Method for inhibiting Fusarium graminearum by symbiotic fermentation of Trichoderma and Metarhizium

Abstract

The present invention provides a method for inhibiting Fusarium graminearum by symbiotic fermentation of Trichoderma and Metarhizium. The present invention obtains the best co-culture combination of dark green Trichoderma (Trichoderma atroviride) CJCCSJ-F-KY10039 and Metarhizium anisopliae (Metarhizium anisopliae) B81755 by screening different Trichoderma and Metarhizium, and the fermentation liquid obtained by symbiotic fermentation culture in liquid can inhibit Fusarium graminearum, the inoculation order of Trichoderma and Metarhizium is simultaneous inoculation, the two bacteria are inoculated at an inoculation ratio of 1:1, the chitinase activity in the fermentation liquid is 242.72IU / L, the chitosanase activity is 269.86U / L, the β-glucanase activity is 497.53U / L, and the inhibition rate of Fusarium graminearum is 63.3%.

Description

Technical Field

[0001] The invention relates to the technical field of biocontrol bacteria, and relates to a method for inhibiting Fusarium graminearum by symbiotic fermentation of Trichoderma and Metarhizium anisopliae, and specifically to a method for increasing the production of active substances in symbiotic fermentation liquid of Trichoderma and Metarhizium anisopliae to inhibit Fusarium graminearum. Background Art

[0002] Corn (Zea mays L.) is widely planted around the world. Corn root rot can be caused by infection with a variety of Fusarium species alone or in combination. Among the Fusarium species, Fusarium graminearum and Fusarium pseudoverticillium are the main pathogens. After infecting corn, Fusarium graminearum will produce a variety of mycotoxins such as deoxynivalenol (DON), nivalenol (NIV) and zearalenone (ZEN), which seriously endanger the health and life safety of humans and livestock. Studies have shown that DON toxins can cause reduced feed intake, vomiting, muscle bleeding and decreased reproductive performance in pigs. The intake of NIV will affect growth, the immune system and the female reproductive system. ZEN and its metabolites will interfere with estrogen function and affect the reproductive capacity of animals. Therefore, it is very necessary to prevent and control diseases caused by Fusarium graminearum.

[0003] Many studies have shown that in integrated pest management (IPM), the use of microorganisms for biological control has attracted more and more attention as a sustainable innovative product. Although single biocontrol agents have achieved good results, the application of single biocontrol agents still has disadvantages such as unstable control effects, narrow control spectrum, and low stress resistance. The combined use of multiple microorganisms enhances the stability of biological control performance and control effects, increases the broad spectrum of control, and multi-target control reduces the possibility of resistance of pests and diseases, which has a good application prospect. Therefore, the study of combined antimicrobial and its mechanism is a trend in plant disease control.

[0004] Trichoderma and Metarhizium anisopliae are important biocontrol bacteria. Because their preparations are harmless to the ecological environment and have good effects in plant protection, they are widely used in disease prevention and insect control respectively. However, they are currently limited to single use or simple compounding of the two. Although single biocontrol agents have achieved good results, the application of single biocontrol bacteria still has disadvantages such as unstable control effect, narrow control spectrum, and low stress resistance. The compound use of multiple microorganisms enhances the stability of biological control performance and control effect, increases the broad spectrum of control, and multi-target control reduces the possibility of resistance of pests and diseases, and has a good application prospect. Therefore, combined antimicrobial control of plant diseases is the trend of control.

[0005] In the process of microbial co-cultivation, the interaction between bacteria runs through the whole process. The composite microbial agent formed by the co-cultivation method can increase the types and yields of antagonistic substances, growth-promoting substances, etc. Studies have shown that the biocontrol function of some microorganisms after co-cultivation is better than that of single culture: Q Ma et al. found that the mixed culture fermentation liquid of amylolytic spores and long-branched Trichoderma was significantly more effective in controlling tomato wilt than the single culture fermentation of long-branched Trichoderma and amylolytic spores; Chen Kai et al. used Trichoderma HB20111 and Trichoderma TW21990 to co-cultivate and ferment wheat seedling stem rot, which was significantly higher than that of a single strain. Shobha et al. co-cultivated Trichoderma harzianum with two strains of Trichoderma reesei and three strains, and found that their products could block the activity of the protein by first binding to the target protein of rice bacterial blight. Tingting Li et al. co-cultured Bacillus B22 and Trichoderma 3403, and the antifungal and plant growth-enhancing metabolites in the fermentation broth increased. The metabolites were able to more significantly reduce the production of gibberellins (GA), deoxynivalenol (DON) and zearalenone (ZEN) of Fusarium graminearum; the co-cultured Trichoderma-Bacillus had a significant effect on Fusarium graminearum infection by triggering the expression of host plant defense genes (including PR1, PR3, PR4, PR5, ACS and SOD). The fermentation broth obtained after co-culture of Trichoderma and Bacillus can also prevent and control tobacco black shank disease. Compared with single culture, the control effect is significantly improved, which can inhibit the growth of Fusarium graminearum and increase the activity of pathogenic G protein and mitogen-activated protein kinase (MAPK).

[0006] The insecticidal function of Metarhizium is mainly produced by its conidia, but when it comes to preventing and controlling plant diseases, researchers focus on the fermentation liquid of Metarhizium and find that the volatile gases produced by the fermentation filtrate of Metarhizium may have an inhibitory effect on plant pathogens.

[0007] Trichoderma and Metarhizium are widely used biological control microorganisms, each with its own advantages. Trichoderma has been studied for the prevention and treatment of corn root rot. Although Metarhizium is mainly used to control underground pests, there are also literature reports that it also has a control effect on diseases. There is almost no relevant research on the synergistic improvement of the effects of biological control and plant promotion through the co-cultivation of the two microorganisms, and there is no in-depth research on the relationship between the co-cultivation metabolites and their effects on Fusarium. There are also some unresolved issues, such as: (1) Trichoderma and Metarhizium have different growth rates, making co-cultivation difficult; (2) Trichoderma has different degrees of inhibitory effects on the growth of Metarhizium; (3) The changes in metabolites after the co-cultivation of Trichoderma and Metarhizium and the reasons for the inhibition are unclear. The co-cultivation of the two has laid the foundation for the creation of a new type of microbial fungicide for the co-cultivation of the two, and provided a new idea for green control to overcome the problem of sustainable development of corn root rot prevention and control. Summary of the invention

[0008] The present invention aims at the deficiencies in the prior art and provides a method for inhibiting Fusarium graminearum by symbiotic fermentation of Trichoderma and Metarhizium. The Trichoderma and Metarhizium screened out by the present invention and their co-culture conditions are adopted, and the co-culture fermentation liquid has no influence on the normal growth and development of the spores of the two bacteria, and the activities of chitinase, chitosanase, beta-glucanase, etc. in the co-culture fermentation liquid can be improved, which are significantly higher than the activities of Trichoderma or Metarhizium cultured alone, and the inhibition rate of the co-culture fermentation liquid on Fusarium graminearum is also significantly higher than the effect of culturing them alone.

[0009] The present invention is achieved through the following technical solutions:

[0010] In the first aspect, the present invention provides a method for inhibiting Fusarium graminearum by symbiotic fermentation of Trichoderma and Metarhizium, wherein the Trichoderma is Trichoderma atroviride CJCCSJ-F-KY10039, with a preservation number of CCTCC No.M20221135, and the Metarhizium is Metarhizium anisopliae B81755.

[0011] In a second aspect, the present invention provides a microbial fungicide, which contains the symbiotic fermentation product of Trichoderma and Metarhizium anisopliae; the Trichoderma is Trichoderma atroviride CJCCSJ-F-KY10039, and its preservation number is CCTCC No.M 20221135; the Metarhizium anisopliae is Metarhizium anisopliae B81755.

[0012] The active ingredients in the microbial fungicide contain symbiotic fermentation products of dark green Trichoderma and Metarhizium anisopliae.

[0013] Preferably, the symbiotic fermentation comprises the following steps:

[0014] S1. preparing spores of Trichoderma and Metarhizium anisopliae into spore suspensions respectively;

[0015] S2. Take the spore suspensions of Trichoderma and Metarhizium and inoculate them into the culture medium for cultivation.

[0016] Preferably, the inoculation order of Trichoderma and Metarhizium in step S2 is simultaneous inoculation.

[0017] Preferably, the concentration of the spore suspension is 10 6 -2×10 6 spores / mL.

[0018] Preferably, the inoculation ratio of the inoculation in step S2 is 1:1.

[0019] Preferably, the method for preparing the spore suspension comprises: rubbing the tip of a pipette on a plate on which Trichoderma and Metarhizium are grown respectively, so that the tip is covered with spores, and placing the tip covered with spores into sterile water to obtain a spore suspension.

[0020] Preferably, the culture conditions include: a culture temperature of 25-30°C and a rotation speed of 180-250 r / min.

[0021] Preferably, the culture medium comprises PD medium.

[0022] Preferably, the inoculation amount of the inoculation is 1 / 100.

[0023] Preferably, the culture time is 5-10 days.

[0024] Preferably, compared with the single culture, the level of antibacterial active substances in the co-culture fermentation broth is upregulated, and the antibacterial active substances include isothiocyanates, osthole, spirocyclam and other substances.

