A purple-producing tularensis strain, its fermentation liquid and its application in controlling corn leaf blight

Abstract

The present application relates to the field of microbial technology, and in particular to a strain of Purple Basilisk, its fermentation liquid and its application in preventing and controlling corn leaf blight. The present application provides a biocontrol fungus fermentation liquid for preventing and controlling corn leaf blight and its application, and its strain preservation number is CGMCC No.41911. The strain and its fermentation liquid can completely inhibit a variety of pathogens. The active substances in the fermentation liquid are relatively stable, have a long effective period of activity, strong systemic absorption, are not easy to degrade, are resistant to high temperatures, and are resistant to microwave radiation. It can significantly improve the antibacterial effect of chemical agents, and when mixed with chemical pesticides, it can significantly reduce the use of drugs and increase their efficacy, thereby significantly reducing environmental safety issues and the development of drug resistance caused by chemical pesticides.

Description

Technical Field

[0001] The present application relates to the field of microbial technology, and in particular to a fermentation liquid of a biocontrol fungus strain for preventing and controlling corn leaf blight and its application. Background Art

[0002] The widespread use of chemical pesticides in agriculture has led to significant problems, including resistance in pathogens and pests, residues in soil, and declining soil productivity. Consequently, beneficial microorganisms are gaining increasing attention as safer alternatives in agriculture. The discovery of new species of Talaromyces provides additional resources for biocontrol. Talaromyces and their metabolites are widely used in agriculture for biocontrol.

[0003] Talaromyces purpureogenus belongs to the phylum Ascomycota, class Euroticulariales, order Euroticulariales, family Trichomycetes, and genus Talaromyces. Originally, Talaromyces was a teleomorph of the genus Penicillium, with the asexual stage called Penicillium and the sexual stage called Talaromyces. Considering nomenclature precedence and the single nomenclature system, Samson et al. transferred most accepted species of the subgenus Biverticillium to the genus Talaromyces in July 2011. The development and utilization of Talaromyces provides new resources for biological control of plant diseases. Talaromyces flavus and Talaromyces purpureogenus can be used for biological control of insect pests in agricultural and forestry plants. The yellow Talaromyces isolated from ginseng seeds by Kim et al. has antagonistic effects against plant pathogens such as Fusarium oxysporum, Rhizoctonia solani, Sclerotinia nivalis, Botrytis cinerea, and Phytophthora capsici. Purple Talaromyces also exhibits significant antibacterial activity against potato scab and saffron stem rot. However, there are no reports on the inhibitory effect of purple Talaromyces against northern corn leaf blight. Northern corn leaf blight is caused by infection with Exserohilum turcicum (Pass.) Leonard et Suggs. Current prevention and control strategies for northern corn leaf blight focus on breeding resistant varieties and chemical pesticides, but both approaches face significant challenges. Regarding the use of disease-resistant varieties, the highly differentiated physiological races of Setosoma zea make resistance easily overcome. This, coupled with the long breeding cycle for disease resistance, often leads to regional declines in actual disease control effectiveness. In the field of chemical control, mainstream triazole fungicides such as propiconazole and difenoconazole can quickly suppress the disease, but they carry multiple risks, including a short application window; pathogens that easily develop resistance through genetic mutations; and long-term use leading to a decrease in soil microbial diversity and associated ecological safety concerns such as excessive pesticide residues. Summary of the Invention

[0004] In view of this, the present application provides a fermentation liquid of a biocontrol fungal strain for preventing and controlling corn leaf blight and its application. The strain and its fermentation liquid can effectively inhibit a variety of pathogens. The active substances in the fermentation liquid are relatively stable, have a long active period, strong systemic properties, are not easy to degrade, are resistant to high temperatures and microwave radiation, and can significantly improve the antibacterial effect of chemical agents. When mixed with chemical pesticides, it has a significant effect of reducing the amount of medicine and increasing the efficacy.

[0005] In order to achieve the above-mentioned invention objectives, this application provides the following technical solutions:

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

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

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

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

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

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

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

[0013] The present application also provides the use of the above-mentioned purple-producing T. virescens or the above-mentioned fermentation broth in any of the following:

[0014] (i) Antagonism against pathogens;

[0015] (ii) control of corn leaf spot;

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

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

[0018] (I) the above-mentioned purple-producing fungus or the above-mentioned fermentation liquid;

[0019] (II) tebuconazole or pyraclostrobin.

