). The inhibition rates of the five fungicides in combination with Trichoderma on Fusarium proliferatum were all greater than those when used alone, indicating that there was a certain synergistic effect after the combination of the fungus and the drug (Table 21).

[0108] After the five fungicides were used in combination with Trichoderma atroviride CK1A, the number of treatments with a synergy coefficient S < 1 for Fusarium proliferatum accounted for 15% of the total, indicating that the combination of the fungus and the drug in these three treatments had a certain antagonistic effect; the number of treatments with 1 ≤ S < 1.5 accounted for 85% of the total, indicating that the combination of the fungus and the drug in these treatments had an additive effect; there was no treatment with S ≥ 1.5, indicating that there was no synergistic effect after the combination of the five fungicides and Trichoderma atroviride CK1A, that is, Trichoderma atroviride CK1A was not suitable for use in combination with fungicides.

[0109] The number of treatments with a synergy coefficient of 1 ≤ S < 1.5 for prochloraz in combination with strain YP7, strain YP8 and Trichoderma atroviride CK1A accounted for 100% of the total, indicating that the combination of prochloraz and biocontrol bacteria had an additive effect.

[0110] After hymexazol was used in combination with strain YP7 and strain YP8 respectively, the synergy coefficient 1 ≤ S < 1.5 for Fusarium proliferatum accounted for 70%, indicating that the combination of the fungus and the drug in these treatments had an additive effect; the number of treatments with a synergy coefficient S ≥ 1.5 accounted for 30% of the total, indicating that the combination of the fungus and the drug in these three treatments of hymexazol had a synergistic effect. Among them, the synergy coefficient of 50 μg·mL -1 of hymexazol and strain YP7 was as high as 1.62, indicating that 50 μg·mL -1Hymexazol is suitable for combined use with Trichoderma longibrachiatum YP7.

[0111] After pyraclostrobin was combined with strain YP7 and strain YP8 respectively, the number of treatments with a synergy coefficient of 1 ≤ S < 1.5 for Fusarium proliferatum accounted for 70% of the total, indicating that the combination of the bacteria and the fungicide in these treatments had an additive effect; the treatments with S ≥ 1.5 accounted for 30% of the total, indicating that the combination of the bacteria and the fungicide in these three treatments had a synergistic effect. Among them, 0.01 μg·mL -1 and 0.1 μg·mL -1 The synergy coefficients of pyraclostrobin and strain YP7 were as high as 1.60 and 1.58 respectively, indicating that it was suitable for combined use and the combination of the bacteria and the fungicide had a synergistic effect.

[0112] After carbendazim was combined with strain YP7 and strain YP8 respectively, the number of treatments with a synergy coefficient of 1 ≤ S < 1.5 for Fusarium proliferatum accounted for 90% of the total, indicating that the combination of the bacteria and the fungicide in these treatments had an additive effect; 0.001 μg·mL -1 The synergy coefficient of carbendazim and strain YP7 reached 1.54, indicating that it was suitable for combined use and the combination of the bacteria and the fungicide had a synergistic effect.

[0113] After difenoconazole was combined with strain YP7 and strain YP8 respectively, its synergy coefficient changed with the change of the concentration of the fungicide. 0.1 μg·mL -1 , 0.5 μg·mL -1 and 1 μg·mL -1 The S values of difenoconazole at 0.1 μg·mL, 0.5 μg·mL and 1 μg·mL after combined use with strain YP7 were ≥ 1.5. The S values of difenoconazole at 0.5 μg·mL and 1 μg·mL after combined use with strain YP8 were ≥ 1.5, indicating that the combination of the bacteria and the fungicide in these five combinations had a synergistic effect; while the S value was < 1 after the combined use of 5 μg·mL -1 and 1 μg·mL -1 difenoconazole, indicating an antagonistic effect with the biocontrol strain. Therefore, 0.5 - 1 μg·mL -1 difenoconazole was suitable for combined use with strain YP7 and strain YP8. -1

[0114] Table 21 Synergistic effect of the combination of Trichoderma and fungicide on the inhibition of Fusarium proliferatum

[0115] 3. Determination of the field control effect of the combination of Trichoderma and fungicide on maize sheath rot (1) Preparation of Trichoderma spore suspension The Trichoderma was inoculated on a PDA plate and cultured for 5 days. A small amount of distilled water was used to wash the Trichoderma conidia on the culture medium plate, and the spore concentration was adjusted to 1×10 6 CFU·mL -1 .

[0116] (2) Preparation of the spore suspension of Fusarium oxysporum f. sp. vasinfectum The test strain was cultured on a PDA plate for 5 days. A puncher was used to punch discs (Φ = 0.6 cm) at the edge of the Fusarium oxysporum f. sp. vasinfectum colony, which were then inoculated into a triangular flask containing 100 mL of sodium carboxymethyl cellulose liquid medium. The mixture was cultured with shaking at 160 r·min -1 at 25 °C for 72 h, and then formulated into a spore suspension with a spore concentration of 1×10 6 CFU·mL -1 .

