Talaromyces purpurogenus, strain fermentation liquor thereof and application of talaromyces purpurogenus in prevention and treatment of northern corn leaf blight

Abstract

The invention relates to the technical field of microorganisms, in particular to talaromyces purpurogenus, strain fermentation liquor of the talaromyces purpurogenus and application of the talaromyces purpurogenus to prevention and treatment of northern corn leaf blight. The invention provides biocontrol fungus fermentation liquor for preventing and treating northern corn leaf blight and application of the biocontrol fungus fermentation liquor. The preservation number of a strain of the biocontrol fungus fermentation liquor is CGMCC No.41911. The strain and the fermentation liquor thereof can completely inhibit various pathogenic bacteria, active substances in the fermentation liquor are relatively stable, the active validity period is long, the systemic property is relatively strong, the strain is not easy to degrade, high temperature resistance and microwave radiation resistance are realized, the antibacterial effect of chemical agents can be remarkably improved, and the effects of pesticide reduction and synergism are remarkably achieved when the strain and the fermentation liquor are mixed with chemical pesticides; therefore, the environmental safety problem and the drug resistance caused by chemical pesticides are obviously reduced.

Description

Technical Field

[0001] The present application relates to the field of microbial technology, and particularly to a fermentation broth of a biocontrol fungal strain for preventing and controlling northern leaf blight of maize and its application. Background Art

[0002] Currently, the widespread use of chemical pesticides in agriculture has led to significant problems such as the resistance of pathogenic bacteria and pests, residues in the soil, and the continuous decline of soil productivity. Therefore, beneficial microorganisms are receiving increasing attention as a safer alternative in the agricultural field. The exploration of new species of Talaromyces has brought more resources for biological control. The genus Talaromyces and its metabolites are widely used in biological control in agriculture.

[0003] Talaromyces purpureogenus belongs to the Ascomycota, Eurotiomycetes, Eurotiales, Trichocomaceae, and Talaromyces genus. Initially, the genus Talaromyces was the teleomorphic genus of the genus Penicillium. The anamorph stage was called Penicillium, and the teleomorphic stage was called Talaromyces. Considering the naming priority and the single-name nomenclature, since July 2011, Samson et al. have transferred most of the accepted species of the subgenus Biverticillium of Penicillium to the genus Talaromyces. The development and utilization of Talaromyces provide a new reserve force for the biological control of plant diseases. Talaromyces flavus and Talaromyces purpureogenus can be used for the biological control of agricultural and forestry plant pests. Kim et al. isolated Talaromyces flavus from ginseng seeds and it has antagonistic effects against plant pathogens such as Fusarium oxysporum, Rhizoctonia solani, Sclerotinia nivalis, Botrytis cinerea, and Phytophthora capsici; Talaromyces purpureogenus has good antibacterial effects against the pathogen of potato common scab and the pathogen of saffron stem rot. However, there is no report on the inhibitory effect of Talaromyces purpureogenus on Exserohilum turcicum. Northern corn leaf blight is caused by Exserohilum turcicum (Pass.) Leonard et Suggs. The current prevention and control system for northern corn leaf blight takes the breeding of resistant varieties and chemical agents as the core strategies, but both of these measures face significant challenges. In the application of disease-resistant varieties, the highly differentiated characteristics of physiological races of Exserohilum turcicum lead to the easy breakthrough of variety resistance, and coupled with the long breeding cycle of disease-resistant breeding, the actual prevention and control effect often shows regional decline. In the field of chemical control, although mainstream agents such as triazole fungicides like propiconazole and difenoconazole can quickly inhibit diseases, there are multiple risks such as a short application window period; pathogens are prone to generate drug resistance through gene mutations; long-term use leads to a decrease in soil microbial diversity and ecological safety problems such as excessive pesticide residues. Summary of the Invention

[0004] In view of this, the present application provides a fermentation broth of a biocontrol fungal strain for controlling northern corn leaf blight and its application. The strain and its fermentation broth can efficiently inhibit a variety of pathogens. The active substances in the fermentation broth are relatively stable, with a long active validity period, strong systemicity, not easily degradable, heat-resistant, and resistant to microwave radiation, and can significantly improve the antibacterial effect of chemical agents. Mixing with chemical pesticides has a significant effect of reducing pesticides and increasing efficiency.

[0005] In order to achieve the above invention purposes, the present application provides the following technical solutions:

[0006] The present application provides Talaromyces purpureogenus with a preservation number of CGMCC No. 41911.

[0007] The present application also provides a fermentation broth of the above-mentioned Talaromyces purpureogenus.

[0008] In some specific embodiments of the present application, the method for preparing the above fermentation broth includes: inoculating the above-mentioned Talaromyces purpureogenus in PDB for cultivation, filtering the obtained culture solution, and taking the filtrate to obtain the fermentation broth.

[0009] In some specific embodiments of the present application, for the above fermentation broth, the temperature of the cultivation is 26 °C, 27 °C, 28 °C, 29 °C or 30 °C.

[0010] In some specific embodiments of the present application, for the above fermentation broth, the rotation speed of the cultivation is 160 rpm, 170 rpm, 180 rpm, 190 rpm or 200 rpm.

[0011] In some specific embodiments of the present application, for the above fermentation broth, the cultivation time is 5 days, 6 days, 7 days, 8 days or 9 days.

[0012] In some specific embodiments of the present application, for the above fermentation broth, the pore size of the filter membrane for filtration is 0.22 µm.