[0020] The present application also provides the use of the above-mentioned purple-producing fungus, the above-mentioned fermentation liquid or the above-mentioned composition in the preparation of a biocontrol agent for preventing and controlling corn leaf blight.

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

[0022] (a) the above-mentioned Talaromyces purpurogenus or the above-mentioned fermentation liquid;

[0023] (b) The above composition.

[0024] The present application also provides a method for preventing and controlling corn leaf blight, comprising using any of the following:

[0025] (A) The above-mentioned purple-producing tularensis;

[0026] (B) the above fermentation broth;

[0027] (C) the above composition;

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

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

[0030] As described in the background technology, chemical control has many defects, while biological control not only has an inhibitory effect on pathogens, but is also environmentally friendly and pollution-free. The present application provides a biocontrol fungal strain fermentation liquid for preventing and controlling corn leaf blight and its application. At the same time, it systematically reveals the biocontrol functional characteristics of the biocontrol fungal strain fermentation liquid for the first time, providing a breakthrough solution for the green prevention and control of corn leaf blight. Specifically, by comparing the differences between the purple-producing fungus FVV4 and FV11 strains, the present application found that the FVV4 strain can produce strong antibacterial substances, which can cause the contents of the opposing hyphae cells to flow out in the confrontation culture to form fan-shaped protrusions, and the hyphae break and die in the later stage and lose vitality. This phenomenon is the first discovery. At the same time, the FVV4 strain fermentation liquid can completely inhibit a variety of pathogens. The active substances in the fermentation liquid are relatively stable, have a long active period, strong systemic absorption, are not easy to degrade, are resistant to high temperatures, and are resistant to microwave radiation. The above-mentioned characteristics of the FVV4 strain fermentation liquid have not been reported, and this application is the first report. Plate inhibition tests showed that FVV4 fermentation broth can significantly enhance the antibacterial efficacy of chemical pesticides. Field trials also demonstrated that a mixture of FVV4 fermentation broth and chemical pesticides was more effective than the chemical pesticides, significantly reducing the need for additional pesticides and increasing their effectiveness. This is also the first time that FVV4 fermentation broth has been shown to reduce the need for additional pesticides.

[0031] Biological Deposit Description

[0032] Biological material: FVV4, taxonomic name: Talaromyces purpureogenus, deposited on April 8, 2025, at the General Microbiology Center of China Culture Collection Administration, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; deposit number: CGMCC No. 41911.

[0033] The FVV4 described in this application is the strain with the above-mentioned deposit number CGMCC No. 41911. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0035] Figure 1 shows the FV11 multigene phylogenetic tree;

[0036] Figure 2 Shown is the FVV4 multigene phylogenetic tree

[0037] Figure 3 The plate confrontation between FVV4 strain and Botrytis cinerea is shown. The first row shows the colony morphology on the front of the plate confrontation, and from left to right are the colony morphology on the 4th to 8th day of the plate confrontation; the second row shows the colony morphology on the back of the plate confrontation, and from left to right are the colony morphology on the 4th to 8th day of the plate confrontation;

[0038] Figure 4 The plate confrontation between FV11 strain and Botrytis cinerea is shown. The first row shows the colony morphology on the front of the plate confrontation, and from left to right are the colony morphology on the 4th to 8th day of the plate confrontation; the second row shows the colony morphology on the back of the plate confrontation, and from left to right are the colony morphology on the 4th to 8th day of the plate confrontation;

[0039] Figure 5 The hyphae morphology of Botrytis cinerea and FVV4 strains on the plate under a 4x microscope is shown. From left to right, the hyphae morphology on the 4th, 5th, and 6th day of confrontation on the plate and the normal Botrytis cinerea hyphae morphology are shown.

[0040] Figure 6 The hyphae morphology of Botrytis cinerea and FVV4 strains in a confrontation plate under a 10x microscope is shown. From left to right, the hyphae morphology on the 4th, 5th, and 6th day of confrontation on the plate and the normal Botrytis cinerea hyphae morphology are shown.

[0041] Figure 7 The hyphae morphology of Botrytis cinerea and FV11 strains on the plate under a 10x microscope is shown. From left to right, the hyphae morphology on the 4th, 5th, and 6th day of confrontation on the plate and the normal Botrytis cinerea hyphae morphology are shown.