[0117] (3) Field inoculation method and pesticide application method for maize leaf sheaths At the early flowering stage of maize, a sterilized puncture device was used to puncture the maize leaf sheath, and then a pipette gun was used to inject 10 μL of the mixed spore suspension of maize sheath rot pathogen into the hole. Maize sheath spraying treatments were carried out on the 3rd and 5th days after inoculation, respectively. A total of 11 treatments were set (Table 22). Each treatment had one plot, with 50 inoculated leaf sheaths. 500 mL of the liquid medicine was sprayed on each plot (the spore suspension of Trichoderma and the fungicide were mixed at a ratio of 1:1). Each treatment was repeated 3 times, and spraying sterile water was used as the control. The disease situation was investigated 10 days after the spraying treatment.

[0118] Table 22 Treatments for the combined field control effect of bacteria and pesticides

[0119] (4) Field disease investigation method The disease situation of field maize was investigated according to the disease grading standard in Table 23. The disease index of maize leaf sheath infection was calculated using formula (8), and the disease control effect was calculated using formula (9).

[0120] Table 23 Disease grading standard for maize sheath rot

[0121] Disease index = ∑(disease level × number of leaf sheaths at this level) / (highest disease level × total number of investigated leaf sheaths) × 100 (8) Disease control effect / % = (control disease index - treatment disease index) / control disease index × 100% (9) According to the indoor determination results of the combined use of bacteria and pesticides, combinations with high inhibition rates and high synergy coefficients were selected for field efficacy tests (as shown in Table 24).

[0122] The field control effects of combined treatment with bacteria and fungicides on maize sheath rot were measured on the 3rd and 5th days after inoculation, respectively. It was found that when the spraying treatment was carried out on the 3rd day after inoculation, the disease index was less than that of the spraying treatment on the 5th day after inoculation, and the control effect on the 3rd day was greater than that on the 5th day. For example, 50 μg·mL -1 Hymexazol + 1×10 6 CFU·mL -1 The combined use of Trichoderma longibrachiatum YP7 had a control effect of 72.08% on the 3rd day and 61.45% on the 5th day; 1 μg·mL -1 Pyraclostrobin had a control effect of 52.54% on the 3rd day and 47.91% on the 5th day, indicating that the control effect was better when the control treatment was carried out 3 days after inoculation. At the same time, it was obvious that the combined control effect of the fungicide and Trichoderma was better than that of the fungicide and Trichoderma alone. For example, on the 5th day, 1 μg·mL -1 Difenoconazole + 1×10 6 CFU·mL -1 The control effect of Trichoderma longibrachiatum YP8 reached 73.96%, while the control effect of 1 μg·mL -1 Difenoconazole treatment was only 40.62%, and 1×10 6 CFU·mL -1 The control effect of Trichoderma longibrachiatum YP8 was 33.45%, indicating that the combined use of bacteria and fungicides had a synergistic effect.

[0123] On the 3rd day, there was no significant difference in the control effects of the 4 combined bacteria and fungicide combinations. On the 5th day, among them, 1 μg·mL -1 Difenoconazole + 1×10 6 CFU·mL -1 Trichoderma longibrachiatum YP8 had the best control effect, which was 73.96%, and there was no significant difference from the control effect of 1 μg·mL -1 Pyraclostrobin + 1×10 6 CFU·mL -1 Trichoderma longibrachiatum YP7, and there was a significant difference from the control effects of the other 2 combined bacteria and fungicide combinations, indicating that the combined control effects of these two combinations of bacteria and fungicides were the best.

[0124] Table 24 Field control effects of combined use of Trichoderma and fungicides on maize sheath rot

[0125] In this embodiment, the Trichoderma longibrachiatum YP7 strain isolated and screened from corn leaves is applied to the prevention and control of corn sheath rot in Shanxi Province. Native Trichoderma has strong adaptability in native applications and is more likely to play a role in biological control. By using a fungicide in combination with Trichoderma longibrachiatum YP7, the control effect is significantly better than that of using the fungicide and Trichoderma alone, indicating that the combination of the bacterium and the drug has a synergistic effect. In addition, the biological bactericide with the YP7 strain as the active ingredient is pollution-free, does not cause environmental pollution, and can also reduce the usage amount of chemical fungicides.

[0126] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A long-branch Trichoderma YP7, characterized in that: The long-branch Trichoderma YP7 was deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration on November 13, 2024, and was classified and named Trichoderma longibrachiatum YP7, the deposit number is CGMCC NO.41627.

2. The use of Trichoderma longibrachiatum YP7 as claimed in claim 1, characterized in that: The long-branch Trichoderma YP7 is used in combination with a fungicide to prevent and control corn sheath rot.

3. The use of Trichoderma longibrachiatum YP7 according to claim 2, characterized in that: The fungicide is fludioxonil, prochloraz, difenoconazole, pyraclostrobin, meconazole or carbendazim.

4. The use of Trichoderma longibrachiatum YP7 as claimed in claim 1, characterized in that: The long-branch Trichoderma YP7 is used to inhibit the growth of plant pathogens.

5. The use of Trichoderma longibrachiatum YP7 according to claim 4, characterized in that: The pathogen is Fusarium pseudoverticillium ( Fusarium verticillioides )、Fusarium spp.( Fusarium proliferatum ).

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