[0025] Preferably, compared with single culture, the expression levels of chitin synthase-related genes in Fusarium graminearum after treatment with co-culture fermentation filtrate are downregulated, and the expression levels of cellular P450 monooxygenase-related genes are upregulated.

[0026] Preferably, the β-glucanase and chitosanase activities of the co-culture fermentation filtrate are increased compared to the mono-culture.

[0027] In some embodiments, the chitinase activity in the co-culture fermentation broth is 242.72 IU / L. The chitosanase activity in the co-culture fermentation broth is 269.86 U / L. The β-glucanase activity in the co-culture fermentation broth is 497.53 U / L. The inhibition rate of the co-culture fermentation broth on Fusarium graminearum is 63.3%.

[0028] The dark green Trichoderma CJCCSJ-F-KY10039 is preserved in the China Center for Type Culture Collection, the preservation address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, the preservation number is CCTCC No.M 20221135, and the preservation date is July 19, 2022.

[0029] In a third aspect, the present invention provides a use of the above-mentioned microbial fungicide in inhibiting Fusarium graminearum.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] (1) The present invention screened and obtained strains of Trichoderma and Metarhizium that have a certain antagonistic effect on Fusarium graminearum. Based on the growth rates of Metarhizium and Trichoderma, the affinity of plate confrontation and liquid co-culture, and the inhibition rate of Fusarium graminearum, a combination of the fastest growing Metarhizium and the slower growing Trichoderma, the affinity of the two on the plate, and the antibacterial effect of the co-culture fermentation filtrate on Fusarium graminearum was selected. It was found that the co-culture combination of dark green Trichoderma (Trichoderma atroviride) CJCCSJ-F-KY10039 and Metarhizium anisopliae (Metarhizium anisopliae) B81755 was better than other combinations, and the inhibition rate of the co-culture fermentation broth on Fusarium graminearum reached 63.3%;

[0032] (2) The present invention co-cultivates Trichoderma and Metarhizium anisopliae and finds that antibacterial active substances in the co-cultivation fermentation broth are upregulated, such as isothiocyanate, osthole, spirocyclam and the like. These substances are metabolites with antibacterial functions reported in the literature and have an antibacterial effect on Fusarium graminearum.

[0033] (3) The combination was selected according to the growth rates of the two strains, which prevented Trichoderma from fully utilizing the space and nutrients during the culture process, thereby preventing the normal growth of Metarhizium;

[0034] (4) In order to avoid growth inhibition of the two during co-culture, the inoculation order of Trichoderma and Metarhizium was determined, and the inoculation ratio of the two bacteria was 1:1, which played a crucial role in creating a new co-culture fermentation filtrate;

[0035] (5) Transcriptome sequencing analysis screened out 1029 genes that were affected by the treatment of Fusarium graminearum with co-culture fermentation filtrate specific to other treatment groups. It was found that the expression levels of related genes such as chitin synthase and cellular P450 monooxygenase in Fusarium graminearum treated with co-culture fermentation filtrate changed compared with those in single culture, indicating that the co-culture fermentation filtrate may inhibit Fusarium graminearum by affecting related biological functions such as chitin synthesis, mycelial growth and development, and MAPK signaling pathway.

[0036] (6) In addition to chitin, the basic components of fungal cuticles also include chitosan and β-glucan. After chitosan is degraded by chitosanase, it becomes small molecular chitosan oligosaccharides with antibacterial ability. After β-glucanase degrades β-glucan, the oligosaccharides produced can also induce the production of other disease-resistant enzymes, thereby improving the disease resistance of plants. Enzyme activity assays found that the activities of β-glucanase and chitosanase in the co-culture fermentation filtrate were 497.53U / L and 269.86U / L, respectively, which were higher than those of Trichoderma and Metarhizium cultured alone. Therefore, the increased activities of chitinase, β-glucanase and chitosanase after co-culture have a synergistic effect on antibacterial activity.

[0037] (7) In addition to antibacterial substances, in the fermentation filtrate of Trichoderma monoculture as the control group and the co-culture group as the treatment group, it was found that the synthesis of zeatin in the metabolic pathway had undergone significant changes. Studies have shown that zeatin can be used as a plant growth regulator to help crops increase yields and resist diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings:

[0039] Figure 1 is the growth rate of different Trichoderma on PDA plate;

[0040] Figure 2 It is the Trichoderma-Metarhizium M3 plate interaction;

[0041] Figure 3 This is the morphological observation of Trichoderma strain D1;

[0042] Figure 4 is the phylogenetic tree of Trichoderma strain D1;

[0043] Figure 5 The spore content of Metarhizium anisopliae at different inoculation ratios;

[0044] Figure 6 The spore content of Trichoderma D1 at different inoculation ratios;

[0045] Figure 7 The effect of Trichoderma D1 fermentation filtrate on the growth of Metarhizium anisopliae M3;

[0046] Figure 8 The effect of the fermentation filtrate of Metarhizium anisopliae M3 on the growth of Trichoderma;

[0047] Fig. 9 The effect of Trichoderma-Metarhizium co-culture on chitinase activity;

[0048] Fig.10 The effect of Trichoderma-Metarhizium co-culture on chitosanase activity;

[0049] Fig.11 The effect of Trichoderma-Metarhizium co-culture on β-glucanase activity;

[0050] Fig.12 OPLS-DA analysis diagram of M3_D1 VS M3, where (A) is M3_D1 VS M3 positive ion OPLS-DA, and (B) is M3_D1 VS M3 negative ion OPLS-DA;

[0051] Fig.13OPLS-DA analysis diagram of M3_D1 VS D1, where (A) is the positive ion OPLS-DA of M3_D1 VS D1, and (B) is the negative ion OPLS-DA of M3_D1 VS D1;

[0052] Fig.14 The differential metabolites in the negative ion mode of M3_D1 VS M3;

[0053] Fig.15 It is the differential metabolite of M3_D1 VS M3 positive ion mode;

[0054] Fig.16 The differential metabolites in the negative ion mode of M3_D1 VS D1;

[0055] Fig.17 It is the differential metabolite of M3_D1 VS D1 positive ion mode;

[0056] Fig.18 Bubble diagram for KEGG enrichment analysis of M3_D1 VS M3;

[0057] Fig.19 Bubble diagram for KEGG enrichment analysis of M3_D1 VS D1;

[0058] Fig. 20 Statistics of differentially expressed genes;

[0059] Fig.21 The Venn diagram of differentially expressed genes;

[0060] Fig. 22 Volcano plot of differentially expressed genes;

[0061] Fig.23 Comparison chart of qRT-PCR and RNA-Seq results. DETAILED DESCRIPTION

[0062] The present invention is described in detail below in conjunction with specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, several variations and improvements may be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0063] Example 1 Co-cultivation combination of Trichoderma and Metarhizium

[0064] 1. Test strains

[0065] As shown in Table 1, the Metarhizium of this embodiment was purchased from Ningbo Mingzhou Biological Company and the Institute of Microbiology, Chinese Academy of Sciences; as shown in Table 2, the Trichoderma of this embodiment was isolated from the forest soil of Xishuangbanna, Yunnan (99°56′~101°50′E, 21°08′~22°36′N) and stored in a laboratory refrigerator at -80°C. Trichoderma and Fusarium graminearum were provided by the Culture Collection Center of Shanghai Jiaotong University.

[0066] Table 1 Information on tested Metarhizium strains

[0067]

[0068] Table 2 Information of the tested Trichoderma strains

[0069]

[0070]

[0071]

[0072] D2 has been recorded in the literature "Preparation of High Water-Soluble Trichoderma Co-Culture Metabolite Powder and Its Effects on Seedling Emergence Rate and Growth of Crops. J. Fungi 2023, 9, 767. https: / / doi.org / 10.3390 / jof9070767", D3 has been recorded in the literature "Transcriptome Analysis of T. asperellum GDFS 1009 Revealed the Role of MUP1 Gene on the Methionine-Based Induction of Morphogenesis and Biological Control Activity. J. Fungi 2023, 9, 215. https: / / doi.org / 10.3390 / jof9020215", and D4 has been recorded in the literature "Effects of Trichoderma atroviride SG3403 and Bacillus subtilis 22 on the Biocontrol of Wheat Head Blight..J.Fungi 2022, 8, 1250. https: / / doi.org / 10.3390 / jof8121250, D7 has been recorded in the literature "Degradation effects on dichlorvos by a biocontrol strain, Trichoderma atroviride T23. Journal of Integrative Agriculture 2023, 22(9): 2746–2758", and D8 has been recorded in the literature "Development of a multiplex Q-PCR to detect Trichoderma harzianum Rifai strain T22 in plant roots. Journal of Microbiological Methods 2016, 121, 44-49, DOI: 10.1016 / j.mimet.2015.12.014".

[0073] 2. Screening of Metarhizium anisopliae (Plate confrontation test between Metarhizium anisopliae and Fusarium graminearum)

[0074] Use a sterilized 0.5 cm diameter pipette tip to take bacterial discs from the most vigorous mycelial growth of Metarhizium anisopliae and Fusarium graminearum activated for 5 days, inoculate 1 bacterial cake of Metarhizium anisopliae on PDA medium, let it grow for 7 days, and then inoculate Fusarium graminearum, with the two bacterial cakes 5 cm apart. Take a single Fusarium graminearum bacterial disc as a control, and culture it under light at a temperature of 28°C for 4 days. After 4 days, measure the colony area of ​​Fusarium graminearum and calculate the antibacterial rate of Fusarium graminearum. Each treatment is repeated 3 times.