[0042] Figure 8 The figures show the mycelial morphology of T. maydis in the confrontation culture, wherein CK shows the aerial mycelia and the base mycelia of T. maydis, A shows the aerial mycelia of T. maydis on the 3rd day of confrontation and the base mycelia of T. maydis on the 5th day, and B shows the aerial mycelia of T. maydis on the 4th day of confrontation and the base mycelia of T. maydis on the 6th day;

[0043] Figure 9 Shows the confrontation culture of FVV4 strain and U. maydis;

[0044] Figure 10 The morphology of the hyphae of Setospora maydis after soaking was observed under a microscope. From left to right, the hyphae of Setospora maydis after soaking in sterile water at 40 times the magnification, the hyphae of Setospora maydis after soaking in the fermentation liquid of FVV4 strain at 40 times the magnification (including aerial hyphae and basal hyphae), the broken basal hyphae after soaking in the fermentation liquid of FVV4 strain at 40 times the magnification, the broken aerial hyphae after soaking in the fermentation liquid of FVV4 strain at 40 times the magnification, and the hyphae gradually degraded after soaking in the fermentation liquid of FVV4 strain at 40 times the magnification;

[0045] Figure 11 The mycelial morphology of Setospora maydis after soaking was observed under a microscope, which is the mycelial morphology of Setospora maydis after soaking in sterile water and FVV4 strain fermentation liquid observed under a 10x microscope;

[0046] Figure 12 The figure shows the growth of mycelia of Tussostrea maydis after being soaked in the fermentation liquid of the FVV4 strain and sterile water on PDA plates. The three plates in the figure represent three replicates.

[0047] Figure 13 The antibacterial effect of the fermentation broth of the FVV4 strain is shown in the figure. The first row, from left to right, shows Botrytis cinerea and the Botrytis cinerea treated with the fermentation broth, and Setospora maydis and the Setospora maydis treated with the fermentation broth; the second row, from left to right, shows Pyricularia oryzae and the Pyricularia oryzae treated with the fermentation broth, and Sclerotium sojae and the Sclerotium sojae treated with the fermentation broth;

[0048] Figure 14 The figure shows the antibacterial effect of FVV4 fermentation broth with different concentrations;

[0049] Figure 15 Figures 2 and 3 show the antibacterial effects of the FVV4 fermentation broth after high-temperature treatment. Figure A shows the inhibition of Botrytis cinerea and Gibberellic acid on the FVV4 fermentation broth after treatment at 120°C; Figure B shows the inhibition of Botrytis cinerea and Gibberellic acid on the FVV4 fermentation broth after treatment at 80°C; and Figure C shows the inhibition of Botrytis cinerea and Gibberellic acid on the FVV4 fermentation broth after treatment at 60°C.

[0050] Figure 16 The figure shows the antibacterial effect of the fermentation broth of FVV4 strain after being treated with proteinase K;

[0051] Figure 17 The antibacterial effect of the FVV4 strain fermentation broth after being treated with DNA adsorption column is shown;

[0052] Figure 18 The antibacterial effect of the FVV4 strain fermentation broth after simultaneous treatment with proteinase K and DNA adsorption column is shown;

[0053] Figure 19 The figures show the antibacterial effect of the fermentation liquid of FVV4 strain after being treated with microwave oven, among which A shows the fermentation liquid treated with microwave oven for 1 min; B shows the fermentation liquid treated with microwave oven for 2 min;

[0054] Figure 20 The figure shows the pH change of the fermentation liquid of FVV4 strain;

[0055] Figure 21 The figures show the inhibition of FVV4 strain fermentation broth mixed with chemical pesticides on corn leaf blight pathogen. A shows the inhibition of the control (CK), 25% pyraclostrobin alone, and the mixture of FVV4 strain fermentation broth and 25% pyraclostrobin from left to right; B shows the inhibition of the control (CK), 40% tebuconazole mixture, and the mixture of FVV4 strain fermentation broth and 40% tebuconazole from left to right.

[0056] Figure 22 The results show the field control effect of mixing FVV4 strain fermentation broth with chemical pesticides. DETAILED DESCRIPTION

[0057] The present application discloses a fermentation liquid of a biocontrol fungal strain for preventing and treating corn leaf blight and its application. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application 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" includes individually each of the items recited after the expression and various combinations of two or more of the recited items, unless otherwise apparent from the context and usage. The expression "and / or" in conjunction with three or more recited items should be understood to have the same meaning, unless otherwise apparent from the context.