[0013] The present application also provides the use of the above-mentioned Talaromyces purpureogenus or the above fermentation broth in any one of the following:

[0014] (i) Antagonizing pathogenic bacteria;

[0015] (ii) Preventing and controlling Exserohilum turcicum;

[0016] The pathogenic bacteria include Botrytis cinerea, Magnaporthe grisea, Exserohilum turcicum or Sclerotinia Sclerotiorum.

[0017] The present application also provides a composition, comprising:

[0018] (I) The above-mentioned Talaromyces purpureogenus or the above fermentation broth;

[0019] (II) Syringic acid tebuconazole or pyraclostrobin.

[0020] The present application also provides the use of the above-mentioned Talaromyces purpureogenus, the above fermentation broth or the above composition in the preparation of a biocontrol agent for preventing and controlling Exserohilum turcicum.

[0021] The present application also provides a biocontrol agent, comprising any one of the following:

[0022] (a) The above-mentioned Talaromyces purpureogenus or the above-mentioned fermentation broth;

[0023] (b) The above-mentioned composition.

[0024] The present application also provides a method for controlling Exserohilum turcicum, comprising using any one of the following:

[0025] (A) The above-mentioned Talaromyces purpureogenus;

[0026] (B) The above-mentioned fermentation broth;

[0027] (C) The above-mentioned composition;

[0028] (D) The above-mentioned biocontrol agent.

[0029] The present invention has the following beneficial effects:

[0030] As described in the background art, there are many defects in chemical control. Biocontrol not only has an inhibitory effect on pathogenic bacteria, but also is environmentally friendly and pollution-free. The present application provides a fermentation broth of a biocontrol fungal strain for controlling Exserohilum turcicum and its application, and at the same time systematically reveals the biocontrol functional characteristics of the fermentation broth of this biocontrol fungal strain for the first time, providing a breakthrough solution for the green prevention and control of Exserohilum turcicum. Specifically, by comparing the differences between Talaromyces purpureogenus strains FVV4 and FV11, it is found that the FVV4 strain can produce strong antibacterial substances, can cause the outflow of the contents of the confrontation hyphae cells to form a fan-shaped bulge in the confrontation culture, and the hyphae break and die in the later stage and lose their vitality. This phenomenon is discovered for the first time. At the same time, the fermentation broth of the FVV4 strain can completely inhibit a variety of pathogenic bacteria. The active substances in this fermentation broth are relatively stable, have a long active validity period, strong systemic absorption, are not easily degraded, are resistant to high temperature, and are resistant to microwave radiation. The above characteristics of the fermentation broth of the FVV4 strain have not been reported, and this application is the first report. The plate antibacterial test shows that the fermentation broth of the FVV4 strain can significantly improve the antibacterial effect of chemical agents. The field test shows that the control effect of the mixture of the fermentation broth of the FVV4 strain and chemical pesticides is higher than that of chemical agents, significantly playing the role of reducing the dosage of pesticides and increasing the efficiency. The role of reducing the dosage of pesticides and increasing the efficiency of the fermentation broth of the FVV4 strain is also discovered for the first time.

[0031] Biological deposit description

[0032] Biological material: FVV4, taxonomic name: Talaromyces purpureogenus, deposited with the China General Microbiological Culture Collection Center on April 8, 2025. The address of the deposit center is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing; the deposit number is CGMCC No. 41911.

[0033] In this application, FVV4 mentioned above refers to the strain with the preservation number of CGMCC No. 41911. Description of the Drawings

[0034] In order to more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art.

[0035] Figure 1 Showing the phylogenetic tree of the FV11 multi-gene

[0036] Figure 2 Showing the phylogenetic tree of the FVV4 multi-gene

[0037] Figure 3 Showing the confrontation between the FVV4 strain and Botrytis cinerea on a plate. Among them, the first row is the frontal colony morphology of the plate confrontation, and from left to right are the colony morphologies on the 4th to 8th days of the plate confrontation; the second row is the back colony morphology of the plate confrontation, and from left to right are the colony morphologies on the 4th to 8th days of the plate confrontation.

[0038] Figure 4 Showing the confrontation between the FV11 strain and Botrytis cinerea on a plate. Among them, the first row is the frontal colony morphology of the plate confrontation, and from left to right are the colony morphologies on the 4th to 8th days of the plate confrontation; the second row is the back colony morphology of the plate confrontation, and from left to right are the colony morphologies on the 4th to 8th days of the plate confrontation.

[0039] Figure 5 Showing the hyphal morphology of Botrytis cinerea and the FVV4 strain in the confrontation plate under a 4× microscope. Among them, from left to right are the hyphal morphologies on the 4th, 5th, and 6th days of the plate confrontation and the hyphal morphology of normal Botrytis cinerea.

[0040] Figure 6 Showing the hyphal morphology of Botrytis cinerea and the FVV4 strain in the confrontation plate under a 10× microscope. Among them, from left to right are the hyphal morphologies on the 4th, 5th, and 6th days of the plate confrontation and the hyphal morphology of normal Botrytis cinerea.

[0041] Figure 7 Showing the hyphal morphology of Botrytis cinerea and the FV11 strain in the confrontation plate under a 10× microscope. Among them, from left to right are the hyphal morphologies on the 4th, 5th, and 6th days of the plate confrontation and the hyphal morphology of normal Botrytis cinerea.