[0075] Antibacterial rate calculation formula:

[0076]

[0077] Functional evaluation was performed based on the growth of different strains and their antibacterial activity against Fusarium graminearum. By comparing the antibacterial rates of seven strains of Metarhizium against Fusarium graminearum, it was found that, as shown in Table 3, the inhibition rate of Metarhizium M3 against Fusarium graminearum was 64.21%, which was significantly higher than that of other Metarhizium strains, indicating that from the evaluation of antibacterial ability, Metarhizium M3 had better antibacterial ability than other strains under the same conditions.

[0078] Table 3 Inhibition rate of Metarhizium anisopliae on Fusarium graminearum

[0079] serial number Antibacterial rate% serial number Antibacterial rate% M1 31.37±0.2587bc M5 35.7±7.866b M2 44.05±0.1262b M6 19.55±13.57c M3 64.21±8.365a M7 40.34±3.902b M4 40.62±4.933b

[0080] 3. Screening of Trichoderma

[0081] (1) Evaluation of Trichoderma growth ability

[0082] The thirty strains of Trichoderma in Table 2 were taken from the most vigorous mycelial growth place on the PDA medium using a sterilized pipette tip with a diameter of 0.5 cm and transferred to the PDA medium. They were cultured under light at a temperature of 28°C for 5 days, with general light at a light intensity of 200 Lux. The colony diameter was observed and recorded at 30 hours.

[0083] Due to the large difference in growth rates between Metarhizium and Trichoderma, it is necessary to consider the balance of their growth rates when used in conjunction. Thirty strains of Trichoderma with biocontrol functions were selected for evaluation of their growth ability and affinity with Metarhizium plates.

[0084] Figure 1 is the growth rate of different Trichoderma on PDA plate, Figure 1 It can be seen that there are significant differences in the growth rates of the 30 strains of Trichoderma on the plate. Among them, A1, B5, and C4 grow faster, while A4, D4, B4, D5, C2, A6, D6, B2, and D1 grow slower.

[0085] (2) Plate affinity test of Trichoderma and Metarhizium anisopliae

[0086] Use a sterilized pipette tip with a diameter of 0.5 cm to take a bacterial disc from the most vigorous growth of Trichoderma and Metarhizium mycelium after activation for 5 days, inoculate 1 bacterial cake of Metarhizium on PDA medium, let it grow for 7 days before inoculating Trichoderma, the two bacterial cakes are 5 cm apart, and use a single Trichoderma and Metarhizium bacterial disc as a control, and culture at a temperature of 28℃ for 4 days, with general light and a light intensity of 200Lux. Observe whether there is an antibacterial zone for both. Each treatment is repeated 3 times.

[0087] The 30 strains were subjected to plate confrontation experiments with Metarhizium anisopliae M3 to observe their affinity. Figure 2 It can be seen that there is no obvious inhibition zone between Metarhizium M3 and A1, A4, A7, A8, B2, B3, B4, B5, B7, B8, C6, D1, D6, and D7, and the plate shows non-antagonism; there is an obvious inhibition zone between Metarhizium M3 and A2, A6, B1, D2, and D5, and the plate shows antagonism.

[0088] 4. Screening of co-culture combination strains

[0089] Through the plate affinity test of Trichoderma and Metarhizium, five groups of Trichoderma-Metarhizium combinations showing antagonism on the plates and five groups of Trichoderma-Metarhizium combinations showing affinity on the plates were screened out. The optimal combination was evaluated and screened out based on the inhibition rate of ten Trichoderma strains against Fusarium graminearum and the plate inhibition rate of ten combinations in liquid culture against Fusarium graminearum.

[0090] (1) Plate confrontation test between Trichoderma and Fusarium graminearum

[0091] Use a sterilized 0.5 cm diameter pipette tip to take bacterial discs from the most vigorous places of Trichoderma and Fusarium graminearum hyphae growth after 4-5 days of activation, inoculate Trichoderma and Fusarium graminearum at the same time, with the two bacterial cakes 5 cm apart, and use a single Fusarium graminearum bacterial disc as a control, and culture at 28°C for 4 days under light conditions, with general light and light intensity of 200 Lux. After 4 days, measure the colony area of ​​Fusarium graminearum and calculate the antibacterial rate. The method is the same as above.

[0092] Table 4 shows the inhibition rate of Trichoderma against Fusarium graminearum. It can be seen from Table 4 that D5, A6, B1, D1, and A4 have better inhibition effects on Fusarium graminearum (inhibition rate>60%), among which A4 has the best inhibition effect, with an inhibition rate of 75.53%.

[0093] Table 4 Inhibition rate of Trichoderma against Fusarium graminearum

[0094] serial number Inhibition rate % serial number Inhibition rate % A4 75.53±0.04a D6 44.47±0.01cdef D1 69.30±0.11ab B2 41.20±0.04def A6 64.87±0.11abc A8 30.03±0.11efg D5 61.33±0.13abcd B8 28.60±0.09efg B5 59.77±0.02abcd A1 27.87±0.05fg B1 58.13±0.09abcd C6 27.60±0.03fg A2 56.27±11abcd B3 17.70±0.00g D2 51.33±0.02bcde

[0095] (2) Preparation of fermentation filtrate

[0096] Select five groups of Trichoderma-Met. anisopliae combinations with obvious antibacterial bands and affinity from the results of the plate affinity experiment. Use a sterilized 0.5 cm pipette tip to rub on the plate with Trichoderma and Met. anisopliae to make the tip full of spores. Put the tip full of spores into an ep tube containing 2 mL of sterile water to prepare a spore suspension. Adjust the concentration of the spore suspension to 10 6 spores / mL for subsequent inoculation. Take 1 mL of each of Trichoderma and Metarhizium seed fungi and culture them in 100 mL of sterile PD liquid culture medium at 28°C and a rotation speed of 180 r / min. Use the single Trichoderma and Metarhizium corresponding to the combination as controls.

[0097] (3) Antibacterial experiment

[0098] This study focuses on the metabolites in the co-culture of Trichoderma and Metarhizium, so it is necessary to filter out the spores. Take a certain amount of co-culture fermentation filtrate in a 50mL centrifuge tube, set the centrifuge speed to 8000rpm, take the supernatant after 5 minutes, and filter the supernatant with a 0.22 micron aqueous filter to remove the mycelium and spores to obtain the fermentation filtrate. Mix the co-culture fermentation filtrate with the liquid PDA medium in a volume ratio of 1:2, and solidify after mixing. Use a sterilized pipette tip to take a bacterial sheet (0.5 cm in diameter) from the most vigorous growth of Fusarium graminearum grown on PDA medium for 5 days and place it on the PDA medium containing filtrate to continue growing. The culture dish is cultured and grown for 3-4 days under the temperature condition of 28°C. General light illumination is adopted, and the light intensity is 200 Lux. The PDA plate with 30% PD medium added is used as a blank to inoculate Fusarium graminearum. After Fusarium graminearum grows for 3-4 days, the colony area of ​​Fusarium graminearum is determined, and the antibacterial rate of Fusarium graminearum is calculated in the same way as above. The inhibition rate of Trichoderma-Metarhizium anisopliae fermentation broth on Fusarium graminearum is shown in Table 5.

[0099] Table 5 Inhibition rate of Trichoderma-Metarhizium anisopliae fermentation broth on Fusarium graminearum

[0100] serial number Antibacterial rate% serial number Antibacterial rate% D1-M3 63.30±5.81a A2-M3 54.39±3.03ab B1 62.53±1.58a D1 51.74±3.63ab B3-M3 62.15±6.02a B2-M3 51.32±14.44ab A2 59.87±6.02a D2 49.87±0.83abc D5 58.71±0.86a D2-M3 37.00±13.29bcd B1-M3 59.42±3.71a B5 28.64±2.24cde D5-M3 57.39±3.15ab B5-M3 28.18±9.01def A6-M3 55.94±10.44ab B2 24.68±4.58def M3 55.32±7.34ab A4-M3 12.69±11.46ef A6 55.28±4.64ab A4 6.92±4.37f

[0101] As shown in Table 5, the antibacterial effects of the sterile fermentation filtrates of different combinations are different. Among the 10 co-culture combinations, the co-culture fermentation filtrates of Metarhizium M3 and Trichoderma D1, B3, B1, and D5 have good antibacterial effects on Fusarium graminearum, reaching 63.3%, 62.15%, 59.42%, and 57.39%, respectively. The antibacterial rate of Metarhizium M3 and Trichoderma D1 against Fusarium graminearum after co-culture is higher than that of Metarhizium M3 and Trichoderma D1 alone.

[0102] Based on the above tests, it can be found that D1 has a slower growth rate and its inhibition rate against Fusarium graminearum is second only to A4; D1 and M3 showed no obvious inhibition zone in the plate confrontation test, and the inhibition rate of the co-culture fermentation filtrate of the two was the highest among the ten combinations. Comprehensive evaluation shows that D1 is an ideal choice for co-culture with M3.