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

[0060] It should be understood that the order of steps or the order in which certain actions are performed is not important as long as the application remains operable. Additionally, two or more steps or actions may be performed simultaneously.

[0061] The use of any and all examples or exemplary language such as "for example" or "including" herein is intended only to better illustrate the present application and does not limit the scope of the present application. No language in this specification should be construed as indicating any non-claimed element is essential to the practice of the present application.

[0062] In addition, the numerical ranges and parameters used to define this application are approximate values. The relevant numerical values in the specific examples have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual testing methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, amounts, values, and percentages used in this disclosure are modified by the word "about." As used herein, "about" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a particular value or range.

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

[0064] The present invention will be further described below with reference to the embodiments.

[0065] Example

[0066] 1. Materials and Methods

[0067] 1.1 Isolation of strains

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

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

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

[0071] The fermentation broth thus obtained was mixed with PDA culture 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 culture medium at a ratio of 25% by volume to prepare a control culture medium, and blocks of U. maydis and Botrytis cinerea were inoculated into the culture medium for antibacterial testing.

[0072] 1.2 Identification of strains

[0073] DNA was extracted from the strains screened for inhibitory activity against Tubulin and Botrytis cinerea. Primers were designed based on the conserved sequences of filamentous fungi, b-Tubulin (BenA), CaM, Rpb2, and ITS, for PCR amplification. A multi-gene phylogenetic tree was constructed to molecularly identify the strains.

[0074] 1.3 Plate confrontation culture

[0075] Using a three-point standoff method, the selected strains were cultured against Botrytis cinerea on PDA plates to observe their inhibitory effects on Botrytis cinerea. The plates were then placed under a microscope to observe changes in hyphae morphology. Strains with the strongest inhibitory effects were selected for the following tests.

[0076] 1.4 Antibacterial activity of FVV4 strain against U. maydis

[0077] Using a three-point standoff method, a T. maydis colony was placed in the center of a PDA plate. FVV4 colonies were placed equidistantly on either side of the T. maydis colony. A control plate containing only T. maydis colony was placed. The plates were incubated in a 28°C incubator for observation and colony diameter measurement. The inhibition rate was calculated. Inhibition rate (%) = [(control colony diameter - treated colony diameter) / control colony diameter] × 100%.

[0078] 1.5 Soaking the mycelium of Rhizoctonia solani with FVV4 fermentation liquid

[0079] 500 μL of the fermentation broth was placed on a plate. Mycelia of the 10-day-old corn leaf blight pathogen were scraped and soaked in the fermentation broth overnight. Mycelia soaked in sterile water served as a control. The soaked mycelia were observed under a microscope. Simultaneously, the soaked mycelia were transferred to a new PDA plate to observe their growth and activity.

[0080] 1.6 Analysis of FVV4 strain fermentation broth characteristics

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

[0082] The culture medium was prepared by mixing the fermentation broth of the FVV4 strain with PDA at a ratio of 50%, 25%, 15% or 5%, and sterile water was used as a control culture medium at the corresponding ratio. The corn leaf blight and gray mold blocks were inoculated into the culture medium for each treatment, and the antibacterial activity of the fermentation broth of the strains in different ratios was observed.

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

[0084] The culture medium was prepared by mixing 25% of the FVV4 fermentation broth with PDA medium. A control medium was prepared by mixing 25% of sterile water with PDA. Subsequently, fungal clumps of Botrytis cinerea, Magnaporthe grisea, Exserohilum turcicum, and Sclerotinia sclerotiorum were placed in the culture medium and incubated in a 28°C incubator. The growth of the fungal clumps was observed. After 15 days of observation, the fungal clumps were removed and replaced with new ones. Growth was further observed to determine the shelf life of the fermentation broth.

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

[0086] The fermentation broth of strain FVV4 was treated at 120℃, 80℃ or 60℃ for 30 min, and then the treated fermentation broth was subjected to the above-mentioned antibacterial test. Botrytis cinerea and Urocera maydis were used as target strains to observe the antibacterial activity of the treated fermentation broth.

[0087] 1.6.4 Proteinase K treatment of FVV4 fermentation broth

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

[0089] 1.6.5 DNA adsorption column treatment of FVV4 strain fermentation broth

[0090] Since DNA adsorption columns can intercept macromolecular substances such as proteins, DNA adsorption columns are used to treat the fermentation broth of the strain, and the treated fermentation broth is subjected to an antibacterial test to observe the antibacterial activity of the treated fermentation broth.