[0042] Figure 8 Showing the hyphal morphology of Exserohilum turcicum in the confrontation culture. Among them, CK shows the aerial hyphae and substrate hyphae of Exserohilum turcicum, A shows the aerial hyphae of Exserohilum turcicum on the 3rd day of confrontation and the substrate hyphae on the 5th day, and B shows the aerial hyphae of Exserohilum turcicum on the 4th day of confrontation and the substrate hyphae on the 6th day.

[0043] Figure 9 Show the confrontation culture of strain FVV4 and Exserohilum turcicum;

[0044] Figure 10 Show the hyphal morphology of Exserohilum turcicum after soaking observed under a microscope. Among them, from left to right are the hyphae of Exserohilum turcicum soaked in sterile water under a 40× microscope, the hyphae of Exserohilum turcicum soaked in the fermentation broth of strain FVV4 (including aerial hyphae and substrate hyphae) under a 40× microscope, the broken substrate hyphae of Exserohilum turcicum soaked in the fermentation broth of strain FVV4 under a 40× microscope, the broken aerial hyphae of Exserohilum turcicum soaked in the fermentation broth of strain FVV4 under a 40× microscope, and the hyphae gradually degraded after being soaked in the fermentation broth of strain FVV4 under a 40× microscope;

[0045] Figure 11 Show the hyphal morphology of Exserohilum turcicum after soaking observed under a microscope, which is the hyphal morphology of Exserohilum turcicum soaked in sterile water and the fermentation broth of strain FVV4 observed under a 10× microscope;

[0046] Figure 12 Show the growth of the hyphae of Exserohilum turcicum soaked in the fermentation broth of strain FVV4 and sterile water in a PDA plate. The three plates in the figure are three replicates;

[0047] Figure 13 Show the antibacterial situation of the fermentation broth of strain FVV4. Among them, in the first row from left to right are Botrytis cinerea and Botrytis cinerea after being treated with the fermentation broth, Exserohilum turcicum and Exserohilum turcicum after being treated with the fermentation broth; in the second row from left to right are Magnaporthe oryzae and Magnaporthe oryzae after being treated with the fermentation broth, Sclerotinia sclerotiorum and Sclerotinia sclerotiorum after being treated with the fermentation broth;

[0048] Figure 14 Show the antibacterial situation of the fermentation broth of strain FVV4 at different concentrations;

[0049] Figure 15 Show the antibacterial situation of the fermentation broth of strain FVV4 after high-temperature treatment. Among them, A shows that the fermentation broth of strain FVV4 inhibits Botrytis cinerea and Exserohilum turcicum after being treated at 120 °C; B shows that the fermentation broth of strain FVV4 inhibits Botrytis cinerea and Exserohilum turcicum after being treated at 80 °C; C shows that the fermentation broth of strain FVV4 inhibits Botrytis cinerea and Exserohilum turcicum after being treated at 60 °C;

[0050] Figure 16 Show the antibacterial situation of the fermentation broth of strain FVV4 after being treated with proteinase K;

[0051] Figure 17 Show the antibacterial situation of the fermentation broth of strain FVV4 after being treated with a DNA adsorption column;

[0052] Figure 18 Show the antibacterial situation of the fermentation broth of strain FVV4 after being treated with proteinase K and a DNA adsorption column simultaneously;

[0053] Figure 19 Show the antibacterial situation of the fermentation broth of strain FVV4 after being treated by a microwave oven. Among them, A shows the fermentation broth treated by the microwave oven for 1 min; B shows the fermentation broth treated by the microwave oven for 2 min;

[0054] Figure 20 Show the change of the pH value of the fermentation broth of strain FVV4;

[0055] Figure 21 Show the inhibitory effect of the mixture of the fermentation broth of strain FVV4 and chemical pesticides on Exserohilum turcicum. Among them, A shows, from left to right in sequence, the control (CK), the single agent of 25% pyraclostrobin, and the antibacterial situation of the mixture of the fermentation broth of strain FVV4 and 25% pyraclostrobin; B shows, from left to right in sequence, the control (CK), the mixture of 40% syringa and tebuconazole, and the antibacterial situation of the mixture of the fermentation broth of strain FVV4 and 40% syringa and tebuconazole;

[0056] Figure 22 Show the field control effect of the mixture of the fermentation broth of strain FVV4 and chemical pesticides. Detailed implementation mode

[0057] This application discloses a biocontrol fungal strain fermentation broth for controlling Exserohilum turcicum and its application. Those skilled in the art can draw on the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all regarded as included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and those related can obviously make changes or appropriate alterations and combinations to the methods and applications described herein without departing from the content, spirit and scope of this application to implement and apply the technology of the present invention.

[0058] It should be understood that the expression "one or more of..." individually includes each object recited after the expression and various different combinations of two or more of the recited objects, unless otherwise understood from the context and usage. The expression "and / or" combined with three or more recited objects should be understood to have the same meaning, unless otherwise understood from the context.

[0059] The terms "comprising", "having" or "containing", including the use of their grammatical synonyms, should generally be understood as open and non-restrictive, for example, not excluding other unrecited elements or steps, unless otherwise specifically stated or understood from the context.

[0060] It should be understood that as long as this application can still be operated, the order of steps or the order of performing certain actions is not important. In addition, two or more steps or actions can be carried out simultaneously.