[0103] 5. Identification of Trichoderma

[0104] (1) Morphological identification of Trichoderma

[0105] Use a sterilized pipette tip with a diameter of 0.5 cm to pick up a bacterial plate from the area where mycelium is growing vigorously, transfer the bacterial plate to a new PDA medium for further growth, culture at 28°C for 3 days, and observe the morphological characteristics of the colonies.

[0106] like Figure 3 , where (A) is 400 times spore observation, (B) is the front of the colony, and (C) is the back of the colony. Morphological observation showed that the Trichoderma strain D1 covered the culture dish in 72 hours, and the colony color was green. It was white in the early stage of growth and then produced green spores. After 3 days of culture, the conidia were white and aggregated in the center. The surface of the colony formed radial, green granular conidia clusters after 5 days of culture. There were many conidia clusters on the edge, which were cushion-shaped, and the back was white. The hyphae were white, branched, smooth, and septate. The conidia cells were solitary, opposite, or in whorls on the conidiophores. The conidia cells were flask-shaped, narrow at the base, wide in the middle, tapering upwards, and colorless. There was an aromatic smell. Thick-walled spores were not seen.

[0107] (2) DNA extraction of Trichoderma species

[0108] The DNA of Metarhizium was extracted using the Fast Pure DNA extraction kit (purchased from Novozyme Biotechnology Co., Ltd.). In order to ensure the quality of DNA extraction, the mycelium of Metarhizium was frozen and machine-broken before DNA extraction: the mycelium of Metarhizium grown for 5 days was collected with a sterilized pipette tip with a diameter of 0.5 cm and placed in a 2 mL sterile tube with two sterilized grinding beads added in advance, immediately placed in liquid nitrogen for quick freezing, and then ground and machine-broken for DNA extraction.

[0109] (3) Molecular identification of Trichoderma species

[0110] The PCR reaction of ITS uses DNA as a template and the reaction system is shown in Table 6.

[0111] The sequence of primer F is: 5'-TCCTCCGCTTATTGATATGC-3' (SEQ ID NO. 1);

[0112] The sequence of primer R is: 5'-GGAAGTAAAAGTCGTAACAAGG-3' (SEQ ID NO. 2).

[0113] Table 6 Polymerase chain reaction system for amplifying Trichoderma sequences

[0114] Reagents Dosage (μL) template 0.5 Premix 12.5 Primer F 1(10μmol / L) Primer R 1(10μmol / L) <![CDATA[ddH 2 The]]> 10 Total 25

[0115] The DNA of D1 was mixed with universal primer ITS for PCR reaction. After sequencing, Blast comparison was performed on the NCBI website. The ITS standard provided by the website was selected to compare the strain sequence and construct a phylogenetic tree ( Figure 4 ), the results showed that strain D1 had the smallest distance with Trichoderma atroviride, indicating that the strain was relatively closely related to Trichoderma atroviride. Combined with its morphological characteristics, the strain was identified as Trichoderma atroviride.

[0116] The dark green Trichoderma (Trichoderma atroviride) CJCCSJ-F-KY10039 was deposited in the China Center for Type Culture Collection (CCTCC) on July 19, 2022. The preservation address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China Center for Type Culture Collection, Wuhan University, and the preservation number is: CCTCC No.M 20221135.

[0117] 6. Effect of inoculation ratio on the filtrate of Trichoderma-Metarhizium co-culture fermentation

[0118] Use a sterilized pipette tip to rub on the plate with Trichoderma and Metarhizium to make the tip full of spores. Place the tip full of spores into an eppendorf tube containing 2 mL of sterile water to prepare a spore suspension. Adjust the spore concentration in the spore suspension to 10 6 / mL for subsequent inoculation. Trichoderma and Metarhizium anisopliae were inoculated into sterilized PD medium at inoculation rates of 1:1, 1:100, 1:200, and 1:500 to prepare co-culture medium, with the corresponding single Trichoderma and Metarhizium anisopliae as controls, and cultured at 28°C and 180r / min for 5 days.

[0119] The inhibition rate of Fusarium graminearum was determined by inoculating Trichoderma D1 and Metarhizium anisopliae M3 at different ratios. Table 7 shows the inhibition rate of co-cultured fermentation broth inoculated at different ratios on Fusarium graminearum. It can be seen from Table 7 that when the inoculation ratio is 1:1, D1-M3 has the highest inhibition rate on Fusarium graminearum, with an inhibition rate of 63.3%; when the inoculation ratio is 1:200, the inhibition rate on Fusarium graminearum is the lowest, with an inhibition rate of 31.6%.

[0120] Table 7 Inhibition rate of co-culture fermentation broth inoculated at different ratios on Fusarium graminearum

[0121] serial number Antibacterial rate% serial number Antibacterial rate% 1:1 63.3±5.81a 1:500 45.39±1.54bcd M3 55.32±7.34ab 1:100 36.30±2.97cd D1 51.73±3.63abc 1:200 31.60±10.47d

[0122] Figure 5 The spore content of Metarhizium anisopliae at different inoculation ratios; Figure 6 The spore content of Trichoderma D1 at different inoculation ratios. Figure 5 and Figure 6 The results showed that different inoculation ratios had an effect on the spore production of Metarhizium and Trichoderma. When the inoculation ratio was 1:200, the spore production of Metarhizium was the largest ( Figure 5 ), when the inoculation ratio is 1:1, the spore production of Trichoderma is the largest ( Figure 6 ).

[0123] Based on the comprehensive inhibition rate and the effect of co-culture on the spore quantity of the two, the inoculation ratio of the two was determined to be 1:1, and subsequent co-culture experiments were carried out.

[0124] The present invention screened and obtained strains of Trichoderma and Metarhizium that have a certain antagonistic effect on Fusarium graminearum. Starting from the respective growth rates of Metarhizium graminearum and Trichoderma, the affinity of plate confrontation and liquid co-culture, and the antibacterial rate to Fusarium graminearum, a combination of the fastest growing Metarhizium, the slower growing Trichoderma, the affinity shown on the plate of the two strains, and the antibacterial effect of the co-culture fermentation liquid filtrate on Fusarium graminearum was selected. The main purpose of screening the combination according to the growth rate of the two strains is to try to avoid Trichoderma from fully utilizing the space and nutrients during the culture process, thereby causing Metarhizium graminearum to be unable to grow normally. When screening the affinity of the two strains, this study not only connected Trichoderma and Metarhizium graminearum to the plate for affinity observation, but also co-cultured the two in liquid to screen the strain combination suitable for co-culture. When evaluating the antibacterial rate of the co-culture combination fermentation broth, five combinations of plate-showing antagonism (two bacteria have antibacterial bands) and plate-showing affinity (two bacteria have no obvious antibacterial bands) were considered. The results showed that the co-culture fermentation broth filtrate of the combination of Trichoderma D1 and Metarhizium M3, which showed affinity on the plate, had the highest antibacterial rate.

[0125] From the above test results, it can be seen that Trichoderma D1 and Metarhizium M3 are the best combination of bacteria for co-culture.

[0126] Inoculation order and inoculation ratio are often considered in mixed fermentation. After determining the strain combination, this study inoculated Trichoderma and Metarhizium anisopliae at different times and Trichoderma-Metarhizium at different inoculation ratios to maximize the inhibition rate of Fusarium graminearum after co-culture of the two and to explore the growth of spores of the two during co-culture. After inoculation at different times, although the inhibition rate of Fusarium graminearum was the highest when Trichoderma was inoculated 24 hours after Metarhizium inoculation, the difference in inhibition rate of Fusarium graminearum with simultaneous inoculation was not significant. From the perspective of actual production, simultaneous inoculation can reduce the possibility of operation and contamination in production.

[0127] Example 2 Effect of Trichoderma-Metarhizium co-culture fermentation broth on the normal growth and development of the spores of the two fungi

[0128] 1. Effects of Trichoderma fermentation filtrate on spore production and growth of Metarhizium anisopliae

[0129] (1) Effect of Trichoderma fermentation filtrate on the number of spores produced by Metarhizium anisopliae

[0130] Mix the Trichoderma fermentation filtrate and sterilized PD medium in a ratio of 1:2, and use a blank PD medium with sterile water as a control, and transfer to a 50mL conical flask (30mL / 50mL). Rub the sterilized pipette tip on the plate with Metarhizium anisopliae to make the tip full of spores, and put the pipette tip full of spores into an ep tube containing 2mL of sterile water to make a spore suspension. Adjust the spore concentration in the spore suspension to 10 6 / mL for subsequent inoculation. Take 1mL and add it to the culture medium mixed with Trichoderma fermentation filtrate-PD. After culturing for 2-3 days at a temperature of 28°C and a rotation speed of 180rpm, count the number of Metarhizium spores. Each treatment was repeated 3 times to determine whether the Trichoderma fermentation broth had an effect on the spore production of Metarhizium.

[0131] The Trichoderma fermentation filtrate can affect the number of spores produced by Metarhizium. After two days of culture, the number of Metarhizium spores was counted using a hemocytometer. The number of Metarhizium spores in the mixed liquid of Trichoderma fermentation filtrate and PD medium reached 1.25×10 5 spores / mL, which is higher than the spore content of Metarhizium anisopliae in the mixed liquid of sterile water and PD medium (4×10 4 spores / mL), indicating that the fermentation filtrate of Trichoderma can promote the spore production of Metarhizium.