[0091] 1.6.6 Simultaneous Treatment of FVV4 Fermentation Broth with Proteinase K and DNA Adsorption Column

[0092] The fermentation liquid of the strain treated with proteinase K was then treated with a DNA adsorption column, and then the fermentation liquid of the treated strain was subjected to an antibacterial test to observe the antibacterial activity of the fermentation liquid.

[0093] 1.6.7 Microwave Treatment of FVV4 Fermentation Broth

[0094] The fermentation liquid of the strain was placed in a microwave oven and treated for 1 min and 2 min respectively. The treated fermentation liquid of the strain was then subjected to an antibacterial test to observe the antibacterial activity of the treated fermentation liquid.

[0095] 1.6.8 pH Analysis of FVV4 Fermentation Broth

[0096] FVV4 strain cakes were placed in 100 mL of PDB medium and shake cultured for 5 days. The fermentation broth was sampled and pH measured at 24, 48, 72, 96, and 120 hours of shaking culture. Each treatment was replicated three times to monitor changes in the pH of the FVV4 fermentation broth.

[0097] 1.7 Inhibition of Setospora turcica by mixing FVV4 fermentation broth with chemical pesticides

[0098] The following six treatments were set up to inhibit the fungus Setospora turcica in the plate:

[0099] ① Mix 1 mL of FVV4 fermentation broth with 19 mL of PDA to prepare the culture medium;

[0100] ② Add 10 μL of 25% pyraclostrobin (1000 times diluted) into 20 mL of PDA medium;

[0101] ③ Take 15 μL of 40% tauconazole (1000 times diluted) and add it to 20 mL of PDA culture medium;

[0102] ④ Take 1 mL of FVV4 strain fermentation broth and 10 μL of 25% pyraclostrobin (1000 times diluted) and add them to 19 mL of PDA medium;

[0103] ⑤ Take 1 mL of FVV4 strain fermentation broth and 15 μL of 40% tauconazole (1000 times diluted) and add them to 19 mL of PDA culture medium;

[0104] ⑥ Add 1 mL of sterile water to 19 mL of PDA culture medium as a control treatment;

[0105] Inoculate Pseudomonas maydis colonies in the culture medium of each treatment, with three replicates per treatment. The inhibition rate was observed and the diameter of the pathogen was measured. The inhibition rate (%) was calculated using the formula: (control colony diameter - treatment colony diameter) / control colony diameter × 100%.

[0106] 1.8 FVV4 fermentation liquid mixed with chemical pesticides to control corn leaf blight in the field

[0107] 1.8.1 Overview of the test site

[0108] This experiment was conducted in a corn experimental field at the Gongzhuling Branch of the Jilin Academy of Agricultural Sciences. The experimental field was managed under conditions consistent with local fields, where northern corn leaf blight occurs year-round. The corn variety Xianyu 335, which is highly susceptible to northern corn leaf blight, was planted.

[0109] 1.8.2 Experimental Design

[0110] The following three treatments are set up for field control of corn leaf spot:

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

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

[0113] ③Clear water was used as control;

[0114] Each treatment area is 40 m 2 The first spraying was carried out on July 5, 2024, and the second spraying was carried out on July 15, 2024. A backpack electric sprayer was used for uniform spraying. At this time, large spot diseased leaves were already seen in the field.

[0115] 1.8.3 Prevention effectiveness investigation

[0116] The disease condition of leaves was investigated 25 days after application. Samples were taken at 5 points in each plot, and 5 corn plants were selected at each point. The proportion of lesions on all leaves was investigated and the severity of the disease was graded: Level 0, no lesions on leaves; Level 1, lesions or only sporadic lesions on leaves, accounting for 5% or less of the leaf area; Level 3, a small number of lesions on the leaves below the ear, accounting for 6% to 10% of the leaf area, and sporadic lesions on the leaves above the ear; Level 5, more lesions on the leaves below the ear, accounting for 11% to 30% of the leaf area, and more lesions on the leaves above the ear; Level 7, a large number of lesions on the leaves below or above the ear, which were connected, accounting for 31% to 70% of the leaf area; Level 9, the leaves of the entire plant were basically covered with lesions, and the leaves died. The disease index and the disease control efficacy relative to the clear water control were thus obtained. The calculation formulas for the disease index and 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] Two strains with significant antibacterial effects were obtained through antibacterial tests. ITS sequence Blast comparison and multi-gene phylogenetic tree analysis revealed that both strains were purple-producing fungi (see Figure 1 、 Figure 2 ), and named them strains FV11 and FVV4, respectively.