[0061] Any and all examples or exemplary language in this document, such as the use of "for example" or "including", are merely intended to better illustrate the present application and do not limit the scope of the present application. No language in this specification should be construed as indicating that any unclaimed element is essential for the practice of the present application.

[0062] In addition, the numerical ranges and parameters used to define the present application are approximate values. The relevant values in the specific embodiments have been presented as precisely as possible herein. However, any numerical value inherently contains an inevitable standard deviation due to the individual test methods. Therefore, unless otherwise clearly stated, it should be understood that all ranges, quantities, numerical values, and percentages used in this disclosure are modified by "about". Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specific numerical value or range.

[0063] Unless otherwise specified, the raw materials, reagents, consumables, and instruments involved in this application are all ordinary commercially available products and can be purchased from the market.

[0064] The present invention will be further described below in conjunction with embodiments.

[0065] Embodiment

[0066] 1. Materials and Methods

[0067] 1.1 Isolation of Strains

[0068] The strains were isolated from soil samples collected from Changchun City, Jilin Province, China (43°43′N, 125°19′E). 1 g of soil sample was collected in 100 mL of sterile distilled water and thoroughly mixed in a shaker at 120 rpm for 30 min. A total of 1 mL of the suspension was serially diluted until a 1×10 5 -fold dilution was obtained. The diluted sample (200 µL) was spread onto potato dextrose agar (PDA) supplemented with 100 µg / mL ampicillin. After incubation at 25°C for 7 to 14 days, typical strains were selected based on the size, shape, and color of the colonies. The colonies were picked and purified. The plates containing the pure cultures were transferred to slant agar and stored at 4°C.

[0069] The strains isolated from the soil were subjected to fermentation and toxicity bioassays.

[0070] The method for preparing the fermentation broth of the strains was as follows: The fungus was fermented in a 0.25 L flask containing 0.1 L of potato dextrose broth (PDB, Chinook, CN230573 - 250g). After incubation in a constant temperature rotary shaker at 28°C and 180 rpm for 5 days, the mycelium was filtered off, and the remaining liquid was filtered through a 0.22 µm membrane to obtain the fermentation broth.

[0071] The obtained fermentation broth was mixed with PDA medium at a ratio of 25% by volume to prepare a culture medium (i.e., the fermentation broth accounted for 25%), and sterile water was mixed with PDA medium at a ratio of 25% by volume to prepare a control culture medium. Corn northern leaf blight fungus and Botrytis cinerea mycelial blocks were inoculated in the culture medium for antibacterial tests.

[0072] 1.2 Identification of strains

[0073] The strains screened for inhibitory activity against corn northern leaf blight fungus and Botrytis cinerea were subjected to DNA extraction. Primers were designed based on the conserved sequences of filamentous fungi, b-Tubulin (BenA), CaM, Rpb2, and ITS, and PCR amplification was carried out to construct a multi-gene phylogenetic tree for molecular identification of the strains.

[0074] 1.3 Plate confrontation culture

[0075] Using the three-point confrontation method, the screened strains were subjected to confrontation culture with Botrytis cinerea in a PDA plate to observe the inhibitory effect of the screened strains on Botrytis cinerea. At the same time, the confrontation plate was placed under a microscope to observe the morphological changes of the confrontation hyphae. Strains with stronger antibacterial effects were selected for the following experimental contents.

[0076] 1.4 Antibacterial activity of FVV4 strain against corn northern leaf blight fungus

[0077] Using the three-point confrontation method, the corn northern leaf blight fungus mycelial block was placed in the middle of a PDA plate, and FVV4 strain mycelial blocks were placed at equal distances on both sides of the corn northern leaf blight fungus mycelial block. A control with only the corn northern leaf blight fungus mycelial block was set, and it was placed in an incubator at 28 °C for cultivation and observation, and the colony diameter was measured. The antibacterial rate was calculated. Antibacterial rate (%) = [(control colony diameter - treated colony diameter) / control colony diameter] × 100%.

[0078] 1.5 Soaking corn northern leaf blight fungus hyphae with FVV4 strain fermentation broth

[0079] Take 500 μL of the strain fermentation broth and place it in a plate. Scrape the mycelia of the corn northern leaf blight pathogen cultured for 10 days and soak them in the fermentation broth overnight. Set the soaking of the corn northern leaf blight pathogen mycelia with sterile water as a control, and observe the soaked mycelia under a microscope. At the same time, transfer the soaked mycelia to a new PDA plate and observe the growth and activity of the mycelia.

[0080] 1.6 Analysis of the characteristics of FVV4 strain fermentation broth

[0081] 1.6.1 Antibacterial activity of different concentrations of FVV4 strain fermentation broth

[0082] The fermentation broth of strain FVV4 was mixed with PDA at ratios of 50%, 25%, 15% or 5% to prepare the culture medium, and the control culture medium was made with sterile water at the corresponding ratios. The mycelial blocks of Exserohilum turcicum and Botrytis cinerea were inoculated into the culture media of each treatment to observe the antibacterial activities of the fermentation broth of the strain at different ratios.

[0083] 1.6.2 Determination of the persistence period of the fermentation broth of strain FVV4

[0084] The fermentation broth of strain FVV4 was mixed with PDA medium at a ratio of 25% to prepare the culture medium. Meanwhile, sterile water was mixed with PDA at a ratio of 25% to make the control culture medium. Then, the mycelial blocks of Botrytis cinerea, Magnaporthe grisea, Exserohilum turcicum, and Sclerotinia sclerotiorum were placed in the culture medium and cultured in an incubator at 28 °C to observe the growth of the mycelial blocks. After observing for 15 days, the mycelial blocks were taken out and new mycelial blocks were placed again to continue observing the growth of the mycelial blocks to clarify the persistence period of the fermentation broth of the strain.