[0132] (2) Effect of Trichoderma fermentation filtrate on the growth of Metarhizium anisopliae spores

[0133] Take a 2 mL sterile tube, add 1 mL of PD medium, and then add 1 mL of Trichoderma fermentation filtrate, sterile water and PD medium respectively, and mix well. 6A spore suspension of Metarhizium anisopliae with spores / mL was added to the culture medium containing Trichoderma fermentation filtrate. After culturing for 2-3 days (28°C, 180rpm), the spore germination and mycelial growth of Metarhizium anisopliae on the slide were observed. Each treatment was repeated 3 times.

[0134] Figure 7 This is the result of observation under a 400x microscope. Figure 7 As shown, in both treatments, the conidia of Metarhizium anisopliae were able to germinate and form hyphae, indicating that Trichoderma metabolites had no obvious inhibitory effect on the spore germination of Metarhizium anisopliae.

[0135] 2. Effects of Metarhizium anisopliae fermentation filtrate on the spore production and growth of Trichoderma

[0136] Perform the same operation as above, exchanging Metarhizium anisopliae and Trichoderma spp.

[0137] (1) Effect of Metarhizium anisopliae fermentation filtrate on the number of spores produced by Trichoderma

[0138] The culture fluid of Metarhizium anisopliae may have affected the normal growth and development of Trichoderma. The spore content of Trichoderma cultured in PD medium with the addition of Metarhizium anisopliae fermentation filtrate was 3.55×10 5 spores / mL, which is slightly lower than the spore content of Trichoderma spores cultured in PD medium (1.05×10 6 spores / mL), indicating that the fermentation filtrate of Metarhizium anisopliae has a certain degree of inhibition on the spore production of Trichoderma. This may be because the Metarhizium anisopliae metabolic filtrate produces secondary metabolites that inhibit the spore production of Trichoderma or the pH of the Metarhizium anisopliae fermentation filtrate is not suitable for the growth of Trichoderma.

[0139] (2) Effect of Metarhizium anisopliae fermentation filtrate on Trichoderma spore germination

[0140] Effect of Metarhizium anisopliae fermentation filtrate on Trichoderma sporulation Figure 8 In both treatments, the conidia of Trichoderma could germinate and form hyphae, indicating that the metabolites of Metarhizium anisopliae had no adverse effect on the spore germination of Trichoderma.

[0141] Example 3 Determination of enzyme activity in Trichoderma-Metarhizium co-culture fermentation broth and co-culture fermentation filtrate

[0142] The method of Example 1 was used to prepare Trichoderma D1-Metarhizium M3 co-culture fermentation broth, Trichoderma single culture fermentation filtrate and Metarhizium single culture fermentation filtrate, and the assay was performed according to the method described in the specification. The chitosanase activity in the fermentation broth was assayed by Beijing Zhongke Optical Analysis Chemical Technology Research Institute (Chemical Laboratory). The β-glucanase activity was assayed according to the method described in the specification.

[0143] 1. Determination of chitinase activity in the fermentation filtrate of Trichoderma-Metarhizium co-culture

[0144] Chitinase extraction kit (purchased from SolarBio) was used to determine the chitinase activity in the fermentation filtrate according to the instructions. Fig. 9 As shown, the chitinase activity of the Trichoderma-Metarhizium co-culture fermentation filtrate was significantly higher than that of the Trichoderma monoculture fermentation filtrate and the Metarhizium monoculture fermentation filtrate (242.72 IU / L>226.15 IU / L>222.95 IU / L).

[0145] 2. Determination of chitosanase activity in the filtrate of Trichoderma-Metarhizium co-culture fermentation

[0146] like Fig.10 As shown, the chitosanase activity of the fermentation filtrate of Trichoderma-Metarhizium co-culture was higher than that of Metarhizium culture alone (269.86U / L>246.62U / L>234.52U / L).

[0147] 3. Determination of β-glucanase activity in Trichoderma-Metarhizium co-culture fermentation filtrate

[0148] like Fig.11 As shown, the β-glucanase activity in the metabolic fluid of Trichoderma-Metarhizium co-culture was significantly higher than that in Trichoderma culture alone (497.53U / L>484.57U / L>464.05U / L).

[0149] In summary, chitinase is an enzyme that can destroy the fungal cell wall composed of chitin compounds. The fermentation filtrate after co-culture is significantly higher than that of Trichoderma and Metarhizium alone, indicating that it may achieve better antibacterial effect by degrading the cell wall of Fusarium graminearum. In addition to chitin, the basic components of fungal cuticle also include chitosan and β-glucan. After chitosanase is degraded, chitosan is a small molecule chitosan oligosaccharide with antibacterial ability. The oligosaccharide produced by β-glucanase after β-glucanase degrades β-glucan can also induce the production of other disease-related enzyme systems, thereby improving the disease resistance of plants. Enzyme activity assays found that the activities of β-glucanase and chitosanase in the co-culture fermentation filtrate were higher than those of Trichoderma and Metarhizium alone. Therefore, the increased activities of chitinase, β-glucanase and chitosanase after co-culture have a synergistic effect on antibacterial activity.

[0150] Example 4 Metabolome sequencing analysis of Trichoderma-Metarhizium co-culture fermentation filtrate

[0151] 1. Test strains

[0152] Metarhizium anisopliae B81755 was purchased from Ningbo Mingzhou Biological Company and Institute of Microbiology, Chinese Academy of Sciences. Trichoderma atroviride CJCCSJ-F-KY10039 was isolated from forest soil in Xishuangbanna, Yunnan (99°56′~101°50′E, 21°08′~22°36′N), stored in a laboratory refrigerator at -80°C, and deposited in China Center for Type Culture Collection (CCTCC) on July 19, 2022. The deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China Center for Type Culture Collection, Wuhan University, and the deposit number is: CCTCC No.M 20221135. Trichoderma and Fusarium graminearum were provided by the Culture Collection Center of Shanghai Jiao Tong University.

[0153] 2. Preparation of co-culture fermentation broth

[0154] Use a sterilized pipette tip to rub on the plate with Trichoderma and Metarhizium to make the tip full of spores. Place the tip full of spores into an eppendorf tube containing 2 mL of sterile water to prepare a spore suspension. Adjust the spore concentration in the spore suspension to 10 6 / mL for subsequent inoculation. Take 1mL and 100mL of sterile PD culture medium, the inoculation order of Trichoderma and Metarhizium is simultaneous inoculation, the two bacteria are inoculated at a 1:1 inoculation ratio, and the inoculation volume is 1mL; the culture conditions are 28℃, 180r / min, and after 5 days of culture, the co-culture fermentation liquid is placed in a centrifuge tube, and the supernatant is taken after centrifugation. The supernatant is filtered with a 0.22 micron aqueous phase filter membrane to completely remove the bacteria to prepare the Trichoderma-Metalrhizium co-culture fermentation liquid (M3_D1) for use.

[0155] 3. Preparation of control and treated fermentation broth

[0156] The preparation methods of Trichoderma monoculture fermentation filtrate (D1), Metarhizium monoculture fermentation filtrate (M3), and blank control (30% PD culture medium) are the same as above.

[0157] The samples obtained above were rapidly cooled with liquid nitrogen and stored in a refrigerator at -80°C. The obtained samples were sent to Shanghai Paisono Biotechnology Co., Ltd. for metabolome sequencing analysis.

[0158] 3. Metabolome sequencing analysis of Trichoderma-Metarhizium co-culture fermentation filtrate

[0159] (1) OPLS-DA analysis

[0160] Fig.12OPLS-DA analysis diagram of M3_D1 VS M3, where (A) is M3_D1 VS M3 positive ion OPLS-DA, and (B) is M3_D1 VS M3 negative ion OPLS-DA; Fig.13 OPLS-DA analysis diagram of M3_D1 VSD1, where (A) is the positive ion OPLS-DA of M3_D1VS D1, and (B) is the negative ion OPLS-DA of M3_D1 VS D1. Fig.12 The square in the middle represents M3_D1, and the circle represents M3; Fig.13 The square represents M3_D1, and the circle represents D1. Fig.12 and Fig.13 As shown in the figure, the co-culture fermentation filtrates were distributed in different quadrants from the single culture fermentation filtrates. The inter-group variation of M3_D1 VS M3 and M3_D1 VS D1 was large, and the intra-group variation was small. Therefore, the variable weight value (VIP value) obtained by the OPLS-DA model has the influence strength and explanatory power for the classification and discrimination of each group of samples. The OPLS-DA VIP>1 and P value<0.05 can be used as the screening criteria for significant difference metabolites.

[0161] (2) Analysis of differential metabolites

[0162] Table 8 Metabolome sequencing screening results

[0163] Comparison samples Total number of significantly different metabolites Total number of significantly upregulated metabolites Total number of metabolites significantly downregulated M3_D1 VS M3 241 91 150 M3_D1 VS D1 78 63 15

[0164] As shown in Table 8, Metarhizium anisopliae was used as the control group and co-culture was used as the treatment group. A total of 241 differential metabolites (DEMs) with VIP greater than 1 and P less than 0.05 were screened, including 91 up-regulated metabolites and 150 down-regulated metabolites; Trichoderma was used as the control group and co-culture was used as the treatment group. A total of 78 differential metabolites (DEMs) with VIP>1 and P<0.05 were screened, including 63 up-regulated metabolites and 15 down-regulated metabolites.