[0122] 2.2 Plate confrontation culture

[0123] 2.2.1 Plate confrontation with Botrytis cinerea

[0124] The three-point standoff method was used to place the FV11 and FVV4 strains against Botrytis cinerea at three points (see Figure 3 and Figure 4 ), from the fourth day of confrontation, the lateral growth of Botrytis cinerea was significantly inhibited, and red pigment began to appear on the confrontation surface of FV11 and FVV4 strains; as time went on, FV11 and FVV4 strains began to cover Botrytis cinerea strains on a large area, and the red pigment also continued to increase with time, but the coverage area and pigment production of FV11 were significantly lower than those of FVV4 strain, and FVV4 strain showed stronger antibacterial activity.

[0125] 2.2.2 Microscope observation of the opposing plates

[0126] Place the above plate under a microscope (4x or 10x magnification) to observe the mycelial development. Figure 5 、 Figure 6 On the fourth day of the plate confrontation, the mycelia of Botrytis cinerea on the confronting side underwent morphological distortion, resulting in a significant inhibition of lateral mycelial growth. Fan-shaped swellings appeared at the tips of the mycelia, and the swellings were light brown. On the fifth day of the confrontation, the mycelia of the FVV4 strain developed numerous red-headed hyphae accompanied by excessive secretion of pigments and toxins. This led to an increase in the number of fan-shaped swellings at the tips of the Botrytis cinerea on the confronting side, and their color changed from light brown to dark brown. On the sixth day of the confrontation, the Botrytis cinerea hyphae and FVV4 hyphae became intertwined. With the continuous secretion and accumulation of pigments and toxins by the FVV4 strain, the distortion of the Botrytis cinerea tips worsened, and the fan-shaped swellings at the tips increased in number and size, and their color changed from dark brown to bright red. This demonstrated the strong antibacterial activity of the FVV4 strain. The lateral growth of the gray mold in opposition to the FV11 strain was also significantly inhibited, but no obvious fan-shaped expansion was formed at the tips of the hyphae on the opposing side. As the FV11 strain secreted pigment, the opposing hyphae were also dyed red, but no obvious fan-shaped expansion was formed until the late stage of culture (see Figure 7), did not cause significant morphological distortion of Botrytis cinerea hyphae, indicating that the FVV4 strain produced a large amount of antifungal substances. Therefore, the FVV4 strain was selected for all subsequent experiments.

[0127] 2.3 Antibacterial activity of FVV4 strain against U. maydis

[0128] The antibacterial activity of FVV4 strain against T. maydis was detected by three-point confrontation method. No obvious inhibitory effect was observed on the third day of confrontation culture. However, microscopic observation revealed fan-shaped swellings at the tips of T. maydis hyphae (see Figure 8 On the 4th day of confrontation culture, the horizontal growth of T. maydis stopped, and microscopic examination revealed that the number of swelling bodies at the tips of the hyphae of T. maydis increased significantly; on the 5th day of confrontation culture, the vertical growth of T. maydis stopped, and microscopic examination revealed that fan-shaped swelling bodies also formed at the tips of the hyphae within the base of T. maydis; on the 6th day of confrontation culture, the FVV4 strain obviously covered the hyphae of T. maydis, and microscopic examination revealed that the hyphae of T. maydis were broken; on the 10th day of confrontation culture, the FVV4 strain completely covered the T. maydis mass (see Figure 9 When the control strain U. maydis grew completely on the plate, the average diameter of the opposing colonies was 1.48 ± 0.06, and the inhibition rate was 81.5% ± 0.86.