[0085] 1.6.3 High-temperature resistance characteristics of the fermentation broth of strain FVV4

[0086] After the fermentation broth of strain FVV4 was treated at 120 °C, 80 °C or 60 °C for 30 min, the treated fermentation broth was subjected to the above antibacterial test using Botrytis cinerea and Exserohilum turcicum as the target strains to observe the antibacterial activities of the treated fermentation broth.

[0087] 1.6.4 Treatment of the fermentation broth of strain FVV4 with proteinase K

[0088] 100 μL of proteinase K (20 mg / mL) was added to 20 mL of the fermentation broth of strain FVV4. The fermentation broth treated with proteinase K was then subjected to the above antibacterial test to observe the antibacterial activities of the treated fermentation broth.

[0089] 1.6.5 Treatment of the fermentation broth of strain FVV4 with a DNA adsorption column

[0090] Since the DNA adsorption column can retain macromolecular substances such as proteins, the fermentation broth of the strain was treated with a DNA adsorption column, and the treated fermentation broth was subjected to the antibacterial test to observe the antibacterial activities of the treated fermentation broth.

[0091] 1.6.6 Simultaneous treatment of the fermentation broth of strain FVV4 with proteinase K and a DNA adsorption column

[0092] The fermented broth of the strain treated with proteinase K was further treated with a DNA adsorption column, and then the treated fermented broth of the strain was subjected to an antibacterial test to observe the antibacterial activity of the fermented broth after treatment.

[0093] 1.6.7 Microwave treatment of the fermented broth of strain FVV4

[0094] The fermented broth of the strain was placed in a microwave oven and treated for 1 min and 2 min respectively. Then, the treated fermented broth of the strain was subjected to an antibacterial test to observe the antibacterial activity of the fermented broth after treatment.

[0095] 1.6.8 pH value analysis of the fermented broth of strain FVV4

[0096] The bacterial cake of strain FVV4 was placed in 100 mL of PDB medium for shaking culture for 5 d. The fermented broth was taken at 24 h, 48 h, 72 h, 96 h, and 120 h of shaking culture for pH value determination. Each treatment was set with 3 replicates to detect the change in the pH value of the fermented broth of strain FVV4.

[0097] 1.7 Inhibiting Exserohilum turcicum with the mixture of the fermented broth of strain FVV4 and chemical pesticides

[0098] The following six treatments were set for inhibiting Exserohilum turcicum in the petri dish:

[0099] ① Take 1 mL of the fermented broth of strain FVV4 and mix it with 19 mL of PDA to make a medium;

[0100] ② Take 10 μL of 25% pyraclostrobin (1000-fold solution) and add it to 20 mL of PDA medium;

[0101] ③ Take 15 μL of 40% syringic acid azoxystrobin tebuconazole (1000-fold solution) and add it to 20 mL of PDA medium respectively;

[0102] ④ Take 1 mL of the fermented broth of strain FVV4 and 10 μL of 25% pyraclostrobin (1000-fold solution) and add them to 19 mL of PDA medium;

[0103] ⑤ Take 1 mL of the fermented broth of strain FVV4 and 15 μL of 40% syringic acid azoxystrobin tebuconazole (1000-fold solution) and add them to 19 mL of PDA medium;

[0104] ⑥ Take 1 mL of sterile water and add it to 19 mL of PDA medium as a control treatment;

[0105] The mycelial blocks of Exserohilum turcicum were inoculated into the medium of each treatment. Each treatment had three replicates. Observe the antibacterial situation and measure the diameter of the pathogen, and calculate the inhibition rate. The calculation formula is inhibition rate (%) = (control colony diameter - treated colony diameter) / control colony diameter × 100%.

[0106] Field control of Exserohilum turcicum on maize by mixing the fermentation broth of strain FVV4 with chemical pesticides

[0107] 1.8.1 General situation of the test field

[0108] This experiment was carried out in a maize experimental field of the Gongzhuling Branch of Jilin Academy of Agricultural Sciences. The management conditions of the experimental field were the same as those of the local large fields, and Exserohilum turcicum occurred frequently all year round. The maize variety Xianyu 335, which is highly susceptible to Exserohilum turcicum, was planted.

[0109] 1.8.2 Experimental design

[0110] The following three treatments were set up for field control of Exserohilum turcicum on maize:

[0111] ① Take 50 mL of 25% pyraclostrobin per 667 m 2 Add it to 50 L of water;

[0112] ② Take 35 mL of 25% pyraclostrobin per 667 m 2 Add it to 20 L of the fermentation broth of strain FVV4 and 30 L of water and mix well;

[0113] ③ Use clear water as the control;

[0114] The area of each treatment plot was 40 m 2 , and 3 replicates were set. The first spraying was carried out on July 5, 2024, and the second spraying was carried out on July 15, 2024. A knapsack electric sprayer was used for uniform spraying. At this time, Exserohilum turcicum-infected leaves had been seen in the field.