[0165] It can be found that the differential metabolites screened out with Metarhizium as the control group are more than those with Trichoderma as the control group, which indicates that the co-cultivation process has a greater impact on Metarhizium.

[0166] In order to facilitate the understanding of which differential metabolites were produced by co-culture compared with mono-culture, the significantly differential metabolites of the control group, with Metarhizium anisopliae M3 mono-culture fermentation and Trichoderma dermatidis D1 mono-culture fermentation, and the co-culture group as the treatment group were normalized, and the cluster heat map was created using Origin software. Figure 14-17 This is the result diagram of OPLS-DA analysis.

[0167] Depend on Fig.14 , Fig.15The changes in the metabolites of M3_D1 VS M3 were intuitively illustrated: in the negative ion mode, 37 significantly up-regulated metabolites were detected, including azacitidine, 2'-hydroxy-4'-methoxyacetophenone, trans-1,4-cyclohexanedicarboxylic acid, and isothiocyanate, and 40 significantly down-regulated metabolites were detected, including clofibric acid acyl-β-D-glucuronide, mycophenolic acid, podophyllotoxin acetate, and rhamnetin; in the positive ion mode, 54 significantly up-regulated metabolites were detected, including short bromamine, O-toluidine, cyclohexylamine, indole, γ-aminobutyric acid, adenosine, N-α-acetyl-L-ornithine, and lupine, and 110 significantly down-regulated metabolites were detected, including fenugreek alkaloids and pretilachlor.

[0168] In positive ion mode: indole can be used as an antibacterial substance against bacterial wilt; 4-aminobutyric acid (Gaba) can be obtained through plant enrichment method, microbial fermentation method and other methods, and can be used as a plant growth regulator to help plants resist adversity; osthole, as a mature pesticide currently put on the market, can prevent and control peach brown rot, rice sheath blight, rice blast, wheat powdery mildew, and cucumber powdery mildew; penicillic acid has a growth inhibitory effect on wheat sheath blight pathogenic fungi and rice sheath blight pathogenic fungi; studies have shown that the addition of exogenous betaine can improve apple's resistance to apple leaf blight, and preliminarily show that betaine plays a certain role in participating in watermelon's resistance to wilt.

[0169] Depend on Fig.16 , Fig.17 The changes in the metabolites of M3_D1 VS D1 were shown as follows: in the negative ion mode, 23 metabolites such as benzoic acid, 2'-hydroxy-4'-methoxyacetophenone, and cyclohexanesulfamic acid were significantly upregulated, and 4 metabolites such as podophyllotoxin acetate, thymol-β-d-glucoside, DL-maleic acid, and malonic acid were significantly downregulated; in the positive ion mode, 40 differential metabolites such as butacal, lupepine, spiroxamine, deoxyadenosine, γ-aminobutyric acid, and dodecacyclic morpholine were significantly upregulated, and 11 metabolites such as tert-butylamino and isopentanin were significantly downregulated.

[0170] In negative ion mode: the metabolite isothiocyanate is upregulated. Data show that isothiocyanate, a key decomposition product of glucose in rapeseed, has anti-plant pathogenic fungi. In recent years, there have been many studies on the mechanism of isothiocyanate-induced cell apoptosis. Some key steps in the apoptosis pathway have also been shown to be affected by isothiocyanate: isothiocyanate may induce apoptosis through the JNK / MAPK signaling pathway. The transcriptome sequencing analysis in this chapter also found that the treatment of co-culture fermentation filtrate caused changes in the MAPK signaling pathway of Fusarium graminearum; Liu Feng et al. found that the synergistic application of curcumin and phloretin had a scientific study on the antibacterial effect of Staphylococcus aureus;

[0171] In the positive ion mode, the systemic sterol biosynthesis inhibitor spirocyclamide can be used to control powdery mildew, and dodecacycline can also be used as a fungicide; dodecanoic acid has a significant inhibitory effect on the radial growth of mycelium, the production of zoosporangia, and the formation and germination of zoospores.

[0172] (3) Metabolite pathways and classification annotations

[0173] KEGG pathway annotation: The metabolites of M3_D1 VS D1 and M3_D1 VS M3 were selected for pathway annotation analysis based on the P value using the KEGG Pathway database.

[0174] Depend on Fig.18 It can be seen that the metabolites of M3_D1 VS M3 can be divided into five categories: the first category is metabolism, including 7 second-level categories: global and overview, amino acid metabolism, biosynthesis of other secondary metabolites, carbohydrate metabolism, metabolism of other amino acids, energy metabolism, biodegradation and metabolism of xenobiotics, of which 33 metabolites are involved in the global and overview, 32 metabolites are involved in amino acid metabolism, 19 metabolites are involved in the biosynthesis of other secondary metabolites, 8 metabolites are involved in carbohydrate metabolism, 5 metabolites are involved in the metabolism of other amino acids, 4 metabolites are involved in energy metabolism, and 3 metabolites are involved in the biodegradation and metabolism of xenobiotics. The second category is the tissue system, including 4 second-level categories: digestive system, endocrine system, circulatory system, and nervous system, of which 17 metabolites are involved in the digestive system, 8 metabolites are involved in the endocrine system, 2 metabolites are involved in the circulatory system, and 4 metabolites are involved in the nervous system. The third category is environmental information processing, including three 2-level classifications of membrane transport, signal molecules and interactions, and signal transduction, of which 20 metabolites are involved in membrane transport, 6 metabolites are involved in signal transduction, and 6 metabolites are involved in signal molecules and interactions. The fourth category is genetic information processing, including translation, folding, classification, and degradation, two 2-level classifications, of which 9 metabolites are involved in the translation pathway, and 2 metabolites are involved in the folding, classification, and degradation pathway. The fifth category is cellular processes, including a 2-level classification pathway of cell community-prokaryotes, with 5 metabolites involved in this pathway.

[0175] KEGG enrichment analysis showed that after co-cultivation of Metarhizium and Trichoderma, the following pathways underwent significant changes compared with Metarhizium alone fermentation: metabolic pathways: amino acid biosynthesis, alanine, aspartate and glutamate metabolism, biosynthesis of each secondary metabolite, tropane, piperidine and pyridine alkaloids biosynthesis, histidine metabolism, citric acid cycle (TCA cycle) involved in material and energy metabolism pathways, D-arginine and D-ornithine metabolism, valine, leucine and isoleucine biosynthesis, 2-oxocarboxylic acid metabolism, cysteine ​​and methionine metabolism, D-alanine metabolism, C5-branched-chain dicarboxylic acid metabolism; environmental information processing: ABC transporters, neuroactive ligand and receptor interactions, mTOR signaling pathway, cAMP signaling pathway underwent significant changes; tissue system: mineral absorption, adipocyte lipolysis regulation underwent significant changes; genetic information processing: aminoacyl-tRNA biosynthesis underwent significant changes; cellular process: quorum sensing regulatory system underwent changes.

[0176] The results are as follows Fig.19The metabolites of D1_M3 VS D1 can be divided into three categories: the first category is environmental information processing, including two second-level categories of signal transduction and signaling molecules and interactions, of which three metabolites are involved in signal transduction pathways and four metabolites are involved in cross-signaling molecules and interactions; the second category is organizational systems, including one second-level category of nervous system, of which two metabolites are involved in nervous system; the third category is metabolism, including amino acid metabolism, xenobiotics biodegradation and metabolism, carbohydrate metabolism, metabolism of other aminoacids, and metabolism of terpenoids and polyketides. Global and overview There are 6 second-level classifications including 3 metabolites involved in the metabolic pathways of amino acids, 5 metabolites involved in the biodegradation and metabolic pathways of xenobiotics, 2 metabolites involved in the metabolic pathways of other amino acids, 4 metabolites involved in the global and overview maps, and 2 metabolites involved in the metabolic pathways of terpenoids and polyketides.

[0177] KEGG enrichment analysis showed that after co-cultivation of Metarhizium and Trichoderma, the following "metabolic" pathways underwent significant changes compared with Trichoderma alone: ​​alanine, aspartate and glutamate metabolism, butyrate metabolism, cysteine ​​and methionine metabolism, caprolactam degradation, amino acid biosynthesis, β-alanine metabolism, aminobenzoate degradation, and zeatin biosynthesis; significant changes occurred in the ligand-receptor interaction of neuroactive factors in environmental information processing and the signaling pathway of cAMP; and significant changes occurred in the synaptic vesicle cycle in the tissue system.

[0178] In addition to antibacterial substances, in the control group and the co-culture treatment group using the fermentation filtrate of Trichoderma monoculture as the control group, it was also found that the synthesis of zeatin had undergone significant changes in the metabolic pathway. Studies have shown that zeatin can be used as a plant growth regulator to help crops increase yields and resist diseases.

[0179] Example 5 Transcriptome Sequencing Analysis of Fusarium graminearum

[0180] 1. Screening of simple compounding ratios of Trichoderma and Metarhizium anisopliae single culture fermentation filtrates

[0181] The fermentation filtrates of Trichoderma and Metarhizium monoculture were compounded according to Table 9 (the total amount of liquid in each treatment was 6.93 mL), and each treatment was mixed with a liquid PDA medium (50°C) at a ratio of 1:2. A bacterial disc (0.5 cm in diameter) was taken from the place where the mycelium of Fusarium graminearum activated for 5 days on the PDA plate grew vigorously, and it was inoculated on the PDA plate containing the fermentation filtrate. The PDA plate with 30% PD medium was used as a blank and the Fusarium graminearum cake was inoculated as a control. After culturing at a temperature of 28°C and light for 3-4 days, the inhibition rate of Fusarium graminearum was calculated, and the appropriate compounding ratio was selected according to the inhibition rate.