[0129] 2.4 Soaking the mycelium of T. maydis in the fermentation broth of FVV4 strain

[0130] The hyphae of the corn leaf blight pathogen have two forms: basal hyphae and aerial hyphae. The hyphae protoplast cells can be clearly observed in the basal hyphae; after soaking the hyphae in sterile water, the hyphae cells can be observed to absorb water and germinate; after soaking the hyphae with the fermentation liquid of the FVV4 strain, the intercellular spaces in the basal hyphae absorb the fermentation liquid of the FVV4 strain and turn red; the aerial hyphae absorb the fermentation liquid of the FVV4 strain and undergo plasmolysis, and the protoplast cells in the hyphae turn red and sausage-shaped; the sausage-shaped hyphae break and the hyphae of the corn leaf blight pathogen gradually disappear. Figure 10 shown by Figure 11 It can be seen that under low-power microscope observation, almost all the mycelia of corn leaf blight in a field of view absorbed the fermentation liquid of the FVV4 strain and turned into red mycelia, which was obviously affected by the fermentation liquid of the FVV4 strain. At the same time, the mycelia soaked in the fermentation liquid of the FVV4 strain and the mycelia soaked in sterile water were transferred to the same new PDA plate, and three replicates were made on each plate. The mycelia soaked in sterile water could grow normally in the PDA plate, while the mycelia soaked in the fermentation liquid of the FVV4 strain did not show obvious growth (see Figure 12 ).

[0131] 2.5 Analysis of FVV4 strain fermentation broth characteristics

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

[0133] PDA culture media containing different proportions of FVV4 strain fermentation broth were prepared to analyze the inhibitory effects of different concentrations of fermentation broth on Botrytis cinerea, Magnaporthe grisea, Exserohilum turcicum, and Sclerotinia sclerotiorum. When the fermentation broth ratio in the plate was 50%, 25%, and 15%, no significant growth was observed for all strains (see Table 1 for some inhibitory effects). Figure 13 ); When the volume ratio was 5%, the growth of all strains was significantly inhibited, with an average inhibition rate of 53% (see Figure 14 ).

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

[0135] Botrytis cinerea, Magnaporthe grisea, Exserohilum turcicum, and Sclerotinia sclerotiorum were placed in culture medium and incubated at 28°C. The growth of the clods was observed for 15 days, after which the clods were removed and replaced with new ones. After 10 days of incubation, the new clods of Botrytis cinerea, Magnaporthe grisea, and Sclerotinia sclerotiorum showed obvious hyphal growth, whereas no significant growth was observed for Exserohilum turcicum. Therefore, the FVV4 fermentation broth has an effective lifespan of at least 25 days.

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

[0137] The FVV4 fermentation broth was treated at 120°C, 80°C, or 60°C for 30 min, and then mixed with PDA at a ratio of 25% to inhibit Botrytis cinerea and Tuberculia zeae. When Botrytis cinerea and Tuberculia zeae fully grew on the plate, no significant growth of the fungi was observed after each treatment, indicating that the FVV4 fermentation broth has high temperature resistance (see Figure 15 , 4 plates per group, including 1 control and 3 treatment replicates).

[0138] 2.5.4 Proteinase K treatment of FVV4 fermentation broth

[0139] Proteinase K is a highly effective enzyme that can effectively break down all proteins in cells. Therefore, proteinase K was used to treat the FVV4 fermentation broth to degrade the proteins within. Antibacterial tests showed that when Botrytis cinerea and Tuberculia zeae overran the plate, no significant growth was observed in the treated bacterial masses. This suggests that the antibacterial substance in the FVV4 fermentation broth is likely not protein, and further indicates that its antibacterial substance is not easily degraded (see Figure 16 , 4 plates per group, including 1 control and 3 treatment replicates).

[0140] 2.5.5 Treatment of FVV4 Fermentation Broth with DNA Adsorption Column

[0141] The antibacterial effect of the FVV4 strain fermentation broth was not affected after treatment with DNA adsorption column (e.g. Figure 17 As shown, 4 plates per group, including 1 control and 3 treatment replicates), once again demonstrated that the antibacterial substance in the fermentation broth of the FVV4 strain was probably not protein, and the antibacterial effect was stable.

[0142] 2.5.6 Simultaneous Treatment of FVV4 Fermentation Broth with Proteinase K and DNA Adsorption Column

[0143] The FVV4 strain fermentation liquid was treated with proteinase K and DNA adsorption column simultaneously, so that the protein in the fermentation liquid was completely decomposed or trapped in the DNA adsorption column. The antibacterial results showed that no obvious mycelium growth was observed in the FVV4 strain fermentation liquid after treatment (see Figure 18 , 4 plates per group, including 1 control and 3 treatment replicates). This indicates that the antibacterial substance in the FVV4 strain fermentation broth is not protein, and the antibacterial substance is not easily degraded and is relatively stable.