[0115] 1.8.3 Investigation of control efficacy

[0116] The disease incidence of leaves was investigated 25 days after spraying. Five sampling points were taken in each plot, and 5 maize plants were selected at each point to investigate the proportion of disease spots on all leaves, and the disease severity was classified: grade 0, no disease spots on the leaves; grade 1, there were disease spots or only sporadic disease spots on the leaves, accounting for 5% or less of the leaf area; grade 3, there were a small number of disease spots on the leaves below the ear position, accounting for 6% - 10% of the leaf area, and sporadic disease spots on the leaves above the ear position; grade 5, there were more disease spots on the leaves below the ear position, accounting for 11% - 30% of the leaf area, and there were also more disease spots on the leaves above the ear position; grade 7, there were a large number of disease spots on the leaves below or above the ear position and they were connected, accounting for 31% - 70% of the leaf area; grade 9, almost all the leaves of the whole plant were covered with disease spots and the leaves withered. Thus, the disease index and the disease index control efficacy relative to the clear water control were obtained. The calculation formulas for the disease index and the control efficacy are shown in Formula I and Formula II respectively:

[0117] (Formula I);

[0118] (Formula II).

[0119] 2. Results and Analysis

[0120] 2.1 Identification of Strains

[0121] A total of 2 strains with obvious antibacterial effects were obtained through antibacterial tests. Through Blast alignment of ITS sequences and analysis of the multi-gene phylogenetic tree, it was found that the 2 obtained strains were both Talaromyces purpureogenus (see Figure 1 , Figure 2 ), and they were named strain FV11 and FVV4 respectively.

[0122] 2.2 Plate Confrontation Culture

[0123] 2.2.1 Plate Confrontation with Botrytis cinerea

[0124] Using the three-point confrontation method, strains FV11 and FVV4 were respectively confronted with Botrytis cinerea in three points (see Figure 3 and Figure 4 ). Since the fourth day of confrontation, the lateral growth of Botrytis cinerea was significantly inhibited, and red pigments also began to appear on the confrontation surfaces of strains FV11 and FVV4; as time passed, strains FV11 and FVV4 began to cover the Botrytis cinerea strain in a large area, and the red pigments also continuously increased over time, but the coverage area and the amount of pigment production of FV11 were significantly lower than those of strain FVV4, and strain FVV4 showed stronger antibacterial activity.

[0125] 2.2.2 Microscopic Observation of the Confrontation Plate

[0126] The above-mentioned plate was placed under a microscope (4 times or 10 times) to observe the hyphal development. See Figure 5 , Figure 6 . On the 4th day of plate confrontation, the hyphae of Botrytis cinerea on the confrontation surface underwent morphological distortion, resulting in significant inhibition of the lateral growth of the hyphae. Fan-shaped swellings appeared at the hyphal tips, and the swellings were light brown; on the 5th day of confrontation, a large number of red-headed hyphae appeared in the mycelium of strain FVV4, accompanied by excessive secretion of pigments and toxins, which in turn led to an increase in the fan-shaped swellings at the hyphal tips of Botrytis cinerea on the confrontation surface and the color changed from light brown to dark brown; on the 6th day of confrontation, the hyphae of Botrytis cinerea and the hyphae of FVV4 were intertwined. With the continuous secretion and accumulation of pigments and toxins by strain FVV4, the distortion of the hyphal tips of Botrytis cinerea was aggravated, and the fan-shaped swellings at the hyphal tips increased in number and size, and the color changed from dark brown to bright red. Thus, the stronger antibacterial activity of strain FVV4 was shown. The lateral growth of Botrytis cinerea confronted with strain FV11 was also significantly inhibited, but no obvious fan-shaped swellings were formed at the hyphal tips on the confrontation surface. As the pigments of strain FV11 were secreted, the confrontation hyphae were also dyed red, but no obvious fan-shaped swellings were seen even in the later stage of cultivation (see Figure 7), which did not cause obvious morphological distortion of the hyphae of Botrytis cinerea, indicating that the FVV4 strain produced more antibacterial substances. Therefore, the FVV4 strain was selected for all subsequent experimental contents.

[0127] 2.3 Antibacterial activity of FVV4 strain against Exserohilum turcicum

[0128] The antibacterial activity of the FVV4 strain against Exserohilum turcicum was detected by the three-point confrontation method. No obvious inhibitory effect was observed on the 3rd day of confrontation culture, but it was found by microscopic examination that fan-shaped swellings appeared at the tips of the confronted Exserohilum turcicum hyphae (see Figure 8 ); on the 4th day of confrontation culture, the growth of Exserohilum turcicum in the horizontal direction stopped, and it was found by microscopic examination that the swellings at the tips of the Exserohilum turcicum hyphae increased significantly; on the 5th day of confrontation culture, the growth of Exserohilum turcicum in the vertical direction stopped, and it was found by microscopic examination that fan-shaped swellings also formed at the tips of the substrate mycelia of Exserohilum turcicum; on the 6th day of confrontation culture, the FVV4 strain significantly covered the hyphae of Exserohilum turcicum, and it was found by microscopic examination that the hyphae of Exserohilum turcicum showed a phenomenon of breakage; on the 10th day of confrontation culture, the FVV4 strain completely covered the Exserohilum turcicum colony (see Figure 9 ). When the control Exserohilum turcicum covered the plate, the average diameter of the confrontation colony was 1.48 ± 0.06, and the inhibition rate was 81.5% ± 0.86.