[0182] Table 9 Combination design of different compounding ratios of Trichoderma D1 and Metarhizium anisopliae M3 single culture fermentation filtrate

[0183] deal with Volume ratio 1 Trichoderma fermentation filtrate: Metarhizium fermentation filtrate = 1:1 2 Trichoderma fermentation filtrate: Metarhizium fermentation filtrate = 2:1 3 Trichoderma fermentation filtrate: Metarhizium fermentation filtrate = 1:2 4 Trichoderma fermentation filtrate: Metarhizium fermentation filtrate = 1:5 5 Trichoderma fermentation filtrate: Metarhizium fermentation filtrate = 5:1 6 Trichoderma fermentation filtrate: Metarhizium fermentation filtrate = 1:10 7 Trichoderma fermentation filtrate: Metarhizium fermentation filtrate = 10:1 8 Trichoderma fermentation filtrate: Metarhizium fermentation filtrate = 20:1 9 Trichoderma fermentation filtrate: Metarhizium fermentation filtrate = 1:20 Comparison 6.93 mL of 30% PD

[0184] The monoculture fermentation filtrates of Trichoderma and Metarhizium were simply compounded. Table 10 shows the antibacterial rate of the monoculture fermentation filtrates of Trichoderma and Metarhizium against Fusarium graminearum. It can be seen from Table 10 that when the monoculture fermentation filtrates of Trichoderma and Metarhizium were compounded in a ratio of 1:1, the antibacterial rate against Fusarium graminearum was the highest, which was 61.1%. Therefore, the monoculture fermentation filtrates of Trichoderma and Metarhizium were compounded in a ratio of 1:1 for subsequent transcriptome sequencing.

[0185] Table 10 Inhibition rate of simple compound fermentation filtrate of Trichoderma and Metarhizium against Fusarium graminearum

[0186] deal with Antibacterial rate% deal with Antibacterial rate% 1 61.10±0.25a 3 55.07±1.62ab 7 58.66±0.94a 5 53.08±1.38ab 2 57.21±2.95a 6 50.42±3.43ab 9 56.75±0.73ab 8 39.08±7.41b 4 55.62±16.62ab

[0187] 2. Transcriptome processing settings

[0188] A total of 5 treatments were set up, namely: treatment of Fusarium graminearum with fermentation filtrate of Trichoderma monoculture (abbreviated as: D1, the same below), treatment of Fusarium graminearum with fermentation filtrate of Metarhizium monoculture (abbreviated as: M3, the same below), treatment of Fusarium graminearum with fermentation filtrate of Trichoderma and Metarhizium co-culture (abbreviated as: M3_D1, the same below), treatment of Fusarium graminearum with simple combination of fermentation filtrate of Trichoderma and Metarhizium (abbreviated as: M3+D1, the same below), and blank control with 30% PD culture medium treated with Fusarium graminearum (abbreviated as: CK, the same below).

[0189] 3. Transcriptome Sample Preparation

[0190] A bacterial cake was cut from the edge of Fusarium graminearum cultured on PDA medium for 5 days and inoculated into 50 mL YEPD medium. After culturing at 28°C and 180 rpm for 3 days, 25 mL of 30% PD medium was added to the co-cultured fermentation filtrate of Trichoderma-Metarhizium, the fermentation filtrate of Metarhizium, the fermentation filtrate of Trichoderma, and the simple compound fermentation filtrate of Trichoderma and Metarhizium, and cultured at 28°C and 180 rpm for 2 days. The obtained bacterial liquid was filtered to obtain the bacterial body, which was quickly cooled with liquid nitrogen and stored in a refrigerator at -80°C. Each treatment was repeated three times.

[0191] Thermo Fisher Scientific Total RNA Extraction Kit (TRIzol TM ) Total RNA was extracted from mycelial tissue. The quality of total RNA was detected by Agilent 2100 Bioanalyzer. The specific polyA structure of mRNA was used to purify mRNA in total RNA. The mRNA with polyA structure in total RNA was enriched by oligonucleotide (dT) magnetic beads. RNA was broken into fragments of about 300bp by ion fragmentation and reverse transcribed to obtain cDNA. The fragments were then PCR screened and amplified, and total RNA was extracted from mycelial tissue using a total RNA extraction kit. The quality of total RNA was detected by Agilent 2100 Bioanalyzer. The specific polyA structure of mRNA was used to purify mRNA in total RNA. The mRNA with polyA structure in total RNA was enriched by oligonucleotide (dT) magnetic beads. RNA was broken into fragments of about 300bp by ion fragmentation and reverse transcribed to obtain cDNA. The fragments were then PCR screened and amplified, and finally a library was established. These libraries were sequenced at both ends using next-generation sequencing technology based on the Illumina sequencing platform. Low-quality reads were deleted, and clean reads were used for subsequent analysis and mapped to the Fusarium graminearum reference genome (GCA_900044135.1_GZPH1RResV1_genome). RSEM obtained the read counts of each gene, and based on the expression results, the differentially expressed genes (DEGs) between the comparison groups were obtained. The differential analysis software was DEseq2, and the screening threshold was the absolute value of the FC logarithm with a base of 2 greater than or equal to 1 and a P value greater than 0.05. The databases used to annotate differentially expressed genes and compare the results were Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). TB tools were used to analyze the GO enrichment and KEGG enrichment of DEGs. The data was analyzed using the Paisono Cloud platform.

[0192] 4. Transcriptome sequencing

[0193] After transcriptome sequencing, the sequencing sequences were quality tested (Table 11), where CK1-CK3 were blank controls (30% PD medium treated with Fusarium graminearum). Among them, the percentage of Q30 bases exceeded 93.55%. This indicates that most clean data are consistent with the reference genome of Fusarium graminearum. Useful bases are all above 90%. The above results show that the RNA sequence data quality of all samples is high and can be used for subsequent data analysis.

[0194] Table 11 Summary of transcriptome data for each treatment

[0195] sample Raw readings Quality Control Readings Q30(％) Useful_bases(%) Total_Mapped CK1 49923446 46977368 94.7 94.09 44496275(94.72％) CK2 48077346 45266198 94.4 94.15 43263674(95.58％) CK3 40966990 38314420 95.33 93.52 36381094(94.95％) D1_1 43332918 40757210 95.14 94.05 3899521(95.69％) D1_2 39543324 37419704 94.29 94.62 35561907(95.04％) D1_3 40916244 38691152 94.96 94.56 36957918(95.52％) M3_1 43066492 40635706 94.55 94.35 37624539(92.59％) M3_2 52038492 48939198 94.17 94.04 46095858(94.19％) M3_3 49599758 46552144 94.3 93.85 44290572(95.14％) M3+D1_1 50164672 47218986 94.4 94.12 44989959(95.28％) M3+D1_2 45223330 42488760 93.88 93.95 38757191(91.22％) M3+D1_3 43641874 41155844 93.55 94.3 39203710(95.26％) M3_D1_1 47466252 44684026 94.39 94.13 4390286(96.88％) M3_D1_2 45100536 42518474 94.56 94.27 40818153(96.00％) M3_D1_3 45568082 43032480 94.8 94.43 41296130(95.97％)

[0196] 4. Annotation of differentially expressed genes (DEGs)

[0197] Fig. 20 For the statistics of differentially expressed genes, Fig.21 is the Venn diagram of differentially expressed genes, Figure 20-21It can be seen that compared with CK, 2154 DEGs were detected in the co-culture treatment, of which 1062 were up-regulated and 1092 were down-regulated. CK was used as the control group, M3_D1, M3+D1, M3 and D1 were used as treatment groups, and the differentially expressed genes of each comparison group were analyzed. There were 1029 differentially expressed genes in M3_D1 relative to CK. This study focused on which genes and metabolic pathways were specifically affected after co-culture, so the functions and GO enrichment analysis of the 1029 genes were analyzed. Among the 1029 differentially expressed genes, 384 were down-regulated and 645 were up-regulated, of which 114 had clear functional annotations. These gene functions were related to ATP-dependent RNA helicase, ribosomal protein, tRNA ligase, ribosomal biosynthesis protein, amino acid permease, chitin synthetase, mitochondrial carrier protein, alkaline protease, cytochrome P450 monooxygenase, etc. By screening the gene functions of Fusarium graminearum, 10 genes related to the pathogenicity and detoxification of Fusarium graminearum were found among the 1029 differentially expressed genes, including FGRAMPH1_ 01T12823 (FGSG_03412) was related to amino acid permease, FGRAMPH1_01T07827 (FGSG_10327) was related to chitin synthase, FGRAMPH1_01T19803 (FGSG_06226) was related to mitochondrial carrier protein, FGRAMPH1_01T02019 (FGSG_00806) was related to alkaline protease, and FGRAMPH1_01T04729 (FGSG_01959) was related to cytochrome P450 monooxygenase genes were upregulated. The genes affecting the MAPK signaling pathway include FGRAMPH1_01T11255 (FGSG_08948, FGSG_16939), FGRAMPH1_01T24437 (FGSG_07289), FGRAMPH1_01T20469 (FGSG_10787), and FGRAMPH1_01T19813 (FGSG_16620), among which FGRAMPH1_01T24437 and FGRAMPH1_01T04729 genes were downregulated, while the other genes were upregulated.