[0144] 2.5.7 Microwave treatment of FVV4 fermentation broth

[0145] The antibacterial activity of the FVV4 fermentation broth was not significantly affected after being treated in a microwave oven for 1 min or 2 min (see Figure 19 , 4 plates per group, including 1 control and 3 treatment replicates), indicating that the antibacterial substances in the fermentation broth of the FVV4 strain have the characteristics of being resistant to microwave radiation.

[0146] 2.5.8 pH Analysis of FVV4 Fermentation Broth

[0147] The average pH of the PDB liquid medium was 5.75. The average pH of the FVV4 strain after 24 hours of shaking culture was 3.78, 3.25, 3.09, 3.03, 3.03, and 3.00, respectively. This indicates that the FVV4 strain produces acidic substances during growth, resulting in a low pH in the fermentation broth. The pH stabilized after 96 hours of shaking (see Figure 20 ).

[0148] 2.6 Inhibition of Setospora turcica by mixing FVV4 fermentation broth with chemical pesticides

[0149] The fermentation broth of FVV4 strain and chemical pesticides were mixed in single and mixed forms on plates to inhibit corn leaf blight. The average antibacterial rate of the fermentation broth of FVV4 strain alone was 53%, the average antibacterial rate of 25% pyraclostrobin alone was 56.33%, and the antibacterial rate of the mixture of FVV4 strain fermentation broth and 25% pyraclostrobin was 100%; the average antibacterial rate of 40% tebuconazole alone was 34.67%, and the antibacterial rate of the mixture of FVV4 strain fermentation broth and 40% tebuconazole was 100% (see Figure 21 ). It can be seen that the fermentation broth of FVV4 strain can enhance the antibacterial effect of chemical agents.

[0150] 2.7 FVV4 fermentation broth mixed with chemical pesticides for field control of corn leaf blight

[0151] The disease situation was investigated 25 days after the application of the drug. The disease level of the leaves in the water control group was mostly 3-7, and there were few leaves without disease. The disease level of the leaves treated with the chemical agent was mostly 1, with a few 3, and no leaves with disease level 5 or above were found, and there were many leaves without disease. The disease level of the leaves treated with the FVV4 strain fermentation liquid and the chemical agent was mostly 1, with a few 3, and no leaves with disease level 5 or above were found, and there were many leaves without disease (see Table 1). The control effect of the FVV4 strain fermentation liquid and the chemical agent mixture was higher than that of the chemical agent alone (see Figure 22 ), which plays the role of reducing the amount of medicine and increasing the efficacy.

[0152] Table 1: Control effects of different pesticides on corn leaf spot

[0153]

[0154] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of this application.

Claims

1. Talaromyces purpureogenus, characterized in that The deposit number is CGMCC No.41911.

2. A fermentation broth, characterized in that It is the fermentation broth of the purple tularensis according to claim 1.

3. The fermentation liquid according to claim 2, wherein The preparation method comprises: inoculating the purple-producing tularensis according to claim 1 into PDB for cultivation, filtering the obtained culture solution, and taking the filtrate to obtain a fermentation solution.

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

5. The fermentation broth according to claim 3, wherein The pore size of the filter membrane is 0.22 μm.

6. Use of purple-producing fungi or fermentation broth in any of the following: (i) Antagonism against pathogens; (ii) Control of corn leaf spot; It is characterized by: The purple-producing Talaromyces is the purple-producing Talaromyces according to claim 1, the fermentation broth is the fermentation broth according to any one of claims 2 to 5, and the pathogen is Botrytis cinerea, Magnaporthe grisea, Exserohilum turcicum or Sclerotinia Sclerotiorum.

7. A composition characterized in that Include: (I) the purple-producing T. purpurogenum according to claim 1 or the fermentation broth according to any one of claims 2 to 5; and (II) tebuconazole or pyraclostrobin.

8. Use of purple-producing Talaromyces, fermentation liquid or composition in the preparation of a biocontrol agent for preventing and treating corn leaf blight, characterized in that: The purple-producing Talaromyces is the purple-producing Talaromyces 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. A biocontrol agent, characterized in that Contains any of the following: (a) the purple-producing T. purpurogenum according to claim 1 or the fermentation broth according to any one of claims 2 to 5; (b) The composition according to claim 7.

10. A method for preventing and controlling corn leaf blight, characterized in that: Use any of the following: (A) the purple-producing tularensis of claim 1; (B) the fermentation broth according to any one of claims 2 to 5; (C) the composition according to claim 7; (D) The biocontrol agent according to claim 9.

Images