[0129] 2.4 Soaking the hyphae of Exserohilum turcicum with the fermentation broth of FVV4 strain

[0130] The hyphae of Exserohilum turcicum have two forms: substrate mycelia and aerial mycelia. The protoplast cells of the hyphae can be clearly observed inside the substrate mycelia; after soaking the hyphae of Exserohilum turcicum with sterile water, it can be observed that the hyphal cells absorb water and germinate; after soaking the hyphae with the fermentation broth of the FVV4 strain, the cell gaps inside the substrate mycelia absorb the fermentation broth of the FVV4 strain and turn red; the aerial mycelia absorb the fermentation broth of the FVV4 strain and undergo plasmolysis, and the protoplast cells inside the hyphae are red and sausage-shaped; the sausage-shaped hyphae break, and the hyphae of Exserohilum turcicum gradually disappear, as Figure 10 shown; as Figure 11 can be seen, it was observed under a low-power microscope that almost all the hyphae of Exserohilum turcicum in one field of view absorbed the fermentation broth of the FVV4 strain and became red hyphae, and were significantly affected by the fermentation broth of the FVV4 strain. At the same time, the hyphae soaked with the fermentation broth of the FVV4 strain and the hyphae soaked with sterile water were transferred to the same new PDA plate, with three replicates for each plate. The hyphae soaked with sterile water could grow normally in the PDA plate, while the hyphae soaked with the fermentation broth of the FVV4 strain did not show obvious growth (see Figure 12 ).

[0131] 2.5 Analysis of the characteristics of the fermentation broth of FVV4 strain

[0132] 2.5.1 Antibacterial activity of different concentrations of the fermentation broth of FVV4 strain

[0133] Prepare PDA media with fermentation broths of FVV4 strain at different ratios, and analyze the antibacterial effects of fermentation broths at different concentrations against Botrytis cinerea, Magnaporthe grisea, Exserohilum turcicum, and Sclerotinia sclerotiorum. When the proportion of fermentation broth in the plate was 50%, 25%, and 15%, no obvious growth was observed in all strains (see some antibacterial situations in Figure 13 ); when the volume ratio was 5%, the growth of all strains was significantly inhibited, and the average antibacterial rate was 53% (see Figure 14 ).

[0134] 2.5.2 Determination of the persistence period of the fermentation broth of FVV4 strain

[0135] Place the mycelial blocks of Botrytis cinerea, Magnaporthe grisea, Exserohilum turcicum, and Sclerotinia sclerotiorum in the medium, culture them in an incubator at 28 °C, and observe the growth of the mycelial blocks. After observing for 15 days, take out the mycelial blocks, place new mycelial blocks again, and continue to observe the growth of the mycelial blocks. After culturing the new mycelial blocks of Botrytis cinerea, Magnaporthe grisea, and Sclerotinia sclerotiorum for another 10 days, obvious mycelial growth was observed on the mycelial blocks, while no obvious growth was observed in Exserohilum turcicum. Therefore, the persistence period of the fermentation broth of FVV4 strain is at least 25 days.

[0136] 2.5.3 High-temperature resistance characteristics of the fermentation broth of FVV4 strain

[0137] After treating the fermentation broth of FVV4 strain at 120 °C, 80 °C, or 60 °C for 30 min, then mix the treated fermentation broth with PDA at a ratio of 25% to inhibit Botrytis cinerea and Exserohilum turcicum. When Botrytis cinerea and Exserohilum turcicum covered the plate, no obvious growth was observed in the mycelial blocks after each treatment, indicating that the fermentation broth of FVV4 strain has high-temperature resistance characteristics (see Figure 15 , 4 plates in each group, including 1 control and 3 treatment replicates).

[0138] 2.5.4 Treatment of the fermentation broth of FVV4 strain with proteinase K

[0139] Proteinase K is a highly efficient enzyme preparation that can effectively decompose all proteins in cells. Therefore, Proteinase K was used to treat the fermentation broth of strain FVV4 to decompose the proteins in the fermentation broth. The results of the antibacterial test showed that when the plates were fully covered with Botrytis cinerea and Exserohilum turcicum, no obvious growth of the treated bacterial blocks was observed, indicating that the antibacterial substance in the fermentation broth of strain FVV4 was likely not a protein, and further suggesting that its antibacterial substance was not easily degraded (see Figure 16 , with 4 plates in each group, including 1 control and 3 treatment replicates).

[0140] 2.5.5 Treatment of the fermentation broth of strain FVV4 with a DNA adsorption column

[0141] After the fermentation broth of strain FVV4 was treated with a DNA adsorption column, its antibacterial effect was not affected (as shown in Figure 17 , with 4 plates in each group, including 1 control and 3 treatment replicates), indicating again that the antibacterial substance in the fermentation broth of strain FVV4 was likely not a protein and its antibacterial effect was stable.

[0142] 2.5.6 Simultaneous treatment of the fermentation broth of strain FVV4 with Proteinase K and a DNA adsorption column

[0143] When the fermentation broth of strain FVV4 was simultaneously treated with Proteinase K and a DNA adsorption column, the proteins in the fermentation broth were completely decomposed or retained in the DNA adsorption column. The antibacterial results showed that no obvious growth of the hyphae in the treated fermentation broth of strain FVV4 was observed (see Figure 18 , with 4 plates in each group, including 1 control and 3 treatment replicates). This indicates that the antibacterial substance in the fermentation broth of strain FVV4 is not a protein, and this antibacterial substance is not easily degraded and is relatively stable.