[0198] FGRAMPH1_01T12823 (FGSG_03412) is related to amino acid permease, which is related to amino acid transport. Liu Xinchun studied the biological functions of the amino acid permease genes MoGAP2, MoGAP1, and MoGAP3 of rice blast fungus and found that the mycelial growth and spore germination rate of the rice blast fungus with the MoGAP1 gene were reduced, the total number of spores was reduced, and the pathogenicity of the rice blast fungus lacking MoGAPs was weakened compared with the wild strain. Through the study of the amino acid permease gene GAP3 of corn anthracnose, it was found that it may affect the growth and development of corn anthracnose and the growth of melanin. After the corn anthracnose lacking the GAP3 gene was inoculated with corn, the lesions were few and small. In this study, it was found that after the treatment of Fusarium graminearum with the co-culture fermentation filtrate, the expression of FGRAMPH1_01T12823 was downregulated, which may affect the mycelial growth, spore germination, pigment production and pathogenicity of Fusarium graminearum.

[0199] The expression of FGRAMPH1_01T07827 (FGSG_10327) gene was down-regulated. This gene is related to chitin synthase, which is involved in the synthesis of chitin. Chitin is one of the main components of the cell wall of Fusarium graminearum. There are 8 chitin synthase genes in total. The eight genes may be associated with the chitin content, spore production ability, hyphal growth and development, sensitivity to fungicides and pathogenicity of Fusarium graminearum.

[0200] The expression level of FGRAMPH1_01T19803 (FGSG_06226) was down-regulated, which was related to mitochondrial carrier protein. Mitochondria are the main site of cellular respiration and may affect the growth and development of Fusarium graminearum and other physiological processes;

[0201] The FGRAMPH1_01T02019 (FGSG_00806) gene is related to alkaline protease. Ma Hongxiang et al. found that the pathogenicity of Fusarium asiatica strains was significantly positively correlated with the alkaline protease activity produced by the strains, and the alkaline protease produced by the strains may be related to the pathogenicity of the fungus. Ma Hongxiang et al. found that the pathogenicity of Fusarium asiatica strains was significantly positively correlated with the alkaline protease activity produced by the strains. Whether the downregulation of the expression of this gene in Fusarium graminearum may affect its pathogenicity requires further study; fungi usually use cytochrome P450 monooxygenase to detoxify plant toxins. P450 enzymes play an important role in various types of primary and secondary metabolism and the degradation of xenobiotics in eukaryotic organisms. Shin Ji Young et al. discovered new P450s that are specifically involved in the virulence and asexual and sexual development of Fusarium graminearum.

[0202] Most P450s may play a redundant role in the degradation of exogenous substances by Fusarium graminearum. Transcriptome sequencing analysis found that the expression level of the gene FGRAMPH1_01T04729 (FGSG_01959) related to cytochrome P450 monooxygenase was upregulated, which may indicate that the co-culture fermentation filtrate caused the ability of Fusarium graminearum to degrade exogenous substances.

[0203] 5. Enrichment analysis of differentially expressed genes (DEGs)

[0204] According to the GO database, DEGs were divided into three categories: biological process (BP), cellular component (CC), and molecular function (MF). There were 119 entries involving biological process, 40 entries involving cellular component, and 54 entries involving molecular function. The biological process involved transition metal ion homeostasis, negative regulation of nitrogen compound metabolism, carbohydrate derivative metabolism, organic matter transport, etc.; the cellular component involved vesicle coat, Golgi apparatus, nuclear membrane, ribosomal subunit, etc.; the molecular function involved passive transmembrane transport, active channel activity, and 5'-3' RNA polymerase activity. Fig. 22 It can be seen that the enrichment analysis of 10 genes related to the detoxification and pathogenicity of Fusarium graminearum found in the annotation of differentially expressed genes (DEGs) enriched a total of 26 pathways, of which 15 involved biological processes, namely, the synthesis of cell component organizations or organisms, cellular processes, the biosynthesis of organic nitrogen compounds, the metabolic process of nitrogen compounds, the process of macromolecular metabolism, the synthesis of organic matter, the biosynthesis of macromolecules, the biosynthesis of carbohydrate derivatives, the outer envelope structure organization, the cell wall organization, the primary metabolic process, the organic metabolic process, the metabolic process of carbohydrate derivatives, the cellular process; the anatomical structure within the cell, the organelles within the cell, the binding of the organelle to the inner membrane, the binding of the organelle to the cell membrane, and the organelle cytoplasm; the molecular functional processes include: the binding of organic cyclic compounds, the binding of heterocyclic compounds, the activity of methyltransferases, and the activity of transferases.

[0205] Example 3 RT-qPCR verification of transcriptome results of Fusarium graminearum

[0206] In order to prove whether the results of the transcriptome were correct, 12 genes were randomly selected for RT-qPCR verification. The sample preparation method and the extraction of total RNA from mycelium were the same as above.

[0207] The specific steps of single-stranded cDNA synthesis are as follows:

[0208] The following reaction mixture (Table 12) was added to a test tube in an ice bath.

[0209] Table 12 Reverse transcription reaction system 1

[0210] Reagents system Total RNA 1 μg Oligo(dT)(50μM) 1μL dNTP Mix (10mmol / L) 1μL

[0211] Add RNase free dH 2 0 to 10 μL, mix well and incubate at 65°C for 5 min, then quickly place on ice. Add the following reaction mixture (Table 13) to the test tube in the ice bath:

[0212] Table 13 Reverse transcription reaction system 2

[0213]

[0214]

[0215] The reaction mixture was incubated at 42°C for 30-60 min and then heated at 95°C for 5 min to terminate the reaction and obtain cDNA.

[0216] RT-qPCR primer design and synthesis of primers are shown in SEQ ID NO. 3-28, and the specific primers are shown in Table 14, which were completed by Qingke Biotechnology Co., Ltd.

[0217] Table 14 Primers used for relative fluorescence quantification

[0218]

[0219]

[0220] The prepared PCR reaction system (Table 15) was reacted in a Roche quantitative PCR instrument. Reaction procedure: 95°C pre-denaturation for 300s; 95°C denaturation for 10s, 60°C annealing for 30s, 40 cycles; 95°C melting for 10s, 65°C melting for 60s, 97°C melting for 1s; 37°C cooling for 30s. After the reaction, observe whether the trend of the melting curve is correct. According to the CT value detected by the real-time fluorescence quantitative PCR instrument, the relative expression of the gene of Fusarium graminearum treated with co-cultured fermentation filtrate in different treatments relative to the blank control Fusarium graminearum was calculated according to the 2-△△CT method.

[0221] Table 15 qPCR reaction system

[0222]

[0223] To verify the expression of DEGs in transcriptome sequencing, RT-qPCR was used to evaluate the expression levels of the above 12 genes in co-culture fermentation filtrate-treated Fusarium graminearum and 30% PD medium-treated Fusarium graminearum. The change trend of the 12 genes in RT-qPCR was the same as the up- and down-regulation trend in the transcriptome sequencing results ( Fig.23 ), indicating that the transcriptome data are trustworthy.

[0224] It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may make various changes or modifications within the scope of the claims, which do not affect the essence of the present invention. In the absence of conflict, the embodiments of the present application and the features in the embodiments may be combined with each other at will.

Claims

1. A method for inhibiting Fusarium graminearum by symbiotic fermentation of Trichoderma and Metarhizium anisopliae, characterized in that: The Trichoderma is Trichoderma atroviride CJCCSJ-F-KY10039, and its preservation number is CCTCC No.M20221135; the Metarhizium is Metarhizium anisopliae B81755.

2. A microbial bactericide, characterized in that: The microbial fungicide contains the symbiotic fermentation product of Trichoderma and Metarhizium; the Trichoderma is Trichoderma atroviride CJCCSJ-F-KY10039, and its preservation number is CCTCC No.M 20221135; the Metarhizium is Metarhizium anisopliae B81755.

3. The microbial bactericide according to claim 2, characterized in that: The symbiotic fermentation method comprises the following steps: S1. preparing spores of Trichoderma and Metarhizium anisopliae into spore suspensions respectively; S2. Take the spore suspensions of Trichoderma and Metarhizium and inoculate them into the culture medium for cultivation.

4. The microbial bactericide according to claim 3, characterized in that: The inoculation order of Trichoderma and Metarhizium in step S2 is simultaneous inoculation.

5. The microbial bactericide according to claim 3, characterized in that: The concentration of the spore suspension is 10 6 -2×10 6 spores / mL.

6. The microbial bactericide according to claim 3, characterized in that: The inoculation ratio of the inoculation in step S2 is 1:

1.

7. The microbial bactericide according to claim 3, characterized in that: The culture conditions include: a culture temperature of 25-30° C. and a rotation speed of 180-250 r / min.

8. The microbial bactericide according to claim 3, characterized in that: The culture medium includes PD medium.

9. The microbial bactericide according to claim 3, characterized in that: The inoculation amount of the inoculation is 1 / 100.

10. Use of the microbial fungicide according to any one of claims 2 to 9 in inhibiting Fusarium graminearum.

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