[0144] 2.5.7 Microwave treatment of the fermentation broth of strain FVV4

[0145] After the fermentation broth of strain FVV4 was treated with a microwave oven for 1 min and 2 min respectively, its antibacterial activity was not significantly affected (see Figure 19 , with 4 plates in each group, including 1 control and 3 treatment replicates), indicating that the antibacterial substance in the fermentation broth of strain FVV4 has the property of resistance to microwave radiation.

[0146] 2.5.8 pH value analysis of the fermentation broth of strain FVV4

[0147] The average pH value of the PDB liquid medium is 5.75. The average pH values of the fermentation broth of strain FVV4 after shaking culture for 24 h, 48 h, 72 h, 96 h, and 120 h are 3.78, 3.25, 3.09, 3.03, and 3.00, respectively. It can be seen that strain FVV4 produces acidic substances during growth, resulting in a relatively low pH value in its fermentation broth, and the pH value tends to be stable after 96 h of shaking culture (see Figure 20 ).

[0148] 2.6 Inhibitory effect of the fermentation broth of strain FVV4 mixed with chemical pesticides on Exserohilum turcicum

[0149] The fermentation broth of strain FVV4, single agents, and mixtures of chemical pesticides were separately mixed in Petri dishes to inhibit Exserohilum turcicum. The average inhibitory rate of the fermentation broth of strain FVV4 alone is 53%, the average inhibitory rate of 25% pyraclostrobin alone is 56.33%, while the inhibitory rate after mixing the fermentation broth of strain FVV4 with 25% pyraclostrobin is 100%; the average inhibitory rate of 40% syringapenconazole alone is 34.67%, while the inhibitory rate after mixing the fermentation broth of strain FVV4 with 40% syringapenconazole is 100% (see Figure 21 ). It can be seen that the fermentation broth of strain FVV4 can enhance the inhibitory effect of chemical agents.

[0150] 2.7 Field control of Exserohilum turcicum by mixing the fermentation broth of strain FVV4 with chemical pesticides

[0151] The disease incidence was investigated 25 days after spraying. In the clear water control treatment group, the disease levels of the leaves were mostly 3 - 7, and there were few non - diseased leaves; in the chemical agent treatment group, the disease levels of the leaves were mostly 1, a few were 3, no leaves with disease levels above 5 were seen, and there were more non - diseased leaves; in the treatment group with the mixture of the fermentation broth of strain FVV4 and chemical agents, the disease levels were mostly 1, a few were 3, no leaves with disease levels above 5 were seen, and there were more non - diseased leaves (see Table 1). The control effect of the mixture of the fermentation broth of strain FVV4 and chemical agents is higher than that of the chemical agent alone (see Figure 22 ), playing the role of reducing the dosage of pesticides and increasing the efficiency.

[0152] Table 1: Control effects of different pesticide treatments on Exserohilum turcicum

[0153]

[0154] The above - mentioned are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of this application.

Claims

1. Talaromyces purpureogenus ( Talaromyces purpureogenus ), characterized in that The preservation number is CGMCC No. 41911.

2. Fermentation broth, characterized in that, It is the fermentation broth of Talaromyces purpureogenus described in claim 1.

3. The fermentation broth according to claim 2, wherein, The preparation method includes: inoculating the Talaromyces purpureogenus described in claim 1 into PDB for culture, filtering the obtained culture solution, and taking the filtrate to obtain the fermentation broth.

4. The fermentation broth according to claim 3, characterized in that, The temperature of the culture is 26 - 30 °C, the rotation speed is 160 - 200 rpm, and the time is 5 - 9 days.

5. The fermented liquid according to claim 3, wherein, The pore size of the filter membrane for filtration is 0.22 µm.

6. Use of Talaromyces purpureogenus or the fermentation broth in any of the following: (i) Antagonizing pathogenic bacteria; (ii) Preventing and controlling Exserohilum turcicum; It is characterized in that The Talaromyces purpureogenus is the Talaromyces purpureogenus described in claim 1, the fermentation broth is the fermentation broth described in any one of claims 2 to 5, and the pathogenic bacteria include Botrytis cinerea ( Botrytis cinerea ), Magnaporthe oryzae ( Magnaporthe grisea ), Setosphaeria turcica ( Exserohilum turcicum ), or Sclerotinia sclerotiorum ( Sclerotinia Sclerotiorum ).

7. A composition, characterized in that, Comprising: (I) Talaromyces purpureogenus described in claim 1 or the fermentation broth described in any one of claims 2 to 5; (II) Syringic acid tebuconazole or pyraclostrobin.

8. Use of **Stachybotrys chartarum**, fermentation broth or composition in the preparation of a biological control preparation for preventing and controlling **Setosphaeria turcica**, characterized in that, The Talaromyces purpureogenus is the Talaromyces purpureogenus described in claim 1, the fermentation broth is the fermentation broth described in any one of claims 2 to 5, and the composition is the composition described in claim 7.

9. Biological control agent, characterized in that, Comprising any one of the following: (a) Talaromyces purpureogenus described in claim 1 or the fermentation broth described in any one of claims 2 to 5; (b) The composition described in claim 7.

10. A method for controlling Exserohilum turcicum, characterized in that, Using any one of the following: (A) Talaromyces purpureogenus described in claim 1; (B) The fermentation broth described in any one of claims 2 to 5; (C) The composition described in claim 7; (D) The biocontrol agent described in claim 9.

Images